Program

Scientific Program

Monday July 11th, 2022

12.00-14.00      Registration

 

14.00-14.20      Opening: welcome by Frank Redegeld (Utrecht, The Netherlands) and Francesca Levi-Schaffer (Jerusalem, Israel)

 

14.20-16.15      Session 1: Novel insights in mast cell/basophil development

                        Chairs: Jenny Hallgren and Joana Vitte  

 

14.20-15.15      Blueprint key note lecture 1: Developmental Origin of Mast Cells

Florent Ginhoux (Singapore, Singapore)

 

15.15-15.35      Single-cell molecular of basophil and mast cell development

Joakim Dahlin (Stockholm, Sweden)

 

15.35-15.55      Human Mast Cell Proteome Reveals Unique Lineage, Putative Functions, and Structural Basis for Cell Ablation

Thomas Plum (Heidelberg, Germany)

 

15.55-16.15      Changes in molecular phenotypes and cell-cell interactions of mast cells in disease revealed by single-cell transcriptomics

Martijn Nawijn (Groningen, The Netherlands)                                        

 

16.15-16.45      Coffee break    

 

16.45-17.45      Oral abstract session 1: big data in mast cell/basophil development     

Chairs: Hanneke Oude Elberink and Gunnar Nilsson

 

16.45-16.57      Investigating haematopoiesis of aberrant and normal mast cells through single-cell multi-omics

                        Daryl Zhong Hao Boey (Solna, Sweden)

 

16.57-17.09      Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors     

                        Chenyan Wu (Solna, Sweden)

           

17.09-17.21      Adult mast cell development critically depends on mitochondrial fusion

                        Manuel Stecher (Freiburg, Germany)

 

17.21-17.33      Deciphering human mast cell differentiation by CRISPR/Cas9

                        Jiezhen Mo (Solna, Sweden)

 

17.33-17.45      Cytoskeletal mechanisms organizing tissue-resident mast cell networks

                        Tim Lämmermann (Freiburg, Germany)

 

17.45-19.45      Poster session 1 + network reception

 

Tuesday July 12th, 2022

08.00-08.45      Board meeting EMBRN

 

9.00-11.35        Session 2: Mast cells in health and disease

Chairs: Marcus Maurer and Frank Redegeld

 

09.00-09.30      Introductory lecture: How mast cells make us sick – mechanisms of mast cell activation in mast cell driven diseases                    

                        Laurent Reber (Toulouse, France)

 

09.30-10.15      Cogent Biosciences key note lecture 2: Mast Cells in Irritable Bowel Syndrome

Guy Boeckxstaens (Leuven, Belgium)

 

10.15-10.30      Short break

 

10.30-10.50      Mast cells in cardiovascular diseases

Ilze Bot (Leiden, The Netherlands)

 

10.50-11.10      Mast cell nerve interactions and MRGPRX2

Nicolas Gaudenzio (Toulouse, France)

 

11.10-11.20      Mast cell disorders

Frank Siebenhaar (Berlin, Germany)

 

11.20-12.00      Coffee break

 

12.00-13.00      Oral abstract session 2: Mechanisms of mast cell activation, mast cells in disease                      Chairs: Ilze Bot and Ulrich Blank            

 

12.00-12.12      Rab44 regulates MC-driven anaphylaxis through kinesin-1-dependent secretory granule translocation

                        Gaël Ménasché (Paris, France)

 

12.12-12.24      Insulin-Regulated Aminopeptidase (IRAP) sets the intensity of FcR-mediated inflammation

                      Shamila Vibhushan (Paris, France)

 

12.24-12.36      Substance P targets MRGPRX2 to the secretory granules by autophagy-aided macropinocytosis

                        Pia Lazki-Hagenbach (Tel Aviv, Israel)

 

12.36-12.48      Impact of ageing on the severity of IgE-, Mrgprb2-, and histamine-mediated anaphylaxis in mice

                        Jasper Kamphuis (Toulouse, France)

 

12.48-13.00      Age-induced mast cell activation and antigen presentation in atherosclerosis

                        Marie Depuydt (Leiden, The Netherlands)

 

13.00-14.00      Lunch including “Meet the Professor”

14.00-15.45      Session 3: Novel strategies in mast cell research, therapy & diagnostics

Chairs: Edward Knol and Frank Siebenhaar

 

14.00-14.45      Key note lecture 3: Novel strategies to actively desensitize allergic effector cells              Alexander Eggel (Bern, Switzerland)

 

14.45-15.05      Identification of mast cell degranulation regulators using functional genomics and single-cell imaging

Jelle Folkerts (Stanford, USA)

 

15.05-15.25      Biomarkers of diagnosis and resolution of allergy

Alexandra Santos (London, UK)

 

15.25-15.45      New mast cell therapies on the horizon

Martijn van Doorn (Rotterdam, the Netherlands)

 

15.45-16.15      Coffee break    

 

16.15-17.15      Oral abstract session 3: Novel strategies in mast cell research, therapy & diagnostics                            

Chairs: Jelle Folkerts and Philip Starkl

 

16.15-16.27      Elevated sFcεRI predicts early response to omalizumab treatment in chronic spontaneous urticarial

                        Sherezase Moñino-Romero (Berlin, Germany)

 

16.27-16.39      Effective anti-melanoma immune response by peritumoral delivery of the heat shock protein 70 (Hsp70)

                        Suzanne Kaesler (Munich, Germany)

 

16.39-16.51      Siglec-6 Interacts with KIT/CD117, Recruits Shp Phosphatases and Inhibits SCF-mediated Inflammation

                        Wouter Korver (San Carlos, United States)

 

16.51-17.03      Mast cell-derived FXIIIA contributes to sexual dimorphic defense against Group B Streptococcus

                        Adrian Piliponsky (Seattle, United States)

 

17.03-17.15      Salt-Inducible Kinases (SIKs): Recently identified regulators of mast cell function

                        Nicola Darling (Dundee, United Kingdom)

 

17.15-18.45      Poster session 2 + drinks          

 

19.00-24.00      Social event                  Drinks/Food/Music at:  

Gasthuis Leewenbergh

Servaasbolwerk 1a
3512 NK Utrecht

 

Wednesday July 13th, 2022

 

08.00-08.45      EMBRN assembly: plenary meeting of EMBRN members, Board Update

 

 

09.00- 10.45     Session 4: Joint session EMBRN/International Eosinophil Society

Chairs: Florence Roufosse and Francesca Levi-Schaffer

 

09.00-09.40      Key note lecture 4: The Allergic Effector Unit and its new druggable targets                                Francesca Levi-Schaffer (Jerusalem, Israel)

 

09.40-09.45      Introduction by Florence Roufosse and Francesca Levi-Schaffer

 

09.45-10.20      Eosinophils, mast cells and Siglecs: from biology to drug target

Bruce Bochner (Chicago, USA)

 

10.20-10.45       Eosinophil kinetics in health and disease

Leo Koenderman (Utrecht, The Netherlands)

 

10.45-11.10      Coffee break    

 

11.10-12.25      Oral abstract session 4: Interactions between mast cells, eosinophils and other immune cells.  

Chairs: Bernhard Gibbs and Maud Hermans

 

11.10-11.25      The Role of Mast Cells, Eosinophils and Basophils in Pemphigoid Diseases

Bernhard Gibbs (Oldenburg, Germany)

 

11.25-11.37      Bidirectional activation of human skin mast cells and blood eosinophils

Stefan Frischbutter (Berlin, Germany)

 

11.37-11.49      CT-M8 : a new mouse model reveals the non-redundant role of basophils in lupus-like disease onset

John Tchen (Paris, France)

 

11.49-12.01      Mast cell granules are endogenous CLR ligands skewing DC function towards TH2/TH17 response

                        Johanna Kotrba (Magdeburg, Germany)

 

12.01-12.13      Mast cells trap and cannibalize swarming neutrophils for metabolic and inflammatory fueling

                        Michael Mihlan (Freiburg, Germany)

 

12.13-12.25      T helper-licensed mast cells promote inflammatory Th17/Treg cells imbalance

Edouard Leveque (Toulouse, France)

 

12.25-12.45      Prizes for best poster/oral presentations and farewell by Frank Redegeld

 

Posters

Poster session 1, July 11th 2022

Poster

Title

First Name

Last Name

Country

1

Mast cell chymase regulate ECM-related events in primary human small airway epithelial cells

Xinran O

Zhao

Sweden

2

Human mast cells produce pro inflammatory mediators in response to chlamydia infection

Animamalar

Mayavannan

Canada

3

Mast cells exposed to cyclic hypoxia exhibit increased responsiveness to FcεRI crosslinking

Deisy

Segura

Mexico

4

The Impact of Industrially-made MoS2 on Human Basophils

Hazel

Lin

France

5

Lipid interconversions mediate secretory granule fusion and fission during their biogenesis

Sewar

Omari

Israel

6

Inducible senescence in bone marrow derived mast cells – a novel model for mast cell aging research

Elisabeth

Kleeblatt

Israel

7

Single-cell RNA sequencing of human lung mast cells

Elin

Rönnberg Höckerlind

Sweden

8

All or nothing: What we may learn from the digital response of antigen-triggered mast cells

Marcel

Kuhny

Germany

9

Identification of PKCβ as a novel regulator that controls Rab12 interactions with its effectors

Jana

Omar

Israel

10

Bruton’s tyrosine kinase inhibition to suppress mast cell activation in atherosclerosis

Esmeralda

Hemme

The Netherlands

11

Kinetics of MRGPRX2 upon stimulation with different ligands in human mast cells.

Romy

Kwakernaak

The Netherlands

12

Mast Cell Proteases Promote Diverse Effects on uPAR and Alveolar Repair Responses

Sofia

Mogren

Sweden

13

The role of mast cell cyclin E1 in the inflammatory response

Gina

Bronneberg

Germany

14

The evolution of mast cell-expressed carboxypeptidase A3 in comparison to other carboxypeptidases

Srinivas

Akula

Sweden

15

SH3BP2 silencing downregulates MITF through miRNAs inducing cell death in human mast cell leukemia.

Elizabeth

Proaño

Spain

16

Characterization of mast cells in fetal barrier and extra-embryonic tissues

Shin Li

Chia

United Kingdom

17

Tryptase reference ranges are age-dependent in a large population-based cohort

Marjan

Slot

The Netherlands

18

SR-BI modulates the synergistic enhancement of FceRI-induced cytokine release from murine mast cells

Sandro

Capellmann

Germany

19

Intra-individual biological variation of tryptase in humans is low

Douwe

de Boer

The Netherlands

20

Tryptase contribute to airway remodeling by inducing cell growth properties in lung epithelial cells

Frida

Berlin

Sweden

21

Mast cell tryptase potentiates neutrophil extracellular trap formation

Fabio

Melo

Sweden

22

Mast cell progenitors are activated by IgE-crosslinking

Erika

Mendez Enriquez

Sweden

23

Mast cells enhance airway hyperresponsiveness by releasing serotonin

Jenny

Hallgren Martinsson

Sweden

24

Mcpt8-driven cell depletion reduces lung mast cells in mice with allergic airway inflammation

Perla Abigail

Alvarado Vazquez

Sweden

25

SCF/KIT-evoked phosphoproteomics in skin mast cells: ERK predominates PI3K and capicua represses KIT

Kristin

Franke

Germany

26

Mast cell activation test: useful to discriminate patients with severe anaphylaxis?

Laia

Ollé

Spain

27

Characterization of the IRE1α interactome in the mast cell leukemia cell line HMC-1.2 via TurboID

Nabil

Ahmed

Germany

28

Hydrangea serrata extract ameliorates airway inflammation of CARAS exacerbated mouse model by PM2.5

JUAN

JIN

South Korea

29

Euonymus alatus extract alleviates PM 2.5 exacerbated-allergic airway inflammation

THI VAN

NGUYEN

South Korea

30

Phosphorylation of KIT – an early attenuating mechanism in antigen-triggered mast cell activation

Bastian

Bosch

Germany

31

Higher Basophil Activation Test Performance Flexibility by Prolonged Assay Read-Out of Fixed Cells

Martina

Berchtold

Switzerland

32

The flow of COX to LOX stops at the rhyme; profiling refutes shunting in lung mast cells

Anna-Karin

Johnsson

Sweden

33

The IRE1a inhibitor KIRA6 inhibits LYN and efficiently suppresses IgE-mediated mast cell activation

Thomas

Wilhelm

Germany

 

Poster session 2, July 12th 2022

Poster

Title

First Name

Last Name

Country

34

Mast cells respond to enteropathogenic bacteria in a lifestyle dependent manner

Christopher

von Beek

Sweden

35

Characterizing the impact of proteolytic cleavage of C1-Inhibitor in MC-mediated angioedema

Carolina Elisa

Vera Ayala

Germany

36

Crucial role for mast cell-derived IL-17A in psoriasis

Theresa

Benezeder

Austria

37

Targeting mast-cell plasticity re-shapes the tumor microenvironment of melanoma

Caterina

Iuliano

Germany

38

Variants of the MRGPRX2 gene in patients with immediate hypersensitivity reactions to drugs

Paola

Quan

Spain

39

Effect of exercise on basophil activation and PGE2 secretion in patients with food anaphylaxis

Lucía

García

Spain

40

An exploratory study on lymphocytes during VIT in patients with or without clonal mast cell diseases

Andrea

Teufelberger

Austria

41

Peripheral mast cell-derived RANKL is a prerequisite for lymphocyte egress from distant lymph nodes

Konstantinos

Katsoulis-Dimitriou

Germany

42

Mast cell reduction does not lead to impaired cutaneous wound healing in humans

Maren

Frenzel

Germany

43

A novel intra-lobular airway ex-vivo guinea pig model to study the role of relaxant EP receptors

Mu

Nie

Sweden

44

Characterization of MRGPRX2 as a novel target to address mast-cell mediated disorders

Veena

Viswanath

United States

45

Neuro-immune interactions in milk allergy: Food allergy, hippocampal progenitors, and mental health

Vikki

Houghton

United Kingdom

46

Application of hollow microneedles in models of allergy and CU

Nana

Shi

Germany

47

IgE antibodies promote mast cell-mediated detoxification of bee venom

Philipp

Starkl

Austria

48

BAT interference study on positive controls to pharmaceuticals and abnormal blood conditions

Michael

Gerspach

Switzerland

49

Elevated, FcεRI -dependent MRGPRX2 expression on basophils in chronic urticaria

Ewa

Bartko

Denmark

50

Differential activation of mast cells by human effector T cells and regulatory T cells

Jiajun

He

Germany

51

Increased number of MRGPRX2+ cells in the skin of ISM patients is not linked to symptom severity

Polina

Pyatilova

Germany

52

Systemic mast cell activation does not affect atherosclerosis development in LDLr-/-Rag1-/- mice

Lucie

Delfos

Netherlands

53

The impact of IgE-induced mast cell degranulation on murine auditory hair cells

Bin

Zeng

Germany

54

Anti-FcεRI mAb MAR-1 depletes basophils and cross-reacts with myeloid cells through its Fc Portion

William

Worrall

France

55

Human IgE cross-reacting with major peanut allergens induce mast cell degranulation and anaphylaxis

Cyprien

Pecalvel

France

56

Inhibition of mast cell function through sialylation of peanut allergens

Charlotte

Castenmiller

Netherlands

57

Molecular profiling of basophils and eosinophils in a tropical urban environment for allergy risk

Anand Kumar

Andiappan

Singapore

58

Mouse naturalization — an effective approach for shaping lung tissue distribution of mast cells

Zou

Xiang

Hong Kong

59

The Mast Cell Activation Test is a Useful Tool for Assessing Nut Allergy

Carolin

Steinert

Germany

60

Selenomethionine attenuates human primary mast cells responses in soy allergy

Xiaoli

Zhao

Netherlands

61

A novel approach for studying mast cell: induced pluripotent stem cell derived mast cell

Yanyan

Luo

Germany

62

Elevated IgE levels in the early phase of SARS-CoV-2 infection

Rafael

Saia

Brazil

63

Activated human mast cells impair esophageal barrier: role of a possible bidirectional crosstalk

Mirelle

Kleuskens

Netherlands

64

The long noncoding RNA MALAT-1 regulates responsiveness to FcεRI in human skin mast cells

Gürkan

Bal

Germany

65

Eosinophil-Derived Neurotoxin (EDN) may contribute to mastocytosis diagnosis

Moise

Michel

France

66

Autoimmune CSU have features of autoallergic CSU but not vice versa

Yi-Kui

Xiang

Germany

67

CD300a and mast cells regulate mouse macrophages functionality in allergic inflammation

Prince

Ofori

Israel

68

Developmental origins of melanoma-associated mast cells

Carvalho

Cyril

Scotland

Floorplan
Abstracts

Abstracts Oral Presentations

Oral abstract session 1: mast cell/basophil development, big data

Investigating haematopoiesis of aberrant and normal mast cells through single-cell multi-omics

Boey, Daryl Zhong Hao; Wu, Chenyan; Mo, Jiezhen; Ungerstedt, Johanna; Nilsson, Gunnar; Dahlin, Joakim

Department of Medicine, Solna, Karolinska Institutet, Sweden

Mast cells are instrumental components of the human innate immune response, with functional abnormalities leading to diseases such as systemic mastocytosis (SM). We have employed multi-omics studies of bone marrow from SM, combining single-cell transcriptomic data with oligo-coupled surface antibodies. Harnessing this method, a sorted spectrum of haematopoietic progenitor cells including oligo-tagged mast cells can be sequenced, generating multi-dimensional datasets, which facilitate a variety of exploratory analysis.  Immune cells have traditionally been defined by surface marker expression. By employing a combination of unsupervised clustering of single-cell gene expression data and readouts from surface antibody oligo-tagged cells we can accurately match transcriptionally defined cell subsets to conventionally described cell types. Through integration of gene and surface expression phenotypes, we accurately charted hematopoietic progenitor cell development. We also identified aberrant mast cells and a residual population of normal mast cells in SM bone marrow. This revealed a novel transcriptomic signature for aberrant mast cells. Comparative analysis between these two mast cell subsets uncovered several markers that could provide new mechanistic insight into disease development of SM.

 

 

 

Single-cell transcriptomics reveals the identity and regulators of human mast cell progenitors

Wu, Chenyan; Boey, Daryl; Bril, Oscar; Grootens, Jennine; Vijayabaskar, M. S.; Sorini, Chiara; Ekoff, Maria; Wilson, Nicola K.; Ungerstedt, Johanna S; Nilsson, Gunnar; Dahlin, Joakim S.

Department of Medicine, Solna, Karolinska Institutet, Sweden

Mast cell accumulation is observed in a number of diseases including systemic mastocytosis and allergic asthma. Mast cell activation through IgE-mediated crosslinking of the FceRI receptors heavily contributes to disease pathogenesis. Whether FceRI appears early in mast cell progenitor development or later in maturing mast cells remains controversial. Utilising single-cell transcriptomics, we identified a temporal association between upregulation of mast cell signature genes and FceRI expression in CD34+ progenitors. Furthermore, long-term culture assays of FceRI+ progenitors led to formation of mature and functional mast cells, demonstrating that FceRI can appear at the progenitor stage. Additionally, we identified expression patterns of potential cytokine receptors regulating the mast cell progenitors’ development through analysis of the single-cell transcriptomics data. Culture assays showed that IL-3 and IL-5 had distinct effects on progenitor cell proliferation and survival respectively, whereas IL-33 downregulated FceRI expression. In conclusion, we demonstrated that FceRI appears during the hematopoietic progenitor stage of mast cell differentiation. Furthermore, we showed how external stimuli regulate mast cell progenitor development. Our results provide a source to explore more factors regulating mast cell progenitor development.

 

 

 

Adult mast cell development critically depends on mitochondrial fusion

Stecher, Manuel; Veltum, Vanessa; Mihlan, Michael; Sesaki, Hiromi; Roers, Axel; Büscher, Jörg; Rambold, Angelika; Lämmermann, Tim

Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany

Introduction: Tissue-resident mast cells (MCs) distribute as long-lived myeloid cells throughout many adult tissues. Mitochondria are critical cell organelles that regulate metabolic pathways controlling survival and effector functions of many immune cells. They are highly dynamic organelles, which can distribute as individual units or fused networks. Whether and how mitochondrial fusion and fission regulate MC development and homeostasis in vivo is completely unknown. Objective: We aimed to identify the functional roles of mitochondrial dynamics and metabolic pathways for MC development and homeostasis in mice, with focus on MCs from peritoneal and skin tissue. 

Methods: By using several static and live imaging techniques, we characterize mitochondrial morphologies in endogenous MCs in situ and cultured mouse MCs in vitro. We generated conditional knockout mouse strains for the depletion of key effector molecules of mitochondrial fission (Drp1) and fusion (Opa1, Mfn1, Mfn2) in connective tissue-type MCs (CTMCs). Peritoneal MC (PMC) cultures from these mice underwent metabolic pathway analysis by Seahorse flux analysis and polar metabolomics. 

Results: CTMC-specific loss of mitochondrial fusion diminishes the population of endogenous skin and peritoneal MCs by more than 90%. Surprisingly, the severity of the phenotype occurs exclusively in adult animals, whereas neonatal skin remains densely seeded with MCs. PMC cultures recapitulated impaired MC development and survival in vitro. Pharmacological inhibition or induced genetic interference with mitochondrial fusion resulted in metabolic shifts towards fatty acid oxidation, a downregulation of glycolysis and the pentose phosphate pathway and a downregulation of nucleotide metabolism causing proliferation arrest and subsequent cell death. Interestingly, we observe that other immune cells, including macrophages and dendritic cells, are substantially less affected. 

Discussion: To our knowledge, we highlight for the first time an organelle-controlled checkpoint for adult MC development. Loss of mitochondrial fusion leads to a substantial impairment of MC development, which has previously mainly been reported for deficiencies in KIT signaling or GATA2-controlled transcriptional programs. While other immune cell types can compensate the loss of mitochondrial fusion, MCs cannot adapt to this organelle failure. Thus, adult MCs appear as metabolically inflexible immune cells. Targeting mitochondrial fusion might promise novel therapeutic approaches for MC-related diseases.

 

 

 

 

Deciphering human mast cell differentiation by CRISPR/Cas9

Mo, Jiezhen; Wu, Chenyan; Boey, Daryl; Nilsson, Gunnar; Dahlin, Joakim

Department of Medicine, Karolinska Institutet, Solna, Sweden

CRISPR/Cas9 is a powerful tool for manipulation of genes in cells. However, its application in human mast cell differentiation has yet to be implemented. The low frequency of mast cell progenitors and the difficulty in evaluating gene disruption constitute major challenges. Here, we developed a high-efficiency approach for knocking out genes of interest and a rapid method to assess knock out efficiency in differentiating human mast cells. By optimizing the dose of Cas9-guide RNA ribonucleoprotein complexes, we achieved efficient gene disruption (~82%) of CD34+ primary peripheral blood progenitors. A high frequency of the treated cells survives and differentiates into mast cells even using a small number of starting cells, which enables knocking out genes in as few as 10000 hematopoietic progenitors. Targeting the PTPRC gene, coding for CD45, allowed us to confirm that the CRISPR/Cas9 approach successfully knocked out surface CD45. We also apply a method targeting analysis for genome edits determined by sanger sequencing followed by Interference of CRISPR Edits. In addition, single-cell transcriptomics from CD34+ hematopoietic progenitors reveals a gene increase significantly during mast cell differentiation. With the developed method, we identified the biology role of this gene throughout differentiation. Taken together, our method will expand the application of CRISPR/Cas9 approach to identify potential genes associated with human mast cell differentiation and function.

 

 

 

 

 

Cytoskeletal mechanisms organizing tissue-resident mast cell networks

Kaltenbach, Lukas; Bambach, Sarah; Martzloff, Paloma; Lämmermann, Tim

Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany

Introduction: Many immune cell types migrate in tissues in an amoeboid fashion, i.e. they use rapid cycles of actin polymerization and actomyosin contraction to propel themselves forward. This movement does not require integrin receptors to interact with tissue structures  (Lämmermann et al., Nature 2008). How integrins regulate mast cell (MC) organization in vivo is unknown. Moreover, it is unclear how MCs control their actin dynamics to form tissue-resident cellular networks.    Objective  We sought to identify the functional roles of integrin receptors and dendritic actin for the formation of tissue-resident MC networks in vivo. 

Methods: We generated mouse strains for the depletion of the integrin-activating protein Talin-1, beta1 integrin receptors and Arpc4 (from the Arp2/3 complex) in connective tissue-type MCs (CTMCs). We used several techniques of static and live imaging to investigate MC positioning in relation to dermal SCF expression in vivo, follow the slow movement of MC in situ by genetic fate tracking and analyze actin cytoskeletal dynamics of 3D cultured MCs. 

Results: Mice with CTMC-specific loss of integrins show substantially altered MC distribution in the dermis. Integrin-deficient MCs form clusters and loose periarteriolar alignment. Control MCs perform slow integrin-dependent migration in 3D gels. In contrast, integrin-deficient MCs fail to move away from proliferation clusters, a phenotype that was confirmed in vivo. Moreover, scRNA sequencing revealed a special role for integrins in defining a mature MC phenotype in the periarteriolar tissue space, an SCF-rich anatomical niche. Mice with CTMC-specific depletion of the Arp2/3 protein complex show an unexpected gradual emptying of the dermal MC niche. Lack of Arp2/3 in MCs leads to cell shattering in tissues and loss of these cells in dermis. 

Discussion: Our study identifies a crucial role for integrin-dependent adhesion in controlling slow MC movement, which shapes the SCF-dependent positioning and network-like tissue distribution. This makes MCs unique among immune cells, as their mode of movement stands in clear contrast to the flexible migration strategies of other leukocytes. Moreover, we identify a novel role of Arp2/3-mediated actin branching for protecting MC integrity and survival in living tissues.

 

 

 

Oral abstract session 2: Mechanisms of mast cell activation, mast cells in disease

Rab44 regulates MC-driven anaphylaxis through kinesin-1-dependent secretory granule translocation

Ménasché, Gaël; Longé, Cyril; Bratti, Manuela; Kurowska, Mathieu; Vibhushan, Shamila; David, Pierre; Blank, Ulrich

INSERM/Institut Imagine, Paris, France

Introduction: Mast cells (MCs) are key effectors of the allergic response. Following the cross-linking of IgE receptors (FcRIs), they release crucial inflammatory mediators through degranulation. Although degranulation depends critically on secretory granule (SG) trafficking towards the plasma membrane, the molecular machinery underlying this transport has not been fully characterized. 

Objective: Here, we analyzed the function of Rab44, a large atypical Rab GTPase highly expressed in MC, in MC degranulation process. Methods: Murine KO mouse models (KORab44 and DKOKif5b/Rab44) were used to perform passive cutaneous anaphylaxis (PCA) experiments and analyze granule translocation in derived bone-marrow-derived MCs (BMMCs) during degranulation.

Results: We demonstrate that mice lacking Rab44 (KORab44) in their BMMCs are impaired in their ability to translocate and degranulate SGs at the plasma membrane upon FcRI stimulation. Accordingly, KORab44 mice were less sensitive to IgE-mediated passive cutaneous anaphylaxis in vivo. A lack of Rab44 did not impair early FcεRI-stimulated signaling pathways, microtubule reorganization, or cytokine secretion. Mechanistically, Rab44 appears to interact with and function as part of the previously described kinesin-1-dependent transport pathway. 

Discussion: Our results highlight a novel role of Rab44 as a regulator of SG transport during degranulation and anaphylaxis acting through the kinesin-1-dependent microtubule transport machinery. Rab44 can thus be considered as a potential target for modulating MC degranulation and inhibiting IgE-mediated allergic reactions.

 

 

 

Insulin-Regulated Aminopeptidase (IRAP) sets the intensity of FcR-mediated inflammation

Vibhushan, Shamila; Bratti, Manuela; Longé, Cyril; Koumantou, Despoina; Ménasché, Gael; Benhamou, Marc; Varin-Blank, Nadine; Blank, Ulrich; Saveanu, Loredana; Ben Mkaddem, Sanae

Centre de Recherche sur l’Inflammation Inserm U1149, Université Paris Cité, Paris, France

Introduction: Insulin-Regulated Amino Peptidase (IRAP) is a type II multifunctional membrane protein that distributes between recycling endosomes and the plasma membrane. Besides its proteolytic activity regulating the activity of physiological peptides and antigen presentation, IRAP is also implicated in immune cell signaling regulating the trafficking of intracellular signaling effectors.  Objective: We investigated the role of IRAP in the signaling of Fc receptor (FcR)-mediated inflammatory responses including in in vivo IgE- and IgG-mediated anaphylaxis and IgG-mediated arthritis models. 

Methods: IgE-mediated passive systemic anaphylaxis (PSA), IgG-mediated active anaphylaxis (ASA) and a more chronic collagen antibody-induced arthritis (CAIA) arthritis model were studied in IRAP-deficient and IRAP-deficient/FcγRIIATg mice versus corresponding WT and FcγRIIATg mice. Signaling studies were performed in bone marrow derived mast cells (BMMC) from the WT and IRAP-deficient mice. 

Results: We found that in the context of IRAP-deficiency, mice exhibit a less severe IgE-triggered passive systemic anaphylaxis. Likewise, IgG-triggered active systemic anaphylaxis and arthritis was decreased supporting that IRAP generally affects FcR-induced inflammatory responses. Using BMMC we found that IRAP+ recycling endosomes get rapidly recruited to the plasma membrane after FcεRI triggering. In agreement with the in vivo data, IRAP-deficient BMMC exhibited a reduced degranulation response and cytokine secretion. Examination of receptor-proximal signaling showed that tyrosine-phosphorylation of several effectors and the calcium response were diminished in IRAP-deficient BMMC. In particular, active SykY519/520 was decreased and less prominent at the plasma membrane. Ex vivo analysis confirmed that, in FcγR-stimulated neutrophils and monocytes, pSyk is significantly decreased in IRAP-deficient FcγRIIATg mice. Syk inhibition was not a consequence of early signaling events mediated by Lyn kinase but rather relied on the inactivation of SHP-1 phosphatase as in IRAP-deficient FcεRI-stimulated BMMC and FcγR-stimulated neutrophils and monocytes the inactivation of SHP1 through phosphorylation of S591 was decreased. 

Discussion: Together, these results revealed an important role of IRAP in setting the intensity of FcR-mediated inflammation. Our findings support a role of the IRAP+ recycling compartments in FcR signaling shifting the kinase/phosphatase balance to activating kinase driven signaling and enhancing inflammatory responses.

 

 

 

Substance P targets MRGPRX2 to the secretory granules by autophagy-aided macropinocytosis

Lazki-Hagenbach, Pia; Kleeblatt, Elisabeth; Ali, Hydar; Sagi-Eisenberg, Ronit

Department of Developmental and Cell Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

While best known for their role in adaptive IgE-mediated immunity and allergy, mast cells (MCs) regulate, independently of IgE, innate immune and pseudo-allergic responses. The latter are triggered by a large family of positively charged substances, including FDA-approved drugs and neuropeptides, that bind to Mas-related G protein coupled receptors (Mrgprs). The cellular functions of G protein coupled receptors (GPCRs) are tightly linked with their cellular positioning. However, the trafficking of this class of receptors and its influence by the type of bound ligand are still unknown. Here we focused on the spatiotemporal patterns of MRGPRX2, the human member of this receptor family, when bound to substance P (SP), a neuropeptide that plays a key role in neurogenic inflammation, contributing to itch and pain. We report on two complementary pathways of MRGPRX2 internalization in response to SP: an endocytic, pitstop2-sensitive, pathway and macropinocytosis. Further, deciphering of the post-endocytic trafficking of the receptor demonstrated its distribution between a perinuclear localization reminiscent of the Golgi/TGN and the secretory granules (SGs). The latter surprising positioning prompted us to investigate the underlying mechanism of MRGPRX2 targeting to the SGs. Our results underscore a novel connection between macropinosome resolution and cargo delivery to the SGs, whereby MRGPRX2-containing, macropinosome-derived vesicles fuse with the SGs. This process is stimulated by SP and involves dynamin and the autophagic machinery, more specifically LC3, giving rise to the incorporation of both LC3 and MRGPRX2 into SGs. Further, SP then promotes the acidification of the LC3-associated SGs, presumably by stimulating fusion with lysosomes. Collectively, our results demonstrate a unique mode of MRGPRX2-trafficking that complements endocytosis and macropinocytosis as well as a novel role for autophagy in regeneration of the SGs thus marking SG exocytosis as an alternative mechanism for endocytic recycling of the receptor.

 

 

 

Impact of ageing on the severity of IgE-, Mrgprb2-, and histamine-mediated anaphylaxis in mice

Kamphuis, Jasper; Loste, Alexia – Presenting Author; Worrall, William; Serhan, Nadine; Martin, Jeremy; Gaudenzio, Nicolas; Reber, Laurent

INSERM, Toulouse, France

Introduction: The elderly are disproportionally affected by anaphylaxis, they are overrepresented in number of cases and number of deaths by anaphylactic shock. While frailty and increased exposure to drugs are doubtlessly a reason, the increased shock severity in response to insect stings is less clearly explainable. We hypothesized that changes in mast cell biology with age play a role in the increased sensitivity to anaphylaxis in the elderly, and set out to investigate this in mouse models.

Objective: To assess the impact of ageing on the severity of anaphylactic shock in a mouse model and investigate its possible underlying biological causes.

Methods: The effect of ageing on skin and peritoneal mast cell density was assessed by histology and flow cytometry in mice 3 to 18 months-old. Passive Systemic Anaphylaxis (PSA) was monitored by measuring core temperature loss and survival in mice of 3, 6, 12 and 18 months (1) sensitized intraperitoneally (i.p.) with α-dinitrophenyl (DNP) IgE and challenged i.p. 24 hours later with DNP-BSA, (2) challenged i.p. with the MRGPRB2-agonist ciprofloxacin, and (3) challenged i.p. with histamine. Passive Cutaneous Anaphylaxis (PCA) model was performed by sensitizing the ear skin of 3 vs. 18 months-old mice with α-DNP IgE and challenging i.p. with DNP-BSA 24 hours later, using local swelling of the sensitized ear skin as readout. 

Results: Skin and peritoneal mast cell numbers increase with age, and mast cell size and granularity profiles, as well as KIT and surface IgE expression, are altered with age. We observed an age-dependent increased severity of both IgE-mediated, MRGPRB2-mediated, and histamine-induced PSA, as well as IgE-mediated PCA. 

Discussion: We have indications that changes in mast cell density and phenotype with advancing age have an impact on severity of both IgE- and MRGPRB2-mediated anaphylaxis. The increased reaction of older mice to histamine-induced PSA also shows that ageing increases susceptibility to this key anaphylaxis mediator.

 

 

 

Age-induced mast cell activation and antigen presentation in atherosclerosis

Depuydt, Marie; Smit, Virginia; Simpson, Conor; Postel, Rimke; Verwilligen, Robin; Bernabé Kleijn, Mireia; Schaftenaar, Frank; Kuiper, Johan; Bot, Ilze; Foks, Amanda

Division of BioTherapeutics, LACDR, Leiden University, Leiden, The Netherlands

Aging is an independent and dominant risk factor for atherosclerosis and is associated with a low-grade chronic inflammation termed inflammaging. This age-induced pro-inflammatory environment may have great impact on plaque-residing immune cells, including mast cells. Mast cells have been found to accumulate in the human atherosclerotic plaque upon disease progression and have been associated with plaque instability. In experimental studies, mast cells have been shown to promote atherosclerosis progression. However, it is currently unknown whether an increased age drives the pro-atherogenic effects of mast cells in atherosclerosis. Aortic root sections of male young (8-12 weeks) and old (78-86 weeks) LDLr-/- mice fed either a chow or western type diet were stained with a CAE mast cell staining to identify both resting and activated mast cells. A significant 19.8% increase of mast cell activation was found with age, independent of diet (young 14.3±1.7 vs. old 17.2±1.5 activated mast cells; P=0.015). Furthermore, we cultured bone marrow-derived mast cells of both young and old mice and detected a CD63+activated mast cell population in unstimulated mast cells of old mice indicative of low-grade activation (young 0.13±0.006% vs. old 3.98±0.12%; P=0.05). Next, we aimed to further identify the potential age-related mast cell activation pathways by flow cytometry. The most prominent mechanism of activation was found to be via the classical IgE pathway. Aged mice showed a 2.1-fold increased number of IgE+CD63+ mast cells in the aortic arch (young 37.5±12.5% vs. old 79.1±2.1%; P = 0.007). Finally, we demonstrated that aging increases the amount of MHCII+ mast cells (young 0.9±0.3 vs. old 46.9±10.8; P = 0.004), allowing them to act as antigen-presenting cells that may contribute to CD4+ T cell proliferation in the plaque. In line, single-cell RNA sequencing showed mast cell specific expression of H2-Ab in aged mouse atherosclerotic aortas and of HLADR in human plaques.  In this study, we identified age-induced phenotypic changes of mast cells in atherosclerosis, regarding both activation status and potential antigen presenting capacity, which might be associated with disease progression.

 

 

 

Oral abstract session 3: Novel strategies in mast cell research, therapy & diagnostics

Elevated sFcεRI predicts early response to omalizumab treatment in chronic spontaneous urticaria

Moñino-Romero, Sherezade; Kolkhir, Pavel; Szépfalusi, Zsolt; Metz, Martin; Scheffel, Jörg; Maurer, Marcus; Altrichter, Sabine

Charité – Universitätsmedizin Berlin Institute of Allergology IFA; Fraunhofer ITMP  Immunology and Allergology IA, Berlin, Germany

Introduction: Anti-IgE treatment with omalizumab is effective in chronic spontaneous urticaria (CSU). Markers of type IIb autoimmune CSU predict slow response to omalizumab treatment, but predictors of fast response have not yet been identified. 

Objective: Assess the value of soluble FcεRI (sFcεRI), a marker of IgE-mediated mast cell activation, as a predictor of response to omalizumab in CSU.

Methods: Serum samples from 67 CSU patients obtained before omalizumab treatment were analyzed for sFcεRI levels by ELISA, and 2 ng/mL was used as cutoff for elevated sFcɛRI. Treatment response to omalizumab during the first 4 weeks was assessed by use of the urticaria activity score (UAS7) and the urticaria control test (UCT). Marked response was defined as a UAS7 reduction of >30% or a UCT of ≥12. Complete response was defined as a UAS7 reduction of >90% or a UCT of 16. The time to onset of response was assessed by use of the rolling UAS7 (rUAS7), with rUAS7 ≤6 and =0 indicative of minimal and no disease activity, respectively.

Results: Of the 12 and 35 patients who achieved complete and marked UAS7 response at week 4, respectively, 9 and 22 had elevated baseline sFcɛRI levels, as compared to 24% of non-responders. UAS7 scores were lower in patients with elevated sFcɛRI levels as early as one week after treatment start, reaching statistical significance at week 3 (p<0.05). Patients with elevated sFcɛRI levels achieved rUAS7 ≤6 and =0 earlier than those without, at day 8±1 vs 13±2 and day 12±2 vs 14±2, respectively. Patients with elevated pretreatment sFcɛRI levels also showed higher rates of well or completely controlled disease, i.e. UCT ≥12 or =16, at week 4 (14/40 and 14/40, =35%, respectively) as compared to patients with normal sFcɛRI (6/40 and 6/40, 30% respectively, p<0.05). ROC analysis showed that sFcɛRI of >2 ng/mL predicted UCT ≥12 and =16 at week 4 with 72% and 74% sensitivity, and 56% and 44% specificity, respectively.

Discussion: Elevated baseline sFcεRI levels can be used as a predictor of early response to treatment with omalizumab which might help to better design treatment options for CSU patients.

 

 

 

Effective anti-melanoma immune response by peritumoral delivery of the heat shock protein 70 (Hsp70)

Kaesler, Susanne; Iuliano, Caterina; Shevtsov, Maxim; Hils, Miriam; Biedermann, Tilo

Dermatology, Klinikum rechts der Isar Technical University Munich, Munich, Germany

Immune checkpoint inhibitors increased overall survival for patients with advanced and metastatic melanoma, but long-term treatment responses are still limited to few patients with tumor specific effector T-cells at the tumor sites as predictors for treatment success. Thus, new treatment strategies are required. In preclinical models we could recently show, that targeted activation of melanoma-associated mast cells (MC) with lipopolysaccharide (LPS) induces an effective anti-melanoma immune response in a TLR4-dependent manner. Tumor control was mediated by MC-derived CXCL10, a T cell recruiting chemokine whose expression positively correlates with survival of melanoma patients. Like LPS, Hsp70 can also activate the TLR4 pathway and extracellular Hsp70 was shown to stimulate immune responses in a variety of cancers. We therefore investigated the impact of Hsp70 on melanoma and its environmental immune cells. In in vitro studies we could demonstrate that Hsp70 activates mast cells in a TLR4-dependent manner and leads to secretion of CXCL10. Repeated peritumoral application of Hsp70 in a B16 mouse melanoma model resulted in tumor control with enhanced tumor-infiltrating lymphocytes and in increase of TNF-a and IFNy producing T cells in the draining lymph nodes. The combination of the Hsp70 treatment with anti-CTLA-4 immunotherapy further augmented anti-melanoma efficacy. Similar to our findings with LPS, initial experiments with MC-deficient mice indicate that the Hsp70-induced anti-melanoma immune response is MC dependent. Collectively, combined Hsp70 administration with immune checkpoint therapy represents a highly efficient anti-tumor modality, and is promising for clinical translation.

 

 

 

Siglec-6 Interacts with KIT/CD117, Recruits Shp Phosphatases and Inhibits SCF-mediated Inflammation

Korver, Wouter; Schanin, Julia; Benet, Zachary; Wong, Alan; Chang, Katherine; Leung, John; Luu, Thuy; Freitas, Naomi; Luehrsen, Ken; Youngblood, Bradford

Department of Research, Allakos, Inc., San Carlos, United States

Introduction: Siglec-6 is an inhibitory receptor selectively expressed on human mast cells (MCs). Engagement of Siglec-6 with an antibody has been shown to inhibit IgE-mediated MC activation and represents an attractive therapeutic approach for treatment of allergic diseases. Siglec family members contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that regulate immune cell activation, but exactly how Siglec-6 mediates MC inhibition and whether Siglec-6 can affect IgE-independent MC activation is not fully understood. Here, we report that Siglec-6 interacts with inhibitory phosphatases, the MC activating receptor KIT/CD117 and attenuates stem cell factor (SCF)-mediated MC activation in vivo.  

Objective: To interrogate protein interactions and signaling pathways effecting Siglec-6 inhibition in primary MCs and evaluate the breadth of inhibition of an agonistic Siglec-6 monoclonal antibody (mAb).  

Methods: Interactions of Siglec-6 with activating receptors and signaling molecules were evaluated biochemically and through confocal microscopy using primary MCs. The activity of an agonistic anti-Siglec-6 mAb was evaluated in vivo in an acute model of SCF-mediated inflammation in transgenic mice that express Siglec-6 on mouse MCs. 

Results: Siglec-6 was found to directly interact and colocalize with KIT/CD117 in MCs. In addition, the non-receptor inhibitory phosphatases Shp-1 and Shp-2 associated with Siglec-6 ITIMs upon phosphorylation. Mutation of both the proximal and distal ITIMs reduced phosphorylation and recruitment of Shp phosphatases. Importantly, engagement of Siglec-6 with an agonistic mAb induced microclusters containing inhibitory phosphatases and KIT/CD117, indicative of immunoregulatory synapses. Treatment with a Siglec-6 mAb inhibited SCF-mediated inflammation and the release of MC-specific proteases in Siglec-6 transgenic mice. 

Discussion: These findings expand on our mechanistic understanding of MC inhibition through Siglec-6 and highlight the receptor as an attractive therapeutic target to broadly inhibit MCs in inflammatory diseases.

 

 

 

Mast cell-derived FXIIIA contributes to sexual dimorphic defense against Group B Streptococcus

Piliponsky, Adrian; Sharma, Kavita; Quach, Phoenicia; Brokaw, Alyssa; Nguyen, Shayla; Orvis, Austyn; Saha, Siddhartha; Samanas, Nyssa; Miralda, Irina; Tang, Yu; Mackey, Emily; Bhise, Gauri; Gendrin, Claire; Furuta, Anna; Seo, Albert; Guga, Eric; Coleman, Michelle; Bauml, Charlotte; Imhof, Diana; Moeser, Adam; Rajagopal, Lakshmi

Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, United States

Introduction: Mast cell response to stimuli is sexually dimorphic with female mast cells exhibiting increased ability to produce and release mediators upon IgE-dependent activation. This difference in mast cell function may be reflected in increased innate immune activity in females relative to males, but the precise mediators of this phenomenon have not been fully characterized.  

Objective: To investigate the contribution of mast cell-derived coagulation Factor XIIIA (FXIIIA) to female mouse susceptibility to infection against Group B Streptococcus (GBS), Gram-positive bacteria that cause invasive infections in human neonates and adults.   

Methods: We examined FXIIIA contribution to protection against systemic infection with GBS by using mice with global deficiency in FXIIIA and mice lacking FXIIIA specifically in mast cells, and by FXIIIA administration to wild type mice. We used a specific FXIIIA transglutaminase inhibitor (Tridegin) to investigate whether FXIIIA influences GBS dissemination. We employed male gonadectomized mice and mast cells from testicular feminized (Tfm) mice to examine the role of androgens on susceptibility to GBS infection and FXIIIA production by mast cells, respectively.  

Results and Discussion: Female mice with global and mast cell-specific deficiency in FXIIIA exhibited significantly increased susceptibility to GBS systemic infections. Female wild type mice displayed increased FXIIIA activity levels and were more resistant to GBS infection compared to isogenic male mice. Furthermore, FXIIIA administration to male mice increased host resistance to GBS infection. Male gonadectomized mice exhibited decreased sensitivity to GBS infections, and mast cells from Tfm mice contained increased FXIIIA amounts suggesting a detrimental role for gonadal androgens in infections with GBS. Tridegin administration to female mice decreased host resistance to GBS indicating that FXIIIA promotes GBS entrapment within cross-linked fibrin networks, thereby limiting bacterial dissemination. Accordingly, we found that FXIIIA transglutaminase activity is required for the interaction between the fibronectin binding GBS surface protein ScpB and fibrin protein residues. Moreover, ScpB-deficient GBS displayed decreased entrapment within fibrin clots and exhibited increased dissemination during systemic infections.   Conclusion: Our studies provide novel insight into the effect of sexual dimorphism and mast cells in FXIIIA expression and its interactions with GBS proteins that mediate bacterial dissemination and pathogenesis.

 

 

 

Salt-Inducible Kinases (SIKs): Recently identified regulators of mast cell function

Darling, Nicola; Cohen, Philip

MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom

Inhibiting the Salt-Inducible Kinases (SIKs) suppresses pro-inflammatory signalling in innate immune cells including macrophages and dendritic cells but their role in mast cells had not previously been investigated. Mast cells synthesise and secrete cytokines and chemokines following stimulation with IL-33, which is released in vivo following cell damage or necrosis. We established that SIKs are required for the IL-33-stimulated transcription of il13, gm-csf and tnf in mast cells. Consequently, small-molecule inhibitors of SIKs strongly suppressed the secretion of these cytokines and various chemokines. Furthermore, using mast cells expressing kinase-inactive mutants of one or more SIKs, we established that SIK2 and SIK3 are the key isoforms regulating cytokine secretion following IL-33 stimulation. However, mast cells also store pre-formed inflammatory mediators within granules, ready for release following activation. Notably SIKs are active under basal conditions and thus may have a role in regulating the basal state of the mast cell. We generated foetal liver-derived mast cells (FLMCs) expressing kinase-inactive mutants of SIK2 and SIK3 and this did not affect the cell surface expression of FcεR1, c-kit or the IL-33 receptor subunit ST2 and did not impact cell size. We performed an unbiased quantitative proteomic study to establish the effect of SIK2/3 inactivation in mast cells. Analysis of over 5000 proteins identified in FLMCs provides new insight into the function of SIK2/3 in mast cells. For example, around 40% of the proteins that comprise the mast cell signature were differentially regulated in FLMCs expressing kinase-inactive SIK2 and SIK3 (30% upregulated and 10% down regulated). This highlights a wider role for SIK2 and SIK3 in regulating the basal state of mast cells in addition to the regulation of IL-33-stimulated cytokine production.

 

 

 

 

Oral abstract session 4: Interactions between mast cells, eosinophils and other immune cells. 

The Role of Mast cells, Eosinophils and Basophils in Pemphigoid Diseases

Gibbs, Bernhard F.

 

Department of Human Medicine, Faculty of Medicine and Health Sciences, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany

 

Pemphigoid diseases (PD) are a group of rare but severe autoimmune diseases that are characterized by autoantibodies directed against proteins of the dermal-epidermal junction, leading to the formation of subepidermal blisters. PDs are also often accompanied by intense itch, which is a particular hallmark of the most common PD, bullous pemphigoid (BP). It has long been known that BP is associated with IgE autoantibodies, particularly against the transmembrane protein BP180 (collagen type XVII), and more recently it has been demonstrated that serum levels of these IgE autoantibodies correlate with disease activity. These observations suggest a role for allergic effector cells in BP and possibly other PDs. Indeed, several studies have reported clinical improvement in both BP and mucous membrane pemphigoid using omalizumab, a monoclonal antibody that depletes free (unbound) IgE, strongly suggesting the involvement of allergic effector cells in these autoimmune diseases. Here, we shall explore the evidence thus far, suggesting a differential role for mast cells, eosinophils and basophils in BP and other PDs in terms of pruritus, blister formation and potential resolution of PD-associated inflammation.

 

 

 

Bidirectional activation of human skin mast cells and blood eosinophils

Frischbutter, Stefan; Luo, Xue Ying; Monino-Romero, Sherezade; He, Jiajun; Levi-Schaffer, Francesca; Maurer, Marcus

Institute of Allergology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany; Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Allergology and Immunology, Berlin, Germany

Introduction: In the late and chronic phase of allergies, mast cells (MCs) and eosinophils (Eos) enter in a bidirectional crosstalk leading to cell activation driven by physical contact or soluble mediators. This interaction, termed the ‘‘Allergic Effector Unit’’ (AEU), substantially contributes to chronic allergic inflammation. However, functional features of the AEU are incompletely understood.

Objective: Here, we studied the contribution of individual MC mediators to Eos activation and the physical interaction of primary human skin MC and human blood eosinophils using in vitro and ex vivo models.

Methods: MCs or eosinophils were treated either with cell culture supernatants or co-cultured. Release of β-hexosaminidase or histamine were used as readouts for MC activation. Eosinophil activation was determined via CD69 expression using flow cytometry. Cytokine blocking antibodies were used to assess the contribution of individual MC-derived cytokines to Eos activation. To mimic eosinophil infiltration into skin, we injected blood eosinophils into human skin explants and performed ex vivo skin microdialysis.

Results: Supernatants from both, anti-IgE and cortistatin-activated skin MCs, strongly increased the expression of CD69 on eosinophils (% CD69+ Eos: unstimulated: 3.6±0.8); anti-IgE: 35.2±13.2; cortistatin: 38.3±5.2). By blocking individual cytokines in the MC culture supernatant we identified GM-CSF as a crucial cytokine for Eos activation, as blocking of GM-CSF reduced CD69 to the levels of unstimulated Eos (% CD69+ Eos:  anti-IgE: 59.3±46; anti-IgE + anti-GM-CSF: 34.6±39.9;  unstimulated: 35.2±28.9). Co-culture of unstimulated MCs and eosinophils induced a strong CD69 upregulation on eosinophils (34.4 % ±33.7), which was further increased (to 63.7% ±29.4) in the presence of previously activated MC. Finally, injection of eosinophils into ex vivo skin induced strong histamine release by MCs (no eosinophils: 49 ng/ml ±36.7 vs. injected eosinophils: 132 ng/ml ±46).

Discussion: Bidirectional crosstalk between MCs and Eos i.e. the AEU might result in a reciprocal activation loop that contributes to chronic activation of both cell types contributing to disease pathology. Thus, therapeutic disruption of the AEU for example by targeting GM-CSF or its receptor may be a valuable approach for the treatment of chronic allergic inflammation.

 

 

 

CT-M8 : a new mouse model reveals the non-redundant role of basophils in lupus-like disease onset

Tchen, John; Simon, Quentin; Chapart, Lea; Pellefigues, Christophe; Karasuyama, Hajime; Miyake, Kensuke; Blank, Ulrich; Benhamou, Marc; Daugas, Eric; Charles, Nicolas

INSERM U1149 Centre de recherche sur l’inflammation, France

Tissue-specific mouse models are essential tools to decipher the role of each cell compartment and/or their expressed genes in the pathophysiology of diseases. Here, we describe a new knock-in mouse model allowing expressions of both the fluorescent protein tdTomato and the CRE recombinase specifically in the basophil compartment, under the control of the Mcpt8 gene. These “CTM8” mice did not show any abnormalities in their immune cell distribution nor in their basophil function. CTM8 mice allowed the identification of basophils by immunofluorescence and flow cytometry, and the basophil-specific CRE- mediated floxed gene deletion. Breeding of our CTM8 mice with the ROSA26flox-stop-DTA mice led to the generation of basophil-deficient mice with no detectable abnormalities in other cell compartments. Basophils are involved in systemic lupus erythematous (SLE) pathophysiology by supporting and amplifying autoantibody production. Transient depletion of basophils through DT injection in Mcpt8DTR mice showed promising therapeutic effects on two distinct lupus-like mouse models (Lyn–/– and pristane-induced) and demonstrated the disease-amplifying role of basophils in these models. Here, constitutive basophil deficiency prevented pristane- induced lupus-like disease development by limiting autoantibody titers and renal damages, strongly suggesting that basophils have a non-redundant role in pristane-induced lupus-like disease induction and amplification. Moreover, we describe a new mouse model that will help to further decipher the role of basophils and their expressed genes in health and disease.

 

 

 

Mast cell granules are endogenous CLR ligands skewing DC function towards TH2/TH17 response

Kotrba, Johanna; Lepenies, Bernd; Kahlfuß, Sascha; Dudeck, Anne

Institute for Molecular and Clinical Immunology, Medical Faculty / Otto-von-Guericke University Magdeburg, Magdeburg, Germany

Mast cells (MCs) are known as key effector cells of type I allergy but also play an important role in host defense against pathogens. Despite increasing evidence for a critical impact of MCs on adaptive immunity, the underlying mechanisms are poorly understood. Particularly, the specific relevance of MC granules (MCG) in MC communication with tissue resident immune cells has not yet been studied. We recently reported that dermal dendritic cells (dDCs) engulf MCG exocytosed by MCs upon skin inflammation. MCG-bearing DCs showed an advanced functionality compared to MCG-negative DCs. We consequently highlighted a unique feature of peripheral MCs to impact on adaptive immunity by modifying DC functions. Understanding the uptake mechanism and DC modulating properties of MCG may give rise to therapeutic strategies to either intentionally boost adaptive immunity or dampen elevated immune responses. We found that solely immature DCs take up MCG via macropinocytosis. Nevertheless, the exclusive engulfment of MCG from fully differentiated MCs revealed that sensing of distinct molecular patterns precedes the uptake. We could show that DCs sense and engulf MCG in a Card9-/Syk-dependent manner. Importantly, MCG-bearing DCs modulated their antigen-presenting capacity towards TH2 and TH17 responses. Consistently, Card9 deficiency resulted in a reduced T cell priming/differentiation upon DNFB sensitization and diminished T cell driven adaptive ear swelling response upon elicitation. Questioning the MCG sensing DC surface receptors, we could show that the c-type lectin receptors (CLRs) SIGNR3 and MCL bind to MCG and cooperate in mediating the MCG uptake. Finally, MCL-/- SIGNR3-/- mice showed a reduced peribronchial inflammation during house-dust-mite (HDM) induced acute allergic asthma, accompanied by significantly reduced numbers of cDC2s and TH2 cytokine-producing cells. Collectively, we show herein that MCG are endogenous CLR ligands that translate (MC degranulation inducing) danger signals into adjuvant effects promoting lymph node-borne adaptive immunity.

 

 

 

Mast cells trap and cannibalize swarming neutrophils for metabolic and inflammatory fueling

Mihlan, Michael; Wissmann, Stefanie; Gavrilov, Alina; Lorentz, Axel; Laemmermann, Tim

Cell Dynamics, Max-Planck-Institute for Immunobiology & Epigenetics, Freiburg, Germany

Introduction: Degranulating mast cells (MCs) release inflammatory mediators, including chemokines and chemoattractants, which recruit other immune cells in tissues. Neutrophils and eosinophils can initiate self-amplifying swarming responses via intercellular communication through the lipid leukotriene B4 (LTB4).  

Objectives: The interrelation of MC and granulocyte dynamics in tissues has been poorly investigated. Further, the recovery of exhausted MCs after degranulation is hardly characterized. Here we investigated how MC activation influences neutrophil dynamics during anaphylaxis and analyzed whether and how neutrophils modify the recovery of exhausted MCs.

Material & Methods: We performed two-photon intravital microscopy in mice, confocal live cell imaging and multiple static imaging methods to characterize interaction dynamics between degranulating primary MCs and neutrophils. We used Ribotag precipitation, RNAseq and mass spectrometry analysis of co-cultivated cells to identify the impact of neutrophils on recovering MCs. Finally, we proofed neutrophil impact on mast cell functionality and survival.

Results: We show that degranulating MCs release LTB4 and exploit this attractant by re-directing neutrophils and inducing their swarm formation in vivo. Unexpectedly, neutrophil cluster formation around MCs results in the trapping of living neutrophils inside MC vacuoles in vivo and in vitro. Thus, we identify a novel cell-in-cell structure between MCs and neutrophils, which we term “Mast Cell Intracellular Trap” (MIT). Trapped neutrophils undergo cell death after 12hrs, and MC translatome analysis of MITs revealed improved MC metabolism and recovery. This leads to several benefits for MITs: (1) they can be more efficiently re-stimulated than MCs, (2) they show improved survival under nutrient limitation, and (3) MITs store neutrophil DNA and effector molecules, which can be released after re-stimulation several days after initial neutrophil uptake, modifying the inflammatory profile of MCs.

Conclusions: The invasion of a living cell into the cytoplasm of another cell (entosis) has been described for several cell types. We here show that MCs undergo a previously unappreciated entosis-like process, which promotes their regeneration, functionality and inflammatory potential. In addition, we define a novel role of neutrophils, which supply nutrients and inflammatory molecules to recovering MCs. Our studies may have potential implications for chronically activated MCs in MC-related immune disorders.

 

 

 

T helper-licensed mast cells promote inflammatory Th17/Treg cells imbalance

Leveque, Edouard; Joulia, Regis; Petitfils, Camille; Mas-orea, Xavier; Payros, Gaëlle; Laurent, Camille; Gaudenzio, Nicolas; Dietrich, Gilles; Valitutti, Salvatore; Cenac, Nicolas; Espinosa, Eric

Infinity, u1291, Toulouse, France

CD4+ T helper cells (Th) infiltrate sites of inflammation and orchestrate the immune response by instructing local leukocytes. Mast cells (MCs) are tissue sentinel cells particularly abundant in skin and mucosa. Here, we analyzed the interplay between human MCs and Th cells and, through the application of RNAseq and functional assays, showed that Th cells induced a specific transcriptomic program in helped MCs (named here MCTH) driving them toward an inflammatory phenotype. The gene signature of MCTH indicated that MCs helped by Th cell acquired in turn the capacity to regulate effector T cell response through wide-range of soluble and membrane ligands. Accordingly, we showed that MCTH decreased Treg While promoting Th17 cells and notably an inflammatory subset of Th17, producing both IFN-γ and GM-CSF  through a PGE2 and IL-1β axis. We finally showed that mast cells in Crohn disease are armed to induce pathogenic Th17 and that mast cell deficient mice are less sensitive to DSS-induced colitis, especially in the T cell dependent phase of the disease. Our findings demonstrate that activated effector/memory CD4+ T cells activate and instruct resting MCs toward a specific differentiated pro-inflammatory phenotype endowed with the capacity to speak back to effector T cells and to mold their functions. This interplay can act as a detrimental amplification loop in Th17 mediated inflammatory diseases.

 

 

 

 

 

 

 

 

 

 

Abstracts Poster Presentations

Poster session 1, Monday July 11th, 2022

 

Poster 1

Mast cell chymase regulate ECM-related events in primary human small airway epithelial cells

Zhao, Xinran O; Sommerhoff, Christian P; Paivandy, Aida; Pejler, Gunnar

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

Background: Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells may re-localize to the epithelial layer, and may thereby affect the functional properties of the airway epithelial cells.

Objectives: Activated mast cells release large quantities of proteases from their secretory granules, including chymase and tryptase. Here we investigated whether these proteases may affect airway epithelial cells.

Methods: Primary small airway epithelial cells were treated with tryptase or chymase, and effects on epithelial cell viability, proliferation, migration, cytokine output and transcriptome were evaluated.

Results: Airway epithelial cells were relatively refractory to tryptase. In contrast, chymase had extensive effects on multiple features of the epithelial cells, with a particular emphasis on processes related to extracellular matrix (ECM) remodeling. These included a suppressed expression of ECM-related genes such as matrix metalloproteases, which was confirmed at the protein level. Further, chymase suppressed the expression of the fibronectin gene, and also caused degradation of fibronectin released by the epithelial cells. It was also shown that chymase suppressed the migratory capacity of the airway epithelial cells, and that chymase degraded the cell-cell contact protein E-cadherin on the epithelial cell surface. 

Conclusion: Our findings suggest that chymase may affect the regulation of ECM remodeling events mediated by airway epithelial cells, with implications for the impact of mast cells in inflammatory lung diseases such as asthma. Clinical implication: This study provides novel insight into how mast cells may influence asthma.

 

 

 

Poster 2

Human mast cells produce pro inflammatory mediators in response to chlamydia infection

Mayavannan, Animamalar; Shantz, Emily; Haidl, Ian D.; Wang, Jun; Marshall, Jean S.

Microbiology and Immunology, Dalhousie University, Halifax, Canada

Introduction: Chlamydia trachomatis (Ct) is an obligate intracellular bacterium that causes reproductive tract complications in women including ectopic pregnancies and tubal factor infertility. Certain TLR2 polymorphisms increase risk of Ct–associated infertility. Many aspects of the host immune response to Ct remain unelucidated.

Objectives: We hypothesized that mast cells contribute to inflammatory responses to Chlamydia infection. This study aimed to define and characterize the mechanism of human mast cell responses to Ct.  

Methods: Human cord blood derived mast cells (CBMCs) were exposed to Ct and examined by electron microscopy and flow cytometry. Mast cell degranulation was assessed using β-hexosaminidase assay. Differential expression of inflammation-associated genes was examined by PCR array. Gene expression readouts were validated by qPCR analysis of CBMC mRNA (n=8). Secreted mediators were assayed by Luminex array (n=6). The role of pattern recognition receptors formyl peptide receptors (FPR1 and FPR2) and Toll-like receptor 2 (TLR2) were investigated using defined antagonists and ELISA assays of cytokines. 

Results: Ct bacteria were sparsely taken up and did not replicate efficiently inside CBMCs. Ct activated mast cells did not degranulate and maintained viability but exhibited homotypic aggregation and upregulation of ICAM-1. Ct treated mast cells showed significantly enhanced expression of IL1β, CCL3, NFκB1, CXCL8 and IL6. Elevated levels of inflammatory proteins including TNF, IL-1β, IL-1RA, IL-6, GM-CSF, IL-23, CCL3, CCL5 and CXCL8 were also produced consistent with a classical pattern recognition receptor response. FPR antagonist treatments did not inhibit responses. The IL-6 response to Ct was significantly (p <0.05) reduced in CBMCs treated with soluble TLR2 coated Ct compared with controls. Endocytic blockade resulted in a 61%, reduction in IL6 expression suggesting Ct-mast cell interaction in both extracellular and intracellular locations. Murine mast cells from TLR2-/- mice also demonstrated a reduced IL-6 response to Chlamydia muridarum.

Discussion: Taken together, these results indicate that human mast cells uptake Ct but do not facilitate its replicative life cycle. They respond to Ct with enhanced ICAM-1 expression and the production of proinflammatory mediators partially via a TLR2-dependent and NF-kB mediated pathway.  This work was funded by CIHR and supported by DMRF and IWK Graduate studentships.

 

 

 

Poster 3

Mast cells exposed to cyclic hypoxia exhibit increased responsiveness to FcεRI crosslinking

Segura-Villalobos, Deisy; Lamas, Mónica; González-Espinosa, Claudia

Pharmacobiology Department, Center for Research and Advances Studies, Mexico City, Mexico

Mast cells (MCs) play an important role in tumor development, executing pro or antitumoral functions depending on tumor type and local conditions present in the tumor microenvironment (TME) such as cyclic hypoxia (cyH), which is a distinguishing feature of almost all solid tumors. In this study, we tested the hypothesis that MCs could localize in zones subjected to cyH inside solid tumors and modify, in consequence, their transcriptional profile and activation parameters. Utilizing a marker of hypoxia (Hypoxyprobe) and confocal microscopy, we found that an important number of MCs are preset in cyH zones of melanoma B16-F1tumors. Then, we analyzed transcriptional changes of murine bone marrow-derived mast cells (BMMCs) exposed to an in vitro protocol of cyH, consisting of interleaved cycles of hypoxia and re-oxygenation at 1% and 21% of oxygen, respectively. Utilizing microarray technology, we identified 2512 genes in MCs subjected to cyH whose expression displayed changes compared with normoxic cells. Furthermore, functional enrichment analysis revealed that cyH-related transcriptomic alterations were for genes associated with oxidative phosphorylation and the FcεRI signaling pathway. Interestingly, FcεRI-dependent degranulation, calcium mobilization, and PLC-γ activity were enhanced in cyH-exposed BMMCs compared with cells maintained under normoxic conditions after IgE/antigen (Ag) challenge. In addition, the expression of calcium signaling-related cytokines such as TNF-α, IL-4, and IL-2 was increased in BMMCs exposed to cyH after IgE/Ag challenge. Taken together, these findings indicate that cyH induces transcriptomic modifications in MCs that are translated into a phenotypic change characterized by hyper-responsiveness to IgE/Ag challenge. Our results provide novel evidence of the effect of environmental conditions such as cyH on MC phenotype and strongly suggest that the intratumoral microenvironment acts as a potent modulator of MCs activation which should be considered in the design of inflammation-targeted therapies to control tumor growth. Supported by Grant Conacyt CF-2019-51488 to CG-E, A1-S-25777 to M.L, and scholarship 781556 to DS-V.

 

 

 

Poster 4

The Impact of Industrially-made MoS2 on Human Basophils

Lin, Hazel; Bianco, Alberto

University of Strasbourg, Strasbourg, France

Introduction: 2-Dimensional molybdenum disulfide has been used in biomedical applications such as drug delivery and cancer detection, rendering it crucial to evaluate downstream compatibility in immune cells. Molybdenum itself is a component of stainless steel implants and has previously been implicated in hypersensitivity in stent patients. This makes it important to determine its effect in allergy, such as in basophils, which are a less commonly used  human blood immune cell that can act as a sentinel for allergy.

Objective: Just testing any one type of MoS2 in basophils could result in differing results downstream given the wide range of MoS2 available commercially. We decided to compare industrial MoS2 from two established companies BeDimensional© (Italy) and Biograph Solutions (Spain), manufactured with two different but commonly used methods (the former using deoxycholate surfactant in liquid exfoliation and the latter using glycine in ball-milling exfoliation), to elucidate immunological end-points for MoS2 in general, and to demonstrate the need for biological experimental verification for downstream users who may require a change of supplier.

Methods: Primary human basophils were isolated from healthy donor PBMCs using a commercial kit. ELISA and flow cytometry were used to measure surface activation markers and cytokine production. The use of sodium molybdate was included as control to rule out any effect due to the molybdate ion generated by the oxidation of MoS2. Deoxycholate was also used to verify any potential surfactant effects.

Results: Higher histamine production was observed in primary human blood basophils with both industrial MoS2, with no impact on surface basophil activation markers CD63 and CD203c, ROS production or cell viability, but having different cytokine production patterns. Briefly, IL-6 and IL-1β but not TNF and GM-CSF were increased for both MoS2. Biograph Solutions MoS2 increased IL-4, while BeDimensional© MoS2 decreased IL-4 and increased IL-13. Deoxycholate surfactant on its own decreased viability long-term and increased ROS upon basophil activation. Molybdate ion itself had minimal effects, only increasing IL-1β and IL-4.

Discussion: These results prove the relative safety of MoS2 in human basophils and demonstrate the importance of considering manufacturer additives and variability when selecting 2D materials for biological applications.

 

 

 

Poster 5

Lipid interconversions mediate secretory granule fusion and fission during their biogenesis

Omari, Sewar; Roded, Amit; Fukuda, Mitsunori; J. Galli, Stephen; Sagi-Eisenberg, Ronit

Cell and Developmental Biology, Tel Aviv University, Tel Aviv, Israel

Homotypic fusion is an intermediate step in the biogenesis of secretory granules (SGs) in professional secretory cells, including endocrine and exocrine cells, and cells of the immune system, such as the mast cells (MCs). Yet, the underlying mechanisms of this process are poorly understood. In MCs, part of their inflammatory mediators is preformed and stored in SGs, that by releasing their content in response to external stimuli, mediate MCs innate and allergic immune responses. Previous work has identified Rab5, a small GTPase known for its involvement in endocytic pathways, as a novel regulator of SG fusion. Here we demonstrate that SG fusion is accompanied by an increase in the SG levels of the inositol phospholipids, PI(3)P, PI(4)P and PI(3,4,5)P3. We show that Rab5-stimulated activation of phosphatidylinositol 3 kinases (PI3Ks) and the consequent formation of PI(3,4,5)P3 are required for the activation of the non-receptor protein tyrosine phosphatase PTPN9 (PTP-MEG2), that mediates SG fusion. We further show that SG fusion with endosomes, that is mediated by the tetraspanin protein CD63 and PI4K type II, is a prerequisite for SG homotypic fusion. Finally, we show that MC SGs also undergo fission, that is mediated by dynamin recruitment to SG PI(4,5)P2. Taken together, our results identify PI3 and PI4 lipid kinases, CD63 and PTPN9 as Rab5 downstream mediators of SG fusion, and dynamin as a regulator of their fission. Our results also reveal that fusion and fission events, that control the size and number of the SGs during their biogenesis, are regulated by lipid interconversions.

 

 

 

Poster 6

Inducible senescence in bone marrow derived mast cells – a novel model for mast cell aging research

Kleeblatt, Elisabeth; Frenkel, Dan; Sagi-Eisenberg, Ronit

Sackler Faculty of Medicine, Department of Cell and Developmental Biology, Tel Aviv University, Tel Aviv, Israel

Mast cells (MCs) are mainly known for their role in mediating allergic reactions. However, MCs are additionally involved in many age-related diseases, including neurodegenerative diseases and cancer. Research on age-related changes in the immune system is critical for the understanding of aging-associated diseases. Yet, deciphering the influence of aging on MC function has been hampered by the difficulty of retrieving tissue MCs from old mice. Therefore, a common model for MC study is based on in vitro differentiation of progenitor cells originating from the bone marrow of young mice (BMMCs), leaving the need for a model for “old” MCs unanswered. To overcome this problem, we relied on the fact that aging is linked with senescence and have established a model of inducible senescence in cultured MCs. The model is based on the in vitro differentiation of MCs from the bone marrow of transgenic mice that carry a tet-on-system of p16, an inhibitor of the cell cycle, the upregulation of which causes senescence. We have confirmed that BMMCs derived from these mice upregulate p16 after doxycycline application and stop dividing. Further characterization of these cells revealed that they express increased amounts of mRNA of members of the senescence-associated secretory phenotype, such as IL-1β and IL-10, and have lower autophagic activity. Senescence also enhances the proinflammatory responses of MCs to both adaptive, allergic triggers (i.e. IgE/antigen), and to an innate immune trigger, such as LPS. Interestingly, the senescent MCs express less FcεRI on their cell surface. However, their triggered secretion of  β-hexosaminidase is unchanged, while exteriorization of their proteoglycans is increased. Taken together, our results suggest that aging reprograms MCs towards a pro-inflammatory phenotype.

 

 

 

Poster 7

Single-cell RNA sequencing of human lung mast cells

Rönnberg Höckerlind, Elin; Ravindran, Avinash; Mazzurana, Luca; Säfholm, Jesper; Lorent, Julie; Defhlefsen, Olga; Orre, Ann-Charlotte; Al-Ameri, Mamdoh; Adner, Mikael; Dahlén, Sven-Erik; Dahlin, Joakim; Mjösberg, Jenny; Nilsson, Gunnar

Medicine (Solna), Karolinska Institutet, Stockholm, Sweden

Introduction: Human lung mast cells are distributed in all compartments of the lung, both ont the central and distal parts of the airways. These cells are involved in the pathology of different airway diseases, where particulary the role of mast cells in asthma has been investigated. Human mast cells are classically divided into the subsets, MCT and MCTC, where MCT express the mast cell protease Tryptase and MCTC in addition express Chymase, Carboxypeptidase A3 (CPA3) and Cathepsin G.  Apart from the disctintion of the MCT and MCTC subsets, little is known about the heterogeniety of human lung. 

Objective: To perform a deep analysis of there heterogeniety of human lung mast cells. 

Methods: Single cell RNA sequencing was performed on sorted human lung mast cells using SMARTseq2. 

Results: The sequenced mast cells showed high expression of classical mast cell markers. However, no sub-populations were identified in the dataset using principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) plot contstruction. Examination of the proteases that are differentically expressed in the classical mast cell subsets showed, to the contrary of the protein, that CPA3 mRNA was highly expressed in all cells, while the gene for Chymase (CMA1) was only decetable in a small proportion of the cells and the expression of Cathepsin G (CTSG) was highly variable. There was a correlation between CMA1 and CTSG expression, while no correlation was seen between CPA3 and CMA1 nor  to CTSG. 

Discussion: In general, the sorted lung mast cells were homogenous in nature with a few highly variable gene sets. Human lung mast cells are predominaly of the MCT subset and the expression of CMA1 was as expected low, and correlated to CTSG. However, to the contrary of the proteins, there was no correlation of CPA3 mRNA to CMA1 and CPA3 was highly detectable in all cells.

 

 

 

Poster 8

All or nothing: What we may learn from the digital response of antigen-triggered mast cells

Kuhny, Marcel; Dickmeis, Julia; Huber, Michael

Biochemistry and Molecular Immunology, Biochemistry and Molecular Immunology, Aachen, Germany

Introduction: Mast cells (MCs) are central effectors in IgE-dependent inflammatory and allergic disorders. Cross-linking of the IgE-bound high-affinity IgE receptor, FcεRI, by multivalent antigens initiates degranulation as well as de novo synthesis of inflammatory mediators like cytokines in a dose-dependent manner. Well-established bulk-analysis methods intuitively suggest a “graded” MC response, i.e., the amount of available antigen dictates the degree of degranulation per cell. However, single-cell resolution methods reveal an “all-or-nothing” response.

Objective: Stimulation via the FcεRI results in two MC populations: one that responds with degranulation and one that does not. The molecular basis for this type of response is yet unknown. 

Methods: We use murine bone marrow-derived MCs and peritoneal MCs in culture and ex vivo to characterize the all-or-nothing response. We rely on flow-cytometry to observe cellular responses, intracellular signaling, and phenotypic changes of antigen-stimulated MCs at single-cell resolution. This will be followed by comparative transcriptomics/proteomics to identify key regulators.

Results: Flow-cytometry-based methods reveal an “all-or-nothing” response: MCs either degranulate after antigen stimulation, or not. The amount of antigen determines the number of MCs that degranulate but does not modulate the extent of granule release. This mode of action is also true for FcεRI-induced TNF production. Sequential stimulations show that at a given antigen concentration degranulating and non-degranulating MCs maintain their respective (un)responsiveness to a second challenge. This suggests a cell-intrinsic bias that pre-determines the propensity of MCs to resist FcεRI-dependent cellular response. Importantly, indistinguishable FcεRI-mediated ERK1/2 activation can be measured in all cells, suggesting equal stimulation of all cells. Discussion: Methods that rely on bulk analyses only allow observations of the entirety of the assayed cells. Their results are by no means wrong, but the conclusions may not accurately describe events at the cellular level. Observing MC degranulation with single-cell resolution unveils an all-or-nothing response where the quantity of the stimulus does not modulate the degree of the degranulation but rather the amount of responding cells. These findings challenge us to revisit the activation mechanism of one of the fundamental MC effector functions and may have implications on how we understand IgE-MC-dependent disorders.

 

 

 

Poster 9

Identification of PKCβ as a novel regulator that controls Rab12 interactions with its effectors

Omar, Jana; Amer-Sarsour, Fatima; Omari, Sewar; Gorzalczany, Yaara; Ansbacher, Tamar; Fukuda, Mitsunori; Gal, Maayan; Ashkenazi, Avraham; Sagi-Eisenberg, Ronit

Cell and Developmental biology, Tel-Aviv University, Tel-Aviv, Israel

Triggered release by exocytosis of inflammatory mediators that are prestored in secretory granules (SGs) is the central mechanism by which mast cells (MCs) elicit their physiological roles in innate immunity and pathological functions in allergic disorders and chronic inflammatory conditions. Previous work in our laboratory has demonstrated that the small GTPase Rab12 regulates the bidirectional transport of MC SGs, which determines the extent of MC degranulation. Our further studies demonstrated that Rab12 binds all three members of the RILP family, RILP, RILP-L1 and RILP-L2. Out of these three effectors, it is the binding of RILP that mediates Rab12 function in negative regulation of MC secretion, by stimulating retrograde transport of the SGs. RILP-L1 and RILP-L2 likely mediate other Rab12 regulated functions. Therefore, the overall functionality of Rab12 is defined by its balanced connectivity. How is Rab12 connectivity regulated is presently unknown. Recent identification of Rab12 as a substrate of LRRK2, a kinase the hyperactivation of which is linked with Parkinson’s disease (PD), led us to propose that Rab12 phosphorylation plays a role in the control of Rab12 connectivity. Our results show that Rab12 phosphorylation is stimulated by innate, but not by an allergic trigger in MCs. Furthermore, using specific inhibitors, we could show that Rab12 is a physiological substrate not only of LRRK2, but also of protein kinase C β (PKCβ). Moreover, we were able to demonstrate that Rab12 phosphorylation by either LRRK2 or PKCβ  bears similar outcomes on Rab12 connectivity, increasing Rab12 binding of its effectors RILP-L1 and RILP-L2. Taken together, our findings show that phosphorylation of Rab12 regulates its connectivity to its effectors, and identify PKCβ as a novel regulator of Rab12 function. We envision that genetic or environmental conditions that increase LRRK2 or PKCβ activity shift the balance of Rab12 interactions with its RILP family effectors and thereby contribute to disease.

 

 

 

Poster 10

Bruton’s tyrosine kinase inhibition to suppress mast cell activation in atherosclerosis

Hemme, Esmeralda; Delfos, Lucie; Depuydt, Marie A. C.; Bernabé Kleijn, Mireia N. A.; Kuiper, Johan; Bot, Ilze

Division of BioTherapeutics, LACDR, Leiden University, Leiden, The Netherlands

Aim: Atherosclerosis is characterized by the accumulation of lipids and immune cells in the vessel wall. Mast cells accumulate within atherosclerotic plaques and activation of mast cells leads to the progression and destabilization via secretion of pro-inflammatory mediators and cytokines. Mast cells can be activated by various stimuli, of which crosslinking of the Fcε receptor I (FcεRI) via IgE-antigen complexes is best known. Bruton’s tyrosine kinase (BTK) is involved in the downstream signaling of FcεRI-mediated mast cell activation and degranulation. In this study, we aimed to assess the effects of the BTK inhibitor ACP-196 on FcεRI-mediated mast cell activation, plaque progression and destabilization in an atherosclerotic mouse model. 

Methods and Results: Male LDLr-/- mice, 7-11 weeks old, were treated with ACP-196 (25 mg/kg p.o., n=15) or control solvent (n=14) three times per week for eight weeks. During treatment, mice were fed a Western-type diet to induce atherosclerotic plaque formation. Plasma total cholesterol levels and body weight did not differ between the control and treatment group during the experiment. After eight weeks, mice were sacrificed and hearts were isolated to determine plaque size and stability by histology. Aorta, spleen, blood and mediastinal lymph nodes were harvested to examine mast cell activation status and other immune cells by flow cytometry. After eight weeks of ACP-196 treatment, a significant 59% reduction in the frequency of mast cells was observed in aortic plaques of ACP-196 treated mice (0.24±0.06%) compared to control mice (0.57±0.08%, p<0.05). However, the number and activation status of adventitial mast cells in the aortic root was not affected. Additionally, ACP-196 treatment (3.6±0.2% mature splenic B cells) inhibited B cell maturation systemically compared to control treated mice (4.7±0.2% mature splenic B cells, p<0.005). However, these effects on immune cells did not translate into effects on atherosclerosis, as ACP-196 treatment (size:12.3±2%) did not significantly affect atherosclerotic plaque size,  collagen content and monocyte/macrophage content when compared to control mice (size:11.5±1.4%).

Conclusions: Conclusively, these findings suggest that ACP-196 treatment leads to reduced mast cells frequencies in aortic plaques of LDLr-/- mice, but does not directly affect mast cell activation and initial atherosclerotic lesion development.

 

 

 

Poster 11

Kinetics of MRGPRX2 upon stimulation with different ligands in human mast cells.

Kwakernaak, Romy; van de Meerendonk, Sanne; Folkerts, Jelle; Pyatilova, Polina; duToit, Michelle; Dik, Willem; Hermans, Maud

Internal Medicine, Erasmus MC, Rotterdam, The Netherlands

Introduction: Mas-related-G-protein-coupled-receptor-X2 (MRGPRX2) forms an important route of mast cell degranulation. However, human mast cells express low levels of MRGPRX2 at their surface, especially in comparison to FcεR1. We hypothesized that MRGPRX2 is mainly localized intracellular in resting human mast cells.

Objective: To study the kinetics of MRGPRX2 in human mast cells upon stimulation with different ligands.

Methods: The ROSA-WT mast cell line and primary human mast cells (HuMC; generated from CD34+ myeloid progenitor cells) were exposed to the MRGPRX2 specific ligands compound 48/80 (C48/80) or substance P (SP), and to anti-(a)IgE and calcium ionophores (CI) for different time periods. Expression of MRGPRX2 and CD63 was determined by flowcytometry. Immunofluorescent staining was used to assess the localization of MRGPRX2 before and after exposure to the aforementioned stimuli. Lastly, RNA sequencing was employed to evaluate the mRNA expression of MRGPRX2 upon stimulation.

Results: MRGPRX2 surface membrane expression was significantly lower than that of FcεR1 on unstimulated ROSA and HuMC. However, both cell types expressed high levels of cytoplasmic MRGPRX2. The membrane expression of MRGPRX2 was already upregulated from 9.7% to 47% of cells upon incubation with C48/80 for 5 minutes, and further increased to 98% after 4 hours of incubation with C48/80. Although SP also induced CD63 upregulation, no significant changes in MRGPRX2 membrane expression were observed. MRGPRX2 membrane expression did not alter upon FcεR1 activation nor incubation with CI. MRGPRX2 mRNA expression remained unchanged upon 1 hour stimulation with C48/80, SP, a-IgE or CI. Similar effects were observed for ROSA and HuMC throughout all experiments.

Discussion: MRGPRX2 is predominantly localized in the cytoplasm of resting human mast cells. Its membrane expression increases strongly upon activation of mast cells with C48/80, whereas SP does not induce significant MRGPRX2 membrane expression, for unknown reasons. It is also yet unclear whether MRGPRX2 is shed off the cells afterwards or is internalized. 

Conclusion: MRGPRX2 is predominantly intracellularly expressed in resting human mast cells, and translocates to the surface membrane upon stimulation with C48/80, but not SP, a-IgE or CI. These findings might have implications for the future development of drugs targeting MRGPRX2.

 

 

 

Poster 12

Mast Cell Proteases Promote Diverse Effects on uPAR and Alveolar Repair Responses

Mogren, Sofia; Berlin, Frida; Tufvesson, Ellen; Van Der Burg, Nicole; Andersson, Cecilia

Faculty of Medicine, Lunds University, Lund, Sweden

Introduction: Tissue damage, epithelial alterations, and intraepithelial and alveolar presence of mast cells (MCs) are characteristics of asthma pathogenesis. The asthma associated receptor, urokinase plasminogen activator receptor (uPAR), has been shown to regulate bronchial epithelial repair responses. Increased uPAR expression, as seen in patients with asthma, leads to attenuated wound repair which may contribute to the progression of airway remodelling in asthma. Previously, we have shown that tryptase increases cell growth and wound gap closure in bronchial epithelial cells. However, the impact of MC proteases, tryptase and chymase, on functional properties and expression of uPAR in alveolar epithelial cells is not fully investigated. 

Aim: To investigate the levels of soluble uPAR (suPAR) in bronchoalveolar lavage (BAL) fractions from asthmatic patients and the impact of MC proteases on the alveolar epithelial uPAR expression and alveolar repair responses.   

Methods: Alveolar epithelial cell migration and wound healing were investigated using holographic live cell imaging in a scratch model post stimulation with tryptase or chymase. The expression of uPAR was investigated on protein and gene level. Levels of suPAR was measured in BAL fluid from mild-moderate allergic asthmatics patients.  

Results: We found that tryptase increased wound gap closure (p<0.005), cellular migration (p<0.01), membrane bound uPAR expression (p<0.01) and MMP9 expression (p<0.05) in alveolar epithelial cells compared to non-stimulated cells. While chymase altered alveolar epithelial morphology (p<0.05), reduced wound gap closure (p<0.0001) and cellular migration (p<0.05) compared to controls. However, chymase induced suPAR release in a time dependent manner (p<0.001) compared to controls. In allergic asthmatics, the levels of suPAR were increased in BAL fluid from the alveolar compartment compared to bronchial fractions, indicating a higher release in peripheral lung compared to central regions (p<0.05).  

Conclusion: Our results show that MC proteases have a significant impact on wound gap closure and the uPAR pathway in alveolar epithelial cells. The data also indicate a different regulatory role of tryptase and chymase in events related to uPA-uPAR expression, ECM degradation and barrier function. This pathway and how it is regulated by MCs may therefore be a potential therapeutic target for asthma treatment.

 

 

 

Poster 13

The role of mast cell cyclin E1 in the inflammatory response

Bronneberg, Gina; Kuo, Chao-Chung; Huber, Michael

Institute of Biochemistry and Molecular Immunology University Clinic, RWTH Aachen, University, Aachen, Germany

E-type cyclins (cyclin E1[CCNE1] and E2) are part of the cell cycle machinery expressed during the G1 phase of the cell cycle. Together with its catalytic partner, the cyclin-dependent kinase 2 (CDK2), phosphorylation of substrates leads to the expression of S-phase specific genes. Initial experiments using the CDK2 inhibitor Roscovitine already revealed a decreased production of pro-inflammatory cytokines (IL-6 and TNF) in murine bone marrow-derived mast cells (BMMCs) in response to antigen and LPS. In contrast, CDK2 inhibition had no effect on antigen-triggered degranulation. Next, we were interested if the regulatory subunit of the CDK complex, CCNE1, might be involved in the regulation of the inflammatory response in mast cells as well. BMMCs were generated from wildtype and CCNE1 knockout mice and degranulation as well as pro-inflammatory cytokine production was analyzed. In addition, we applied next generation sequencing to analyze CCNE1-dependent gene expression in mast cells. The differentiation of BMMCs from CCNE1-deficient mice was comparable to wildtype mice. Expression of the cell surface proteins KIT and FceRI and proliferation of BMMCs was not altered suggesting that CCNE1 is not essential for differentiation and proliferation of mast cells. Analysis of degranulation revealed no difference between wildtype and CCNE1 knockout BMMCs, indicating no involvement of CCNE1 in immediate mast cell responses. Intriguingly however, dissection of the antigen and LPS induced pro-inflammatory cytokine response revealed attenuated cytokine production in CCNE1-deficient compared to wildtype BMMCs on protein as well as on mRNA level. Initial phospho-analysis of central signaling proteins (e.g. PKB, ERK1/2, p-38 and NFkB) revealed no obvious differences. Thus, we conducted an analysis of the transcriptome of antigen and LPS-stimulated wildtype and CCNE1-deficient BMMCs. The bioinformatic analysis, however, is currently still in progress. Our study revealed an unexpected role of the cell cycle regulator CCNE1 in the pro-inflammatory mast cell response upon antigen and LPS stimulation. Since experiments were done using non-synchronized BMMCs, a direct involvement of the cell cycle can be excluded and a regulatory function of CCNE1 in addition to its interaction with CDK2 might be envisaged.

 

 

 

Poster 14

The evolution of mast cell-expressed carboxypeptidase A3 in comparison to other carboxypeptidases

Akula, Srinivas; Hellman, Lars; Aviles, Francesc; wernsersson, Sara

Department of Anatomy, Physiology and Biochemistry, Swedish university of agricultural sciences, Uppsala, Sweden

Introduction: Metallo-carboxypeptidases are a group of exopeptidases that can be found in all kingdoms of life, from bacteria to mammals. One of these, the carboxypeptidase A3 (CPA3), is expressed and stored in large amounts in cytoplasmic granules of mast cells. Mast cells (MCs) are highly specialized sentinel cells that store significant amounts of bioactive mediators in cytoplasmic granules, including CPA3. CPA3 is a member of the M14A subfamily of metallo-carboxypeptidases, which also contains the digestive enzymes CPA1, CPA2, CPB1, and CPO. A few potential substrates of CPA3 have been identified including the blood pressure regulator endothelin. Several clinical studies have indicated elevated CPA3 levels in asthma patients. However, the physiological relevance of CPA3 and its evolutionary origin have yet to be explored.

Objective: To determine when CPA3 first appeared during vertebrate evolution

Methods: A broad panel of metazoan animals, such as invertebrates and mammals, were studied for their homologous genes, loci, and phylogenetic relationships.

Results: According to phylogenetic analyses and gene loci, CPA3 evolved by a gene duplication from CPB1 during the early stages of tetrapod evolution, as evidenced by the fact that it is only found in tetrapods and not in fish. CPA3 and CPB1 are both present on chromosome 3 in the same place in humans. However, the apparent loss of CPA3 in some tetrapod lineages, such as birds and monotremes, suggests that the CPA3 gene has undergone a complex evolutionary history. In the absence of CPA3, zebrafish MCs express CPA5, for which the most closely equivalent human carboxypeptidase is CPA1, which has a cleavage selectivity similar to CPA3.

Discussion: MC expressed CPs are likely to have evolved in a complex manner. CPB1 and CPO are both digestive enzymes with the closest sequence similarity. CPB1 is a pancreatic secreted enzyme, like CPA1 and CPA2, whereas CPO is a membrane-bound brush border enzyme. Both have completely different cleavage specificities than CPA3. Our collected data concerning the evolution of CPA3 thereby indicates that CPA3 appeared at the base of tetrapod evolution approximately 400 million years ago by a gene duplication of CPB1.

 

 

 

Poster 15

SH3BP2 silencing downregulates MITF through miRNAs inducing cell death in human mast cell leukemia.

Elizabeth, Proaño; Ollé, Laia; Guo, Yanru; Muñoz, Rosa; Martin, Margarita

Biomedicine, University of Barcelona, barcelone, Spain

Introduction: Activating mutations in KIT (CD117) has been associated with several diseases including gastrointestinal stromal tumors and mastocytosis. Previously, we reported that the adaptor molecule: SH3 Binding Protein 2 (SH3BP2) regulates KIT expression at the transcriptional level and Microphthalmia associated-transcription factor (MITF) expression at the post-transcriptional level, in human mast cells, and gastrointestinal stromal tumors (GIST) cell lines. To dissect the SH3BP2 pathway, a miRNA array was performed in the SH3BP2-silenced GIST cell line. Among the most upregulated miRNAs, we found putative candidates for targeting MITF. 

Objective: The purpose of this study was to elucidate the post-transcriptional regulation of MITF by SH3BP2 in human mast cells and demonstrate that MITF is key to the survival of HMC-1, a mastocytosis cell model.

Methods: MiRNAs were validated by qPCR in the human mast cell leukemia cell line (HMC-1). MiRNAs were overexpressed by lentiviral transduction and MITF and MITF-dependent targets were assessed by western blot. MITF-shRNAs and MITF inhibitor ML329 were also used. WST-1 assay was used to assess cell proliferation, and caspases 3/7 to examine apoptosis. Propidium iodide staining was used for the cell cycle analysis by FACS.

Results: MiR1246 and miR5100 overexpression downregulated MITF and MITF-dependent targets. In that context, cell viability was reduced, cell cycle arrested in S-G2/M, and apoptosis was enhanced in HMC-1. Similar results were obtained with specific MITF silencing carried out by shRNAs. Moreover, ML329 treatment confirmed the important role of MITF in cell viability and cell cycle progression in HMC-1. 

Conclusions: Our results highlight the KIT-SH3BP2-MITF pathway for mast cell survival and cell cycle progression.

 

 

 

 

 

 

 

 

Poster 16

Characterization of mast cells in fetal barrier and extra-embryonic tissues

Chia, Shin Li; Morley, Rachel; Patatsos, Katelyn; Gentek, Rebecca

Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom

Introduction: Mast cells (MCs) are amongst the type of immune cells already present in the fetus. They have a dual hematopoietic origin: The first MCs are generated from yolk sac (YS)-derived erythro-myeloid progenitors (EMPs), followed by MCs originating from hematopoietic stem cells (HSCs)[1,2].  Fetal MCs contain intracellular granules filled with effector molecules (e.g. heparin), which suggests that they are already fully functional during these early stages. They have been implicated in the vascular and neuronal fine-tuning in the developing cornea[3]. Additionally, fetal MCs appear to promote the switch from scar-free healing to scar formation in wounded skin[4]. Apart from this, however, MCs remain incompletely characterized during development, and their physiological functions remain unclear at these early life stages. 

Objective: We hypothesize that fetal MCs might contribute to safeguarding fetal development. We thus studied MCs in tissues that can potentially sense the maternal environment, i.e. the barrier (skin, gut, and lung) and extraembryonic tissues. Here, we addressed if fetal MCs can be found in these tissues, characterized them phenotypically and determined their hematopoietic origins.  

Methods: Flow cytometry was used to identify and characterize fetal MCs during late gestation. To identify their origins, the Cdh5-CreERT2 x RosatdT fate mapping model was used, where 4-hydroxy-tamoxifen was administered at E7.5 or E10.5 to label YS- or HSC-derived MCs respectively.  

Results: We confirmed the presence of fetal MCs (c-kit+ ST2+ or c-kit+ avidin+) in the barrier tissues (skin, gut, lung) and also identified them in extraembryonic tissues (amniotic fluid, umbilical cord and fetal membrane, but not placenta). They uniformly express CD16/32, CD200R and CX3CR1, while the frequency of integrin α4β7 expression varies between tissues and developmental stages. Preliminary data show that extraembryonic MCs are predominantly YS EMP-derived, but also receive some HSC contribution. 

Discussion: Our data confirm the presence of fetal MCs in barrier tissues and identify them in extraembryonic tissues, where they have not previously been characterized and appear mostly of YS origin. Further work will continue to characterize MCs in barrier and extraembryonic tissues and address if they have a role in fetal development.  

References: 1. doi:10.1016/j.immuni.2018.04.025 2. doi:10.1016/j.immuni.2018.09.023 3. doi:10.1038/srep17569 4. doi:10.1038/JID.2011.324

 

 

 

Poster 17

Tryptase reference ranges are age-dependent in a large population-based cohort

Slot, Marjan; Claessen, Luuk; Bons, Judith; Menheere, Paul; Nieuwhof, Chris; de Boer, Douwe

Allergology and Clinical Immunology, Maastricht UMC, Maastricht, The Netherlands

Background. Tryptase is a mast cell specific mediator. Elevated tryptase levels can be an indication for an underlying systemic mastocytosis or a systemic allergic reaction. A few small company-driven studies have determined reference ranges for tryptase, denoting that every laboratory should determine its own reference range. Currently applied upper reference limit is 11.4 µg/L based on these studies.

Methods. In a large population-based cohort in Maastricht, the Netherlands, diagnostic samples for tryptase concentration testing were collected. We used the Bhattacharya method to determine tryptase reference ranges for our laboratory regarding to gender and age. 

Results. All consecutive patients with tryptase testing between December 2002 and January 2020 were included (N=7003). After excluding multiple measurements, the dataset included 5248 measurements. Data was normalized by logarithmic transformation and 97.5th percentile was calculated. Tryptase concentrations were dependent of age but not of gender. Age-based upper limit of the reference range varied from 8.74 µg/L (age 0-9 years) to 13.6 µg/L (age 70+ years) with lowest levels at age 10-19 years (7.28 µg/L).

Conclusions. Reference ranges for tryptase are generally lower than previously suggested, especially in young adults. These observations may also explain the published reports that show the risk of severe anaphylaxis is associated with tryptase levels >8 ng/mL. We recommend implementing our age-based reference ranges in general practice to prevent under- or over-diagnosis of mastocytosis or systemic allergic reactions.

 

 

 

Poster 18

SR-BI modulates the synergistic enhancement of FceRI-induced cytokine release from murine mast cells

Capellmann, Sandro; Huber, Michael

Institute of Biochemistry and Molecular Immunology, University Hospital RWTH Aachen, Aachen, Germany

The high-affinity receptor for IgE, FceRI, is responsible for antigen (Ag)-dependent mast cell (MC) activation triggered via multivalent Ag binding to IgE and subsequent crosslinking of multiple FceRI monomers. This induces rapid degranulation of pre-formed granule-stored mediators and secretion of arachidonic acid-derived lipid mediators and proinflammatory cytokines. As distinct from the T or B cell receptors, for both of which many accessory proteins modulating the receptor’s response have been identified, there is not much known about FceRI-associated proteins. We found that the HDL receptor scavenger receptor class B member I (SR-BI, gene name: Scarb1) is expressed in different murine and human MC models. SR-BI is associated with the FceRI at the plasma membrane (PM), which we could prove by co-internalization studies using murine bone marrow-derived MCs (BMMCs). SR-BI can mediate transport of cholesterol from the PM onto acceptor proteins in the surrounding milieu and vice versa, thereby regulating the PM cholesterol content. The SR-BI selective inhibitor BLT-1 reduced proinflammatory cytokine production in response to Ag and IL-33 in BMMCs as determined by ELISA. Knockout of Scarb1 in BMMCs revealed minor effects on degranulation measured by beta-hexosaminidase release or LAMP1 externalization. However, Scarb1-/- BMMCs exhibited reduced cytokine production upon IL-33 stimulation, as well as upon co-stimulation of Ag with stem cell factor (SCF), prostaglandin E2 (PGE2) or adenosine. The D4 domain of the exotoxin Perfringolysin O of the Gram-positive bacterium Clostridium perfringens selectively binds to cholesterol-rich regions in cellular membranes. Using the D4 domain coupled to GFP (GFP-D4) as a probe for high focal concentrations of cholesterol, we could show that Scarb1-/- BMMCs have reduced focally enriched cholesterol in the PM under basal conditions. Mimicking PM cholesterol reduction in wildtype BMMCs using cholesterol oxidase or methyl-beta-cyclodextrin revealed reduced activation of PLCg1 and the PI3K/AKT pathway. As both of these pathways have been described to be important for the synergistic activation of MCs by PGE2/Ag and SCF/Ag, respectively, we suppose that SR-BI is an important regulator of FceRI signaling contributing to the enhancement of proinflammatory cytokine production by affecting PLCg1 and/or PI3K/AKT activation.

 

 

 

Poster 19

Intra-individual biological variation of tryptase in humans is low

Slot, Marjan; Bons, Judith; Menheere, Paul; Nieuwhof, Chris; de Boer, Douwe

Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands

Introduction: For monitoring mastocytosis and the diagnosis of anaphylaxis the basal tryptase level and the individual reference range of tryptase in blood are important. However, the intra-individual biological variation (intra-BV) of tryptase is of equal importance. 

Objective: Establish the intra-BV of tryptase in patients suffering from mastocytosis and anaphylaxis.

Methods: To assess the intra-BV the tryptase data of 98 patients as measured in serum samples since 2005 were included. Data were fitted in iterative mathematical model based on a polynomial function, meaning after the removal of statistical outliers using Median Absolute Deviation, data were refitted until no outliers remained. Inclusion of patients required a data set of at least 6 remaining individual data points. Intra-BV was obtained by correcting the individual total observed variation (total-CV) for the analytical variation (anal-CV) according to following function: intra-BV = SQRT(SQR[total-CV2] – SQR[anal-CV]). Anal-CV was based on the analysis of internal quality control samples. Because of the slight right-skewness of the data, the overall intra-BV was calculated with a lognormal approach.

Results: Data sets of included patients (85 mastocytosis patients and 13 others) contained an average of 9 points collected over a mean period of 7.5 years for mastocytosis and 12 points over 2.8 years for others. Anal-CV was 5.2%. Median overall intra-BV and its confidence interval (CI) for mastocytosis was 5.6% (CI 4.8%-6.4%) and for other 6.0% (CI 5.4%-6.8%).

Discussion. The adequate method of determining intra-BV requires selecting a group of individuals in steady-state and measuring tryptase at regular intervals over a period which will reflect intra-BV. Important statistical considerations include determining amongst others significant sample size, outlier removal, and confidence intervals. This study fulfills those requirements and as such is a solid source for intra-BV for monitoring mastocytosis and the diagnosis of anaphylaxis.

Conclusions: The overall intra-BV of tryptase indicates that the biological variation is low and similar between different patients and similar to the analytical variation. This is important for interpretating results from patients who are monitored for the progress of their mastocytosis status or from patients who show possible elevated tryptase concentrations in case of suspected anaphylaxis.

 

 

 

Poster 20

Tryptase contribute to airway remodeling by inducing cell growth properties in lung epithelial cells

Berlin, Frida; Mogren, Sofia; Andersson, Cecilia

EMV, Lund University, Lund, Sweden

Introduction: Bronchial and alveolar remodelling and impaired function are characteristics of asthmatic disease. In these patients, increased number of mast cells (MCs) positive for serine proteases tryptase and chymase infiltrate the epithelium and alveolar parenchyma. However, the interaction between MCs and epithelial cells remains unknown.  

Objectives:  The study aim is to investigate if MC proteases tryptase and chymase have implications on bronchial and alveolar remodelling. Since asthmatics have chronic airway inflammation with viral induced exacerbation, we wanted to study these parameters in a pro-inflammatory and viral environment. 

Methods: Human bronchial and alveolar epithelial cells were treated with MC tryptase and/or chymase and co-stimulated with toll-like receptor agonist TLR3 to mimic a pro-inflammatory setting. Cell growth and division rates were assessed using holographic live cell imaging and a profiler gene array associated with cell survival. Data was confirmed with qPCR, western blot and immunocytochemistry. The epithelial release of vascular epithelial growth factor (VEGF) and fibroblastic growth factors (FGF) was analysed.   

Results: Tryptase enhanced cell growth and cell division rate in bronchial and alveolar cells when compared to non-stimulated cells, both in normal and pro-inflammatory/viral condition. The release of growth factors FGF and VEGF as well as the expression of anti-apoptotic proteins BIRC3 and BFL-1 were increased in tryptase treated cells, both at gene and protein level. No alterations were found in chymase treated cells compared to non-stimulated cells. 

Discussion: In this study, we show that MC tryptase, but not chymase, increases cell growth, anti-apoptotic gene and protein expression as well as release of growth factors in both bronchial and alveolar epithelial cells. These effects remained elevated in a pro-inflammatory and viral setting. Thus, our data implies that intraepithelial and alveolar MC release of tryptase may play a critical role in airway epithelial and alveolar homeostasis by affecting cell growth-death regulation. The tryptase induced release of growth factors may contribute to epithelial and submucosal remodelling and can further have implications on other structural cells. Apart from advancing the concept of the role of MCs in asthma, this observation may provide important indications regarding how to improve treatment strategies by targeting tryptase.

 

 

 

Poster 21

Mast cell tryptase potentiates neutrophil extracellular trap formation

Melo, Fabio; Alanazi, Sultan; Grujic, Mirjana; Adler, Jeremy; Olsson, Anna-Karin; Sommerhoff, Christian; Pejler, Gunnar

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

Previous research has indicated an intimate functional communication between mast cells and neutrophils during inflammatory conditions, but the nature of such communication is not fully understood. Activated neutrophils are known to release DNA-containing extracellular traps (NETs) and, based on the known ability of tryptase to interact with negatively charged polymers, we here hypothesized that tryptase might interact with NET-contained DNA and thereby regulate NET formation. In support of this, we show that tryptase markedly enhances NET formation in phorbol myristate acetate (PMA)-activated human neutrophils. Moreover, tryptase was found to bind vividly to the NETs, to cause proteolysis of core histones and to cause a reduction in the levels of citrullinated histone-3. Secretome analysis revealed that tryptase caused increased release of numerous neutrophil granule compounds, including gelatinase, lactoferrin and myeloperoxidase. We also show that DNA can induce the tetrameric, active organization of tryptase, suggesting that NET-contained DNA can maintain tryptase activity in the extracellular milieu. In line with such a scenario, DNA-stabilized tryptase was shown to efficiently degrade numerous pro-inflammatory compounds. Finally, we show that tryptase is associated with NET formation in vivo in a melanoma setting, and that NET formation in vivo is attenuated in mice lacking tryptase expression. Altogether, these findings reveal that NET formation can be regulated by mast cell tryptase, thus introducing a novel mechanism of communication between mast cells and neutrophils.

 

 

 

 

Poster 22

Mast cell progenitors are activated by IgE-crosslinking

Mendez Enriquez, Erika; Salomonsson, Maya; Janson, Christer; Malinovschi, Andrei; Hallgren, Jenny

Uppsala University, Uppsala, Sweden

Introduction. Mast cells are immune cells known for their involvement in asthma and allergy. They are activated through the crosslinking of IgE on FcRI with an antigen. This mode of activation induces immediate degranulation, followed by a rapid generation of lipid mediators and the synthesis and release of various cytokines. Mast cells are tissue-resident cells which develop from mast cell progenitors. During acute lung inflammation in mice, mast cell progenitors equipped with the FcεRI receptor are recruited to the inflammatory site. 

Objective. Because FcεRI-expressing mast cell progenitors are the dominating mast cell type during acute allergic lung inflammation in vivo, we hypothesized that they are activated by IgE cross-linking. 

Methods. Mouse peritoneal and human peripheral blood cells were sensitized and stimulated with antigen or stimulated with anti-IgE, and the mast cell progenitor population was analyzed for signs of activation by flow cytometry. Isolated peritoneal mast cell progenitors were studied before and after anti-IgE stimulation at a single-cell level by time-lapse fluorescence microscopy. Lung mast cell progenitors were analyzed for their ability to produce IL-13 by intracellular flow cytometry in a mouse model of ovalbumin-induced allergic airway inflammation. 

Results. Sensitized mouse peritoneal mast cell progenitors demonstrated increased levels of phosphorylation of tyrosines on intracellular proteins (total tyrosine phosphorylation), and spleen tyrosine kinase (Syk) phosphorylation after antigen exposure. Anti-IgE induced cell surface-associated lysosomal-associated membrane protein-1 (LAMP-1) in naive mast cell progenitors and prompted loss of fluorescence signal and altered morphology of isolated cells loaded with lysotracker. In human mast cell progenitors, anti-IgE increased total tyrosine phosphorylation, cell surface-associated LAMP-1, and CD63. Lung mast cell progenitors from mice with ovalbumin-induced allergic airway inflammation produced IL-13. 

Discussion. In summary, we show that IgE cross-linking induces activation of mouse and human mast cell progenitors. We suggest that these cells play an active role in acute allergic inflammation by producing cytokines such as IL-13.

 

 

 

Poster 23

Mast cells enhance airway hyperresponsiveness by releasing serotonin

Mendez-Enriquez, Erika; Alvarado-Vazquez, Perla Abigail; Abma, Willem; Simonsson, Oscar; Rodin, Sergey; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Malinovschi, Andrei; Janson, Christer; Adner, Mikael; Hallgren, Jenny

Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

Introduction: In allergic asthma, mast cells (MCs) accumulate at different lung sites and release mediators that are thought to play a vital role in the pathology. The expansion of lung MCs in experimental asthma is due to the recruitment of MC progenitors to the lung. MCs have been implicated in airway hyperresponsiveness (AHR) using MC deficient mice, and different antigens and strategies to induce allergic airway inflammation.  

Objective: To study the role of MCs in asthma using a mouse model that replicates the increase of MCs in the lung smooth muscles and epithelium. 

Methods:  Mice were sensitized repeatedly with house dust mite (HDM) extract to induce allergic airway inflammation. Cpa3cre/+ mice which lack MCs, and antagonists to block serotonin and muscarinic receptors were used to determine the role of MCs in antigen-induced bronchoconstriction and AHR to methacholine. Mouse lung-derived MCs, LAD-2, and human lung MCs were studied in vitro. 

Results:  The major MC population in the acute phase of HDM-induced experimental asthma were MC progenitors. The HDM protocol also induced accumulation of MCs in the lung epithelium and smooth muscles, and release of mMCP-1. HDM-induced airway contraction was lost in Cpa3cre/+ mice, by blocking serotonin receptors, and in mice lacking activating Fc receptors. HDM-induced AHR to methacholine measured as airway resistance in vivo was reduced by blocking serotonin receptors in Cpa3cre/+ mice. Airway contraction ex vivo, and lung-associated serotonin levels were reduced in Cpa3cre/+ mice with HDM-induced asthma. Primary mouse and human lung mast cells express muscarinic M3 receptors. Mouse lung mast cells store serotonin intracellularly, and methacholine induces the release of serotonin from lung-derived mouse mast cells and Ca2+ flux in LAD-2 cells.  

Discussion: Our data demonstrate that MCs are required for HDM-induced bronchoconstriction via Fc-receptor-mediated release of serotonin, causing serotonin receptor-mediated release of acetylcholine from nerves, which in turn promotes smooth muscle contraction. Importantly, we establish for the first time the significance of MC-derived serotonin for the enhancement of AHR. We suggest a mechanism in which methacholine directly activates M3-receptors on lung MCs, causing serotonin release, which induces serotonin receptor-mediated release of acetylcholine, and enhancement of airway contraction.

 

 

 

Poster 24

Mcpt8-driven cell depletion reduces lung mast cells in mice with allergic airway inflammation

Alvarado Vazquez, Perla Abigail; Cardenas, Eduardo I.; Das, Aviji; Hallgren, Jenny

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

Introduction: Genetically engineered mouse models have exploited the expression of mast cell protease-8 (mMCP-8), a known basophil marker, to study basophil function in vivo. However, mast cell (MC) progenitors also express Mcpt8 transcripts upon allergic airway inflammation.

Objective: To determine whether genetic depletion of mMCP-8-expressing cells impacts lung MC populations in mice with allergic airway inflammation.

Methods: For allergic airway inflammation induction, mice were sensitized intranasally with house dust mite (HDM) extract 7 times over 18 days. The expression of mMCP-8 was investigated in lung basophils and MCs from HDM-sensitized Basoph8 mice, which express a yellow fluorescent protein (YFP) in an Mcpt8-dependent manner. The effect of Mcpt8-driven deletion on lung MCs was investigated in the offspring of Basoph8 x Rosa-DTA mice or Basoph8 littermates by flow cytometry and by quantifying the number of tryptase expressing (mMCP-6+) MCs by immunofluorescence microscopy.

Results: At day 19, the lungs of HDM-sensitized Basoph8 mice contained around 97% YFP+ basophils. However, 37% of MC progenitors, 10% of the induced MCs, and 2% of mature MCs were also YFP+. In Basoph8 x Rosa-DTA mice, where Mcpt8-expressing cells were deleted, there was a 96% reduction in lung basophils and a 50% decrease of MC progenitors compared with Basoph8 littermates. In addition, mMCP-6+ lung cells were decreased by 28% on day 26, suggesting that a reduction in MC progenitors can translate into a decrease in total MCs at later time points. Unexpectedly, Basoph8 mice had a 5-fold increase in lung basophils compared to wild-type littermates.  

Discussion: Our results demonstrate that Mcpt8-driven cell ablation reduces the lung MC populations during allergic airway inflammation. Thus, MCs could be affected in other settings, which needs to be considered when using the Basoph8 mice to address basophil function. The mechanism behind the heightened infiltration of basophils observed in HDM-sensitized Basoph8 mice is still unknown. However, the high basophil numbers in the Basoph8 mice might exacerbate the importance of basophils in inflammatory models. To conclude, while it has been previously established that MC deficient strains have decreased basophil populations, the Basoph8 strain has decreased MC populations upon allergic airway inflammation.

 

 

 

Poster 25

SCF/KIT-evoked phosphoproteomics in skin mast cells: ERK predominates PI3K and capicua represses KIT

Franke, Kristin; Kirchner, Marieluise; Mertins, Philipp; Zuberbier, Torsten; Babina, Magda

Charité-Universitätsmedizin Berlin, Berlin, Germany

Introduction: The SCF/KIT axis represents a vital receptor tyrosine kinase (RTK) of mast cells (MCs). Despite considerable research into KIT, a comprehensive atlas of phosphorylation events downstream of this RTK is unavailable.

Objective: To evaluate global changes in phosphoproteins in human skin MCs elicited by the SCF/wildtype-KIT-axis, to couple early signaling events with cellular outcomes, and to identify novel regulators of the KIT network.

Methods: MCs were isolated from human skin. Mass spectrometry technique was performed to detect global phosphoproteomic changes upon SCF. Immunoblot was used to verify prominent findings. Different assays served to study basic cellular programs (survival, proliferation), while gene induction was studied by RT-qPCR and cytokine production by ELISA. Besides pharmacological inhibition of different kinases, siRNA-mediated knock-down was applied. CIC-specific siRNA served to investigate its role in downstream programs, while its translocation and degradation were inspected upon cellular fractioning.

Results: Out of over 10,500 class I phosphosites, around 5,400 sites (i.e. 51.5 %) were regulated upon SCF-induced KIT dimerization and we found ≈400 high-confidence sites not detected previously in any other cell type. Extensive validations of the mass spectrometry analysis confirmed the MEK/ERK cascade to be highly inducible by SCF surpassing STAT5>PI3K/Akt>p38>JNK. Direct comparison between MEK/ERK’s and PI3K’s support of basic programs (apoptosis, proliferation) revealed equipotency with substantial redundancy between the modules. In functional outputs, ERK showed dominance over PI3K regarding cytokine stimulation and gene expression. Additionally, the massive SCF-induced phosphorylation of capicua (CIC) led us to identify its function as KIT repressor, which potently interfered with SCF-mediated signaling, functional programs and cell survival. SCF induced CIC translocation and degradation in an ERK-dependent manner.

Discussion: Our study reveals the utility of an unbiased interrogation into the phosphoproteome to determine signaling networks downstream of a crucial RTK and provides a first-rate resource for scientists interested in general signal transduction and MC biology. Its comprehensive nature is excellently suited to experimentally connect signaling components to functional programs and uncover new influential entities. In fact, we uncover a negative circular relationship between KIT and CIC. CIC deregulation may well contribute to MC hyperplasia and mastocytosis.

 

 

 

Poster 26

Mast cell activation test: useful to discriminate patients with severe anaphylaxis?

Ollé, Laia; García, Lucía; Bartra, Joan; Pascal, Mariona; Roca-Ferrer, Jordi; Martín, Margarita; Muñoz-Cano, Rosa

Hospital Clinic de Barcelona, Barcelona, Spain

In the last decades, the incidence of food anaphylaxis has increased at an important rate and its risk is unpredictable so understanding the molecular regulation of anaphylactic shock is becoming essential in developing prevention and therapy. Traditionally, mast cells have been considered the main effector cells in patients with allergic reactions. For this reason, we sought to develop a mast cell activation test (MAT) to improve the current diagnostic techniques. Patients with food allergy to LTP (Lipid protein-transfer) were recruited and classified in three groups: group A (patients diagnosed with cofactor-exacerbated LTP allergy with a history of systemic reaction (anaphylaxis)), group B (patients diagnosed with allergy to LTP without cofactors involved, with history of systemic reaction (anaphylaxis)) and group X (patients sensitized to LTP who are asymptomatic or have mild local symptoms). Volunteers with no respiratory or food allergies were also recruited as control individuals for the study. Mast cells from each patient and healthy volunteers (huMCs) were obtained from peripheral blood and processed following the protocol previously described. Four patients from each group (A, B and X) were selected to create the serum pools to perform MAT. huMCs, from patients, sensitized with sera from pools A and B resulted in a higher CD63+ expression than those cells sensitized with sera from pool X after activation with peach LTP (Pru p 3) allergen extract. We obtained similar results with LAD2 cells. Our data suggest that the MAT response is dependent on the serum specific IgE levels and the IgE affinity. We conclude that MAT could discriminate patients that are in risk of develop severe symptoms among those that are sensitized to LTP.

 

 

 

Poster 27

Characterization of the IRE1α interactome in the mast cell leukemia cell line HMC-1.2 via TurboID

Ahmed, Nabil; Wilhelm, Thomas; Preisinger, Christian; Huber, Michael

Institute of Biochemistry and Molecular Immunology, Medical Faculty, RWTH Aachen University, Aachen, Germany

 

Mast cell leukemia (MCL) is the most aggressive form of systemic mastocytosis (SM). We have shown previously that proliferation/survival of the MCL cell line HMC-1.2 is dependent on the unfolded protein response (UPR).  The UPR is activated by accumulation of misfolded proteins in the ER and includes several mechanisms to maintain protein homeostasis such as optimized protein folding and degradation.  The ER stress sensor inositol-requiring enzyme 1α (IRE1α) contains a luminal N-terminal domain, and serine/threonine kinase and endoribonuclease domains in its cytoplasmic C-terminus. While the kinase domain activates signaling pathways like NF-κB and JNK, the endoribonuclease domain unconventionally splices an mRNA encoding the transcription factor XBP1. Depending on the strength/duration of ER stress, IRE1α forms dimers or oligomers, which affect the function of IRE1α.  We performed a proteomic-based screen using the optimized BirA biotin-ligase TurboID to identify interacting proteins involved in the regulation of IRE1α. For this analysis, ER stress was induced by Tunicamycin in HMC-1.2IRE1α-/- cells stably transduced with IRE1α or IRE1β constructs C-terminally fused to the biotin-ligase TurboID and compared to non-treated controls. Pulldowns identified biotin-positive partner proteins using streptavidin beads followed by LC-MS/MS analysis. Subsequently, principal component analysis (PCA) and hierarchical clustering were performed to identify and quantify the biotinylated proteins. Gene ontology (GO) analysis revealed that 50% of proteins are located in the cytosol. These proteins included those known to interact with activated IRE1α, such as protein translocation Sec63 and protein folding HSP90. Furthermore, some proteins like vesicle-trafficking protein SEC22 and coatomer subunit beta COPB2 were related to the ER-Golgi vesicle-mediated transport suggesting a role in mediating the transition to the trans-Golgi network. In addition, proteins involved in lysosome biogenesis like syntaxin-5 (STX5) and vesicle transport protein USE1 were identified as participating in the targeting and fusion of Golgi-derived retrograde transport vesicles, and may play a role in granule forming and loading in mast cells. We are currently confirming a direct interaction of the identified proteins by immunoprecipitation and confocal microscopy. We propose that our approach using IRE1α-TurboID could help to understand regulatory mechanisms affecting the function of IRE1α.

 

 

 

Poster 28

Hydrangea serrata extract ameliorates airway inflammation of CARAS exacerbated mouse model by PM2.5

JIN, JUAN; JIANG, YUNA; Nguyen, Thi Van; YU, ZHENNAN; Chai, Ok Hee; Song, Chang Ho

Anatomy, Jeonbuk National University, Jeonju, South Korea

Particulate matter (PM) is a significant health hazard globally, especially for the development of respiratory disorders. Chronic exposure to PM2.5 increases the risk of developing allergic rhinitis (AR) and asthma. When AR and asthma occur simultaneously, the actual terminology is combined AR and asthma syndrome (CARAS), a concept of single airway disease. Anti-histamines, decongestants and nasal corticosteroids used to treat airway disorders are partially effective and are frequently associated with side effects such as throat discomfort, headache, and dry eyes. Thus, a safer and natural compound with minimal side effects is needed at this juncture. Hydrangea serrata has therapeutic potential against airway inflammation, however, its influence on the OVA-induced CARAS exacerbated by the administration of PM2.5 stimulation is uncertain.  Hence, we assessed the effect of H. serrata extract (HSE) on airway inflammation and attempted to identify its mode of action using the CARAS mouse model. Here, CARAS mice were randomly segregated into 7 groups. Group 1, control; Group 2, CARAS; Group 3, CARAS with PM2.5; Group 4, 5, and 6 were CARAS mice exposed to PM2.5 and treated with 50, 100, and 200 mg/kg HSE, respectively. Group 7 consists of CARAS mice exposed to PM2.5 and treated with dexamethasone (Dex). All the test compounds were administered through oral gavage. We assessed nasal symptoms, inflammatory cells, OVA-specific immunoglobulins, cytokine production, mast cell activation and nasal histopathology to infer the influence of HSE on CARAS/PM2.5.  HSE substantially attenuated the degranulation of rat peritoneal mast cells by compound 48/80 in vitro. Also, HSE reduced nasal symptoms like rubbing and sneezing incidences dose-dependently. Moreover, HSE down-regulated OVA-specific IgE and up-regulated OVA-specific IgG2a in serum. HSE alleviated mucosal thickness, goblet cell hyperplasia, eosinophil and mast cell infiltration in nasal tissues. Further, HSE positively regulated allergic responses by reducing the accumulation of inflammatory cells and improving nasal and lung histopathological characteristics. Together, the administration of HSE effectively regulated the airway inflammation in CARAS exacerbated mice by PM2.5, suggesting the therapeutic potential of HSE in suppressing airway diseases.

 

 

 

Poster 29

Euonymus alatus extract alleviates PM 2.5 exacerbated-allergic airway inflammation

NGUYEN, THI VAN; YU, ZHENNAN; JIN, JUAN; Song, Chang Ho; Chai, Ok Hee

Anatomy, Medical school of Jeonbuk National University, Jeonju-si, South Korea

Exposure to particulate matters (PMs) can lead to an acute exacerbation of allergic inflammatory airway diseases, increasing the severity of symptoms and mortality. Combined allergic rhinitis and asthma syndrome (CARAS) recently has been defined as one allergic inflammatory condition in both upper and lower airway system. Euonymus alatus has been reported to have anti-inflammation, anti-hyperlipidemia and anti-cancer properties. However, there is no study about E.alatus extract (EAE) on an allergic airway inflammatory disease. Here, an OVA-induced CARAS exacerbated mouse model by PM 2.5 was used to evaluate the anti-inflammatory and anti-allergic effects and possible mechanism of EAE. EAE administration, especially EAE 200mg/kg dose, could improve airway inflammation by reducing inflammatory cells and mast cell infiltration, Th2 cytokines (IL-4, IL-5) and eotaxin in both NALF and BALF, depressing serum anti-OVA IgE and histamine levels, improving lung histopathologic changes. Also, EAE treatment significantly decreased MMP-9, increased the expression of E-cadherin and ocludin proteins in the epithelium, improved the epithelial barrier function.  Furthermore, EAE relieved the activation of MAPK signaling in the CARAS as well. Therefore, EAE may be an efficacious agent for treating allergic airway inflammatory disease.

 

 

 

Poster 30

Phosphorylation of KIT – an early attenuating mechanism in antigen-triggered mast cell activation

Bosch, Bastian; Preisinger, Christian; Gast, Mathias; Huber, Michael

Institute of Biochemistry and Molecular Immunology, University Clinic RWTH Aachen, Aachen, Germany

Introduction: Immune cell activation is under cooperative control of functionally interacting receptors. Two central receptors on mast cells (MCs) are the high-affinity receptor for IgE (FcεRI) and the receptor tyrosine kinase KIT. While the IgE-bound FcεRI is activated by crosslinking via multivalent antigen, KIT is dimerized/activated by stem cell factor (SCF). Combined activation with antigen and SCF results in synergistic pro-inflammatory MC activation. Determining protein tyrosine phosphorylations after anitgen stimulation using mass spectrometry (MS), KIT was found to be basally phosphorylated at several tyrosine residues; interestingly, KIT Y719 phosphorylation was slightly upregulated upon Ag stimulation.  

Methods and Results: Transient KIT Y719 phosphorylation upon antigen stimulation was verified in bone marrow derived MCs (BMMCs) by phospho-specific Western blotting. Using LYN-deficient BMMCs, we could reveal that KIT Y719 phosphorylation upon antigen stimulation is dependent on LYN. This was corroborated using the tyrosine kinase inhibitors imatinib and dasatinib, which suppress KIT and LYN/KIT, respectively. This suggests that KIT Y719 is not auto-phosphorylated after Ag stimulation, but might serve as a transmembrane adaptor phosphorylated by LYN. In line, KIT was co-internalized with FcεRI upon antigen stimulation, possibly allowing for the necessary vicinity of FcεRI-bound LYN and KIT. For the analysis of KIT-modified FcεRI-mediated effector functions, KIT-deficient BMMCs were differentiated in IL-3-only medium using BM from KitW-sh/W-sh mice and respective wildtype mice. These BMMCs showed increased degranulation upon antigen stimulation compared to WT BMMCs; no impact was observed on cytokine production suggesting an immediate role for KIT downstream of FcεRI aggregation. Fitting, antigen-triggered transcription of the immediate-early response gene Atf3 was stronger in KIT-deficient compared to wildtype BMMCs. Therefore, we performed a complete transcriptome analysis to compare transcriptional changes of WT vs. KIT-deficient BMMCs after 20 and 90 minutes of antigen treatment. The data is currently in bioinformatic evaluation. Next, a combination of KIT immunoprecipitation and MS analysis will be applied to decipher differences in the KIT interactome in antigen vs. SCF-stimulated MCs. 

Conclusion: We hypothesize that KIT besides its important receptor tyrosine kinase functionality also serves as a transmembrane adaptor organizing an attenuating signaling complex upon antigen-triggered MC activation.

 

 

 

Poster 31

Higher Basophil Activation Test Performance Flexibility by Prolonged Assay Read-Out of Fixed Cells

Berchtold, Martina; Vogt, Dominik; Melone, Anna; Romano, Michele; Shaw, Collin; Beck, Nathanael; Schuster, Thomas; Schneider, Michael; Gerhold, Christian-Benedikt; Gerspach, Michael

Product Development, BÜHLMANN Laboratories AG, Schönenbuch, Switzerland

Background: Basophil Activation Tests (BAT) have gained increasing importance in the field of allergy diagnostics, supported by growing scientific evidence for the higher accuracy and clinical relevance over other allergy tests. BAT is a functional assay relying on live basophils and detected by the low-mid throughput flow cytometry technology. Therefore, the appropriate conditions for the specimen storage and the management of the acquisition and analysis of processed samples are crucial for the spread use of the test. 

Objective: To improve logistics, practicability and time management of BAT testing, a novel version of the BÜHLMANN Basophil Activation Test which includes a stabilizing agent has been investigated for stimulated and processed basophils.

Methods: Two separated studies were performed with EDTA whole blood from four normal blood donors, using a mAb anti-FcƐRI stimulant. First, the specimen stability of unprocessed EDTA whole blood was assessed by storing the blood after drawing at different temperatures (2-8°C and 28°C) for 0 to 4 days before performing the BAT assay. Second, the stability of processed samples was assessed at different temperatures (2-8°C and 28°C) and measured at different time points (0 to 10 days) after cell stimulation and fixation with a new version of the BÜHLMANN Basophil Activation Test kit. 

Results: Unprocessed EDTA whole blood samples of all four donors were stable for 48 hours when stored at 2-8°C and for 24 hours stored at 28°C before basophil activation with at least 80% recovery compared to the initial basophil activation directly after blood collection. Processed and fixed basophils remained stable for the flow cytometry acquisition for at least 10 days at 2-8°C (min. 80% recovery), while for 28°C, 48 hours stability could be shown. 

Conclusion: Enabling trustable blood sample logistic, EDTA whole blood samples stored at 2-8°C gives a window of 48 hours until performing a BAT. The novel stabilizing agent prolonged the stability of the activated and fixed basophils prior of the flow cytometry acquisition up to 10 days at 2-8°C and to 2 days at room temperature. This significantly facilitates time management and hence practicability of BAT testing at laboratories that perform flow cytometry measurements.

 

 

 

 

 

Poster 32

The flow of COX to LOX stops at the rhyme; profiling refutes shunting in lung mast cells

Johnsson, Anna-Karin; Rönnberg, Elin; Fuchs, David; Kolmert, Johan; Wheelock, Craig E.; Nilsson, Gunnar; Dahlén, Sven-Erik

Karolinska Institutet, Solna, Sweden

Introduction: Lipid mediators, also termed oxylipins, are metabolites of ω-3 and ω-6 polyunsaturated fatty acids (PUFAs). Among them, the most well-known to play an important part of mast cell biology are the prostaglandins and leukotrienes, but in theory hundreds of different lipid mediators may be synthesized from arachidonic acid and other PUFAs. 

Objective: A lot of what is known about mast cell lipid mediator release has been gained from animal models, however, there are large variations between species. We therefor set out to profile the lipid mediator metabolome of human lung mast cells (HLMC).

Method: Using targeted UPLC-MS/MS with 113 analytes included, we have profiled the lipidmediator metabolome secreted by HLMC under steady state and after IgE-receptor activation. In order to examine further the enzymatic origin and interdependencies of lipid mediator production, we blocked the COX and 5-lipoxygenase pathways. 

Result: This revealed that COX-1 is the predominant COX enzyme in HLMC prostanoid production, that shunting occurs within the COX-1 pathway and that the COX-1 and 5-LOX pathways are disconnected. We also show that 15-HETE, often used as a marker for 15-lipoxygenase activity, is in HLMC in fact not generated by the 15-lipoxygenase pathway. 

Discussion: Using this large and sensitive mass spectrometry panel, we have an unbeatable tool to look at the broad and detailed picture of lipid mediator production, which can define the flow of lipid mediators and their metabolites along their biosynthetic routes.

 

 

 

Poster 33

The IRE1a inhibitor KIRA6 inhibits LYN and efficiently suppresses IgE-mediated mast cell activation

Wilhelm, Thomas; Wunderle, Veronika; Boukeileh, Shata; Margreiter, Michael; Sakurov, Roman; Bronneberg, Gina; Capellmann, Sandro; Martin, Christian; Schubert, Thomas; Levi-Schaffer, Francesca; Rosetti, Giulia; Tirosh, Boaz; Huber, Michael; Gossen, Jonas

Medical Faculty, RWTH Aachen University, Institute of Biochemistry and Molecular Immunology, Aachen, Germany

Antigen (Ag)-induced production of pro-inflammatory cytokines in mast cells (MCs) is controlled by several mechanisms. The stress sensor IRE1a promotes protein homeostasis during endoplasmic reticulum (ER) stress by unconventional splicing of Xbp1 mRNA leading to the formation of the transcription factor XBP1 that targets protein folding genes, but is also able to bind to the promoters of Il6 and Tnf. We initially used the IRE1a inhibitor KIRA6 to address the role of IRE1a for the production of pro-inflammatory cytokines in murine bone marrow-derived MCs (BMMCs). Unexpectedly, in addition to Ag-induced cytokine production KIRA6 completely blocked degranulation, Ca2+-mobilization, substrate tyrosine phosphorylation, and activation of various signaling pathways, strongly suggesting an IREa-independent effect. This was confirmed by KIRA6 titration, revealing a greater sensitivity for reduction of Ag-triggered cytokine production compared to ER-stress-induced Xbp1 splicing. Recently, we have described a comparable phenotype in Lyn-deficient BMMCs and therefore analyzed the effect of KIRA6 on LYN activity. Indeed, direct inhibition of LYN could be demonstrated using an in-vitro kinase assay. Moreover, applying microscale thermophoresis, direct binding of KIRA6 to recombinant LYN could be verified. Importantly, Lyn-deficient BMMCs lacked the KIRA6-dependent reduction of degranulation and cytokine production.  KIRA6 was also able to inhibit FceRI-mediated activation of the human MC line ROSA KIT WT and of cord blood-derived MCs. Next, the effect of KIRA6 on allergen-induced bronchoconstriction was analyzed in rat precision-cut lung slices, which was inhibited by KIRA6 in a concentration-dependent manner. In addition, computational molecular modeling emphasized the ability of KIRA6 to bind and block the active center of the kinase.  Our data clearly demonstrate that KIRA6 is an efficient inhibitor to block LYN activity for the prevention of FceRI-mediated release of MC mediators. Interestingly, we additionally could show that KIRA6 inhibits the activation of the receptor tyrosine kinase KIT, hence enabling a co-operative inhibition of two central MC receptors involved in pro-inflammatory MC activation.  Our data strongly suggest that the molecular structure of KIRA6 could serve as pharmacophore for development of efficient combined high-affinity inhibitors of FceRI and/or KIT-mediated MC activation.

 

 

 

Poster session 2, Monday July 12th, 2022

 

Poster 34

Mast cells respond to enteropathogenic bacteria in a lifestyle dependent manner

von Beek, Christopher

IMBIM, Uppsala University, Uppsala, Sweden

 

Mast cells can be found in many host-pathogen interfaces such as skin or the intestinal mucosa. Historically, their responses to bacterial pathogens have mainly been attributed to toll-like receptors (TLRs). However, the last decade revealed evidence that although TLR ligands and bacteria do elicit responses in mast cells, their potency is low compared to the mast cell’s answer to pathogen-specific virulence activities. In addition, while diverse bacteria are detected by TLRs, mast cells can differentiate pathogens from harmless bacteria by specific ways of detecting assaults on cellular integrity, such as membrane disturbance.  Since we and others established that mast cells recognize and respond to membrane stress by pore-forming toxins, secreted by e.g. Listeria, E. coli or Streptococci, we were curious if the needle-like type three secretion system (T3SS) that enteropathogens use to inject effector proteins into and invade host cells, causes a similar mast cell activation by membrane perturbance.  We measured immunomodulatory (il6, il13, tnf) responses of bone marrow-derived mast cells infected with Salmonella enterica serovar Typhimurium (S. Tm) or Shigella flexneri on transcript and protein level. By utilizing different T3SS-mutants (ΔinvG and ΔsipAsopBEE2) of S. Tm, we found that while mast cells respond slowly in a TLR4-mediated manner, full-blown mast cell activation requires secreted effector proteins rather than just membrane docking by the T3SS. Consistently, infection with S. flexneri without T3SS (ΔmxiD) resulted in a similar mast cell activation compared to S. Tm without T3SS or effectors. However, while S. Tm WT induced IL-6 secretion to a much higher extent than the noninvasive mutants, S. flexneri WT infection lead to a 2-fold reduction of IL-6 release compared to the mutant.  Overall, our findings contribute to the growing evidence of pathogen-specific immune responses in mast cells, rather then general reactions to bacteria. The differentiation between “guest” and “intruder” protects the body from unnecessary and potentially harmful immune responses.

 

 

 

Poster 35

Characterizing the impact of proteolytic cleavage of C1-Inhibitor in MC-mediated angioedema

Vera Ayala, Carolina Elisa; Maurer, Marcus; Scheffel, Jörg

Institute of Allergy / Charité & Fraunhofer, Berlin, Germany

Angioedema is a potentially life threatening disease, which is defined as a transient swelling of the gastrointestinal or respiratory tract mucosa or the skin due to increased vascular permeability induced by vasoactive mediators such as histamine or bradykinin. Three forms of angioedema – with various subclasses – have been proposed, I) a mast cell mediator-related form (also referred to as histaminergic form), II) a bradykinin-related form, and III) an idiopathic form (with potential involvement of bradykinin. One of the features in mast cell (MC)-associated chronic inflammation is the release of mediators including histamine, prostaglandins, leukotrienes and various proteases such as tryptase and chymase. These proteases are known to cleave a large array of proteins and are suggested to contribute to the development of recurrent AE. In some patients, occurrence of AE has been proposed to be due to the cleavage of C1-Inhibitor. Therefore, we assessed the effects of the MC proteases tryptase and chymase on C1-Inhibitor. Supernatant obtained from degranulated, cultured human MCs cleaved purified as well as recombinant C1-Inhibitor, resulting in a protein with reduced size as shown by SDS-PAGE and coomassie staining. Using purified human tryptase and chymase, we confirmed that chymase cleaves C1-Inhibitor, potentially at the N-terminus of the molecule. Importantly, chymase-cleaved C1-Inhibitor showed reduced inhibitory capacity in a chromogenic FXIIa/kallikrein activity assay. These findings demonstrate that C1-Inhibitor is a potential target of MC chymase. Cleavage by chymase results in decreased efficacy of C1-Inhibitor to control the contact system.  Based on these findings we hypothesize that the release of chymase by activated mast cells in patients with CSU may contribute to kallikrein-activation and the generation of bradykinin by proteolytic cleavage of C1-Inhibitor thereby reducing its inhibitory function. This process may contribute to angioedema formation in patients with CSU and other forms of mast cell mediator-related angioedema.

 

 

 

Poster 36

Crucial role for mast cell-derived IL-17A in psoriasis

Benezeder, Theresa; Painsi, Clemens; Zhan, Qian; Lange-Asschenfeldt, Bernhard; Clark, Rachael; Wolf, Peter

Department of Dermatology, Medical University of Graz, Graz, Austria

The proinflammatory cytokine IL-17A plays a major role in the inflammatory cascade of psoriasis. The current perception is that Th17 cells are the major source of IL-17A, however, immunostaining has shown that neutrophils and mast cells also express IL-17A in psoriasis. Transcriptional analyses revealed that IL-17A mRNA is not expressed by mast cells and thus it was hypothesized, that mast cells do not produce the cytokine themselves, but rather store it in their granules. We performed immunostaining of T cells (CD3), neutrophils (myeloperoxidase), mast cells (tryptase), combined with IL-17A staining, in samples from psoriatic skin and neonatal foreskin. Cell counts for IL-17A positive mast cells were significantly increased in lesional psoriatic skin compared to non-lesional and healthy skin. At baseline, mast cells comprised the majority of IL-17A expressing cells (83%), while some neutrophils were IL-17A positive (14%) and only very few T cells were IL-17A positive (3%). In resolved lesions (after topical treatment with dithranol), IL-17A positive mast cell counts remained high, whereas all other IL-17A expressing cells had nearly completely vanished. In fact, mast cells can express IL-17A in absence of T cells, as evidenced by immunostaining of neonatal foreskin containing hardly any T cells.  Our results show that mast cells comprise the majority of IL-17A positive cells in psoriatic skin and persist in clinically resolved lesional skin. Mast cell-derived IL-17A might be essential to restart the inflammatory feedback loop that leads to recurrence of psoriasis.

 

 

 

Poster 37

Targeting mast-cell plasticity re-shapes the tumor microenvironment of melanoma

Iuliano, Caterina; Kaesler, Susanne; Biedermann, Tilo

Department Allergy and Immunology, Klinikum Rechts der Isar, Dermatologie am Biederstein, Munich, Germany

Introduction: The incidence of malignant melanoma is steadily rising. Due to its metastatic potential the cancer has a poor prognosis and is responsible for 80% of patients with skin cancer dying. The treatment of metastatic melanoma was revolutionized by the development of new immunotherapies, however long-term treatment responses are still limited to few patients and complete remissions are rare due to therapy resistance. Therefore, there is need for new therapeutic options. Resistance to therapy in malignant melanoma is linked to an increase of phenotypic heterogeneity of melanoma cells in their responsiveness to pro-inflammatory signals from the tumor microenvironment (TME). Within the TME of melanoma, an accumulation of MCs has been found, especially in areas of regression. We have recently demonstrated that LPS-activated tumor-resident MCs initiate effective anti-melanoma immune control.  Objective: Since MCs are plastic in regard to their functions and can secrete various mediators, we are currently investigating the impact of endogenous factors that are found in the TME of melanoma and how they impact MC and thereby mediate the control of melanoma.   Methods: To decipher the role of MC plasticity in melanoma progression, we isolated MC precursors from murine femur (BMMCs) and matured them in medium containing IL-3 and stem cell factor. Maturity of MCs was determined by flow cytometry, gating for CD117 (c-kit) and high-affinity IgE receptor (FcεRI) positive cells. To simulate the endogenous TME, the cells were stimulated with the alarmin IL-33 in combination with TLR-agonists. Results: RNA Sequencing showed a strong enrichment in genes involved in cytokine-mediated signaling pathways in BMMC stimulated with IL-33. The combinations of stimuli acted antagonistically or synergistically on MC regarding the secretion of cytokines that were detected in the supernatant. Upon the highest concentration of detected cytokines were CCL3 and CCL22, which play a crucial role in the recruitment of immune cells. Supernatant from stimulated MC also showed an inhibitory effect on B16 cells in vitro. In vivo experiments using the B16 melanoma model in mast-cell deficient Mcpt5-cre RDTA mice showed that the combined treatment of tumors with IL33R-TLR2/6 inhibits tumor progression in a mast-cell dependent manner, by modulating the TME.

 

 

 

Poster 38

Variants of the MRGPRX2 gene in patients with immediate hypersensitivity reactions to drugs

Quan, Paola Leonor; Guo, Yanru; Sabaté-Brescó, Marina; Ollé, Laia; Munoz-Cano, Rosa; Laguna, Jose Julio; Doña, Inmaculada; Martín, Margarita; Gastaminza, Gabriel

Department of Allergy and Clinical Immunology, Clínica Universidad de Navarra, Pamplona, Spain

Introduction: Management of drug hypersensitivity reactions (DHRs) is challenging, especially when evidence of an IgE-mediated mechanism is missing. Characterizing alternative pathways will facilitate risk-stratification and treatment in affected individuals. The novel Mas-related G-protein coupled receptor-X2 (MRGPRX2), selectively expressed in connective tissue mast cells (MC), has been identified as a target for quinolones and neuromuscular blocking agents. Furthermore, our group has shown that degranulation responses induced by morphine, vancomycin and cisatracurium are decreased in MRGPRX2-silenced MCs. In this study, we sought to analyze whether patients with reactions to known receptor ligands showed variants in the MRGPRX2 gene. 

Methods: We prospectively recruited patients who suffered immediate hypersensitivity reactions that met specific criteria and that were induced by quinolones, neuromuscular blocking agents, opiates, or vancomycin. Patients with chronic urticarias or NSAID hypersensitivity were excluded. An IgE-mediated mechanism was explored 4 to 8 weeks after the reaction and only patients who had negative skin testing to the index drug and to latex, chlorhexidine, and other suspected inducers were selected. Venous blood samples were collected to perform whole-exome sequencing (WES). Intracellular calcium concentrations in HEK-293 cells transfected with the selected variants were measured after stimulation with substance P. 

Results: A total of 13 patients (mean age: 46.5 +/- 4.7 years) met the specified inclusion criteria. Nine patients suffered reactions to quinolones, 3 had perioperative reactions, and 1 reacted to vancomycin. Five of the reactions met clinical criteria for anaphylaxis. A total of 3 exonic nonsynonymous variants of the MRGRPX2 gene, found in 4 of the studied patients, were detected. Calcium influx responses in HEK-293 cells transfected with one of the variants, or with the other two variants combined, were higher than those observed in cells expressing wild-type MRGPRX2.  

Discussion: So far, gain-of-function variants for MRGRPX2 have yet to be reported in the context of DHRs. In this analysis of patients with immediate reactions to target drugs, preliminary in vitro data show that some of the variants detected in the MRGPRX2 gene potentially augment activation responses in transfected cells. This finding provides insights into the factors which could predispose individuals to reactions mediated by the receptor.

 

 

 

Poster 39

Effect of exercise on basophil activation and PGE2 secretion in patients with food anaphylaxis

García, Lucía; Casas, Rocío; Pérez, María; Ollé, Laia; Martín, Margarita; Bartra, Joan; Pascal, Mariona; Roca-Ferrer, Jordi; Muñoz-Cano, Rosa

Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

Introduction/Objective: Cofactors, such as exercise, have been shown to modulate the severity of food anaphylaxis (FA) but the mechanism underlying remains unknown. It has been reported that serum PGE2 increases in response to exercise as a physiological response. Our aim was to analyze the effect of exercise on both PGE2 production and basophil activation in FA patients.

Methods: FA patients (N=10) and healthy subjects (HS, N=6) were recruited. All underwent an exercise stress test following a cycle ergometer-based model. Blood samples were collected pre-exercise (T0) and 30 minutes after finishing the exercise, post-exercise (T30). A basophil activation test (BAT) (activation measured by CD63 expression by flow cytometry) after stimulation with anti-FcεRI (SC) was performed at both timepoints. PGE2 concentration in both serum and BAT supernatant was measured by ELISA at T0 and T30. Results were expressed as median (25th-75th percentile).

Results: Post-exercise serum PGE2 showed an increasing trend in HS (T0=75.9[34.1-156.8]pg/ml; T30=190.6[42.9-374.9]pg/ml; p=0.063) with no increase in FA (T0=82.8[51.5-159.8]pg/ml; T30=68.2[43.6-92.5]; p=0.688). PGE2 levels in BAT supernatant of post-exercise SC stimulated basophils showed a decreasing trend in FA (T0=31.0[23.2-47.3]pg/ml; T30=26.8[19.9-44.1]pg/ml; p=0.074) with no decrease in HS (T0=28.1[21.8-36.7]pg/ml; T30=27.8[20.8-42.5]pg/ml; p=0.563). Basophil activation post SC stimulation was increased but not different when comparing HS with FA. Indeed, there were not differences in pre and post-exercise in none of the groups.

Conclusions: Exercise had no effect on basophil activation measured by means of CD63 expression. However, exercise trends to increase serum PGE2 in HS, but no effect was observed in FA. Therefore, a deficiency in PGE2 production in response to exercise may partially explain the cofactor effect.

 

 

 

Poster 40

An exploratory study on lymphocytes during VIT in patients with or without clonal mast cell diseases

Teufelberger, Andrea R.; Bokanovic, Danijela; Cerpes, Urban; Laipold, Karin; Dan, Andrada R.; Kränke, Birger; Wolf, Peter

Dermatology and Venerology, Medical University Graz, Graz, Austria

Introduction: Loss of tolerance towards Hymenoptera venom after termination of venom immunotherapy (VIT) is a well-known phenomenon for patients with clonal mast cell diseases (CMDs) – in contrast to non-CMD patients – which results in a recommended life-long VIT. It is, however, not known which factors lead to this loss of tolerance.

Objective: We compared CMD and non-CMD patients to determine whether there are differences in T and B cell subsets, induced by the VIT treatment, which could explain the consequent loss of tolerance in CMD patients.

Methods: Blood samples of a total of 15 CMD and 17 non-CMD patients were included in this first exploratory study phase. All patients had experienced anaphylaxis (Ring and Messmer grade III or IV) after a wasp or bee sting and were sampled either before or during VIT treatment. Serum tryptase, KIT D816V mutation, total IgE, and wasp- and bee venom specific IgE were measured in all patients to determine the disease status. PBMCs were analyzed by flow cytometry to quantify T and B memory- and T and B regulatory cell subsets, and stimulated with wasp venom to measure their reactivity by proliferation.

Results: CMD patients had significantly elevated serum tryptase levels compared to non-CMD patients, which was expected. CMD patients, who underwent VIT for more than three years, had significantly lower cell numbers of CD4+ T effector memory cells (TEMs), than CMD patients before VIT treatment, while TEM cell numbers in the respective treatment groups of non-CMD patients did not differ. Wasp venom stimulation of PBMCs induced proliferation of TEMs, which was more pronounced in CMD than in non-CMD patients.

Discussion: Our preliminary findings indicate that TEMs could be relevant in tolerance development during VIT. Furthermore, TEMs of CMD patients might differ in their responsiveness towards wasp venom from non-CMD patients. To substantiate these findings, more patient samples will need to be gathered in the future.

 

 

 

Poster 41

Peripheral mast cell-derived RANKL is a prerequisite for lymphocyte egress from distant lymph nodes

Katsoulis-Dimitriou, Konstantinos; Dudeck, Anne

Institute for molecular and clinical immunology, Clinical University Magdeburg, Magdeburg, Germany

Introduction: Mast cells (MC) are key initiators of vasoactivation and immune cell infiltration upon allergic contact dermatitis. We recently identified MCs as a prominent source of receptor activator of NFκB (RANKL), which is mostly known for being involved in bone resorption, with more information coming to light about its qualities as an immune regulator. However, the relevance of MC-derived RANKL in skin inflammation is completely unknown. 

Methods: Using a conditional RANKL knockout in connective tissue type MCs mouse line, we identified a crucial role of MC-derived RANKL in contact hypersensitivity. 

Results and discussion: Surprisingly, mice lacking MC-derived RANKL displayed massive lymphocyte hyperplasia in inguinal lymph nodes (LN) 24h after DNFB challenge, accompanied by profound blood lymphopenia. This was a temporal effect, suggesting that lymphocyte egress is delayed in the absence of MC-derived RANKL. However, despite an increased immune cell infiltration at 48h after challenge, skin inflammation remained markedly dampened. Strikingly, MC depletion and reconstitution with RANKL deficient MCs only locally in the ear skin resembled inguinal LN hyperplasia, blood lymphopenia and reduced lymphocyte infiltration to inflamed skin 24h post DNFB challenge. Importantly, the phenotype of lymph node hyperplasia and blood lymphopenia could be rescued by intravascular injection of Shingosine-1-phosphate (S1P) 2h after challenge. 

Conclusion: Consequently, RANKL release by peripheral skin MCs upon DNFB challenge exerts a long-distance effect, involving S1P signalling, that is a prerequisite for the timely lymphocyte egress from remote LNs, which is in turn critical for the onset and severity of allergic skin inflammation.

 

 

 

Poster 42

Mast cell reduction does not lead to impaired cutaneous wound healing in humans

Frenzel, Maren; Kiefer, Lea Alice; Grekowitz, Eva Maria; Hawro, Tomasz; Metz, Martin; Altrichter, Sabine; Alvarado, Diego; Crowley, Elizabeth; Heath-Chiozzi, Margo; Maurer, Marcus; Terhorst-Molawi, Dorothea

Charité – Universitätsmedizin Berlin, Berlin, Germany

Introduction: Several previous studies in mice indicate a role for skin mast cells in murine wound healing. As of now, the role and relevance of MCs in human skin wound healing remains unknown. Barzolvolimab (CDX-0159) is a humanized monoclonal antibody (mAb) that inhibits SCF-dependent KIT activation, and is shown to reduce clinical activity and deplete cutaneous mast cells in patients with chronic inducible urticaria (CIndU) providing a unique opportunity to study the contribution of mast cells to human wound healing. 

Objective: To characterize the effects of MC depletion on the healing of cutaneous wounds in humans.  

Methods: CIndU patients enrolled in a phase 1 trial (NCT04548869) received a single intravenous dose of barzolvolimab (3 mg/kg). Full thickness skin wounds were induced on the upper inner arm (3 mm biopsy punches) predose and 1, 4, 8, and 12 weeks after treatment (n=14). Wound areas were determined by quantitative morphometry of digital images obtained daily. Skin MC numbers at the site and time of wounding were quantified on KIT-stained sections.  Results Skin MC numbers were significantly reduced after treatment with barzolvolimab, from 9.7±3.8 MCs/mm2 at baseline to 4.4±2.8 (-55%), 1.3±1.6 (-87%), 0.9±2.1 (-91%), and 1.2±1.7 (-87%) MCs/mm2 at week 1, 4, 8, and 12, respectively. Skin wounds induced before MC depletion showed an average reduction of wound size to 47±12% (n=14), on day 7 after wounding, with complete closure by day 19±6. Wound healing in MC-depleted skin was very similar, with an average time to complete closure of 17±4, 17±4, 18±5, and 20±4 days in wounds induced 1, 4, 8, and 12 weeks post-treatment, respectively. The average wound size reduction on day 7 after wounding, in wounds induced 1, 4, 8, and 12 weeks after dosing, was 53±8%, 49±11%, 51±16%, and 53±14%, respectively. 

Discussion: In humans, the depletion of skin MCs does not affect the speed or duration of wound healing. This indicates that human MCs are dispensable for normal healing of small cutaneous wounds, different from skin wound healing in mice.

 

 

 

Poster 43

A novel intra-lobular airway ex-vivo guinea pig model to study the role of relaxant EP receptors

Nie, Mu; Liu, Jielu; Säfholm, Jesper; Adner, Mikael

The Institute of Environmental Medicine, Karolinska Institutet, Solna, Sweden

Introduction: Postmortem bronchoconstriction, the phenomenon that occurs in guinea pigs soon after their death, makes it impossible to inflate or deflate the lungs rendering functional investigations impossible. Mast cells associated contractile mediators such as leukotrienes, histamines, and prostanoids have been suggested to play a role in this irreversible constriction. Prostaglandin E2 has a powerful effect on the basal tone in the guinea pig trachea by activating EP receptors. However, the effect of the EP receptors in guinea pig isolated small airways are still unknown.

Objective: The aim of this study was to create an intra-lobular small airway ex-vivo model by preventing postmortem bronchoconstriction to investigate the relaxant role of EP receptors in small airways.

Methods: Six male albino guinea pigs (Dunkin-Hartley, 479-785g) were sacrificed with an overdose of pentobarbital following a procedure described in the result section. Functional experiments were performed using myographs recording isometric tension and force was normalized to contraction generated by 60 mM potassium chloride. To study the relaxant role of EP receptors, segments were pre-contracted by carbachol to 75% of maximal contractility and then exposed to either the EP2 receptor agonist ONO-AE1-259 or EP4 receptor agonist TCS2510.  

Results: To generate a model without the postmortem bronchoconstriction, the lungs were, directly after death, filled with Krebs-Henseleit buffer containing a cocktail of pharmacological agents including histamine 1 receptor antagonist pyrilamine, FLAP inhibitor MK-886, TP receptor antagonist SQ-29538, and β2 agonist salbutamol. After dissection, the small airways segments were incubated in a DMEM-F12 medium without the pharmacological agents over a period of 48 hours which successfully allowed the segments to respond to both contractile and relaxant mediators. To investigate the role of the relaxant EP receptor, both ONO-AE1-259 and TCS2510 caused a concentration dependent relaxation (46.95±13.98% and 16.75±9.37%, respectively) of pre-contracted segments.

Conclusion: This ex-vivo animal model blocked the effect of mast cell mediators in combination with a general broncho-relaxant therapy to prevent bronchiole collapse, which provide a new animal model for the investigation of intralobular small airways in guinea pigs. Both EP2 and EP4 receptor induced a relaxation, but the EP2 receptor was more efficacious.

 

 

 

Poster 44

Characterization of MRGPRX2 as a novel target to address mast-cell mediated disorders

Viswanath, Veena; Boehm, Marcus; Pittner, Richard; Dvorak, Lisa; Napora, Jim; Vest, Alan; Charlot, Brittney; Vasquez, Alexis; Freeman, David; Pisacane, Corine; Anderson, Samantha; Kim, Andrew; Villescaz, Christiane; Solomon, Michelle; Wollam, Joshua

Translational Biology, Escient Pharmaceuticals, San Diego, United States

Introduction: Mast cells are innate immune cells involved in the pathophysiology of many diseases, including disorders of the skin, airways, and gastrointestinal tract.  Mas- Related G-Protein Coupled Receptor X2 (MRGPRX2) (mouse ortholog Mrgprb2) is a promiscuous receptor expressed on mast cells that mediates non-histaminergic, IgE-independent mast cell activation and degranulation in response to a wide variety of agonists including endogenous neuropeptides such as substance P, PACAP etc., microbial defense peptides, and also a number of pseudo-allergic drugs. 

Objective: To support the development of MRGPRX2 antagonists, we aimed to characterize the biology of MRGPRX2 in vitro and in vivo, including the ability of structurally diverse MRGPRX2 agonists to induce mast cell degranulation and the interaction between the MRGPRX2 and IgE pathways.  

Methods: The downstream effects of selected agonists to activate human MRGPRX2 were studied utilizing commercially engineered overexpressing cell lines, LAD2 mast cells as well as primary human mast cells derived from peripheral stem cells (PSCMCs).  We also generated Mrgprb2 knockout mice to evaluate functional consequences of mast cell activation in vivo. In addition, we created a novel, functional human MRGPRX2 knockin transgenic mouse model to enable in vivo evaluation of optimized human MRGPRX2-specific antagonists. 

Results: MRGPRX2 agonists were found to activate both MRGPRX2 and Mrgprb2 overexpressing cell lines with differing efficacy and potency.  We have extensively characterized MRGPRX2 agonists and show that they induce degranulation of LAD2 mast cells and PSCMCs.  Furthermore, we demonstrate that the MRGPRX2 and IgE pathways act independently to induce mast cell degranulation in LAD2 mast cells.   Additional measures of cytokines and tryptase release from mast cells have added to our understanding of the effects of MRGPRX2 activation.  We expand these significant findings in vivo to demonstrate that MRGPRX2 is the primary receptor mediating agonist-induced mast cell degranulation and vascular leakage in our proprietary transgenic knockout and knockin animals.

Discussion: Taken together, these findings expand our understanding of MRGPRX2 biology that supports MRGPRX2 as a promising target for inhibiting IgE-independent mast cell degranulation and point to (oral) MRGPRX2 antagonism as an attractive, highly differentiated approach to the treatment of mast cell mediated disorders.

 

 

 

Poster 45

Neuro-immune interactions in milk allergy: Food allergy, hippocampal progenitors, and mental health

Houghton, Vikki; Kwok, Matthew; Lee, Hyunah; Thuret, Sandrine; Santos, Alexandra

King’s College London, London, United Kingdom

Background: Food allergy has no curative treatment and current management strategies predominantly focus on allergen avoidance. These strategies have been associated with higher anxiety in children, and food allergic reactions themselves may lead to a PTSD-like syndrome in some patients, yet the underlying neurobiological mechanisms are poorly understood. Owing to its relationship with a range of psychiatric disorders, one potential mechanism is hippocampal neurogenesis (HN). HN occurs along a highly vascularised niche and thus can be influenced by blood-borne factors circulating in the systemic environment. 

Objective: To investigate the association between food allergy and hippocampal neurogenesis. 

Methods: Blood samples from 60 patients (10 milk- and 10 egg allergic children who experienced severe reactions; 10 milk and 10 egg allergic who experienced non-severe reactions and 10 non-milk and 10 non-egg allergic children) were tested in two in vitro models: (i) Human mast cells, sensitised in patients’ plasma, are used to quantify CD63+ cells, following allergen stimulation; and (ii) HN readouts are quantified via immunocytochemistry, using human hippocampal progenitor cells exposed to patients’ serum.  

Results: Egg allergic children had a higher proportion of CD63+ mast cells compared to non-allergic children, and severe allergic more so. The HN assay demonstrated that egg allergy status accounted for ~34% of the variance in neurogenic outcomes. Specifically, severe egg-allergic patients exhibited increased apoptosis (%CC3+ cells) during both proliferation and differentiation, increased proliferation (%Ki67+ cells) and increased %MAP2+ neurons during differentiation, compared with non-allergic patients. Despite decreased number of DAPI+ cells during the proliferative stage, severe allergic children samples demonstrated no difference in cell count during differentiation compared with non-allergic children, suggesting no changes to net neurogenesis.  

Discussion: These data demonstrate a relationship between food allergic and HN readouts for egg-allergic children, and the milk-allergy data to come will inform whether this is an allergen-specific effect, or a wider effect of food allergy in general. Further investigation will be conducted to (i) compare food allergic reactions and HN readouts to measures of anxiety and cognition in children, and (ii) explore the underlying biological mechanisms, including the effects of mast cell degranulation on hippocampal progenitor cell fate.

 

 

 

 

Poster 46

Application of hollow microneedles in models of allergy and CU

Shi, Nana

Institute of Allergology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany

Introduction: The pathomechanisms of chronic inflammatory skin diseases such as urticaria, psoriasis, or and atopic dermatitis are complex and challenging to investigate. Histomorphometric analyses of skin biopsies combined with the analysis of blood, serum and skin microdialysis (SMD) samples are gold-standard tests in skin research. However, these tests are either invasive, or samples are difficult to obtain and with the risk of infection. Moreover, locally produced tissue biomarkers are often diluted in the circulation below detection thresholds of routinely used assays.  

Objective: To improve diagnostic options and facilitate biomarker discovery we have developed a dermal interstitial fluid (ISF) extraction method using sharp hollow microneedle chips (hMNs). 

Methods and Results: Microneedle chips with 37 hollow microneedles of 420 µm length were manufactured from monocrystalline silicon wafers and applied to human abdominal ex vivo skin for ISF extraction. On average, a volume of 12.6 µl could be extracted with hMNs by application of -70 kpa subpressure (skin soaked in PBS for 2 h). The recovery of biomolecules such as histamine or immunoglobulins in the ISF was enhanced when extracted with hMNs compared to sampling by SMD. In addition, we could demonstrate that this method can be applied in ex vivo skin-serum transfer models for allergy and inducible urticaria, e.g. cold urticaria (ColdU) and symptomatic dermographism (SD). Mast cell degranulation, as indicated by an increase of histamine in the ISF, could be detected in response to injection of serum of an allergic patient followed by injection of the respective allergen, whereas this was not the case when serum of a non- allergic donor was injected. Similarly, injection of SD or ColdU serum followed by application of the respective trigger, i.e. cooling and rewarming or scratching of the skin, induced a detectable increase of histamine in the ISF. 

Discussion: In summary, we have developed a novel, accessible, efficient, and minimally invasive tool for sampling ISF and used it successfully in ex vivo models of allergy and inducible urticaria to detect MC degranulation. Thus, analysis of ISF extracted by hollow microneedles is a promising approach for research and diagnosis of various skin diseases.

 

 

 

Poster 47

IgE antibodies promote mast cell-mediated detoxification of bee venom

Starkl, Philipp; Gaudenzio, Nicolas; Marichal, Thomas; Reber, Laurent L.; Sibilano, Riccardo; Watzenboeck, Martin; Fontaine, Frédéric; Mueller, André C.; Tsai, Mindy; Knapp, Sylvia; Galli, Stephen J.

Dept. of Medicine I, Medical University of Vienna, Vienna, Austria

The reason for the evolutionary conservation of IgE antibodies and mast cells – despite their dangerous and detrimental function in allergic diseases – is one of the biggest riddles of immunology. One controversial explanation was provided by Margie Profet’s theoretical concept “the Toxin Hypothesis”, proposing important functions of allergic reactions in host defense against noxious substances. Recent findings in mice support this idea, indicating critical contributions of IgE antibodies and effector mast cells in acquired host defense against whole honeybee venom or venom components. In the current study, we dissected the specific effects of IgE antibodies on mast cell sensitivity to honeybee venom and explored the mechanisms of venom detoxification.  Using live cell imaging and flow cytometry approaches, we found that honeybee venom-specific serum IgE antibodies markedly increase the venom sensitivity, signal transduction and extent of degranulation of primary mast cells in vitro. IgE-activated mouse or human mast cells showed highly efficient, heparin- and protease-dependent venom detoxification capacity in cell-based toxicity assays. Proteomics analysis revealed specific mast cell-mediated degradation of numerous honeybee venom constituents, including major allergens.  Our results highlight the intriguing venom detoxification potential of IgE-sensitized mast cells and underline the importance of their preformed granule-stored mediators.

 

 

 

Poster 48

BAT interference study on positive controls to pharmaceuticals and abnormal blood conditions

Gerspach, Michael

Special Products Development, BÜHLMANN Laboratories AG, Schönenbuch, Switzerland

Introduction: Basophil Activation Test (BAT) has emerged as a new diagnostic test in the field of allergy and as a research tool for new drug development for kinase inhibition (PI3K, SYK, BTK) or allergy treatment. 

Objective: For diagnostic testing as well as comparison studies for drug development using basophil activation it is crucial, that pharmaceuticals, or abnormal blood conditions of patients and normal whole blood donors do not lead to a systematic bias on the stimulation controls (positive controls) of the BAT. 

Methods: The study was performed with EDTA whole blood from four normal blood donors, spiked with the potential interference substance. A total of 13 substances were investigated, including eight (8) pharmaceuticals (antiallergic drugs, mast cell stabilizers, leukotriene antagonists, cortisone medication and anti-IgE antibody omalizumab), four abnormal blood conditions (triglyceride, bilirubin, and hemolysis) and one sample additive (K-EDTA). The interference study was performed according to CLSI guideline EP07-A2. Bias in results exceeding 20 % for stimulation control anti-FcεRI mAb (PC1) and 20 % CD63+ (absolute) for stimulation control FMLP (PC2) was considered as an interference. 

Results: For PC1 the observed difference (dobs) ranged between -16 % and +12 % (relative), which was within the stated acceptance criteria of ±20 % relative difference in CD63+ cells and were thus considered as noninterfering. One exception was found for K-EDTA, for which one donor exhibited a -108 % difference. All other donors were between -8 % to -4 % for K-EDTA, i.e. less than ±20 % deviation. For the PC2, dobs ranged between -17 % and +9 % CD63+ and was also considered as noninterference according to the stated acceptance criteria of ±20 % absolute difference in CD63+ cells. Omalizumab was as well tested with both stimulation controls on four (4) donors with no indication of interference. 

Discussion: No interferences were detected at the tested concentrations on the stimulation controls anti-FcεRI mAb and FMLP, except for excess K-EDTA (short venipuncture draw) for one donor. Although no interference was found for the substances (incl. omalizumab) on the stimulation controls, interference with BAT on diagnostic results cannot be excluded.

 

 

 

Poster 49

Elevated, FcεRI -dependent MRGPRX2 expression on basophils in chronic urticaria

Bartko, Ewa

LMA, Rødovre, Denmark

Background: Chronic urticaria (CU) is a skin condition driven by mast cells and basophils. The exact responsiveness profile of these cells, especially regarding the anti-IgE treatment, Omalizumab, is not fully investigated. We sought to characterize surface activation profile of basophils in CU during Omalizumab treatment and their responsiveness to IgE and non-IgE stimulation. 

Methods:  Whole blood basophils from 11 CSU patients and 10 healthy controls were simultaneously stained and stimulated with either medium, 4 – 4000ng/mL anti-IgE, 0.5 µg/mL fMLP, 1000 ng/mL C5a or 10 µM Substance P for 30 min at 37°C, followed by flow cytometric readout.

Results:  CU patients showed broad range of basophil count. Statistically increased number of unstimulated CD69+, but not CD63+ basophils was observed in CU groups in comparison to healthy. The expression of CD203c and CD200R were comparable between the groups, whilst the FcεRI was significantly reduced with the treatment. Both IgE and non-IgE mediated stimulation upregulated CD63, CD203c and CD200R, but not CD69 in all groups, however no statistical significance between the groups was observed. The resting cell expression of MRGPRX2 was significantly higher in the after treatment than in healthy group. The anti-IgE increased the % of MRGPRX2+ basophils that was significantly elevated in the CU groups in contrary to healthy. The fMLP and C5a stimulations had the same effect, however the % of MRGPRX+ basophils triggered by a stimulant was lower than with IgE mediated stimulation. SP did not activate basophils.

Conclusion: CU basophils show increased expression of MRGPRX2 after IgE and non-IgE stimulation.

 

 

 

Poster 50

Differential activation of mast cells by human effector T cells and regulatory T cells

He, Jiajun; Frischbutter, Stefan; Maurer, Marcus

Institute of Allergology, Charité – Universitätsmedizin Berlin, Berlin, Germany

Introduction: Skin-infiltrating or tissue-resident T-effector cells (Teff) and their secreted mediators are known to be involved in MC recruitment (e.g. IL-9), activation (e.g. IL-4, IL-5, IL-13) and survival (e.g. IL-3). In contrast, regulatory T cells (Treg) have been described to dampen or enhance mast cell activation depending on the experimental condition. Thus, T cells modulate MC responses and might contribute to chronic mast cell (MC)-driven diseases such as chronic spontaneous urticaria. However, previous studies on MC-Tcell interactions mainly used murine cells or knock-out mouse models while comprehensive studies investigating the influence of different human T cells subsets (i.e. Teff and Treg) on primary human MCs are lacking. 

Objective: In this preliminary study, we aimed to dissect the response of primary human skin MCs after direct contact with peripheral Teff and Treg and their respective secreted mediators.

Method: Freshly isolated human primary CD4+ Teff and Treg from human peripheral blood were activated overnight using antibodies against CD3 and CD28, and cell culture supernatant was collected. Primary human skin MC were primed with IgE overnight and then activated with anti-IgE in the presence or absence of Teff (ratio 1:1) or Treg (ratio: 1:1) or their respective culture supernatants. MC activation was assessed after 30 min by measuring CD63 surface level by flow cytometry.

Results: Mast cell CD63 levels increased after IgE-mediated activation in the presence of Teff or their supernatant as compared to anti-IgE stimulation alone (%CD63: anti-IgE: 45.2 ± 9.3; anti-IgE +Teff: 55.83 ± 9.3; anti-IgE +Teff-supernatant: 71.8 (n=2)). Interestingly, MC-Treg co-culture increased CD63 (anti-IgE +Treg 50.4 % ± 7.8), whereas Treg-supernatant decreased CD63 level (anti-IgE +Treg-supernatant 24.45% (n=2)).

Discussion: We observed a differential influence of primary human T cell subsets on skin MC activation, which should encourage attempts to identify and characterize the underlying molecular mechanisms of these effects.  Targeting these mechanisms and MC-Tcell interaction could benefit the development of novel treatments of diseases where MC and Tcell, together, contribute to the pathogenesis.

 

 

 

Poster 51

Increased number of MRGPRX2+ cells in the skin of ISM patients is not linked to symptom severity

Pyatilova, Polina; Ashry, Tameem; Luo, Yanyan; He, Jiajun; Bonnekoh, Hanna; Jiao, Qingqing; Moñino-Romero, Sherezade; Hu, Man; Scheffel, Jörg; Frischbutter, Stefan; Hermans, Maud; Youngblood, Bradford; Maurer, Marcus; Siebenhaar, Frank; Kolkhir, Pavel

Institute of Allergology, Charite, Berlin, Germany

Background: Recently, expression of the mast cell (MC) receptor Mas-related G protein–coupled receptor X2 (MRGPRX2) has been detected in lesional skin of adult patients with cutaneous mastocytosis. As of yet, little is known about the clinical relevance of MRGPRX2 and its agonists in patients with mastocytosis, including indolent systemic mastocytosis (ISM). 

Methods: MRGPRX2 and MRGPRX2 agonists, cortistatin (CST) and major basic protein, were analyzed in lesional and nonlesional skin of patients with ISM and skin of healthy controls by immunohistochemistry. Co-localization of MRGPRX2 and MRGPRX2-mRNA with the MC marker tryptase was assessed by immunofluorescence microscopy and in situ hybridization, respectively. In addition, MRGPRX2, CST, substance P and eosinophilic cationic protein (ECP) levels in blood were analyzed by ELISA. We assessed clinical, demographic and laboratory data, including mastocytosis activity score (MAS), serum tryptase and KIT D816V mutation allele burden. 

Results: The number of MRGPRX2-expressing (MRGPRX2+) cells, MRGPRX2-mRNA+ MCs and CST-expressing (CST+) cells was significantly higher in lesional skin as compared to nonlesional skin and/or skin of healthy controls (all p<0.05). In ISM patients, median levels of MRGPRX2 and the MRGPRX2 agonists in blood, i.e. ECP, CST and substance P, were significantly lower or comparable to those in healthy controls. Increased numbers of MRGPRX2+ cells, MRGPRX2-mRNA+ MCs, CST+ cells and eosinophils as well as blood levels of MRGPRX2 and its agonists were not associated with clinical and laboratory features of ISM, including disease severity, evidence of anaphylaxis and tryptase levels.  

Conclusions: Skin lesions of patients with ISM showed high numbers of MRGPRX2+ cells, although they as well as blood levels of MRGPRX2 agonists were not linked to symptom severity. Clinical relevance of the MRGPRX2-mediated pathway of mast cell activation in ISM remains unclear and should be investigated in further studies.

 

 

 

Poster 52

Systemic mast cell activation does not affect atherosclerosis development in LDLr-/-Rag1-/- mice

Delfos, Lucie; Foks, Amanda C.; Bernabé Kleijn, Mireia N.A.; Kuiper, Johan; Bot, Ilze

Division of BioTherapeutics, LACDR, Leiden University, Leiden, The Netherlands

Introduction and objective: Acute cardiovascular syndromes are the main cause of death in the Western society, of which atherosclerosis is the underlying disease pathology. In atherosclerosis, plaques are formed due to the accumulation of lipids and immune cells, such as macrophages, mast cells and T cells, in the vessel wall. Mast cells are implicated in atherosclerotic plaque development via the release of amongs others proteases and cytokines, such as IL-6, TNF-α and IFNγ, but may also contribute to plaque development through interactions with adaptive immune cells such as T- and B cells. The relative contribution of these indirect cellular mechanisms to atherosclerotic lesion development is however unknown. In this study, we therefore studied the effects of mast cell activation on atherosclerosis in the absence of T- and B cells. 

Methods: Male LDLr-/-Rag1-/- mice (3 months old), deficient for T- and B cells, were fed a Western-type diet for 12 weeks. During these 12 weeks mice (n=15) were injected intraperitoneally once weekly with 1 µg αDNP-IgE on day 1 and intravenously with 0.5 mg DNP on day 2 to induce a systemic mast cell activation. Control mice (n=15) were injected with PBS. The mice were sacrificed in week 12 to determine atherosclerotic plaque size and morphology in the aortic root. Serum cytokine levels were measured with a Legendplex assay. 

Results: Plasma total cholesterol levels and total body weight were not affected by systemic mast cell activation during the experiment. At sacrifice, plasma IL-6 levels were significantly higher in the mast cell activated group (IgE: 88.4±18.1 pg/mL) compared to the control group (controls: 41.6±8.3 pg/mL, p<0.05), indicating that the systemic mast cell activation protocol was successful. In the aortic root, plaque size did not differ between the groups (IgE: 32±6*103μm2 versus controls: 38±6*103 μm2). Also, the relative MOMA-2 content, which indicates the presence of macrophages was similar in both groups (IgE: 38.2±3.2% versus controls: 40.2±3.2%). 

Discussion: Mast cell activation did not affect atherosclerotic plaque development and macrophage content, in LDLr-/-Rag1-/- mice, suggesting that mast cells contribute to early atherosclerosis at least partly via T- and B cell interactions.

 

 

 

Poster 53

The impact of IgE-induced mast cell degranulation on murine auditory hair cells

Zeng, Bin; Fuchs, Julia; Hegend, Olga; Petzhold, Friederike; Monino-Romero, Sherezade; Scheffel, Joerg; Szczepek, Agnes; Siebenhaar, Frank

Institute of Allergology, Charité – Universitätsmedizin Berlin, Berlin, Germany

Introduction: Mast cells (MC) are important immune cells of the myeloid lineage and are proven to be present in the murine cochlea. Studies have suggested that Immunoglobulin E (IgE)-mediated hypersensitivity may contribute to the development of inner ear diseases, including hearing disorders. However, MC and MC mediators’ role in the inner ear remains unknown.

Objective: We investigated the effect of MC-derived mediators released from MCs upon IgE/anti-IgE stimulation on the auditory outer and inner hair cells (HC).

Methods and Results: As an ex vivo model of the inner ear, we used the organ of Corti (OC) explants isolated from p3-5 C57BL/6 mice of both genders. The explants were cultured for 24h, followed by 24h incubation with supernatants derived from degranulated bone marrow-derived MCs induced by IgE/anti-IgE treatment for 1h. Outer and inner HCs in the cochlea were identified by phalloidin staining and their presence and morphology were assessed by fluorescence microscope imaging. We observed a significant decrease (*p<0.05, Student’s t-test) in the number of HCs after incubation with MC supernatant (the equivalent of 10 MCs) in the experimental group (n=13) compared to the control group incubated with supernatant of unstimulated MCs (n=12). In addition, cell morphology differed between the experimental and control groups. The inner hair cell number decreased from a mean of 11.67±0.45 (SD) to 4.45±0.50 in the apical part, 11.50±0.47 to 6.15±0.23 in the middle, and 11.92±0.43 to 6.38±0.45 in the basal part of the cochlea. The outer hair cell number decreased from 41.58±0.80 to 20.30+1.46 in the apical part, 42.08±0.99 to 23.77±1.38 in the middle part, and 46.83±0.67 to 23.92±1.42 in the basal part of the cochlea.

Discussion: We demonstrated that MC degranulation leads to the loss of murine auditory HC, providing an insight into the effects of MC on HC function, homeostasis, and pathology. Future studies are needed to explore the relationship between the resident inner ear MC and their functional relevance in patients affected by MC-driven diseases.

 

 

 

Poster 54

Anti-FcεRI mAb MAR-1 depletes basophils and cross-reacts with myeloid cells through its Fc Portion

Worrall, William; Kamphuis, Jasper; Stackowicz, Julien; Mougel, Aurélie; Mauré, Émilie; Pecalvel, Cyprien; Brûlé, Sébastien; Bruhns, Pierre; Guilleminault, Laurent; Reber, Laurent

Asthma Allergy and Immunotherapy Research Group, Toulouse Institute of Infectious and Inflammatory Diseases, Toulouse, France

Introduction: MAR-1 is a prominent anti-mouse FcεRI Armenian hamster IgG antibody used to stain FcεRI on mast cells and basophils both in vitro and in vivo. It has also been used to induce FcεRI-mediated anaphylaxis as well as deplete basophils in mice to examine their role in disease. It is reported that MAR-1 cross-reacts with FcγRI and IV, however, the nature of this cross-reactivity is not clear.  

Objective: To investigate the mechanism of MAR-1 interaction with FcγRs, and the consequences of such interaction on MAR-1-induced anaphylaxis and basophil depletion in vivo. 

Methods: We have compared the binding profiles of MAR-1 and a recombinant version of MAR-1 unable to engage FcRs through its Fc portion (“Fc silent” format) on different cell types of wildtype or FcεRI-deficient mice by flow cytometry. We have compared FcεRI-crosslinking-induced systemic anaphylaxis and basophil depletion by intraperitoneal injection of MAR-1 or Fc silent MAR-1 in wildtype, in mast cell-deficient, and in mFcεRI-deficient mice. 

Results: MAR-1 marks mast cells and basophils through its interaction with FcεRI. However, we show that MAR-1 also recognises myeloid cells such as monocytes and macrophages in an FcεRI-independent manner, by engaging IgG receptors (FcγRI and FcγRIV) through its Fc portion. Basophil depletion is dependent on the Fc portion of MAR-1, while MAR-1-induced systemic anaphylaxis is mostly dependent on FcεRI-crosslinking on mast cells. 

Discussion: MAR-1 is an important tool and the only readily available antibody for the murine FcεRI. We recommend using an Fc silent format of this antibody to avoid confounding FcεRI expression with the non-specific binding of FcγRs. Although MAR-1 can be used to deplete basophils we propose that genetic and inducible models of basophil depletion be used to verify and corroborate results obtained with this antibody, because to the many off-target effects. Our results demonstrate that the expression of FcεRI is restricted to mast cells and basophils in mice, and call into question prior results obtained using MAR-1.

 

 

 

Poster 55

Human IgE cross-reacting with major peanut allergens induce mast cell degranulation and anaphylaxis

Pecalvel, Cyprien; Leveque, Edouard; Casanovas, Natacha; Kamphuis, Jasper B.J; Bruhns, Pierre; Balbino, Bianca; Guilleminault, Laurent; Michelet, Marine; Reber, Laurent L

Asthma Allergy and Immunotherapy Inserm UMR1291, Toulouse, France

Background: Peanut allergy is the leading pediatric food allergy and the major cause of fatal or near-fatal anaphylaxis. Recently, conserved high-affinity IgE clonal families with cross-reactivity to major peanut allergens were identified in several peanut allergic subjects. It is possible that the cross-reactive nature of these IgE clones makes them particularly potent at inducing allergic reactions. 

Objective: To characterize the ability of recombinant human IgE clones at inducing mast cell activation and anaphylaxis. 

Methods: We used a highly sensitive bioluminescent IgE detection method (LuLISA) to assess binding of several recombinant monoclonal peanut-specific human IgE to the major peanut allergens Ara h 1, 2, 3 and 6. We tested the ability of these IgE clones to induce human mast cell degranulation in vitro, and anaphylaxis in FcRI humanized mice.  

Results: We identified human IgE clones cross-reacting with all major peanut allergens Ara h 1, 2, 3 and 6. Sensitization of human mast cells with these cross-reactive IgE clones induced strong degranulation of human mast cells upon stimulation with peanut. FcRI humanized mice sensitized with these cross-reactive IgE also developed anaphylaxis upon challenge with peanut extract. 

Conclusion: We identified human IgE clones which cross-react with the major peanut allergens Ara h 1, 2, 3 and 6. These clones are particularly potent at inducing human mast cell activation in vitro, and anaphylaxis in humanized mice. It is thus possible that the severity of peanut anaphylaxis might be linked to the presence of such cross-reactive IgE clones.

 

 

 

Poster 56

Inhibition of mast cell function through sialylation of peanut allergens

Castenmiller, Charlotte

Experimental Immunology, Amsterdam UMC, Amsterdam, The Netherlands

Mast cells are one of the primary effector immune cells in IgE-mediated food allergy. Cross-linking of FcεRI via binding of IgE and allergens results in release of mediators that induce pathological responses. Although for some allergies immunotherapies are available, there are still major drawbacks, including long treatments and risk on severe side effects. Therefore, novel therapies are required. Here, we studied the potential of inhibiting mast cell functions through modification of the peanut allergen Ara h 2 with sialic acid. Sialic acids binds to sialic acid-binding immunoglobulin-type lectins (Siglecs), of which some contain an intracellular immunoreceptor tyrosine-based inhibitory motif. In this study, we used mast cells (hMCs) derived from human CD34+ stem cell progenitors. Siglec expression analysis by flow cytometry revealed the expression of a wide array of Siglecs, including Siglec-2, -3, -5, -6, -7, -8 and -10. Furthermore, hMCs were loaded with serum from peanut allergic patients and subsequently exposed to Ara h 2 or α2,3-sialic acid Ara h 2. Sialylation of Ara h 2 resulted in a decrease in β-hexosamindase release and IL-8 secretion. Altogether, these data indicate that modification of allergens with sialic acid reduces mast cell function and is a promising strategy for innovative immunotherapies.

 

 

 

Poster 57

Molecular profiling of basophils and eosinophils in a tropical urban environment for allergy risk

Andiappan, Anand Kumar

Allergy biomarkers, Singapore Immunology Network, Singapore, Singapore

Introduction: Allergies in tropical urban environments are on the rise and in Singapore we report nearly 50% of children report allergic disease. Allergies are also associated with increased incidence of obesity and adversely impacts mental well-being. We have reported a highly dominant mono-sensitisation of house dust mites in our population and thus hope to identify key pathways underlying the immunopathobiology of airway allergies such as asthma and allergic rhinitis (AR) .

Objective: Here we aim to understand the role of allergic immune cells such as eosinophils and basophils by profiling their transcriptomic landscape and single cell proteomics. 

Methods: We use whole transcriptomic sequencing, single cell proteomics and functional assays to evaluate the immune mechanisms and pathways underlying the immunopathophysiology of allergic diseases.  

Results: We identified candidate ENPP3 (CD203c) as a marker of basophil anergy and associated with reduced risk of allergic rhinitis. The CD203c levels at steady state correlated with CD63 degranulation on stimulation with allergens. Furthermore we also found an association of SNPs in this gene to expression levels. For the eosinophils we found an enrichment of counts in allergic patients compared to controls while basophils was not significantly different. Interestingly, we also isolated eosinophils from whole blood and then associated to allergies and found HES1 and HRASLS5 as potential candidates for further evaluation. Interestingly we also found that IL5RA, OLIG2 and P2RY2 are associated to AR with concomitant asthma. At the pathway level we also identified activation and proliferation of eosinophils as key enriched mechanisms and  associated to disease severity. Currently we are performing single cell sequencing to evaluate the immune sub-clusters in these allergic mediator cells in blood and nasal tissues.  

Discussion: Basophil degranulation and eosinophil activation are critical for allergic pathogenesis and disease severity. However, clear biomarkers and mechanistic pathways for therapeutic interventions need further validation. Hence we use a highly sensitised urban environment of Singapore to understand the role of basophils and mast cells in allergic airway diseases and evaluate if there are any potential candidates which can be further exploited for diagnosis or intervention.

 

 

 

 

 

Poster 58

Mouse naturalization — an effective approach for shaping lung tissue distribution of mast cells

Xiang, Zou; Yeh, Yu-Wen – Presenting Author; Liu, Shuwei; Zewdie, Olifan; Sen Chaudhuri, Arka; Boysen, Preben

Department of Health Technology and Informatics, Hong Kong Polytechnic University, Hong Kong, Hong Kong

It is well documented that laboratory mice (including C57BL/6 or BALB/c) profoundly lack lung parenchymal tissue mast cells, which stands in clear contrast to the abundant presence of mast cells in human lungs. Therefore, the clinical relevance of conventional mouse asthma models has been challenged. Accumulating evidence suggests that wild mice, which display a richer microbiota composition compared to ultra-hygienic specific pathogen-free (SPF) laboratory mice, may be a more relevant model to mimic human immunity. We recently reported the distribution of mast cells at a substantial density in the lung tissues of wild mice trapped in Oslo, Norway and Hemtabad, India. Consistently, wild mice also expressed higher pulmonary levels of stem cell factor. Having speculated that microbiota could have impacted the lung tissue residency of mast cells, we next bred C57BL/6 laboratory mice in purposefully built enclosures with bedding material regularly brought in from the natural environment with wild rodent activity, in order to normalize the microbiota of these laboratory mice to that of the wild mice. Interestingly, the offspring which were born and spent their entire life (eight weeks) in this environment did develop lung mast cells at an appreciable density. However, adult C57BL/6 mice transferred from a clean SPF barrier failed to develop lung mast cells even if they had spent the same amount of time in this “dirty” environment. These findings supported a critical neonatal period for the modulation of microbiota composition. We obtained similar results using natural bedding materials in two different locations, Norway and Hong Kong. As wild populations of mice present various problems as a research tool (e.g., unreliable supply, large individual variation, etc.), our “naturalization” or “re-wilding” approach may provide a practical solution to the establishment of mouse models harbouring lung mast cells, which are expected to more closely mimic human asthmatic responses.

 

 

 

Poster 59

The Mast Cell Activation Test is a Useful Tool for Assessing Nut Allergy

Steinert, Carolin; Scheffel, Jörg; Dölle-Bierke, Sabine; Worm, Margitta; Beyer, Kirsten; Maurer, Marcus; Altrichter, Sabine

Institute of Allergology, Charité – Universitätsmedizin Berlin, Berlin, Germany

Introduction:  Routine diagnostic tools of IgE-mediated food allergy include skin prick test and specific IgE (sIgE) measurements. However, these tests only assess sensitization status and not true clinical reactivity. The basophil activation test has been developed to assess clinical allergic reactivity of patients. However, using basophils has several limitations including fresh blood sampling and challenging timely analysis. As mast cells are accepted as key players in allergy, the mast cell activation test (MAT) has been suggested as an alternative. 

Methods: To assess the usefulness of the MAT in peanut and hazelnut allergy (hereafter: nut allergy), peripheral CD34+ stem cell-derived mast cells (PSCMCs) as well as skin derived mast cells (hsMCs) were passively sensitized with serum samples from well characterized adults diagnosed either as 1) nut allergic (n=31) or 2) nut sensitized but clinical tolerant (n=9) or 3) non-allergic (n=7).  After stimulation with nut extract, activation was assessed by CD63 surface staining and mast cell mediator release.  

Results: Allergen stimulation of mast cells sensitized with serum from nut allergic patients resulted in a dose-dependent activation. This was not observed for mast cells that were either sensitized with serum from tolerant or non-allergic subjects. Specifically, maximal PSCMC and hsMC activation measured as CD63 upregulation significantly differed between allergic and tolerant patients (PSCMCs: median (allergic) = 25.5%, median (tolerant) = 3.3%, p = 0.0133; hsMCs:  median (allergic) = 9%,  median (tolerant) = 3.2%, p = 0.0003). Maximum mast cell activation was strongly correlated with serum levels of sIgE against allergen extract (r(PSCMCs) = 0.7404, p = 0.0003; r(hsMCs) = 0.9071, p < 0.00031), i.e. the higher the serum levels the stronger the activation of mast cells.  

Discussion: The MAT is suitable for measurements of functional IgE in nut allergic patients. Two different mast cell populations can be used, including PSCMCs, which are readily available. The MAT should be considered for development as a tool for routine clinical practice.

 

 

 

Poster 60

Selenomethionine attenuates human primary mast cells responses in soy allergy

Zhao, Xiaoli; Blokhuis, Bart; Redegeld, Frank; Chang, Iris; Dunham, Diane; Chen, Hongbing; Nadeau, Kari; Garssen, Johan; Knippels, Leon; Hogenkamp, Astrid

Pharmacology, Utrecht University, Utrecht, The Netherlands

Background: Selenium is an essential micronutrient that plays a crucial role in immune response. Selenomethionine (SeMet), an organic form of selenium, is the main nutritional source of selenium for animals and humans. We previously demonstrated that dietary SeMet supplementation reduced murine mast cell protease-1 (mMCP-1) levels in a murine model of whey-induced food allergy. The mechanism underlying this finding has not been elucidated yet, but direct effects of SeMet on mast cell degranulation may be involved. 

Aim: In this study, we investigated whether SeMet could affect mast cell responses in an in vitro model for soy allergy.

Methods: Soy protein isolate (SPI), β-conglycinin (7S) and glycinin (11S) were isolated from soy from the USA and China. Primary human mast cells (PHMC) were preincubated with SeMet, sensitized with sera from healthy donors or sera from soy allergic patients and challenged with SPI. β-hexosaminidase (β-hex) release, interleukin 8(IL-8)production, calcium flux, high-affinity immunoglobulin (Ig)E receptor (FcεRI) expression and IgE binding ability were measured. 

Results: Soy protein isolated from USA soy induced more mast cell degranulation than soy protein isolated from Chinese soy and degranulation was greater in cells incubated with SPI compared to 7S and 11S. Furthermore, our data suggest that SeMet prevented IgE binding to FcεRI rather than affecting crosslinking of IgE by the allergen.

Conclusion: These results show that selenium can attenuate mast cell activation and degranulation, demonstrating that adequate levels of micronutrients should not be overlooked in the treatment of allergic disease.

 

 

 

Poster 61

A novel approach for studying mast cell: induced pluripotent stem cell derived mast cell

Luo, Yanyan; Fernandez-vallone, Valeria; He, Jiajun; Frischbutter, Stefan; kolkhir, pavel; Moñino-Romero, Sherezade; Stachelscheid, Harald; Maurer, Marcus; Siebenhaar, Frank; Scheffel, Jörg

Institute of Allergology, Charité – Universitätsmedizin Berlin, Berlin, Germany

Introduction: To study functions of mast cells (MCs) in vitro, human primary MCs are isolated directly from several tissues or differentiated from hematopoietic progenitors. However, these techniques bear several disadvantages and challenges including low proliferation capacity, donor-dependent heterogeneity, and the lack of a continuous cell source.

Objective:  To address this, we developed a novel strategy for the rapid and efficient differentiation of MCs from human induced pluripotent stem cells (hiPSC).

Methods: A four-step protocol for the generation of hiPSC derived MCs (hiPSC-MCs), based on the use of three hiPSC-lines, was established and validated by comparison with human skin MCs (hsMCs) and peripheral hematopoietic stem cell-derived MCs (PSCMCs).

 Results: hiPSC-MCs share phenotypic and functional characteristics of hsMCs and PSCMCs. They are MCTC (tryptase and chymase double positive MC), display stable expression of the MC-associated receptors CD117, FcεRIα, MRGPRX2 and degranulate in response to IgE/anti-IgE and substance P.  

Conclusion: This novel hiPSC-based approach provides a sustainable and homogeneous source for a rapid and highly productive generation of phenotypically mature, functional MCs and its principle allows for the investigation of disease- and patient-specific MC populations.

 

 

 

Poster 62

Elevated IgE levels in the early phase of SARS-CoV-2 infection

Simone Saia, Rafael; Giusti, Humberto; Luis-Silva, Fabio; Bonicenha Pedroso, Karina; Auxiliadora-Martins, Maria; Morejon, Karen; Degiovani, Augusto; Cadelca, Marcia; Basile-Filho, Anibal

Physiology, University of Sao Paulo, Ribeirão Preto, Brazil

Introduction: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the causative agent of the most recent pandemic, which promotes the activation of the host immune response. Immunoglobulin E (IgE) participates on the immune hypersensitivity, allergy, asthma and parasitic infections. However, respiratory viral infections also induce the production of IgE and it may contribute to the pathophysiology of airway dysfunction. 

Objectives: To evaluate the systemic levels of IgE on the course of the SARS-CoV-2 infection. 

Methods: SARS-CoV-2 infected patients were enrolled in five medical centres in the São Paulo State, Brazil. At admission (n=213), 7th (n=131) and 14th day (n=71) of hospitalisation, plasma samples were collected and their IgE concentrations were measured by ELISA. The exclusion criteria were subjects younger than 18 years old, HIV positive, alcohol and drugs addictions and those with degenerative disorders. Healthy volunteers (HV; n=30) from our Institution (Ribeirão Preto Medical School, Brazil) and their family members were also invited. None of these exhibited any clinical symptoms of infection, they were seronegative for the presence of anti-SARS-CoV-2 IgM and IgG antibodies and none were vaccinated. 

Results: At admission, SARS-CoV-2 patients displayed increased plasma IgE levels (753.9 ± 864.7 ng/mL) when compared to the HV (263.2 ± 168.2 ng/mL) (p<0.01). However at the 7th (624.6 ± 759.6 ng/mL) and 14th day (619.3 ± 740.7 ng/mL) of hospitalisation, plasma IgE concentrations were not different in relation to the HV. IgE levels did not differ in critically ill patients and nonsurvivors compared to those mild cases and survivors, respectively.

Discussion: Our results revealed hyper IgE response in the early phase of the SARS-CoV-2 infection, which is not associated to illness severity or poor prognosis. The increased IgE levels may lead to exacerbation of several syndromes, mast cell degranulation and aggravation of respiratory chronic diseases.

 

 

 

Poster 63

Activated human mast cells impair esophageal barrier: role of a possible bidirectional crosstalk

Kleuskens, Mirelle; Bek, Marie; Al Halabi, Youmna; Blokhuis, Bart; Haasnoot, Laura; Bredenoord, Arjan; Garssen, Johan; van Esch, Betty; Redegeld, Frank

Pharmacology, Utrecht University, Utrecht, The Netherlands

Introduction: Recent studies suggest a major role for mast cells in the pathogenesis of eosinophilic esophagitis (EoE), a chronic allergic disorder of the esophagus that is accompanied by type 2 inflammation and esophageal barrier dysfunction. Mast cells accumulate in the esophageal epithelium, but the effects of their interaction with epithelial cells in the disease pathogenesis is not well understood.  

Objective: To study the crosstalk between human mast cells and a barrier of human esophageal epithelial cells (EPC2).   

Methods: Human peripheral blood mononuclear cell-derived mast cells (HMC) were cocultured in the basolateral compartment of differentiated EPC2 cultured on semi-permeable membranes under air-liquid interface (ALI) conditions. Interleukin (IL) 13 stimulation of EPC2 ALI cultures was used as a positive inflammatory control. Barrier function was monitored by transepithelial electrical resistance (TEER), FITC-dextran paracellular flux (FITC flux), and qPCR of barrier proteins following IgE-mediated HMC activation. Crosstalk between HMC and EPC2 was further examined by proteomic analysis of 45 cytokines in mono and coculture supernatants.  

Results: IgE-activated HMC significantly impaired EPC2 barrier function to a similar degree as IL-13 after 4 days of coculture. This was demonstrated by a 20% decrease in TEER and a 14% increase in FITC flux compared with non-activated cocultures. In addition, mRNA transcripts for the barrier proteins desmoglein-1 (5.8-fold), filaggrin (2.3-fold) and involucrin (2-fold) were decreased following coculture with activated HMC. Proteomics revealed that coculture with activated HMC increased the levels of multiple pro-inflammatory cytokines including IL-1β, IL-18, oncostatin M, M-CSF and GM-CSF, and the type 2 cytokine IL-13 compared with monocultures and non-activated cocultures.  

Discussion: Our results demonstrate that (i) activated HMC impair EPC2 barrier function via the downregulation of barrier proteins, and (ii) EPC2 enhance the production of HMC-derived pro-inflammatory mediators. This bidirectional crosstalk may contribute to the perpetuation of local inflammation in EoE.

 

 

 

Poster 64

The long noncoding RNA MALAT-1 regulates responsiveness to FcεRI in human skin mast cells

Bal, Gürkan; Franke, Kristin; Shin, Jay W.; de Hoon, Michiel J. L.; Kasukawa, Takeya; Zuberbier, Torsten; Babina, Magda

Institute of Allergology, Charité – Universitätsmedizin Berlin, Berlin, Germany

Introduction: MALAT-1 (metastasis-associated lung adenocarcinoma transcript-1) represents one of the most widely studied long-noncoding RNAs. It is highly conserved and restricted to the nucleus where it regulates genes at the transcriptional and post-transcriptional levels. While abundantly expressed in skin MCs (skMCs), its function in MCs remains unexplored. 

Objective: To investigate the implications of MALAT-1 on functional programs of skMC and its shaping of the transcriptome at baseline and following FcεRI-mediated activation. 

Methods: skMCs were isolated from normal skin and cultured in SCF plus IL-4 for 2-4 weeks. MALAT-1 knockdown was achieved by two distinct anti-sense oligonucleotides (ASO) vs non-target control. Apoptosis was assessed by the PI/YoPro technique. MCs were stimulated by FcεRI-aggregation (AER-37). MC degranulation was assessed by histamine release. Genome-wide transcriptomic profiling was performed by Cap Analysis of Gene Expression sequencing (CAGE-seq) in non-stimulated and stimulated MCs. Gene expression was also studied by RT-qPCR.

Results: Both ASOs suppressed MALAT-1 expression in skMCs with an efficiency of ≈50%.  MALAT-1 knockdown significantly lowered apoptotic cell death (≈10 vs ≈18%). Interestingly, the suppression of MALAT-1 resulted in significantly reduced histamine release upon FcεRI-aggregation. While attenuation of MALAT-1 altered the expression level of few genes only in non-stimulated MCs (encompassing FCER1A, however), it had substantial impact on MC stimulability. In fact, optimal induction of various genes by IgER-crosslinking required unperturbed Malat-1 levels. Interesting representatives were cytokines (CSF1=M-CSF, CCL3), transcription factors (TFs) (REL, NFATC1, MYC, FOSL2, SOX13), cytokine/growth factor receptors (CRLF2=TSLPR, IL-9R) and a miRNA (hsa-miR-132). Attenuated gene expression following FcεRI-aggregation was also found to require intact Malat-1. Some examples of the latter category were PPCDC (involved in the biosynthesis of coenzyme A from pantothenic acid) and GSTM4 (a glutathione S-transferase). Other differentially expressed genes were KRCC1, NXT1, SCARNA2, HIST1H3C, suggesting that MALAT-1 may have an impact on chromatin architecture and nuclear organization. 

Discussion: MALAT-1 influences MC responsiveness to FcεRI-aggregation. It fine-tunes inducibility (or attenuation) of TFs, cytokines, cell surface receptors, and miRNAs. Thereby, MALAT-1 chiefly acts as a positive regulator of FcεRI-driven programs, including degranulation, cytokine induction, and (positive or negative changes) in the transcriptional activity, possibly through altered FcεRI expression.

 

 

 

Poster 65

Eosinophil-Derived Neurotoxin (EDN) may contribute to mastocytosis diagnosis

Abecassis, Anna; Vitte, Joana; Sahli, Wided; Perrier, Robert; Blanchard, Patricia; Arnaud, Camille; Kaplanski, Gilles; Michel, Moise

Immunology lab, CHU Nîmes, Nîmes, France

Introduction: Interactions between eosinophils and mast cells can modify the evolution of pathologies involving these cells. It may be an immunological synapse favoring exchanges and mutual regulation via secreted mediators: tryptase and histamine induce eosinophils degranulation; conversely, eosinophils can activate the mast cell thanks to granular proteins. Mastocytosis is a pathology characterized by the proliferation of abnormal mast cells. Some mastocytosis with a poor prognosis may be accompanied by hypereosinophilia. Eosinophil-derived neurotoxin (EDN) is a protein secreted by eosinophils, which has been recently used as a marker of eosinophilic activation in asthma. 

Objective: We assume that serum EDN may be a suitable biomarker for mast cell pathologies.  

Methods: Serum EDN was determined using an ImmunoCAP platform (Thermo Fisher Scientific, Uppsala, Sweden). We performed a retrospective analysis of eosinophils count, serum basal tryptase (sbT) and EDN/sbT ratio in a cohort of 70 adult patients, including 32 patients with mastocytosis, 10 suspected mastocytosis with elevated sbT, 10 chronic urticaria and 18 control subjects.  

Results:  Serum EDN levels in patients with mastocytosis are higher than in the control group (78.2 vs 32.2 µg/L, p < 0.001), with elevation proportional to disease severity. Using Receiver operating characteristic (ROC) analysis, an EDN level of 49.2 µg/L or higher identifies patients with mastocytosis with a specificity (Sp) of 100 % and a sensitivity (Se) of 65.6 %. This performance is improved using the EDN/sbT ratio (Sp 75 %, Se 90.6 % for a cutoff of 7.8). EDN, and EDN/sbT values were not significantly associated with mast cell degranulation, histological mast cell infiltration or efficacy of antihistamine treatment. 

Discussion: EDN and EDN/sbT ratio are useful in identifying patients with systemic mastocytosis. Longitudinal studies in larger cohorts are needed to evaluate the performance of these biomarkers in the follow-up of mastocytosis.

 

 

 

Poster 66

Autoimmune CSU have features of autoallergic CSU but not vice versa

Xiang, Yi-Kui; Kolkhir, Pavel; Scheffel, Jörg; Maurer, Marcus; Altrichter, Sabine

Institute of Allergology, Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

Introduction: Two endotypes of chronic spontaneous urticaria (CSU) associated with mast cell-activating autoantibodies are known, namely autoallergic CSU (aaCSU) and type IIb autoimmune CSU (aiCSU). The rates of patients with aaCSU, aiCSU, and both are currently unknown. 

Methods: We investigated 111 CSU patients for aiCSU, i.e. +BAT, +ASST, together with +IgG-anti-FceRI/IgE and for aaCSU, i.e. +IgE-anti-TPO/IL24 assessed by ELISA. Clinical and laboratory parameters were compared in patients with aaCSU, aiCSU, and both.

Results: Across 111 patients with CSU, 9 and 65 had aiCSU and partial-aiCSU, 64 had aaCSU. Eight of nine aiCSU patients also had aaCSU, but only 13% (8/64) patients with aaCSU also had aiCSU. In total, 7% (8/111) of patients had both aiCSU and aaCSU, and 41% (46/111) had neither. All 8 patients with aiCSU and aaCSU had IgE-anti-TPO, half had IgE-anti-IL24, and 6 had angioedema. Of note, Patients who had aiCSU, as compared to those who did not, had significantly higher QoL impairment (DLQI7, p = 0.046) as well as higher levels of IgG-anti-TPO (p = 0.02) and ANA (p < 0.001). They also had lower basophil and eosinophil counts (p = 0.015, p = 0.032).  Disease activity was higher (p = 0.067), and total IgE levels were lower (p = 0.186), but without statistical significance. Patients with aaCSU, as compared to those without, had significantly higher serum levels of circulating immune complexes (p = 0.042) and IgG-anti-TPO (p = 0.016). They also had lower total IgE levels (p = 0.027) and leukocyte counts (p = 0.027), as compared to non-aaCSU patients. To determine the impact of coexisiting aiCSU in patients with aaCSU, we compared standalone aaCSU patients (n = 56) with aaCSU patients who also had aiCSU (n = 8). The latter showed markedly higher QoL impairment (p = 0.05), numerically higher disease activity (p = 0.134) and ANA levels (p = 0.001) as compared to patients with standalone aaCSU.

Discussion: Our novel finding that most aiCSU patients also have aaCSU should be confirmed in larger and multicenter patient populations. The clinical relevance of overlapping autoimmunity and autoallergy in CSU should be further investigated.

 

 

 

Poster 67

CD300a and mast cells regulate mouse macrophages functionality in allergic inflammation

Ofori, Prince

Institute of Drug Research, School of Pharmacy, Hebrew university of Jerusalem, Jerusalem, Israel

Introduction: CD300a is an inhibitory receptor (IR) expressed on mast cells (MCs) and eosinophils. We have previously characterized CD300a role in mouse models of allergy, in which absence of CD300a resulted in increased inflammatory features and delayed resolution. However, the exact mechanism of this delayed resolution is unclear. Our hypothesis is that macrophages (Macs), important players in resolution, might be impaired when CD300a is not expressed.

Objective: To investigate CD300a-dependent functionality of mouse Macs.

Methods: Macs were purified from the peritoneum of Wild Type (WT) and CD300a-/- mice naïve and 48h and 96h after challenge with Ovalbumin/Alum. Phenotype switching was analyzed via specific M1-M2 inducers and markers. Macs phagocytotic ability was assessed via Staphylococcus aureus pHrodo-conjugated bioparticles. The influence of MCs on Macs was investigated by incubating WT Macs with supernatants from non-activated and IgE-activated bone marrow-derived MCs and analyzing functional responses.

Results: CD300a-/- Macs purified from AP-induced mice48h after challenge showed decreased Arg1 expression and increased IL-6 release when Macs are purified from AP-induced mice48h after challenge. Similar results were obtained when CD300a-/- Macs were purified 96h after challenge. Naïve CD300a-/- Macs displayed increased sensitivity to activation when treated with LPS. On the other hand, CD300a absence did not affect Macs phagocytosis. WT Macs incubated with supernatants of non-activated and IgE-activated BMMCs exhibited increased iNOS expression and decreased Arg1 levels.

Discussion: The IR CD300a might control the activation state of Macs since its absence could augment the inflammatory state seen in CD300a-/- mice. Moreover, MCs can also influence Macs phenotype switching toward the pro-inflammatory M1 This may partially explain the delayed AI resolution seen in these mice.

 

 

 

 

Poster 68

Developmental origins of melanoma-associated mast cells

Carvalho, Cyril and Rebecca Gentek

University of Edinburgh, Scotland

Mast cells (MCs) are abundantly found in and around solid tumors, including melanomas. Studies on patients and animal models have associated them with both favorable and poor outcomes, whilst others have concluded they do not have any effect on tumor growth. These discrepancies may in part be explained by the heterogeneity of MCs, which is increasingly recognized. For example, MCs can have different developmental origins, being derived either from fetal-restricted yolk sac (YS) progenitors or hematopoietic stem cells (HSC). They share these dual developmental origins with macrophages, which can impact tumor growth in an origin-specific manner. In hepatocellular carcinoma, fetal-derived macrophages engage in selective crosstalk with endothelial cells that have reactivated fetal-like cellular programs and thereby provide a pro-tumorigenic “onco-fetal ecosystem”. Whether similar considerations apply to tumor-associated MCs has not been explored.

 

Here, we sought to address in mouse melanoma models if MCs are developmentally heterogenous, and whether MCs with distinct origins may differentially impact tumor growth.

 

To this end, we grafted BrafV600E Cdkn2a-/- Pten-/- (YUMM, Yale University Mouse Melanoma) into fate mapped mice, in which MCs of either YS or HSC origin are genetically labelled. This revealed that fetal YS-derived MCs comprise on average 40% of melanoma-associated MCs, where they co-exist with MCs originating from HSC. Compared to the skin of the same tumor-bearing mice, this represents am approximately 2-fold enrichment, suggesting that fetal-derived MCs are selectively recruited to melanomas. Bulk RNAsequencing revealed that fetal YS- and HSC-derived MCs are transcriptomically very distinct, in line with potential functional differences. Finally, Diphtheria toxin-mediated depletion of mature MCs prior to melanoma engraftment resulted in reduced tumor growth in FcER1DTR mice, suggesting that pre-existing MCs, like those derived from YS progenitors, may exert pro-tumorigenic functions. We now aim to delineate the respective functions of fetal- and HSC-derived MCs in mouse melanomas.