1^st EMBRN Webinar: “Mast cells as targets for novel treatments”

Chair: Prof. Marcus Maurer

January 11, 2021: 17.00 – 18.30 CET

We thank the presenters of the first EMBRN webinar and all the participants. We are incredibly happy with the interest in this first webinar exceeding our expectations.

The recording is now available for everyone to watch here.

Agenda

Introduction

Aim and scope of the EMBRN webinars

Prof. Francesca Levi-Schaffer, EMBRN President (5 minutes)

Pharmacology & Experimental Therapeutics Unit, Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

Keynote presentation

Mast cells as targets for novel treatments

Prof. Marcus Maurer, EMBRN Past President, webinar chair

(20-25 minutes)

Director of Research, Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin,

Germany

Selected oral presentations (6 min presentation and 4 min Q&A and discussion each)

1. Chalatip CHOMPUNUD NA AYUDHYA (Philadelphia,PA, USA): Role of Tyrosine residues in MRGPRX2 on G protein and β-arrestin-mediated signaling in response to substance P
2. Jessy ELST (Antwerp, Belgium): MRGPRX2 and immediate drug hypersensitivity: insights from cultured human mast cells
3. Jiajun HE (Sichuan, China): Validation of a novel immunomodulatory compound for restoring tolerance
4. Pier PUZZOVIO (Jerusalem, Israel): Revisiting the effect of cromolyn sodium on human and murine mast cells
5. Willem ABMA (Stockholm, Sweden): Prostaglandin D2 inhibits mediator release and antigen induced bronchoconstriction in the guinea pig trachea by activation of DP1 receptors
6. Deisy SEGURA-VILLALOBOS (Mexico City, Mexico): Cyclic hypoxia promotes a hyperresponsive phenotype to FcεRI crosslinking in mast cells

Brief summary, 2^nd webinar announcement, farewell

3rd EMBRN Webinar: "Mast cells in health and disease"

Chair: Prof Francesca Levi-Schaffer

EMBRN President Pharmacology & Experimental Therapeutics Unit, Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

May 3rd, 2021: 14.00 to 15.30 CET

Agenda of the 3rd EMBRN webinar, May 3rd 2021, 2.00-3.30 pm CET

Introduction

Prof. Francesca Levi-Schaffer, EMBRN President
Pharmacology & Experimental Therapeutics Unit, Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

Keynote presentation (15 min)

Dr. Joana Vitte (Marseille, Montpellier, France): Tryptase, a mast cell marker in health and disease
Associate Professor, Aix-Marseille University and IDESP Montpellier, France

Selected oral presentations (6 min presentation and 4 min Q&A and discussion each)

Simon P. Goldie (Southampton, Portsmouth, UK): Staphylococcus aureus within nasal mast cells as a driver of chronic rhinosinusitis: A role for simvastatin in disease management?

Anna-Karin Johnsson (Stockholm, Sweden): Characterization of lipid mediator release in IgE stimulated lung mast cells

Erika Haide Mendez Enriquez (Uppsala, Sweden): Mast cell-derived serotonin enhances methacholine-induced airway hyperresponsiveness in house dust mite-induced experimental asthma

Gunnar Nilsson (Stockholm, Sweden): Immunoprofiling reveals novel mast cell receptors and the continuous nature of human lung mast cell heterogeneity

Irit Shefler (Tel Aviv, Israel): Lung cancer-derived extracellular vesicles:  a possible mediator of mast cell activation in the tumor microenvironment

Pratibha Gaur (Jerusalem, Israel): Dexamethasone and CD300a activation display additive inhibitory effect on human and murine mast cell functions

Nyssa B. Samanas (Seattle, USA): Characterization of the mast cell surface proteome leads to the identification of CD98hc as a critical surface molecule for optimal mast cell function

Brief summary, 4th webinar announcement, farewell

Here you can find the abstracts of the webinar

Basophils and Mast Cells in Health and Disease, chairs:

Chairs: Didier G Ebo (University of Antwerpen, Belgium) and Bernhard F. Gibbs (University of Oldenburg, Germany)

September 27th, 2021: 10-11.30 Israel time, 9-10.30 am CEST (Berlin), 7-8.30 GMT

The recording is now available for everyone to watch here.

Agenda 4th webinar with all abstracts

4th EMBRN webinar: BASOPHILS AND MAST CELLS IN HEALTH AND DISEASE
September 27th 2021, 9.00-10.30 am CEST (Amsterdam, Berlin, Rome, Stockholm, Vienna)

Introduction (5 min)
Prof. Bernhard F. Gibbs, EMBRN Vice-President
Carl von Ossietzky University Oldenburg, Faculty for Medicine and Health Sciences, University Clinics for Dermatology, Oldenburg, Germany

Keynote presentation (20-25 min)
Prof. Didier G. Ebo (Antwerp and Ghent, Belgium): Flow assisted cellular allergy diagnostics: can mast cells beat basophils?
Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Faculty of Medicine and Health Sciences, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium; Department of Immunology, AZ Jan Palfijn Hospital Gent, Ghent, Belgium

Selected oral presentations (6 min presentation and 4 min Q&A and discussion each)

John Tchen (Paris, France)* Mcpt8-CRE-tdT or “CTM8” mice: A new mouse model demonstrates that basophils have a non-redundant role in pristane-induced lupus-like disease development
William Worrall (Toulouse, France)* The anti-FcεRI antibody MAR-1 depletes basophils and cross-reacts with myeloid cells through its Fc portion.
Manuela Bratti (Paris, France)* Intracellular recycling compartment localized Insulin-Regulated Aminopeptidase (IRAP) controls FcR-mediated inflammatory diseases
Jessy Elst (Antwerp, Belgium)* The mast cell activation test in chlorhexidine allergy: a proof of concept
Yu-Wen Yeh (Hong Kong, China)* Wild mice and naturalized laboratory mice express lung parenchymal mast cells
Hadas Pahima (Jerusalem, Israel)* CD48 contributes to the IgE-dependent activation of murine mast cells

Brief summary, announcement, farewell

*Abstract included below (speakers bold)
 
Mcpt8-CRE-tdT or “CTM8” mice: A new mouse model demonstrates that basophils have a
non-redundant role in pristane-induced lupus-like disease development

John TCHEN^1,2 Quentin SIMON^1,2 Léa CHAPART^1,2 Christophe PELLEFIGUES^1,2 Hajime KARASUYAMA³ Kensuke MIYAKE³ Juan HIDALGO PAREJA⁴ Ulrich BLANK^1,2 Marc BENHAMOU^1,2 Eric DAUGAS^1,2,5 Nicolas CHARLES^1,2

¹ Université de Paris, Centre de Recherche sur l’Inflammation, INSERM UMR1149, CNRS
ERL8252, Faculté de Médecine site Bichat, Paris, France
² Université de Paris, Laboratoire d’Excellence Inflamex, Paris, France
³ Department of Immune Regulation, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University (TMDU), Tokyo, Japan
⁴ Universidad Autonoma de Barcelona, Facultad de Biociencias, Unidad de Fisiologia Animal
Bellaterra, Barcelona, Spain
⁵ Service de Néphrologie, Hôpital Bichat, Assistance Publique – Hôpitaux de Paris, Paris,
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.

 
The anti-FcεRI antibody MAR-1 depletes basophils and cross-reacts with myeloid cells through its Fc portion
William Worrall ^{a, #}, Jasper Kamphuisa ^{a, #}, Julien Stackowicz^{b, c}, Aurélie Mougel^a, Emilie Mauré^a, Pierre Bruhns^b, Friederike Jönsson^b, Laurent Guilleminault^{a, d}, Laurent L. Reber^{a, b}

^a Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), UMR 1291, University of Toulouse, INSERM, CNRS, Toulouse, France.
^b Unit of Antibodies in Therapy and Pathology, Institut Pasteur, UMR 1222 INSERM, F-75015 Paris, France.
^c Sorbonne University, ED394, F-75005 Paris, France.
^d Department of Respiratory Medicine, University Hospital Centre of Toulouse, Toulouse, France.
^#These authors contributed equally to this work.

MAR-1 is a monoclonal antibody used to stain mouse high-affinity IgE receptor FcεRI on basophils and mast cells. This clone is also used in vivo to deplete basophils and induce anaphylaxis. However, the use of MAR-1 has been controversial because this mAb can also bind the IgG receptors FcεRI and FcRIV. We have investigated the mechanisms of MAR-1’s interaction with FcRs, depletion of basophils and anaphylaxis. We found that MAR-1 stains mast cells and basophils and induces anaphylaxis through its interaction with FcεRI, independently of FcgRs. However, MAR-1 recognizes myeloid cells such as monocytes and macrophages in a FceRI-independent manner, by engaging FcgRs through its Fc portion. Indeed, an “Fc silent” recombinant version of MAR-1 only recognizes basophils and mast cells. Strikingly, basophil depletion was also mediated by the Fc portion of MAR-1, since the Fc silent MAR-1 was unable to deplete basophils. We propose that Fc silent MAR-1 should be preferred to assess FcεRI expression. However, this Fc silent format cannot be used for basophil depletion experiments.

Intracellular recycling compartment localized Insulin-Regulated Aminopeptidase (IRAP) controls FcR-mediated inflammatory diseases
Manuela Bratti1, Shamila Vibhushan1, Marc Benhamou1, Ulrich Blank1*#, Loredana Saveanu1* and Sanae Ben Mkaddem2*
¹Université de Paris, Centre de Recherche sur l'Inflammation, INSERM UMR1149, CNRS EMR8252, Faculté de Médecine site Bichat, Paris, France ; Laboratoire d'Excellence Inflamex, Paris, France.
²INSERM, U978, Bobigny, France 3Université Paris 13 Sorbonne Paris Nord, UFR SMBH, Bobigny, France
  * co-senior authors; # corresponding author

We investigated the role of Insulin-Regulated Amino Peptidase (IRAP) in IgE receptor (FcεRI) signaling by mast cells (MC). We confirmed that IRAP locates to an intracellular recycling compartment that rapidly recruits to the plasma membrane upon FcεRI stimulation. IRAP-/- versus WT MC exhibited a reduced ability to degranulate and release various chemokines and cytokines. Tyrosine-phosphorylation of several signaling effectors was diminished in IRAP-/- MC. Importantly, we identified Syk, a central tyrosine kinase downstream of FcεRI, as being less phosphorylated at the plasma membrane in IRAP-/- MC. In addition, pSHP-1S591, which positively regulates ITAM signaling, was decreased as well as further downstream events such as intracellular calcium increases. In vivo, IRAP-/- versus WT mice exhibited a less severe IgE-dependent passive systemic anaphylaxis. IgG-triggered active systemic anaphylaxis in humanized FcγRIIA transgenic mice was also reduced in the absence of IRAP and in vivo profiling confirmed decreased phosphorylation of Syk and SHP-1S591 in FcγRIIA-expressing neutrophils and monocytes. In a chronic model of autoimmune arthritis, disease development was reduced in IRAP-/- mice both in a FcγRIIA transgenic or WT background. Our findings support a role of IRAP-positive recycling compartments in FcR signaling, enhancing phosphorylation events at the plasma membrane and FcR-mediated inflammatory diseases.

 
The mast cell activation test in chlorhexidine allergy: a proof of concept
Jessy Elst¹, Marie-Line M. van der Poorten^1,2, Margaretha A. Faber¹, Athina L. Van Gasse^1,2, Lene H. Garvey^4,5, Chris H. Bridts¹, Leander P. De Puysseleyr¹, Christel Mertens¹, Margo M. Hagendorens^1,2, Vito Sabato^1,3, Didier G. Ebo^1,3*

¹ University of Antwerp, Faculty of Medicine and Health Sciences, Department of Immunology, Allergology, Rheumatology and the Infla-Med Centre of Excellence, Antwerp (Belgium) and Immunology, Allergology, Rheumatology, Antwerp University Hospital, Antwerp (Belgium)
² University of Antwerp, Faculty of Medicine and Health Sciences, Department of Paediatrics and the Infla-Med Centre of Excellence, Antwerp (Belgium) and Paediatrics, Antwerp University Hospital, Antwerp (Belgium)
³ Department of Immunology and Allergology, AZ Jan Palfijn Gent, Ghent, Belgium
⁴ Allergy Clinic, Department of Dermatology and Allergy, Gentofte Hospital, Denmark
⁵ Department of Clinical Medicine, University of Copenhagen, Denmark

Background: Conventional confirmatory testing for immediate drug hypersensitivity reactions (IDHRs) includes skin tests and quantification of drug-specific IgE antibodies (sIgE). However, these tests are not absolutely predictive for the clinical outcome and can yield false negative and false positive results.
Aim: To assess whether the mast cell activation test (MAT) could benefit diagnosis of chlorhexidine (CHX) IgE-mediated hypersensitivity.
Methods: Human mast cells (dMCs) were generated from CD34+ progenitor cells and sensitized with patients’ sera to become dMCIgE+ and subsequently incubated with CHX. We compared the outcome of the MAT with serum from patients with and without positive skin test and basophil activation test (BAT) to CHX.
Results: dMC sensitized with sera from patients with a positive skin test and BAT to CHX showed a dose-dependent degranulation upon stimulation with chlorhexidine, determined by upregulation of the degranulation marker CD63. In contrast, dMC sensitized with sera from patients with a negative skin test and BAT to chlorhexidine were unresponsive in the MAT.
Conclusion: The MAT can be used to diagnose IgE-dependent IDHRs. Besides, it shows potential to assess the clinical relevance of drug-sIgE antibodies in their ability to elicit MC degranulation and therefore discriminate between allergy, and merely sensitization.

 
Wild mice and naturalized laboratory mice express lung parenchymal mast cells
Yu-Wen Yeh¹, Arka Sen Chaudhuri¹, Ling Zhou², Yu Fang², Preben Boysen³, Zou Xiang¹

¹Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, Hong Kong, China
²Center for Clinical Laboratory, Affiliated Hospital of Guizhou Medical University, Guiyang, China
³Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo, Norway

Background: Conventional laboratory strains of mice profoundly lack lung parenchymal mast cells, which stands in clear contrast to the abundant presence of mast cells in human lungs. Ultra-hygienic specific pathogen-free (SPF) laboratory mice display reduced richness and complexity of microbiota. Accumulating evidence suggests that wild mice may be a more relevant model to mimic human immune responses. This project aimed to investigate the lung distribution of mast cells in free-living wild mice.
Results: Wild mice were trapped at Oslo, Norway and Hemtabad, India, and their mouse (Mus musculus) identity was confirmed by genotyping. Mast cells were identified at a substantial density in the lung parenchymal tissues of wild mice from both habitats using toluidine blue, tryptase and cKit staining. In contrast, lung mast cells were indeed absent in the conventional C57BL/6 or BALB/c strains of laboratory mice examined in parallel. Consistently, wild mice also expressed higher pulmonary levels of stem cell factor, a critical mast cell growth factor. Higher levels of histamine were recorded in the lung tissues of the wild mice. Having speculated that gut microbiota could have impacted the lung tissue residency of mast cells, we next bred C57BL/6 laboratory mice in a purposefully built, closed environment with bedding material obtained from the natural environment with wild rodent infestation in order to normalize the gut microbiota of these super clean SPF laboratory mice to that of the dirty wild mice. Interestingly, C57BL/6 mice born and spent their entire life in this environment developed lung mast cells at an appreciable density.
Conclusions: 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 with constitutive lung mast cells, which may more closely mimic human asthma.

 
CD48 contributes to the IgE-dependent activation of murine mast cells
Pahima H, Puzzovio PG, and Levi-Schaffer F

Pharmacology & Experimental Therapeutics Unit, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Israel

Background: CD48 is a glycosylphosphatidylinositol (GpI) receptor belonging to the SLAM family expressed on all the hematopoietic cells, including mast cells (MCs) in both mouse and human.  Being a GpI anchored receptor it lacks an intracellular domain molecule. Therefore, CD48 is considered to be a co-activating receptor rather than a bona fide activating receptor. We and others have previously shown its role in bacteria and bacterial toxins-driven MC activation.
Aim: To investigate the role of CD48 in IgE-dependent MC activation systems in vitro and in vivo.
Results: In vitro, IgE-dependent activation (IgE+anti-IgE or IgEanti-DNP+DNP) of CD48-/- bone marrow derived mast cells (BMMCs) resulted in reduced release of both beta-hexosaminidase and TNFα in comparison to WT BMMCs. As expected, a MC-dependent allergic peritonitis model (OVA / S.aureus enterotoxin B(SEB)) induced in Sash-MC deficient mice after i.p. reconstitution with CD48-/- BMMCs, displayed a decreased level of inflammation as measured by total cells and eosinophil numbers in the peritoneum, in comparison to Sash mice reconstituted with WT BMMCs. However in CD48-/- mice OVA/SEB allergic peritonitis induced a significant increase in the level of inflammation as measured in total peritoneal but not in eosinophil numbers.
Conclusion: CD48 has a clear activating/co-activating role on IgE- dependent activation as displayed specifically on MCs. This role is less evident if other cells participating in allergic inflammation are also deficient for this receptor possibly because of arising compensatory factors.