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Serpin regulation of leukocyte proteases in cellular homeostasis and inflammation

English title Serpin regulation of leukocyte proteases in cellular homeostasis and inflammation
Applicant Benarafa Charaf
Number 173137
Funding scheme Project funding (Div. I-III)
Research institution Institut für Virologie und Immunologie IVI Sensemattstrasse 293 3147 Mittelhäusern
Institution of higher education Other Research Institutes - FINST
Main discipline Immunology, Immunopathology
Start/End 01.06.2017 - 30.11.2021
Approved amount 700'000.00
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All Disciplines (3)

Discipline
Immunology, Immunopathology
Biochemistry
Cellular Biology, Cytology

Keywords (6)

Neutrophil; Serpin; Mouse models; Cell death; Proteases; Immunity

Lay Summary (French)

Lead
Les serpines constituent la plus grande famille d’inhibiteurs de protéases que l’on retrouve chez toutes les formes de vies. Chez les vertébrés, une famille de serpines, la clade B, a évolué de nouvelles fonctions qui n’ont pas été explorées de manière exhaustive. Ce projet propose d’en élucider les fonctions en utilisant des outils génétiques et une combinaison d’approches ciblées et non-ciblées.
Lay summary

Contenu et objectifs du travail de recherche

Les leucocytes, cellules du système immunitaire, éliminent les pathogènes en partie grâce à leurs protéases. Nos travaux ont démontré que la serpine B1 protège certains leucocytes d’une mort cellulaire prématurée induite par leurs propres protéases : notamment la cathepsine G (CatG). Dans ce projet, nous utiliserons une approche biochimique classique ainsi qu’une analyse protéomique non-ciblée pour identifier les cibles de la CatG induisant la mort cellulaire. Nous évaluerons également les fonctions des protéases dans l’homéostase d’autres leucocytes (cellules NKT invariantes) dont la biologie est altérée dans les souris déficientes en serpine B1. Les fonctions d’un grand groupe de serpines de la clade B seront déterminées en étudiant des souris déficientes que nous avons générées au travers de l’analyse détaillée du système immunitaire ainsi que par un criblage des fonctions physiologiques majeures en collaboration avec un centre de phénotypage. Ces résultats seront ensuite validés en élucidant les mécanismes dépendant des protéases ainsi qu’en identifiant la serpine impliquée.

Contexte scientifique et social du projet de recherche

Ce projet de recherche fondamentale a pour objectif de mieux comprendre les mécanismes moléculaires régulés par les protéases et leurs serpines inhibitrices dans la réponse aux infections ainsi que dans d’autres domaines physiologiques et pathologiques encore inconnus.

Direct link to Lay Summary Last update: 05.06.2017

Responsible applicant and co-applicants

Employees

Project partner

Publications

Publication
Granule Leakage Induces Cell-Intrinsic, Granzyme B-Mediated Apoptosis in Mast Cells
Burgener Sabrina Sofia, Brügger Melanie, Leborgne Nathan Georges François, Sollberger Sophia, Basilico Paola, Kaufmann Thomas, Bird Phillip Ian, Benarafa Charaf (2021), Granule Leakage Induces Cell-Intrinsic, Granzyme B-Mediated Apoptosis in Mast Cells, in Frontiers in Cell and Developmental Biology, 9, 1-12.
ATG5 promotes eosinopoiesis but inhibits eosinophil effector functions
Germic Nina, Hosseini Aref, Stojkov Darko, Oberson Kevin, Claus Meike, Benarafa Charaf, Calzavarini Sara, Angelillo-Scherrer Anne, Arnold Isabelle C., Müller Anne, Riether Carsten, Yousefi Shida, Simon Hans-Uwe (2021), ATG5 promotes eosinopoiesis but inhibits eosinophil effector functions, in Blood, 137(21), 2958-2969.
Chronic cigarette smoke exposure and pneumococcal infection induce oropharyngeal microbiota dysbiosis and contribute to long-lasting lung damage in mice
Hilty Markus, Wüthrich Tsering M., Godel Aurélie, Adelfio Roberto, Aebi Susanne, Burgener Sabrina S., Illgen-Wilcke Brunhilde, Benarafa Charaf (2020), Chronic cigarette smoke exposure and pneumococcal infection induce oropharyngeal microbiota dysbiosis and contribute to long-lasting lung damage in mice, in Microbial Genomics, 6(12), 1-14.
Serpinb1a Is Dispensable for the Development and Cytokine Response of Invariant Natural Killer T Cell Subsets
Leborgne Nathan G. F., Taddeo Adriano, Freigang Stefan, Benarafa Charaf (2020), Serpinb1a Is Dispensable for the Development and Cytokine Response of Invariant Natural Killer T Cell Subsets, in Frontiers in Immunology, 11, 1-9.
Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils
Ohayon Delphine, De Chiara Alessia, Dang Pham My-Chan, Thieblemont Nathalie, Chatfield Simon, Marzaioli Viviana, Burgener Sabrina Sofia, Mocek Julie, Candalh Céline, Pintard Coralie, Tacnet-Delorme Pascale, Renault Gilles, Lagoutte Isabelle, Favier Maryline, Walker Francine, Hurtado-Nedelec Margarita, Desplancq Dominique, Weiss Etienne, Benarafa Charaf, Housset Dominique, Marie Jean-Claude, Frachet Philippe, El-Benna Jamel, Witko-Sarsat Véronique (2019), Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils, in Journal of Experimental Medicine, 216(11), 2669-2687.
Cathepsin G Inhibition by Serpinb1 and Serpinb6 Prevents Programmed Necrosis in Neutrophils and Monocytes and Reduces GSDMD-Driven Inflammation
Burgener Sabrina Sofia, Leborgne Nathan Georges François, Snipas Scott J., Salvesen Guy S., Bird Phillip Ian, Benarafa Charaf (2019), Cathepsin G Inhibition by Serpinb1 and Serpinb6 Prevents Programmed Necrosis in Neutrophils and Monocytes and Reduces GSDMD-Driven Inflammation, in Cell Reports, 27(12), 3646-3656.e5.
Untangling “NETosis” from NETs
Yousefi Shida, Stojkov Darko, Germic Nina, Simon Dagmar, Wang Xiaoliang, Benarafa Charaf, Simon Hans‐Uwe (2019), Untangling “NETosis” from NETs, in European Journal of Immunology, 49(2), 221-227.
Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production
Amini Poorya, Stojkov Darko, Felser Andrea, Jackson Christopher B., Courage Carolina, Schaller André, Gelman Laurent, Soriano Maria Eugenia, Nuoffer Jean-Marc, Scorrano Luca, Benarafa Charaf, Yousefi Shida, Simon Hans-Uwe (2018), Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production, in Nature Communications, 9(1), 2958-2958.
Transgenic Mice Expressing Human Proteinase 3 Exhibit Sustained Neutrophil-Associated Peritonitis
Martin Katherine R., Pederzoli-Ribeil Magali, Pacreau Emeline, Burgener Sabrina S., Dahdah Albert, Candalh Céline, Lauret Evelyne, Foretz Marc, Mouthon Luc, Lucas Bruno, Thieblemont Nathalie, Benarafa Charaf, Launay Pierre, Witko-Sarsat Véronique (2017), Transgenic Mice Expressing Human Proteinase 3 Exhibit Sustained Neutrophil-Associated Peritonitis, in The Journal of Immunology, 199(11), 3914-3924.

Collaboration

Group / person Country
Types of collaboration
Philip I Bird, Monash University Australia (Oceania)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Véronique Witko/Institut Cochin, Paris France (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Markus Hilty/Institute of Infectious Diseases, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Stefan Freigang/Department of Pathology, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
German Mouse Clinic Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Guy Salvesen / Sanford Burnham Prebys Medical Discovery Institute United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Hans-Uwe Simon/Institute of Pharmacology, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Ulrich auf dem Keller/Technical University of Denmark Denmark (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
- Exchange of personnel

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Invited seminar, IRSET Individual talk Key functions of Serpinb1 and Serpinb6 in neutrophil survival and inflammation 10.02.2020 University of Rennes, Rennes, France Benarafa Charaf;
Graduate School GCB Symposium, University of Bern Poster Unrequired Function of the Cytosolic Serpin Serpinb1a in Homeostasis and Activity of Invariant Natural Killer T Cells 30.01.2020 Bern, Switzerland Benarafa Charaf; Leborgne Nathan;
11th International Proteolysis Society (IPS) General Meeting Poster Unrequired function of the cytosolic serpin Serpinb1a in homeostasis and activity of invariant Natural Killer T cells 29.09.2019 Marienbad, Czech Republic Leborgne Nathan; Benarafa Charaf;
9th International Serpin Symposium (Serpins2019) Poster Generation of a new mouse model to investigate overlapping functions of clade B serpins 19.09.2019 Sevilla, Spain Lindh Debora; Leborgne Nathan; Benarafa Charaf;
9th International Serpin Symposium Talk given at a conference Serpinb1 and Serpinb6 inhibit inflammation by controlling gasdermin D activation by cathepsin G 19.09.2019 Sevilla, Spain Benarafa Charaf;
9th International Serpin Symposium (Serpins2019) Poster Targeting SERPINB1 and SERPINB6 using CRISPR/Cas9 RNP transfection in human myeloid cell lines 19.09.2019 Sevilla, Spain Benarafa Charaf;
9th International Serpin Symposium (Serpins2019) Poster Dispensable role of the cytosolic serpin Serpinb1a in homeostasis and function of invariant Natural Killer T cells 19.09.2019 Sevilla, Spain Benarafa Charaf; Leborgne Nathan;
14th World congress on Inflammation Poster Cathepsin G inhibition by Serpinb1a and Serpinb6a prevents programmed necrosis in neutrophils and monocytes and reduces GSDMD-driven inflammation 15.09.2019 Melbourne, Australia Burgener Sabrina Sofia;
Graduate School GCB Symposium, University of Bern Poster Functions of the cytosolic serpin Serpinb1 in the biology of the invariant NKT cells 31.01.2019 Bern, Switzerland Benarafa Charaf; Leborgne Nathan;
Invited seminar, Karolinska Institute Individual talk Regulation of proteolysis by cytosolic serpins in cell death pathways 05.11.2018 Stockholm, Sweden Benarafa Charaf;
8th Meeting on Cellular Proteolysis - Societé Française de Biochimie et Biologie Moléculaire Talk given at a conference Regulation of proteolysis by cytosolic serpins in cell death pathways 15.10.2018 La Grande Motte, Montpelier, France Benarafa Charaf;
Invited seminar, Institute of Anatomy, University of Bern Individual talk Regulation of proteolysis by cytosolic serpins in cell death pathways 11.10.2018 Bern, Switzerland Benarafa Charaf;
Swiss Apoptosis Meeting Poster Role of cytosolic serpins in the biology of invariant Natural Killer T cells 12.09.2018 Bern, Switzerland Leborgne Nathan; Benarafa Charaf;
Gordon Research Conference - Proteolytic Enzymes and Their Inhibitors Talk given at a conference Serpinb1 and Serpinb6 Control Cathepsin G Cleavage of Gasdermin D: Effects on Cell Death and IL-Beta Release 03.06.2018 Il Ciocco, Italy Benarafa Charaf;
Host Pathogen Interactions (HoPa) research focus group Symposium, Vetsuisse Faculty Talk given at a conference Functions of the cytosolic serpin Serpinb1 in the biology of the invariant NKT cells 04.05.2018 Bern, Switzerland Leborgne Nathan;
Host Pathogen Intercations (HoPa) research focus group Symposium, Vetsuisse Faculty Talk given at a conference Serpinb1a and Serpinb6a protect neutrophils and monocytes from cathepsin G mediated death bit gasdermin D processing by cathepsinG is only required in monocytes 04.05.2018 Bern, Switzerland Burgener Sabrina Sofia;
10th International Proteolysis Society (IPS) General Meeting Poster The chymotrypsin-like serine protease cathepsin G induces cell death through a non-classical mechanism in myeloid cells in absence of Serpinb1a and Serpinb6a 28.10.2017 Banff, Canada Benarafa Charaf; Burgener Sabrina Sofia;
50th Annual Meeting of The Society for Leukocyte Biology “Leukocyte Memory: Health and Disease” Poster Serpinb6a cooperates with Serpinb1a in protecting myeloid cells from cathepsin G-mediated death in vivo 05.10.2017 Vancouver, Canada Benarafa Charaf; Burgener Sabrina Sofia;


Awards

Title Year
Dr. Lutz Zwillenberg-Preis 2021
SNSF Early Postdoc Mobility Fellowship 2019

Associated projects

Number Title Start Funding scheme
149790 Serpin regulation of leukocyte proteases in cellular homeostasis and inflammation 01.12.2013 Project funding (Div. I-III)
127464 Role of serpinB1 in cellular homeostasis in the bone marrow and the lung 01.10.2009 Project funding (Div. I-III)
149790 Serpin regulation of leukocyte proteases in cellular homeostasis and inflammation 01.12.2013 Project funding (Div. I-III)

Abstract

Serine proteases carried in cytoplasmic granules or lysosomes of granulocytes, monocytes, mast cells, cytotoxic lymphocytes and NK cells contribute to both protective and destructive inflammatory and immune responses. A plethora of functions have been attributed to these proteases such as killing pathogens, inactivating toxins, inducing death of infected or cancer cells, modulating cytokine activity and remodeling extracellular matrix proteins. Much progress has been made in the last decade in understanding the functions of intracellular serpins which inhibit granule proteases of immune cells. Importantly, we have established that Serpinb1 is an essential survival factor of neutrophils (PMNs) through the inhibition of their own granule protease CatG. CatG triggers a form of programmed cell death that has features of apoptosis through activation of caspases and of regulated necrosis with rapid loss of plasma membrane integrity and release of inflammatory mediators. Although we have made significant advances, the exact molecular mechanisms leading to cell death and inflammation in the absence of intracellular serpins remain to be identified. In this project, we will use a combination of targeted (hypothesis-driven) and untargeted (degradomics and phenotype screening) methods to address the knowledge gaps in the mechanisms of CatG-mediated cell death and in the functions of Serpinb1, Serpinb6 and Serpinb9 in vivo. In particular, we have taken the high risk, high reward approach of mouse genetics and developed unique models to directly address these important questions. The proposed project will address the following two aims: Our first specific aim is to identify the mechanisms of cell death mediated by cathepsin G in myeloid cells. We will focus on the biology of myeloid cells and further explore specific cell death pathways associated with PMN and monocyte PCD in vitro. We will use a comparative proteomic approach to identify the CatG-specific degradome in PMNs associated with cell death. The identified targets will be validated by genetic targeting, including CRISPR and Cas9 designer nuclease technology. The second specific aim is to investigate the global and individual functions of the 14 functional serpins encoded in the expanded mouse SerpinB6-SerpinB9-SerpinB1 gene cluster on mouse chromosome 13, for which we generated a complete, yet viable knock-out. We will investigate specific defects of innate and adaptive immune cell development, homeostasis in steady state and inflammatory conditions. We will particularly investigate the cell autonomous and non-cell autonomous functions of invariant NKT cell subsets, which appear to be negatively regulated by SerpinB1 and, potentially, also by other intracellular serpins of this cluster. Finally, we will perform a standardized, comprehensive phenotypic screening of mice lacking all clade B serpin genes on mouse chromosome 13. This explorative analysis will be followed by a second stage testing where identified defects will be functionally investigated for individual serpin genes and their known target proteases. Overall, this multipronged project based on animal genetics will identify and functionally validate novel proteolytic targets associated with programmed cell death and will reveal important pathophysiological mechanisms mediated by granule proteases of immune cells and their inhibitors, which will likely have implications for developmental, inflammatory, infectious and proliferative diseases.
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