Biomedical research institute
     

    Research project 1

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    Control of activation and death of neutrophils is crucial for the resolution of inflammation

    It is now well admitted that neutrophils are involved in numerous non infectious conditions including cancer, autoimmune, cardiovascular and allergic diseases.

    Figure 1 : New concepts in neutrophil biology

    During the last decades, new concepts have dramatically modified the image of the neutrophils: (1) Neutrophils are now endowed of plasticity with the generation of new sub-populations of neutrophils with diverses immunomodulatory functions; (2) Neutrophils can do reverse migration;  (3) Neutrophils can communicate and instruct various immune cells; (4) Neutrophil death is complex and is a very important determinant in the immune and inflammatory response (Figure 1).

    Reference: Chatfield et al. Expanding Neutrophil  Horizons: New Concepts in Inflammation. J Innate Immun.  2018;10(5-6):422-431.

     

    It is now evident that there is a tight link between death pathways that are activated in neutrophils and inflammation resolution.

    Historically, neutrophils have been viewed only as professional phagocytic cells that are able to phagocytose and destroy infectious agents. Indeed, they are key anti-infectious actors in host defense but can mediate tissue damages (Witko-Sarsat et al Lab Invest 2000).

    However, it is now clear that the role of neutrophils go far beyond phagocytosis and pathogen killing. Following successive steps of adhesion, chemotaxis and migration, can phagocytose invading pathogens and mobilize their microbicidal effector molecules, including reactive oxygen species, antibiotic proteins and proteinases. This neutrophil activation triggers an apoptosis program to ultimately signal their phagocytosis to macrophages (Witko-Sarsat et al Trends Immunol 2011).

    The recognition and the subsequent engulfment of apoptotic neutrophils by macrophages is a key event of the resolution of inflammation, which can be associated with autoimmunity or inflammatory.  In addition, this safe disposal of apoptotic neutrophils generate anti-inflammatory signals to complete the inflammation resolution (Thieblemont et al seminar Immunol 2017 ; Thieblemont et al Eur J Clin Invest. 2018)

    In contrast, necroptosis, the death induced by death receptors when apoptosis is inhibited, is an inflammatory death. It is mediated by the necrosome, an oligomer composed of RIPK1, RIPK3 and MLKL leading to the cell lysis. More recently, the pyroptosis, another inflammatory death induced by inflammasomes has been characterized.

    Figure 2 : Mechanisms leading to the activation of the inflammasome NLRP3 and to the secretion of interleukine-1beta has been extensively studied in monocytes/macrophages. Less attention has been paid to inflammasome in neutrophils which seem to have specific signaling pathways.

    Reference: Tourneur L, Witko-Sarsat V. Inflammasome activation: Neutrophils go their own way. J Leukoc Biol. 2019 Mar;105(3):433-436.

     

     

     

    FADD : a regulator of apoptosis and other death pathways

    The role of FADD protein is well known to regulate apoptosis-related pathways but its biologic function extends beyond apoptosis to other death pathways. FADD is crucial for numerous biologic processes such as embryogenesis, survival, proliferation, cell cycle progression, autophagy, innate immunity, inflammation and cancer (Tourneur et al. Medical Immunol 2005; Tourneur and Chiocchia. Trends Immunol 2010; Vilmont et al. Rheumatology 2012; Mouasni and Tourneur. Trends Immunol 2018).

    Figure 3 : FADD is involved in different signaling pathways leading to different death pathways

    Reference: Mouasni S, Tourneur L. FADD at the crossroads between cancer and inflammation. Trends Immunol. 2018; 39:1036-1053.  

    In a recent study, the group of Lea Tourneur has shown that FADD was secreted in plasma membrane-derived microvesicles and that this secretion was associated to the inflammatory relapses in rheumatoid arthritis.

    Reference :
    Mouasni S, Gonzalez V, Schmitt A, Bennana E, Guillonneau F, Mistou S, Avouac J, Ea HK, Devauchelle V, Gottenberg JE, Chiocchia G, Tourneur L. The classical NLRP3 inflammasome controls FADD unconventional secretion through microvesicle shedding. Cell Death Dis. 2019 Feb 25;10(3):190.

     

    PCNA (Proliferating Cell Nuclear Antigen) : an unexpected actor in neutrophil survival

    Figure 4: Discovery of Proliferating Cell Nuclear Antigen (PCNA) in neutrophil cytosol

    There are many different mechanisms responsible for the balance between apoptosis and survival of neutrophils and disruption of these mechanisms has the potential to lead to uncontrolled inflammation.

    Our group has identified an unexpected anti-apoptotic role for PCNA. In proliferating cells, PCNA is a critical factor in DNA synthesis and repair. In neutrophils which are devoid of proliferative capacities, PCNA is exclusively cytosolic.

    References:  Witko-Sarsat V, Mocek J, Bouayad D, Tamassia N, Ribeil JA, Candalh C, Davezac N, Reuter N, Mouthon L, Hermine O, Pederzoli-Ribeil M, Cassatella MA. Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival. J Exp Med. 2010 Nov 22;207(12):2631-45 

     

    Figure 5 : Molecular mechanisms involved in PCNA-mediated survival

    In neutrophils, PCNA is a trimeric protein (in blue on the cartoon) which can build a scaffold by its association with several proteins (Witko-Sarsat et al. J Exp Med 2010). In the neutrophil, PCNA is exclusively cytosolic and its scaffold is adapted to the different states of neutrophil physiology: 1) Under the steady state survival (represented in the center), PCNA is associated with “steady-state partners” such as pro-apoptotic proteins (procaspases) 2) Under inflammation or G-CSF treatment or hypoxia, de novo synthesis of “inducible partners” (for exemple p21/waf1) will modify the scaffold to promote survival. 3) Under non-inflammatory conditions, physiologic apoptosis is accompanied by a proteasomal degradation of PCNA with caspase activation. 4) Phagocytosis also modulates the PCNA scaffold but the role of PCNA in neutrophil fate seems to be highly dependent on the type of bacteria phagocytosed.

    References:  Witko-Sarsat V & Ohayon D. Proliferating cell nuclear antigen in neutrophil fate. Immunol Rev. 2016 Sep;273(1):344-56.

     

    PCNA is a new regulator of the NADPH oxidase

    Recently we have uncovered a new function of PCNA in the control of NADPH oxidase : PCNA can bind the p47phox protein thereby controlling the production of ROS (Ohayon et al J Exp Med 2019). This work has shown for the first time that it was possible to obtain a strong anti-inflammatory effect by targeting the association between PCNA and p47phox in neutrophils in a colitis model in mice. Lien news Cochin

    Figure 6: Molecular modelling of the interaction between PCNA and the PX domain of p47phox (collaboration Dominique Housset, Philippe Frachet IBS Grenoble)

     

    PCNA: a new marker of resistance to chemotherapy in leukemia

    We have also shown that the exclusive cytosolic localisation of PCNA results from an active nuclear export controlled by the CRM1 exportin. We have identified the NES in PCNA sequence which is located at the inner face of the trimer and is accessible to the exportin only when PCNA is monomeric. (Bouayad et al J Biol Chem 2012; De Chiara et al J Leukocyte Biol 2013).

    Figure 7 : PCNA has a nuclear export sequence (NES) and is relocalized from nucleus to cytosol at the end of granulocyte differentiation.

    References:

    De Chiara A, Pederzoli-Ribeil M, Mocek J, Candalh C, Mayeux P, Millet A, Witko-Sarsat V. Characterization of cytosolic proliferating cell nuclear antigen (PCNA) in neutrophils: antiapoptotic role of the monomer. J Leukoc Biol. 2013 Oct;94(4):723-31.

    Bouayad D, Pederzoli-Ribeil M, Mocek J, Candalh C, Arlet JB, Hermine O, Reuter N, Davezac N, Witko-Sarsat V. Nuclear-to-cytoplasmic relocalization of the proliferating cell nuclear antigen (PCNA) during differentiation involves a chromosome region maintenance 1 (CRM1)-dependent export and is a prerequisite for PCNA antiapoptotic activity in mature neutrophils. J Biol Chem. 2012 Sep 28;287(40):33812-25.

     

    Figure 8: The promyelocytic HL-60 cells resistant to chemotherapy have an increased cytosolic localization of PCNA connecting NAD+ metabolism, glycolysis and survival.

    This nuclear-to-cytosolic relocalization occurred which occured at the end of granulocyte differentiation under normal conditions, is observed in the promyelocytic HL-60 cells resistant to daunorubicin to favor their survival. Cytosolic PCNA could constitute a novel therapeutic target or a marker of response to treatment.

    Reference :

    Ohayon D, De Chiara A, Chapuis N, Candalh C, Mocek J, Ribeil JA, Haddaoui L, Ifrah N, Hermine O, Bouillaud F, Frachet P, Bouscary D, Witko-Sarsat V. Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia. Scientific Reports 2016 Oct 19;6:35561.

    This study has been supported  by the association pour la recherche  contre le cancer (ARC).