«Les polynucléaires neutrophiles» DU maladies systémiques et autoimmunes

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1 «Les polynucléaires neutrophiles» DU maladies systémiques et autoimmunes Véronique Witko-Sarsat INSERM U1016 Cochin Institute Team: Neutrophils and vasculitis Cochin Hospital, Paris Les neutrophiles dans l inflammation et en auto-immunité 1. Origine et systèmes effecteurs des neutrophiles Migration Phagocytose et bactéricidie 2. Les neutrophiles dans l autoimmunité : spécificité fonctionnelles Les protéines antibiotiques et les NETs dans le lupus érythémateux systémique, la polyarthrite rhumatoïde 3. Interplay des neutrophiles et des monocytes/macrophages phase active de l inflammation résolution de la l inflammation: apoptose des neutrophiles et leur phagocytose par les macrophages 4. Les neutrophiles dans les vascularites à ANCA à la fois des cibles cibles et effecteurs des dommages endothéliaux Page 1

2 Les cellules phagocytaires dans l inflammation et l immunité innée PMN monocyte Endothelium activation 3 PMN chemoattractant s: CXC chemokine : IL8, F-MLP, LPS, LTB4 Lipid mediators Resolvin, protectin, lipoxin Annexin1 macrophage Monocyte recruitment via CC chemokines IL6, TNF-alpha, IL1 Lymphocyte recruitment via CC chemokines (MCP1) 4 Antigen presentation Instruction of the immune response Microbicidal mechanisms Oxidative burst: superoxide anion, H 2 O 2, HOCl Proteinase, antibiotic proteins, defensins Time Phagocytosis of apoptotic PMN Anti-inflammatory process IL10, TGF-beta, IL13 Lipid mediators Lymphocytes within lymphatic circulation Kantari et al. The role of neutrophils and monocytes in innate immunity. Contrib Microbiol Neutrophil is derived from hematopoietic stem cells in bone marrow. Bone marrow Hematopoietic stem cell (HSC) Myeloid pregenitor Granulocyte-monocyte progenitor Blood monocyte Neutrophil 1-6% 50-70% Multilobed nucleus (polymorphonuclear leukocyte (PMN)) Granules (granulocytes) Short lived (only a few days) and non-dividing Page 2

3 Mécanismes effecteurs et microbicides des polynucléaires neutrophiles 1 Chimiotactisme - Adhérence - Migration 4 Mécanismes microbicides indépendants de lʼoxygène dégranulation 2 Phagocytose via des récepteurs spécifiques 3 Mécanismes microbicides dépendants de lʼoxygène Explosion respiratoire Neutrophils are recruited to the sites of inflammation Chemokines produced at the site of injury (e.g IL-8) are released and bind to heparan sulfate on vessel wall, facilitating activation of rolling leukocyte and firm adhesion via integrin mediated actions Induction of adhesion molecules on endothelial cells is induced by an array of inflammatory mediators like TNF, IL-1, histamine and others Page 3

4 Neutrophils migrate through endothelial cells within post-capillary venules. IL8 (or other chemoattractants) gradient guides neutrophils toward the site of infection. Leukocyte Adhesion Deficiency (LAD) LAD-I Patients are susceptible to recurrent and chronic bacterial infections. The patients lack CD18. CD18 is the β subunit of integrin LFA-1. LFA-1 mediates the tight binding of neutrophil to ICAM on endothelial cells during extravasation into inflammation site. CD18 is the β subunit of the integrin complement receptors (CR3 and CR4), which are involved in adhesion and opsonin-mediated phagocytosis. Integrin (LFA-1) Activated integrin ICAM α β CD18 Ig domain tight adhesion, and arrest ICAM Page 4

5 LAD-II Patients are susceptible to severe recurrent bacterial infections. Patients lack Sialyl-Lewis x on the mucin-like CAM (PSGL-1) of neutrophils. Sialyl-Lewis x is the ligand for E-selectin on endothelial cells, and mediates the initial adhesion of neutrophil with endothelial cells during extravasation. PSGL-1 E-selectin PSGL-1 E or P-selectin Sialyl-Lewis x NEJM: Vol. 343: No 23, pp Leukocyte adhesion deficiencies (LAD 1 and 2) Neutrophils Leukocytes unable to aggregate unable to leave the circulatory system Neutrophil counts are commonly twice the normal level even without an ongoing infection (Leukocytosis) Clinical findings: History of delayed separation of umbilical cord Severe peridontitis Recurrent bacterial and fungal infections of oral and genital mucosa (enteric bacteria, staph, candida, aspergillus) Infected foci contain few neutrophils (no pus) and heal poorly LAD 2 immunodeficiency is less severe, however the defect is associated with growth retardation, dysmorphy and neurological deficits Page 5

6 Fc receptors mediate phagocytosis. Récepteurs Fc FcgRII (CD32) FcgRIII (CD16) IgG: IgG1, IgG2, IgG3, IgG4. IgG1 is the most effective opsonin for phagocytosis. Voies de signalisations activées par la phagocytose Page 6

7 Neutrophils have oxidative and non-oxidative mechanisms of killing NADPH oxidase system, a membrane bound enzyme complex, reduces O2 to superoxide anion (02-), hydrogen peroxide (H2O2), and hydroxyl radical (OH) = Oxidative burst Bacteriocidal and cell degrading enzyme contents of lysosomal granules (azurophil- and specific granules) fuse with phagosome to form phago-lysosome H2O-MPO-halide system is thought to be the most efficient bactericidal system (in vitro!!) by catalyzing the formation of bleach (hypochlorous acid = HOCL) from H2O2 and Cl- Assembly of the phagocyte NADPH oxidase NOX2 Bedard, K. et al. Physiol. Rev. 87: ; doi: /physrev Copyright 2007 American Physiological Society Page 7

8 Déficit génétique en NADPH oxydase Granulomatose septique chronique (CGD) Phagocytose normale sans génération d anion superoxyde Déficit de gp91phox lié à l X : 80 % Déficit autosomal récessif P47phox : 30 % P22phox : 2% P67phox : 3% Infections bactériennes et fongiques léthales : pneumonies récurrentes, abcès, infections de la peau Souris knock-out : CGD-p47phox et CGD-gp91phox Jackson et al J Exp Med 1995; Pollock et al Nat Genet 1995 Granulomatose septique (Chronic granulomatous disease: CGD) Germes isolés (catalase positifs): Bactériens : Staphylococcus aureus, Serratia marcesens, Salmonella Fongiques : aspergillus Pas de susceptibilité particulière aux infections virales ou parasitaires Les infections peuvent débuter dès la première année de vie et les localisations sont cutanées, ganglionnaires, pulmonaire, digestives et hépatiques Formations granulomateuses à lʼhistologie Traitements : Antibiothérapie, interféron-gamma Transplantation moelle Thérapie génique : Etudes précliniques chez la souris knock out via retroviral transfert Essais chez lʼhomme: transfection des cellules souches hématopoiétiques avec le gène gp91phox via un vecteur rétroviral (Malechet al J Infect Dis, 2004; Ott et al Nat Med 2006) Page 8

9 Formation du NET : Neutrophil extracellular traps : un piège extra-cellulaire pour bactéries Pendant l activation, les neutrophiles libèrent des protéines granulaires et de la chromatine qui forment des fibres extracellulaires qui lient les bactéries. Les proteases immobilisées sur le NET dégradent les facteurs de virulence des bactéries Brinkmann et al Science 2004 Neutrophil extracellular traps: Is immunity the second function of chromatin? Brinkmann and Zychlinsky J Cell Biol, 2012 Visualizing NETs using chromatin antibodies or DNA-intercalating dyes. Brinkmann V, Zychlinsky A. JCB 2012;198: In autoimmunity settings, what is called NET are either complexes between DNA and granule proteins (or other proteins) and chromatin or extracellular DNA Page 9

10 Implication of NETS in anti-ll37 autoantibodies in lupus Evidence for NET formation in kidney from patients with ANCA-associated vasculitis PR3 or MPO within NET Netting neutrophils in autoimmune small-vessel vasculitis.kessenbrock et al Nat Med Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil antigens to myeloid dendritic cells toward ANCA induction and associated autoimmunity Sangaletti et al. Blood : Page 10

11 Les protéines granulaires des neutrophiles sont la cible des ANCA (anti-neutrophil cytoplasmic antibodies) Perinuclear pattern p-anca Cytoplasmic pattern c-anca anti-mpo Polyangéite microscopique, GN nécrosante à croissant Churg-Strauss (60%) anti-pr3 Granulomatose de Wegener (85 %) Page 11

12 ANCA associated vasculitis Tree of knowledge GPA MPA Clinical fruits Kidney Lung ENT Granuloma Histological Foliage VASCULITIS Autoimmune branching Anti-PR3 Anti-MPO Environment S. aureus PTU Genetical roots PRTN3 AAT Z allele HLA DP HLA DQ Millet et al. Ann Rheum Dis 2013 In vivo pathogenic role of anti-mpo-anca In vivo : murine model of anti-mpo glomerulonephritis (Xiao et al J Clin Invest 2002) Necrotizing and crescentic glomerulonephritis Systemic vasculitis Glomerular immune deposits GN Fibrin IgG Crucial role of neutrophils and alternative pathway of the complement (Xiao et al Am J Pathol 2005, 2007; Schreiber et al JASN 2006) Role of the p38-map kinase (van der Veen et al Ann Rheum Dis 2011) PI3 kinase pathway (Schreiber et al 2010 Kidney Int) Experimental autoimmune vasculitis (EAV) in rat : anti-mpo glomerulonephritis (Little et al Am J Pathol 2009) Page 12

13 Le neutrophile dans les vascularites associées aux ANCA : cellule effectrice de la destruction endothéliale et cible de l autoimmunité Mouthon et al. Presse Med 2012 Proteinase 3 and other neutrophil serine proteinase homologues Serine proteinase from myeloid cells present in mature neutrophil granulocytes and in monocytes Baggiolini et al. Agents and Actions1978 Granular protein stored mainly within azurophilic granules together with its «Serprocidin» homologues : human neutrophil elastase (60 % sequence homology), cathepsin G and azurocidin Degradation of extracellular matrix proteins (collagens, vitronectin, fibronectin) Rao et al. J. Biol. Chem 1996 Known as myeloblastin and involved in the control of myeloid proliferation and differentiation Bories et al. Cell. 1989; Witko-Sarsat et al J Biol Chem 2002; Dublet et al J Biol Chem 2005 Page 13

14 Membrane expression? In contrast to MPO,PR3 is expressed in a subset of resting neutrophils IgG1 MPO 70% PR3 +TNF No relationship with intracellular PR3 content TNF-alpha which mobilizes secretory vesicles at the neutrophil surface, increased PR3 membrane expression Witko-Sarsat et al, Blood1999 Interférence de la PR3 dans les mécanismes de résolution de l inflammation Gabillet et al. J Immunol 2012 Page 14

15 Modification of EC interactions Differentiation program Neutrophil deregulation In AAV Immune program Aberrant expression of autoantigen PR3 MPO Apoptosis Cell death NET-osis Cytokines BAFF Increased synthesis Mislocalization? Proform? Defect of clearance by macrophages Cross-talk with other immune cells Autoimmunity conclusion 1. Les neutrophiles sont des cellules clé de l inflammation et un défaut de leur clearance peut aboutir à un retard de la résolution de l inflammation et à des phénomènes d autoimmunité. 2. Rôle des NET? Complexes protéines cationique et DNA qui peuvent activer les cellules dendritiques Rôle des protéines cationiques antibiotiques qui peuvent être la cible d autoanticorps? 3. Capacité immunomudulatrice en particulier via leur synthèse de BAFF qui active les lymphocytes B 4. Interplay des neutrophiles et des monocytes/macrophages dans la polarisation de la réponse immune aux cellules apoptotiques Interférence par des autoantigènes comme la protéinase 3 dans la granulomatose avec polyangéite Page 15