Pathogens and commensals: War and Peace at mucosal surface Nothing - PowerPoint PPT Presentation

Pathogens and commensals: War and Peace at mucosal surface Nothing in biology makes sense except in the light of evolution Theodosius Dobzhansky Philippe J Sansonetti & collaborators Royal Society, London New Fellows Seminar July 9-10, 2014

Co-evolution has created an « immunological Adaptive immunity conundrum » that forces to conjugate tolerance to commensal bacteria and quick Pathogens recognition, and efficient recognition and elimination of capture, completion of eradication process, protection bacterial pathogens. Symbionts Pathogens Recognition network: PRRs :TLRs,NLRs, Rig1, MDA5… 10 12 /ml in colon Innate immunity Innate immunity Physiological inflammation Pathological inflammation Surveillance/Tolerance Microbe & tissue destruction Regulation Rupture of homeostasis = IBD, Amplification loop: Loss of control obesity, diabetes, cancer ? TREM, HMGB1, Gal3, Sepsis, Sansonetti, 2004, Nature Rev. Immunol. Sansonetti, 2006, Nat. Immunol. Septic shock Sansonetti & Di Santo, 2007, Immunity Sansonetti & Medzhitov, 2009, Cell Sansonetti, 2010, Mucosal Immunology

How does one identify a harmful pathogen hidden amidst a herd of harmless symbionts ?

Bacterial life at mucosal surfaces « Seating on a volcano » Symbionts/commensals: survive at distance or in particular niches, Mucus escape (regulate) innate defenses Pathogens: engage epithelium and subvert immune defenses - Blocking of danger signaling - Post-translational modification of key immune signaling molecules O2, NO, ROS Antimicrobial peptides Lysozyme, proteases, lectins, phospholipases Transmigrating phagocytes sIgA Epithelium Kim et al., PNAS, 2005 Arbibe et al., Nature Immunol., 2007 Sperandio et al., J. Exp. Med., 2008 Cationic antimicrobial peptide hBD3 Marteyn et al., Nature, 2010 Sperandio et al. Konradt et al., Cell Host Microbe, 2011 Puhar et al., Immunity, 2013

Peace: symbiotic bacteria maintain gut homeostatic mechanisms Antimicrobial SYMBIONTS molecules Microbiota QSM MDP Symbionts Absence (limitation) of (hsl) virulence factors Mucus layer OCTN2 PepT1 PAMPs less agonist ? Epithelial apex ? TLR Sequestration, weak activity NLR of TLRs Regulatory cascade Life in biofilms on mucus surface (at distance of Regulatory genes epithelium) Regulatory cytokines, chemokines Controled diffusion and Increase in ratio sampling of PAMPs and Treg/Th1, Th17 prokaryotic signalisation lymphocytes molecules Increase in ratio Immature/mature DC and M Φ

War: pathogenic bacteria disrupt / by-pass gut homeostatic mechanisms PATHOGENS 1 – Mucinases Eradication of microbiota (niche occupancy) K+ Adhesins / Invasins Secretory systems /effectors Caspase-1 Hemolysins K+ activation IL-1 β TLR Massive engagement of PRRs NLRs Pathogenic properties sensed Muropeptides Pro-inflammatory Flagellin cascade as exogenous danger signals DC PMN Pro-inflammatory genes 2 - Release of endogenous danger signals (DAMPs /small molecules) before initiation of Pro-inflammatory cytokines, chemokines proinflammatory transcriptional reprogramming. 3 – Subversion / dampening of PMN Th1 / Th17 Activated DC & M Φ innate (inflammatory) and adaptive immune responses

Pathogenesis of Shigella infection: central role of Type Three Secretory Apparatus (T3SA) Follicle-associated epithelium Defensins and other bactericidal molecules «facilitated mucus translocation» ? ? TTSS PGN Motility, cell to cell M cell IcsA spread (TTSS) Nod1 M Φ Vacuole lysis NF- κ B (TTSS) JNK B Lympho Escape to autophagy Pro-inflammatory genes PNN M Φ IL-8, other cytokines Epithelial cells chemokines pyroptosis CCL-20 Basolateral TTSS/IpaB macropinocytosis (TTSS) DC -Pyroptosis = proinflammatory apoptosis -Activation of caspase-1, Release of IL-1 β and IL-18 - Development of inflammation - Rupture of epithelial barrier - Facilitation of invasion T Lympho - Stimulation of epithelial bactericidal capacities B Lympho

Shigella - danger signaling 1 : cytosolic sensing of PGN by Nod molecules Tri-Tetra-DAP/Nod1 TLRs MDP/Nod2 PAMPs Epithelial cells IRAK TRAF6 MEKK3 U U P U U P PGN IkB IkB P IKK complex P IkB IkB NOD1 CARD NOD LRRs Muramyl-tri/tetrapeptide IkB RICK/Rip2 NOD2 Inflammatory programing JNK based on sensing abnormal CARD CARD NOD LRRs Caspase-9 Muramyl-dipeptide localisation of bacterial cell wall fragments and transcription of innate Pro-inflammatory genes immunity genes Girardin et coll., EMBO Reports, 2001 Girardin et coll.., Science, 2003 Inflammation Girardin et coll., J.Biol.Chem., 2003

Shigella K + Nigericin S.aureus ATP L.monocytogenes Maitotoxin A.hydrophyla - danger signaling 2 : K + Bacterial hemolysins P2X7 pyroptosis Pannexin-1 (K + ) inflammasome IpaC Uric acid TTSS translocator NLRP3 cristals NBD LRR ? Pyd IpaB Flagellin L..pneumophila HSP90 SGT1 NLRP3 S.flexneri Pyd NBD ? NBD NAIP5 NLRP3 inflammasome Flagellin NBD NLRC4/IPAF S.typhimurium P.aeruginosa Caspase NLRC4/IPAF inflammasome Pro-caspase-1 Macrophages Rapid inflammatory Apoptosis programing based on Caspase-1 release of a presynthetized pool of pro-inflammatory cytokines (Il-1 β β , IL-18) β β Pro-IL1 β IL1 β Zychlinsky et al., 1992, Nature IL1 β Pro-IL18 IL18 Inflammation Zychlinsky et al., 1994, J Clin Invest Hilbi H et al., 1998, J Biol Chem IL18

Shigella - danger signal 3 : early epithelial cell release of ATP across connexin-based hemichannels (Danger-associated molecular patterns) HEMICHANNEL (Connexins) x x Rapid x x x x x x x x x x d d inflammatory x x x x d d d d d d d d x x x x d d programing x x d d d x x x x x x d d d d d x x based on release dx x x x d d x x d d d d d x x x x x x of intracellular x x d d x x d d x x x x d x x d d d d x x ATP (DAMPs) d d d d d d x x d d x x d d x x = Inflammasome d d d d d d x x d d d d d activation d d d d = Th17 lymphocytes differentiation Inflammasome activation Inflammation Tran Van Nhieu et al., 2003, Nat Cell Biol Differentiation of naive T cells to Th17 cells Puhar et al., 2013, Immunity

Expression, regulation, subversive function of T3SA effectors after TTSS activation before secretion (target cell recognition) VirB MxiE ipaA, ipaB , ipaC, ipaD, ospB ospD3, ospE1/2, ipgB1, ipgD , icsB, ospF ospG, ospC2/3/4, ospC1 ipaH1/2, ipaH4, ospD1, ospD2 virA ipaH7, ipaH9.8 INVASION MODULATION OF INNATE RESPONSES IpgD : phosphatidyl-inositol phosphatase, hydrolyses P in 4 in IpaB, IpaC, IpaA, Pi(4,5)P2 (Niebuhr et al, 2002, Pendaries et al, 2006 EMBO J.). IpgB1,VirA, IpgD Anti-inflammatory +++ (Puhar et al., in review). INHIBITION OF SECRETION OspG : kinase,binds/blocks ubiquitin transfer protein E2, IpaB:(Mounier et al.,2012. protects I-kB from degradation. Anti-inflammatory +++ (Kim et al., 2005, PNAS). Cell Host & Microbe) OspF : dephosphorylation of Erk1/2, epigenetic regulation of OTHER PHENOTYPES pro-inflammatory genes - i.e. IL-8. Regulates transmigration IcsB: inhibion or autophagy of PMNs through epithelium (Arbibe et al., 2007,Nat.Immunol.). Phosphothreonine lyase (Li et al., 2007, Science). (Ogawa et al., 2005, Science) VirA: inhibition of microtubules, IpaHs : (5 + 5 chromosomal copies): New family of Ubiquitin ligases (E3) (Rohde et al., 2007, Cell Host & Microbes ) facilitates actin-based motility (Yoshida et al., 2006, Science) IpaH9.8 targets NEMO (Ashida et al., 2010, Nat.Cell Biol.)

Shigella T3SA effector IpgD dampens ATP-mediated danger signaling Histopathology, HES Rabbit ileal loop 8 h infection wt S. flexneri ) IpgD- IpgD IpgD HEMICHANNEL x x Pi(5)P (Connexins) x x x x d d x x x x x x x x d d d d x x dx x Inflammation x x d d d d x x d d d d d d d d d d d ATP release in luminal Pi(4,5)P2 Pi(5)P fluid, rabbit ileal loop. IpgD 4h infection Niebuhr et al., Mol. Micro. 2000 Niebuhr et al., EMBO J. 2002 Pendaries et al., EMBO J 2006 Puhar et al., 2013, Immunity

Immunosuppressive environment induced by Shigella (innate) follicle-associated epithelium Suppression of humoral defense mechanisms «facilitated translocation» mucus AMPs ATP TTSS motilité/passage PGN ATP M cell cellule-cellule IcsA (TTSS) Nod1 M Φ lyse vacuole (TTSS) NF- κ B Lympho B TTSS JNK Osp(s) ATP M Φ cellules épithéliales PR pyroptosis IL-8 macropinocytose TTSS/IpaB ATP baso-latérale (TTSS) CCL-20 Suppression of danger signaling - activation of caspase-1 -pyroptosis = pro- PNN inflammatory apoptosis - release of IL-1 β et IL-18 DC Kim et al. 2005. PNAS Arbibe et al. 2007. Nat Immunol Sperandio et al.2008. J Exp Med Suppression of cellular defense mechanisms Bergounioux et al. 2012. Cell Host Microbe Sperandio et al. 2013. Infect Immun Puhar et al. 2013. Immunity,