Philippe J Sansonetti & collaborators

Royal Society, London New Fellows SeminarJuly 9-10, 2014

Nothing in biology makes sense except in the light of evolutionTheodosius Dobzhansky

Pathogens and commensals: War and Peace at mucosal surface

Symbionts

Innate immunityPhysiological inflammationSurveillance/Tolerance

Recognition network:PRRs:TLRs,NLRs,

Rig1, MDA5…

Pathogens

Innate immunityPathological inflammationMicrobe & tissue destruction

Amplification loop:TREM, HMGB1, Gal3, Sepsis,

Septic shock

Regulation

Loss of control

Sansonetti, 2004, Nature Rev. Immunol.Sansonetti, 2006, Nat. Immunol.Sansonetti & Di Santo, 2007, ImmunitySansonetti & Medzhitov, 2009, CellSansonetti, 2010, Mucosal Immunology

Rupture of homeostasis = IBD,obesity, diabetes, cancer ?

Adaptive immunityPathogens recognition,capture, completion of

eradication process, protection

Co-evolution has created an « immunologicalconundrum » that forces to conjugatetolerance to commensal bacteria and quick and efficient recognition and elimination of bacterial pathogens.

1012/ml in colon

How does one identify a harmful pathogen hiddenamidst

a herd of harmless symbionts ?

Bacterial life at mucosal surfaces« Seating on a volcano »

O2, NO, ROSAntimicrobial peptidesLysozyme, proteases, lectins, phospholipasesTransmigrating phagocytessIgA

Epithelium

Mucus

Cationic antimicrobial peptide hBD3Sperandio et al.

Kim et al., PNAS, 2005Arbibe et al., Nature Immunol., 2007Sperandio et al., J. Exp. Med., 2008Marteyn et al., Nature, 2010Konradt et al., Cell Host Microbe, 2011Puhar et al., Immunity, 2013

Symbionts/commensals: survive at distance or in particular niches, escape (regulate) innate defenses

Pathogens: engage epithelium and subvert immune defenses- Blocking of danger signaling- Post-translational modification of key immune signaling molecules

OCTN2 PepT1

QSM (hsl)

MDP

TLR

NLRRegulatorycascade

?

Regulatory genes

Mucus layer

Regulatory cytokines, chemokines

Antimicrobialmolecules

Epithelial apex

Microbiota

Increase in ratioTreg/Th1, Th17lymphocytes

Increase in ratioImmature/matureDC and MΦ

Peace: symbiotic bacteria maintain gut homeostaticmechanisms

SYMBIONTS

Absence (limitation) of virulence factors

PAMPs less agonist ?

Sequestration, weak activityof TLRs

Life in biofilms on mucus surface (at distance of epithelium)

Controled diffusion and sampling of PAMPs and prokaryotic signalisation molecules

Symbionts

Pro-inflammatory

cascade

TLR

Pro-inflammatory genes

PMN

Activated DC & MΦ Th1 / Th17

Pro-inflammatory cytokines, chemokines

PMNDC

K+

K+

NLRs

Caspase-1

activation

IL-1β

Muropeptides

Flagellin

PATHOGENS

1 – MucinasesEradication of microbiota(niche occupancy)Adhesins / InvasinsSecretory systems /effectorsHemolysinsMassive engagement of PRRsPathogenic properties sensedas exogenous danger signals

2 - Release of endogenousdanger signals (DAMPs /smallmolecules) before initiation ofproinflammatory transcriptionalreprogramming.

3 – Subversion / dampening of innate (inflammatory) and adaptive immune responses

War: pathogenic bacteria disrupt / by-pass guthomeostatic mechanisms

Epithelial cells

mucus

Basolateral macropinocytosis (TTSS)

Vacuole lysis(TTSS)

Escape to autophagy

Motility, cell to cellspread (TTSS)IcsA

??

M cell

MΦ

B Lympho

MΦpyroptosis

TTSS/IpaB

-Pyroptosis = proinflammatory apoptosis-Activation of caspase-1, Release of IL-1β and IL-18

DC

«facilitated translocation»

Follicle-associated epithelium

PNN

Nod1

PGN

NF-κBJNK

TTSS

Pro-inflammatorygenes

IL-8, other cytokineschemokines

- Development of inflammation- Rupture of epithelial barrier- Facilitation of invasion- Stimulation of epithelial bactericidal capacities

Defensins and other bactericidalmolecules

CCL-20

B Lympho

T Lympho

Pathogenesis of Shigella infection: central roleof Type Three Secretory Apparatus (T3SA)

NOD LRRsCARD

NOD LRRsCARDCARD

TLRs

NOD1

NOD2

IRAKTRAF6

RICK/Rip2

IkBIkBPP

PP IkBIkBPP

PPU UUU

MEKK3

IKK complex

IkB

PGN

PAMPs

Inflammatory programingbased on sensing abnormallocalisation of bacterial cellwall fragments and transcription of innateimmunity genes

JNKCaspase-9

Girardin et coll., EMBO Reports, 2001Girardin et coll.., Science, 2003Girardin et coll., J.Biol.Chem., 2003

Epithelial cells

Muramyl-tri/tetrapeptide

Muramyl-dipeptide

Inflammation

Pro-inflammatory genes

Tri-Tetra-DAP/Nod1MDP/Nod2

Shigella - danger signaling 1: cytosolic sensing of PGN by Nod molecules

Pyd

HSP90

Pyd NBD

SGT1

LRR

NLRP3

NBD

NLRP3

Caspase

NBD

NBD

NAIP5

NLRC4/IPAF

Pro-caspase-1

NLRC4/IPAFinflammasome

NLRP3inflammasome

Caspase-1

Pro-IL1β

Pro-IL18

IL1β

IL18

Apoptosis

?

P2X7Pannexin-1

(K+)

K+

Bacterialhemolysins

IpaB

IpaCUric acidcristals

TTSS translocator

ATP

FlagellinS.typhimuriumP.aeruginosa

FlagellinL..pneumophila

?S.flexneri

IL1β

IL18

S.aureusL.monocytogenesA.hydrophyla

NigericinMaitotoxin

K+

Zychlinsky et al., 1992, NatureZychlinsky et al., 1994, J Clin InvestHilbi H et al., 1998, J Biol Chem

Macrophages

Shigella- danger signaling 2: pyroptosisinflammasome

Rapid inflammatoryprograming based onrelease of a presynthetizedpool of pro-inflammatorycytokines (Il-1ββββ, IL-18)

Inflammation

Inflammasome activationDifferentiation of naive T cellsto Th17 cells

HEMICHANNEL

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Tran Van Nhieu et al., 2003, Nat Cell BiolPuhar et al., 2013, Immunity

Shigella - danger signal 3: early epithelial cell release of ATP across connexin-based hemichannels (Danger-associated molecular patterns)

Rapidinflammatoryprogramingbased on release of intracellularATP (DAMPs)= Inflammasomeactivation= Th17 lymphocytes differentiation

Inflammation

VirB

ipaA, ipaB, ipaC, ipaD,ipgB1, ipgD, icsB,ospC2/3/4,ospD1, ospD2

ospD3, ospE1/2, ospG,ipaH1/2, ipaH4,ipaH7, ipaH9.8

MxiE

Expression, regulation, subversive function of T3SA effectors

before secretion after TTSS activation(target cell recognition)

INVASION MODULATION OF INNATE RESPONSESIpaB, IpaC, IpaA, IpgB1,VirA, IpgD

IpgD: phosphatidyl-inositol phosphatase, hydrolyses P in 4 in Pi(4,5)P2 (Niebuhr et al, 2002, Pendaries et al, 2006 EMBO J.). Anti-inflammatory +++ (Puhar et al., in review).

OspG: kinase,binds/blocks ubiquitin transfer protein E2, protects I-kB from degradation. Anti-inflammatory +++ (Kim et al., 2005, PNAS).

OspF: dephosphorylation of Erk1/2, epigenetic regulation of pro-inflammatory genes - i.e. IL-8. Regulates transmigrationof PMNs through epithelium (Arbibe et al., 2007,Nat.Immunol.). Phosphothreonine lyase (Li et al., 2007, Science).

IpaHs: (5 + 5 chromosomal copies): New family of Ubiquitinligases (E3) (Rohde et al., 2007, Cell Host & Microbes)IpaH9.8 targets NEMO (Ashida et al., 2010, Nat.Cell Biol.)

OTHER PHENOTYPESIcsB: inhibion or autophagy(Ogawa et al., 2005, Science) VirA: inhibition of microtubules, facilitates actin-based motility(Yoshida et al., 2006, Science)

ospBospFospC1virA

INHIBITION OF SECRETIONIpaB:(Mounier et al.,2012. Cell Host & Microbe)

HEMICHANNEL

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IpgD

Pi(4,5)P2 Pi(5)PIpgD

Pi(5)P

Shigella T3SA effector IpgD dampens ATP-mediateddanger signaling

Niebuhr et al., Mol. Micro. 2000 Niebuhr et al., EMBO J. 2002Pendaries et al., EMBO J 2006

Inflammationx

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ATP release in luminalfluid, rabbit ileal loop.4h infection

Puhar et al., 2013, Immunity

wt S. flexneri) IpgD-

Histopathology, HESRabbit ileal loop8 h infection

IpgD

cellules épithéliales

mucus

macropinocytosebaso-latérale (TTSS)

lyse vacuole(TTSS)

motilité/passagecellule-cellule

(TTSS)IcsAM cell

MΦ

Lympho B

MΦpyroptosis

TTSS/IpaB

- activation of caspase-1-pyroptosis = pro-inflammatory apoptosis- release of IL-1β et IL-18 DC

«facilitated translocation»

follicle-associated epithelium

Nod1

PGN

NF-κBJNK

TTSS

Osp(s)TTSS

IL-8

CCL-20

PNN

Suppression of humoral defense mechanisms

Suppression of cellular defense mechanisms

AMPs

Kim et al. 2005. PNASArbibe et al. 2007. Nat ImmunolSperandio et al.2008. J Exp MedBergounioux et al. 2012. Cell Host MicrobeSperandio et al. 2013. Infect ImmunPuhar et al. 2013. Immunity,

ATP

ATP

Suppression of danger signaling

PR

ATP

ATP

Immunosuppressive environment induced by Shigella (innate)

Laurence ArbibeStéphane GirardinBenoît MarteynF-Xavier Campbell-ValoisMaria MavrisJoëlle MounierClaude ParsotArmelle PhaliponThierry PédronAndrea PuharJohn RohdeBrice SperandioPamela Schnupf

REST OF THE WORLDGuy Tran Van Nhieu et al. (INSERM, Coll. de France)Laurent Combettes (INSERM, Orsay)Bernard Payrastre et al. (INSERM, Toulouse) Chris Tang (University of Oxford)Philippe Jay (INSERM, IGF, Montpellier)Françoise Poirier et al. (Institut Jacques Monod, Paris)Sylvie Robine et al. (Institut Curie, Paris)

Dana PhilpottMarie-C. PrévostJean-Yves CoppéeChristian MuchardtJost EnningaNathalie Sauvonnet