Role of he Inflammasome in fighting against infection. Gabriel Nuñez Department of Pathology University of Michigan

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1 Role of he Inflammasome in fighting against infection Gabriel Nuñez Department of Pathology University of Michigan

2 NLRs and TLRs mediate elimination of pathogens Pathoge n Mammals Plants TLRs LRRs membrane R LRRs TIR NLRs α LRRs Cyt R α /TIR LRRs Host defense against pathogen

3 Takeda and Akira, 2005 QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.

4 The -like Receptor (NLR) FAMILY Apaf-1 CARD WD40Rs 1, IPAF CARD LRRs 2 CARD CARD LRRs Cryopyrin and 13 NLR proteins PYD LRRs NAIP ( ) (DC)-CIITA CARD AD LRRs BIR BIR BIR LRRs 3, 9, 26 LRRs X Plant R proteins (95 in A. thaliana) TIR LRRs Plant R proteins (49 in A. thaliana) α/c LRRs CED-4 (C. elegans) CARD 8 PYD DEFCAP PYD LRRs DT PYDCARD RRS1(A. thaliana) TIR TIR LRRs WRKY Inohara and Nuñez, Nature Rev Immunol, 2003

5 Cryopyrinopathies (MWS, FCAS, CINCA) CIAS1 (skin, eyes, joints) Blau Syndrome/ Early-Onset Sarcoidosis 2 (skin, eyes, joints) Asthma 1 (lungs) Sarcoidosis 1 (lungs) Bare Lymphocyte Syndrome CTIIA (lymphocytes) Crohn s Disease 2 (ileum) Graft vs. Host Disease 2 (systemic, gastrointestinal tract)

6 ie-dap MDP Flagellin Microbial RNA Uric Acid Crystals Nod1 Nod2 Ipaf Cryopyrin RICK RICK ASC Caspase-1 ASC Caspase-1 MAPK IKK Ub P JNK Erk p38 IκBα P IκBα NFκB Pro-IL-1β Pro-IL-18 mature IL-1β IL-18 Cytokines/Chemokines IL-6, IL-8, TNFα, MCP-1, MIP-1α Adhesion Molecules VCAM, ICAM, E-selectin Inflammatory Mediator Enzymes inos, COX-2

7 PAMPs TLRs secretion system pore-forming toxin MyD88/TRIF flagelli n P2X 7 PAMPs NF-κB Ipaf Cryopyrin Pannexin-1 LLO Urate Crystals CARD PyD PyD Nucleus Caspase-1 Pro-IL-1β IL-1β

8 LIGAND-INDUCED PROXIMITY AND ACTIVATION OF NLR PROTEINS LRD NLR proteins Ligands EBD Effector Molecules Effector Domain Receptor-binding Domain Signalling

9 NLR proteins in caspase-1 inflammasomes

10 Salmonella Type III Secretion System SipB Salmonella TLR?? Caspase-1 NF-κB MAPK Pro-IL-1β Pro-IL-18 mature IL-1β IL-18

11 Calvin K. Yip and Natalie C. J Strynadka TRENDS in Biochemical Sciences 31(4) 2006

Ipaf is required for caspase-1 activation and IL-1β secretion Salmonella Salmonella IL-1β(pg/ml) 700 600 500 400 300 200 100 0 Uninf.

12 Ipaf is required for caspase-1 activation and IL-1β secretion Salmonella Salmonella IL-1β(pg/ml) Uninf. WT Ipaf-KO 1/1 1/5 1/10 IL-18(pg/ml) WT Ipaf-KO Uninf 1/ hrs procaspase-1 p20 Ipaf-WT Ipaf-KO

13 Which Salmonella molecule activates IPAF? TLR IPAF proil-1β Caspase-1 IL-1β

Point mutation of flagellin abrogates caspase-1 dependent IL-1β production and cell death. a IL-1β(pg/ml) 700 600 500 400 300 200 Salmonella I411A b procaspase-1 p20 100 0 Uninf.

14 Point mutation of flagellin abrogates caspase-1 dependent IL-1β production and cell death. a IL-1β(pg/ml) Salmonella I411A b procaspase-1 p Uninf. 1/1 1/5 1/10 Time (h) Salmonella I411A c Cell death (%) Salmonella flic-fljb I411A Uninf. 1/1 1/5 1/10 WT d Cell death (%) Salmonella flic-fljb I411A Uninf. 1/1 1/5 1/10 Ipaf-KO

15 Is the activation of IPAF dependent on TLR5? TLR5 IPAF Caspase-1 proil-1β IL-1β

Salmonella-induced caspase-1 activation is TLR5 independent a 700 600 500 WT TLR5-KO b

16 Salmonella-induced caspase-1 activation is TLR5 independent a WT TLR5-KO b procaspase-1 IL-1β (pg/ml) p Uninf. 1/1 1/5 1/10 Time (h) TLR5-WT TLR5-KO

17 Is purified flagellin able to activate caspase-1? FLG TLR5 IPAF Caspase-1 proil-1β IL-1β

18 Flagellin-induced caspase-1 requires IPAF but not TLR5 c d medium FLG DOTAP DOTAP + FLG medium FLG DOTAP DOTAP + FLG medium FLG DOTAP DOTAP + FLG medium FLG DOTAP DOTAP + FLG procaspase-1 procaspase-1 p20 p20 WT TLR5-KO WT IPAF-KO

19 Salmonella In addition: Legionella Pseudomonas Shigella ASC Caspase-1 Type III Secretion System SipB Ipaf FLAGELLIN IκBα / P IKK IκBα NFκ B TLR MAPK Salmonella Pro-IL-1β Pro-IL-18 mature IL-1β IL-18 Franchi et al. Nature Immunol., 2006; Suzuki et al. PLOS Pathogens, 2007; Franchi et al. EJI, in press

20 Legionella pneumophila causes Legionnaire s s Disease Replication Transmission Replication Amoeba Respiratory Tract If host defenses are poor Legionnaires Disease Alveolar macrophages No human to human Transmission

21 Infectious Legionella pneumophila replicate within a specialized phagosome Human monocytes A/J (Naip5) mice-derived macrophages lysosome 8 hrs Type IV secretion mutant Infectious Legionella

22 Ipaf -/- and caspase-1 -/- macrophages allow Legionella replication 7 Log bacterial colony forming unit hr 24 hr 48 hr 72 hr Time after infection IPAF WT IPAF KO TLR2 WT TLR2 KO Cryo WT Cryo KO Caspase-1 KO 2 KO

23 Ipaf and caspase-1 restrict Legionella replication DAPI DAPI/GFP WT Ipaf KO Casp1 KO 24 h after infection

24 Ipaf and caspase-1 regulate the maturation of the Legionella-containing phagosomes

25 Ipaf and caspase-1 regulate the maturation of the Legionella-containing phagosomes

26 The presence of bacterial flagellin restricts Legionella replication in macrophages and in mice Log bacterial colony forming unit hr 24 hr 48 hr 72 hr IPAF WT/ Leg WT IPAF WT/ Leg flaa IPAF KO / Leg WT IPAF KO/ Leg flaa

27 A role for Ipaf in Legionella replication independent of IL-1β and IL-18 Legionella factors (Flagellin) Ipaf No Ipaf Caspase-1 activation No caspase-1 activation Fusion with the lysosome No fusion with the lysosome Legionella degraded Legionella multiply Amer et al, 2007

28 L1007fs G980R R702W Nod2 LRRs CARDs N670K T605P M513T H496L L469F R382E R334W/Q Cryopyrin/Nalp3 LRRs PYD E637G D303N R260W Loss/Reduction of Signaling Uncontrolled Signaling Uncontrolled Signaling Crohn s Disease Blau Syndrome Early-onset Sarcoidosis CINCA FCAS MWS

29 Treatment of MWS patients with IL1ra (Anakinra) NAD FIIND PYD PYD CARD CARD CARD CARD Casp1 Casp1 IL1ra IL1ra IL-1β IL-6 TNF proil-1 Hawkins et al. N Engl J Med. 348:2583-4, 2003 Hawkins et al. Arthritis Rheum Feb;50(2): SAA IL-1 a target for therapy

30 The Two-signal Model for activation of the inflammasome Mariathasan and Monack (2007) Nature Rev Immunol. 7, 31

31 Cryopyrin is required for ATP- and killed bacteris-induced Caspase-1 activation and IL-1β secretion a - ATP + ATP Unst S. t F. t L. p P. a L. m P. g B. s S. a Unst S. t F. t L. p P. a L. m P. g B. s S. a p45 WT p20 Cryo -/- p45 p20 b IL-1β (pg/ml) WT Cryo-KO 0 Unst S. t F. t L. p P. a L. m P. g B. s S. a IL-6 (pg/ml) WT Cryo-KO Unst S. t F. t L. p P. a L. m P. g B. s S. a

32 The Cryopyrin inflammasome is activated independent of TLR signaling + ATP p45 p20 MyD88 -/- TRIF -/- TLR4 -/- TLR2 -/- Unst S. t F. t L. p P. a L. m P. g B. s S. a p45 p20 WT p45 p20 p45 p20 p45 p20

Coli RNA Unsti LPS Lipid A Pam3 PGN MDP LTA E. Coli DNA E.

33 Cryopyrin mediates PAMP-induced caspase-1 activation independent of TLRs a - ATP + ATP p45 Cryo -/- WT Unsti LPS Lipid A Pam3 PGN MDP LTA E. Coli DNA E. Coli RNA Unsti LPS Lipid A Pam3 PGN MDP LTA E. Coli DNA E. Coli RNA p20 p45 p20 b WT TLR4-KO Unst LPS LA Unst LPS LA IL-1β (pg/ml) p45 p20 c Un s t LP S LA IL-1β (pg/ml) W T T LR 4

34 The Two-signal Model for activation of the inflammasomes Mariathasan and Monack (2007) Nature Rev Immunol. 7, 31

35 Alternative: The Cytosolic Sensing Model

36 Role of Pannexin-1 in ATP-induced P2X7-mediated large pore formation These experiments confirm results from Pelegrin and Surprenant EMBO J., 2006

37 Critical role of Pannexin-1 in activation of the cryopyrin inflammasome

38 PAMPs TLRs secretion system pore-forming toxin MyD88/TRIF flagelli n P2X 7 PAMPs NF-κB Ipaf Cryopyrin Pannexin-1 LLO Urate Crystals CARD PyD PyD Nucleus Caspase-1 Pro-IL-1β IL-1β

University of Michigan Yun-Gi Kim Thirumala-Devi Kanneganti Mohamed Lamkanfi Luigi Franchi Jong-Hwan Park Amal Amer Christine McDonald Mathias Chamaillard Nesrin Ozören Raul Munoz Noemi Marina-Garcia

39 University of Michigan Yun-Gi Kim Thirumala-Devi Kanneganti Mohamed Lamkanfi Luigi Franchi Jong-Hwan Park Amal Amer Christine McDonald Mathias Chamaillard Nesrin Ozören Raul Munoz Noemi Marina-Garcia Michael Shaw Grace Chen Peter Kuffa Vladimir Galchev Naohiro Inohara Mary O Riordan Millennium Ethan Grant Tony Coyle John Bertin Osaka University Shizuo Akira Koichi Fukase Yale University Richard Flavell Koichi Kobayashi Case W. Res Univ George Dubyak Ghent University Peter Vandenabeele Petra Van Damme Tom Vanden Berghe Kris Gevaert Isabel Vanoverberghe Joël Vandekerckhove Univ of Ryukyus

Pannexin-1-Mediated Recognition of Bacterial Molecules Activates the Cryopyrin Inflammasome Independent of Toll-like Receptor Signaling

Article Pannexin-1-Mediated Recognition of Bacterial Molecules Activates the Cryopyrin Inflammasome Independent of Toll-like Receptor Signaling Thirumala-Devi Kanneganti, 1,4 Mohamed Lamkanfi, 1,4 Yun-Gi