1. Molecular signalling in inflammation 2 lectures 2 nd Med Molecular Medicine Andrew Bowie, School of Biochemistry and Immunology

3. Definition of Inflammation A normally beneficial host response to foreign challenge or tissue injury that leads ultimately to the restoration of tissue structure and function. Normally is self-limiting while prolonged inflammation can lead to disease

6. The molecular basis? The key stages: Initiation… Signal transduction… Altered gene expression… Resolution

7. Initiation of inflammation 1.Infection-induced inflammation now well characterised 2.‘Sterile Inflammation’ is not 1. Rheumatoid Arthritis 2.Atherosclerosis 3.IBD 4.Psoriasis

8. Initiation of inflammation by infection Quasi-infectious stimuli (e.g. LPS) used for decades to model inflammation and sepsis Inflammatory mediators involved, and their effects well- characterised Only recently have the initiating events been described. Involves PRRs recognising PAMPs Three key families of PRRs: –Toll-like receptors (TLRs) –Nucleotide-binding oligomerisation domain proteins (NODs) –RIG-I-like receptors (RLRs)

9. TLRs are PRRs for PAMPs Medzhitov (2001) Nature Rev. Immunol. 1, 135

10. 2. Toll-Like 1. IL-1RI-Like dToll 1-8 hTLR1 hTLR2 hTLR3 hTLR4 hTLR5 hTLR6 hTLR7 hTLR8 hTLR9 hTLR10 mTLR11 mTLR12 mTLR13 N L6 RPPs IL-1RI 1L-1RAcP IL-18R IL-18RAcP IL-1Rrp2 T1/ST2 IL-1RAPL TIGIRR-1 SIGIRR Mammals Plants Insects 3. TIR Adaptors MyD88, Mal, TRIF, TRAM SARM

11. TLRs have a role in both the innate and adaptive immune responses Adapted from Medzhitov (2001) Nature Rev. Immunol. 1, 135 NO TNF, IL-6

12. NODs Implicated in inflammatory diseases Intracellular proteins Some are PRRs NOD domain LRR domain Effector domains (e.g. CARD)

14. Taken from Akira et al., Cell 124, 783-801, 2006 RLRs (and TLRs) recognise viral RNA

15. Sterile inflammation Vs infection-induced Little known about sterile inflammation but probably involves many of the same pathways Similar end-points, e.g. TNF production Therapeutic opportunity: upsteam blockade of signalling rather than targetting individual downstream cytokines Endogenous ligands of TLRs initiate it? ‘Danger signals’

17. Detection of PAMPs and DAMPs by TLRs Karin et al., (2006) Cell 124, 823

18. Overview of gene induction Signalling pathways affect gene induction DNA  mRNA  Protein Transcription Regulation of RNA (stabilisation, splicing) Translation Post-translational modification

19. Signalling by TLRs, NODs &RLS Leads to changes in gene induction These genes encode many inflammatory mediators (N.B. IL-1 and TNF) Transcription factors activated (e.g. NF  B) MAP kinases activated (e.g. p38 MAPK) The inflammasome & caspases activated

21. NF  B Central mediator of immune and inflammatory responses. Activated by diverse stimuli. Role in many physiological and pathological processes. IBIB p50 p65 Activator p65 p50 P IBIB P Degradation Gene Induction ?

22. P P IBIB IBIB p65 p50 p65 IKK complex    Ubiquitination by E3 Ligase Degradation p65 p50 Gene Indn Phosphorylation of IKKs Cyto Nuc

23. P P IBIB IBIB p65 p50 p65 IKK complex    Ubiquitination by E3 Ligase Degradation p65 p50 Gene Indn Phosphorylation of IKKs Cyto Nuc PKC  NIK TAK1 MEKK1 S6 KINASE MEKK 2 + 3 TPL-2 p105 Phosp and degrad p65 Phosp PKA Y42 Phosp PI3KRIP IRAK?

24. Basic signalling paradigm for TLRs leading to NF  B activation TLR MyD88 IRAK TRAF6 IKK NF  B

25. nucleus TRIF TIR NEMO IKK  IKK  TAB2/3 TAK1 RIP1 TRAF6 Uq NF-  BI-  B P P NF-  B P TIR LPS TIR TRAM DD TIR Mal MyD88 IRAK4 IRAK1 IRAK2 How LPS (via TLR4) causes NF  B activation

26. MyD88 TRIF Mal MyD88 TRIF MyD88 MyD88 MD2 TLR2/1 or 2/6 TLR3 TLR4 TLR5 TLR7/8/9 Mal NF  B NF  B IRF3 NF  B TNF  IL6 IL8 TNF  IFN  IL6 RANTES IL8 TNF  IFN  IL6 RANTES IL8 TNF  IL6 IL8 IRAKs TRAF6 RIP1 TBK1 IRAKs TRAF6 IRAKs TRAF6 IRAKs TRAF6 NF  B TNF  IL6 IL8 IRF5/7 IFN  TRAM

27. P38 MAP kinase Activated by TLR pathways TRAF6  TAK1  MKK  MAP kinase P38 phosphorylates some Txn Factors P38 has a role in stabilising cytokine mRNA

28. Caspase 1

29. Caspase 1 is activated by the inflammasome …but what activates the inflammasome??

30. How NODs signal changes in gene expression

31. Aberrant NOD signalling in disease

32. IL-1 Gene upregulated in inflammation Pro-IL-1 cleaved by ICE (Caspase 1) Secreted IL1R1, IL-1R2, IL1ra IL-1 signalling cascade Gene induction (IL-8, CAMs, COX, iNOS)

33. TNF Proinflammatory and apoptotic Key role in RA How it signals How its regulated

35. Regulation of TNF production and function TNF gene expression – NF  B and p38 Membrane bound initially (cleaved by TACE) Receptor shedding Receptor endocytosis SODD (silencer of death domains) TRIP (TRAF interacting protein), A20. NF  B/I  B autoregulation

36. Normally, initiation of inflammation (e.g. by TLR4) is tightly regulated

37. Resolution of inflammation Further gene products and lipid mediators Suppress pro-inflammatory gene expression Inhibit cell trafficking Induce apoptosis of inflammatory cells Injurious stimulus cleared Normal tissue structure and function restored.

38. Reading list 1.Akira & Takeda (2004) Toll-like receptor signalling. Nature Reviews Immunology 4: 499-511 2.Inohara & Nunez (2003) NODs: Intracellular proteins involved in inflammation and apoptosis. Nature Reviews Immunology 3: 371-382 3.Meylan, Tschopp & Karin (2006) Intracellular pattern recognition receptors in the host response. Nature 442: 39-44 4.Kanneganti, Lamkanfi & Nunez (2007) Intracellular NOD-like Receptors in Host Defense and Disease. Immunity 27: 549-559