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Molecular signalling in inflammation 2 lectures 2nd Med Molecular Medicine Andrew Bowie, School of Biochemistry and Immunology 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 The molecular basis? • • • • • The key stages: Initiation… Signal transduction… Altered gene expression… Resolution Initiation of inflammation 1. Infection-induced inflammation now well characterised 2. ‘Sterile Inflammation’ is not 1. 2. 3. 4. Rheumatoid Arthritis Atherosclerosis IBD Psoriasis 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 wellcharacterised • 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) TLRs are PRRs for PAMPs Medzhitov (2001) Nature Rev. Immunol. 1, 135 2. Toll-Like T1/ST2 IL-1RAPL TIGIRR-1 SIGIRR hTLR1 hTLR2 hTLR3 hTLR4 hTLR5 hTLR6 hTLR7 hTLR8 hTLR9 hTLR10 mTLR11 mTLR12 mTLR13 3. TIR Adaptors MyD88, Mal, TRIF, TRAM SARM Plants N L6 RPPs Mammals IL-1RI 1L-1RAcP IL-18R IL-18RAcP IL-1Rrp2 dToll 1-8 Insects Mammals 1. IL-1RI-Like TLRs have a role in both the innate and adaptive immune responses NO TNF, IL-6 Adapted from Medzhitov (2001) Nature Rev. Immunol. 1, 135 NODs • • • • • • Implicated in inflammatory diseases Intracellular proteins Some are PRRs NOD domain LRR domain Effector domains (e.g. CARD) RLRs (and TLRs) recognise viral RNA Taken from Akira et al., Cell 124, 783-801, 2006 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’ Detection of PAMPs and DAMPs by TLRs Karin et al., (2006) Cell 124, 823 Overview of gene induction • • • • • • Signalling pathways affect gene induction DNA mRNA Protein Transcription Regulation of RNA (stabilisation, splicing) Translation Post-translational modification 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. NFkB) • MAP kinases activated (e.g. p38 MAPK) • The inflammasome & caspases activated NFkB •Central mediator of immune and inflammatory responses. •Activated by diverse stimuli. •Role in many physiological and pathological processes. Activator P ? p50 IkBa P Degradation p65 IkBa p50 p65 Gene Induction Phosphorylation of IKKs IKK complex g a b p50 p65 IkBa Cyto Nuc p50 p50 p65P IkBa p65 P Ubiquitination by E3 Ligase Degradation Gene Indn RIP IRAK? NIK TAK1 PKCz MEKK1 S6 KINASE TPL-2 MEKK 2 + 3 Phosphorylation of IKKs p105 Phosp and degrad IKK complex g Cyto a b p50 p65 IkBa Nuc p50 p50 p65P IkBa p65 P Y42 Phosp p65 Phosp PI3K Ubiquitination by E3 Ligase PKA Degradation Gene Indn Basic signalling paradigm for TLRs leading to NFkB activation TLR MyD88 IRAK TRAF6 IKK NFkB LPS TRAM How LPS (via TLR4) causes NFkB activation Mal MyD88 TRIF TRAF6 Uq RIP1 TAB2/3 TAK1 NEMO IKKa IKKb P P I-kB NF-kB P NF-kB nucleus TLR2/1 or 2/6 TLR3 TLR4 TLR5 TLR7/8/9 MD2 Mal MyD88 TRIF Mal MyD88 TRIF MyD88 MyD88 TRAM IRAKs IRAKs RIP1 TBK1 TRAF6 NFkB NFkB TNFa IL6 IL8 TNFa IL6 IL8 IRF3 IFNb RANTES TRAF6 NFkB TNFa IL6 IL8 TBK1 IRF3 IFNb RANTES IRAKs IRAKs TRAF6 TRAF6 NFkB NFkB TNFa IL6 IL8 TNFa IL6 IL8 IRF5/7 IFNa 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 Caspase 1 Caspase 1 is activated by the inflammasome …but what activates the inflammasome?? How NODs signal changes in gene expression Aberrant NOD signalling in disease 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) TNF • • • • Proinflammatory and apoptotic Key role in RA How it signals How its regulated Regulation of TNF production and function • • • • • • • TNF gene expression – NFkB and p38 Membrane bound initially (cleaved by TACE) Receptor shedding Receptor endocytosis SODD (silencer of death domains) TRIP (TRAF interacting protein), A20. NFkB/IkB autoregulation Normally, initiation of inflammation (e.g. by TLR4) is tightly regulated 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. Reading list 1. 2. 3. 4. Akira & Takeda (2004) Toll-like receptor signalling. Nature Reviews Immunology 4: 499-511 Inohara & Nunez (2003) NODs: Intracellular proteins involved in inflammation and apoptosis. Nature Reviews Immunology 3: 371-382 Meylan, Tschopp & Karin (2006) Intracellular pattern recognition receptors in the host response. Nature 442: 39-44 Kanneganti, Lamkanfi & Nunez (2007) Intracellular NOD-like Receptors in Host Defense and Disease. Immunity 27: 549-559