Toll-like Receptor Signaling 1 Professor of Medicine University of Massachusetts Medical School, Worcester, MA, USA Why do we need innate immunity? Pathogens multiply very fast We literally swim in viruses and other microbes E. coli doubles every 30 minutes = 2x10 143 bugs in 3 days But adaptive immunity requires 3-14 days to respond 2 Innate immune recognition of microbial products: pathogen associated molecular patterns Microbes contain or release a variety of products that are recognized by the host as foreign These are components unique to microbes (not normally produced by the host) Often they are invariant between microbes of a different class Essential for microbial survival :> excellent targets for innate immune recognition 3 The screen versions of these slides have full details of copyright and acknowledgements 1
Gram-negative lipopolysaccharide (LPS, endotoxin): one of the best studied activators of innate immunity a. Bacterial cell (E. coli) b. cell-wall organization Lipopolysaccharide (endotoxin) Outer membrane Peptidoglycan Inner membrane d. Structure of lipid A c. Architecture of lipopolysaccharide O-specific chain Core region Lipid A LPS comprises 3-4% (dry weight) of E. coli - is located in the outer membrane: highest potential for recognition; Most Gram negatives are not viable without LPS 4 Nat Rev Immunol. 2003; 3 (2): 169-76 Gram-negative lipopolysaccharide (LPS, endotoxin): one of the best studied activators of innate immunity (2) 5 Nat Rev Immunol. 2003; 3 (2): 169-76 How is lipopolysaccharide (LPS, endotoxin) recognized? Lipopolysaccharide binding protein (LBP) binds LPS and enhances its activity LBP transfers LPS onto CD14 CD14 transfers LPS to a signaling receptor 6 LPS induces transcription of hundreds of immune response genes How? The screen versions of these slides have full details of copyright and acknowledgements 2
Cloning of Drosophila Toll Dorsal Toll mutant Ventral Lemaitre et al., Cell 1996; 86: 973-83 Mutations in Toll predispose to fungal infections Aspergillus fumigatus 7 Leucine-rich repeats (LLR) in TLRs 19-25 repeats Consensus sequence: each LLR contains XLXXLXLXX and XØXXØX 4 FXXLX motifs (L=leucine, F=phenylalanine, Ø=hydrophobic, X=any) TIR (Toll-IL1R-Resistance) domain: LRRs: ligand interaction TIR: signal transduction (adapter molecule interactions) Akira 2004 Nat Rev Immunol 8 The TLR/IL-1R superfamily 9 The screen versions of these slides have full details of copyright and acknowledgements 3
LPS hypo-responsive C3H/HeJ mice 10 Ligand induced activation of TLR3 The dsrna interacts with both N-terminal and C-terminal sites on the lateral side of the convex surface of TLR3 The positively charged residues of the termini of TLR3 interact with the sugar-phosphate backbones of the dsrna ligand dsrna-protein interaction is the main driving force for TLR3 dimerization Science, 2008; 320 (5874): 379-81 11 Pattern recognition, MD-2 Small, soluble extracellular protein Associates with TLR4 Structurally related to lipid binding proteins including the dust mite allergen Essential for LPS response 12 The screen versions of these slides have full details of copyright and acknowledgements 4
MD-2 has a pocket for LPS C 13 Mouse MD-2 The TLR4-MD-2-LPS complex Nature, 2009; 458 (7242): 1191-5 14 The TLR reportoire: all infectious diseases involve TLRs 15 Nature Reviews Immunology; 5: 446-458 The screen versions of these slides have full details of copyright and acknowledgements 5
Localization of TLRs 16 Nucleic acid sensing TLRs (TLR3, 7, 8 and 9) are sequestered in the endosomal compartment Endosomal localization of TLR9 prevents recognition of extracellular DNA 17 Nature Immunology, 2006; 7: 13-15 Heterologous expression of TLR4 enables LPS responses in HEK293 cells HEK293-TLR4/MD2 NFkB-p65 Overlay MD-2 18 The screen versions of these slides have full details of copyright and acknowledgements 6
Generic model of signaling by all PRRs Receptor - TLR Adaptors - MyD88 S/T kinase - IRAK E3 ligase - TRAF MAP3K - TAK1 Txn factors - NF-kB 19 LPS triggers activation of NFkB-driven cytokines 20 21 The screen versions of these slides have full details of copyright and acknowledgements 7
MyD88 was critical for turning on expression of inflammatory genes (TNF, IL-1, COX-2); In the absence of MyD88, expression of a number of genes was still induced at normal levels (IP-10, GARG16, IRG1) These genes contain ISRE elements within their promotors Cells treated with Lipid-A but not with the TLR-2 ligand MALP, led to formation of a complex that could bind the ISRE elements An IRF-3 antibody supershifted the complex, indicating that the complex contains IRF-3 22 23 TLRs signal through TIR domain adapter molecules 24 The screen versions of these slides have full details of copyright and acknowledgements 8
TLRs signal through recruitment of distinct TIR domain adapter molecules 25 Nature Reviews Immunology9: 535-542 LPS triggers two major signaling pathways 26 27 The screen versions of these slides have full details of copyright and acknowledgements 9
28 29 Post-translational modifications of TRAM CFP YFP Overlay 30 The screen versions of these slides have full details of copyright and acknowledgements 10
31 32 Sorting vs. Signaling adapters 33 Nature Reviews Immunology9: 535-542 The screen versions of these slides have full details of copyright and acknowledgements 11
Delivery of TLR4 to phagosomes occurs via a recycling endosome intermediate - controlled by the GTPase Rab11a 34 Immunity,2010; 33 (4): 578-80 Signaling downstream of TRIF/TRAM 35 Phosphorylation induced activation of the Interferon Regulatory Factor-3 36 The screen versions of these slides have full details of copyright and acknowledgements 12
IKK-related kinase TBK1, a central mediator of IRF-3 signaling 37 Control of TLR signaling by soluble TLRs TLR induced inflammatory gene products have the potential to elicit tissue damage TLR signaling must be tightly regulated Regulation occurs at multiple levels a b 38 Nature Reviews Immunology, 2005; 5: 446-458 Transmembrane and intracellular regulators of TLR signaling 39 Nature Reviews Immunology, 2005; 5: 446-458 The screen versions of these slides have full details of copyright and acknowledgements 13
40 The screen versions of these slides have full details of copyright and acknowledgements 14