Immunology - Lecture 2 - Pattern Recognition Receptors A - 3 TLR signalling pathways: . Ligand induces formation of dimer or conformational changes in pre-formed TLR dimer . X-ray crystal structures show at least mammalian TLRs make direct contact with microbial ligands · Direct contact between ligand and LRR domain > dimerise > brings them in close proximity > conformation change in TIR domain > recruit MyD88 > signal propagation · TLRs activate NFKB, AP-1, IRF transcription factors > induce expression inflammatory cytokines, chemotactic factors, antimicrobial peptides and type 1 IFNs (alpha and beta) > TLR 4 as a model for PRR activation: · Lipid A = biologically active component of LPS * Yersinia: cleaves acyl group > down to 5 > decrease immunostimulatory power · LPS binds LPS binding protein > concentrates · LPS-LBP associates CD-14 · CD-14 presents LPS-LBP complex to TLR4-MD-2 compplex · TLR 4 dimerises > activates pro-inflammatory genes · Subtle structural differences in LPS > human recognises different types of LPS with variability > E.coli best, S. typhimurium, penta-acylated, hexa-acylated · MD-2 = crucial binding partner Different species > different electrical surface interactions affecting binding · hTLR4 blocked by Lipid IVa which blocks MD-2 binding pocket * Lipid IVa can block LPS responses in humans > chlamydia infected macrophages exp # Because it only has 4 acyl groups, need at least 5 for an immune response · Single molecule studies: total internal reflection microscopy: Gazinger · Photobleaching Analysis: Hopkins, Claire Bryant Elife Cells with TLR4 knockout * Virally transduce SNAP-tagged TLR4 gene > fluorescently labelled, fixed > bleached X Stimulation cells with LPS > shine a laser> then fixation > to determine to order of oligomerisation upon activation > looking at a single TLR4 molecule > bleach by single photons > bleach partially, 50% fluorescence > shine a second photon > complete bleaching > determined by step function * Number of photons required directly proportional to number of monomers/molecules present * Decrease in intensity vs. time in steps * Shows presence of dimer
· Gay et al (2014): cell permeable peptides that inhibit interaction of TRIF and TRAM in TLR4 pathway > effective in mouse model of sepsis Candidate for human endotoxic shock · Husebye et al (200ff): Endocytic pathways regulate Toll-like receptor 4 signalling and link innate and adaptive immunity 1 TLR3/4 - TRIF - TRAM signalling: · Gay et al (2014): TRIF TIR domain binds to active TLR3 + TLR4-TRAM This releases TRIF-NTD from complex # Enables binding of TBK1 and TRAF3 > activation IRF3 and/or IRF7 * T1 IFN > anti-intracellular effect > breakdown membranes to expose bacteria > generate more powerful responses # Compartmentalisation: intracellular bacteria > chlamydia > avoid inflammasomes * TRIF pathway also has crucial role in regulating adaptive immunity Activation TLR3 in mouse CD8alpha+ myeloid dendritic cells (mDC) > required croos-presentation exogenous antigens in dying, virally infected cells taken up by phagocytosis " TRIF signal > activation cytotoxic CD8+ T-cells > not cross tolerance " TRIF signalling in