1.Dr. Tushar B. Patil, MD Senior ResidentDepartmet of NeurologyKing Georges Medical University , Lucknow, India

2. Introduction TLRs are germline-encoded pattern recognition receptors Sense conserved molecular structures produced bymicroorganismsPlay essential role in host defence to microbial infection.Activate intracellular signalling pathwaysInduce genes involved in immune responses andinflammation.Act as a bridge between Innate and Adaptive immunity bymediating dendritic cell maturation and activation ofpathogen-specific T lymphocytes. 3. Introduction TLRs recognize pathogens and generates an immediate defenceresponse. Induce cytokines which destroy or limit invading pathogens. Activation of APCs & expression of MHC and co-stimulatorymolecules like CD40, CD89, CD86 and CD70. Activation and differentiation of naive T cells into Th1, Th2,Th3 and Th17 cells or T-regs, facilitating cell mediatedimmune responses. 4. Toll: Origin of the word (1985) The gene in question, when mutated, makes the Drosophila (fruit fly) embryo lookunusual. The researchers were so surprised that they spontaneously shouted out in German"Das ist ja toll!" which translates as "Thats great!".[The Nobel Prize in Physiology or Medicine 1995:Edward B. Lewis, ChristianeNsslein-Volhard, Eric F. Wieschaus] 5. Important milestones in the discovery of Toll receptors. 6. Schematic representation of Toll/TLR pathways in Drosophila and mammals.Chtarbanova S , Imler J Arterioscler Thromb Vasc Biol 2011;31:1734-1738 7. Genes for TLRTLRLocation Phenotype Phenotype MIMno.TLR1 4p14 {Leprosy, protection against} 613223{Leprosy, susceptibility to, 5} 613223TLR2 4q31.3 {Colorectal cancer, susceptibility to}114500{Leprosy, susceptibility to}246300TLR3 4q35.1 Herpes simplex encephalitis, susceptibilityto613002TLR4 9q33.1 Endotoxin hyporesponsiveness, {Colorectal 114500cancer, susceptibility to}{Macular degeneration, age-related, 10} 611488 8. Genes for TLRTLR Locatio PhenotypePhenotype MIMnno.TLR51q41 {Legionaire disease, susceptibility to} 608556 {Systemic lupus erythematosus,601744 resistance to} {Systemic lupus erythematosus,601744 susceptibility to, 1}TLR64p14 ????TLR7Xp22.2 ????TLR8Xp22.2 ????TLR93p21.2 ????TLR10 ?? ???? 9. Cellular Localization of TLRs 10. Cellular Localization of TLRs TLR1, TLR2, TLR4, TLR5, and TLR6 localized on the cell surface andrecognize microbial membrane components. TLR3, TLR7, TLR8, and TLR9 expressed within intracellular vesicles andrecognize nucleic acids. Intracellular vesicles with TLR3, TLR7, TLR8, and TLR9 are localized inendoplasmic reticulum (ER), endosomes, lysosomes, and endolysosomes. Intracellular localization important for avoiding contact with self nucleicacids and risk of autoimmunity. Regulated mechanism is present for TLR mobilization. 11. Structure of TLRs TLRs are type I membraneglycoproteins. Homology in the cytoplasmicregion--- interleukin-1receptors (IL-1Rs)superfamily Extracellular region of TLRscontains leucine-rich repeat(LRR) motifs, & IL 1Rscontains threeimmunoglobulin-like domains 12. Structure of TLRs Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs)have a conserved cytoplasmic domain, that is known as theToll/IL-1R (TIR) domain. The TIR domain is characterized by the presence of threehighly homologous regions (known as boxes 1, 2 and 3). 13. Distribution of TLRs 14. Ligands for TLRs 15. Ligands for TLRs 16. TLR Signaling Pathway1]MyD88 (myeloid differentiation primary-response protein 88) Forms homodimers through DDDD and TIR-domainTIR-domain interactions and exists as a dimer when recruited to the receptor complex. Functions as adaptor linking TLRs/IL-1Rs with downstream signalling molecules that have DDs.2] IRAK family (IL-1-receptor-associated kinases) Four IRAKs IRAK1,IRAK2,IRAK4 and IRAK-M identified The kinase activity of IRAK1 increases strongly following TLR/IL-1R stimulation, and its kinase domain is essential for signalling through nuclear factor-B (NF-B)3] TRAF6 (tumour-necrosis-factorreceptor- associated factor 6) Signalling mediator for both the TNF-receptor superfamily and the TLR/IL-1R superfamily, interacting directly with members of the TNF-receptor superfamily4] NF-B (nuclear factor-B) Promote expression of genes and synthesis of cytokines 17. TLR-signalling pathways 18. TIR-domain-containing MyD88 mediates TLR pathway that activatesIRAKs and TRAF6 Activation of the IKK complex (inhibitor of nuclear factor-B (IB)- kinasecomplex), which consists of IKK-, IKK- and IKK- (also known asIKK1,IKK2 and NF-B and releases NF-B from its inhibitor so NF-B translocates to the nucleus and induces expression of inflammatorycytokines. TIRAP (TIRdomain- containing adaptor protein), is involved in the MyD88-dependent signalling pathway through TLR2 and TLR4. TLR3- and TLR4-mediated activation of interferon (IFN)-regulatory factor3 (IRF3) and the induction of IFN- are observed in a MyD88-independentmanner. 19. TLRs and susceptibility to diseases 20. TLR 11]PROTECTION AGAINST LEPROSY SNP in TLR1, 1805T-G, that results in an ile602-to-ser (I602S) substitutionat the junction of the transmembrane and intracellular domains of TLR1.[Johnson et al. 2007, Misch et al.2008]2] SUSCEPTIBILITY TO LEPROSY Association of an asn248-to-ser (N248S) SNP in the TLR1 gene and leprosy (LPRS5; 613223) in a Bangladeshi population consisting of 842 patients and 543 controls.[Schuring et al. (2009)] Homozygosity for S248 significantly associated with leprosy (OR = 1.34) & heterozygosity was found to be protective against leprosy (OR = 0.78) Homozygous N248 genotype was equally distributed among patients and controls Patients with erythema nodosum leprosum reactions were more likely to have the N248 allele (68%) than were patients who had no reactions (46%) 21. TLR21]Susceptibility to Leprosy Arg677-to-trp polymorphism (R677W; 603028.0001) in the intracellular domainof TLR2 in 10 (22%) of 45 Korean lepromatous leprosy patients [Kang and Chae2001] R677W leads to poor cellular immune response associated with lepromatousleprosy.[Bochud et al. 2003] R677W was undetectable in the Japanese patients [Mikita et al. 2009], similar tothe findings in Indian patients reported by [Malhotra et al. 2005] Bochud et al. (2008 )analyzed 3 TLR2 polymorphisms in 441 patients and 187controls in 3 Ethiopian groups. 597C-T SNP was associated with reduced susceptibility to reversal reaction Homozygous for a 280-bp microsatellite marker had an increased risk of reversalreaction 22. TLR21] Lyme Disease Monocytes and lymphocytes from healthy subjects produced more TNFand IFNG, respectively, in response to high concentrations of Borrelialysate than did healthy subjects heterozygous for an arg753-to-gln (R753Q)SNP. [Schroder et al. 2005] R753Q SNP may protect from development of late-stage Lyme disease dueto reduced signaling via TLR2/TLR1.2] Tuberculosis Increased risk of TB in carriers of a nonsynonymous 2258G-A SNP in the TLR2 gene, which causes the R753Q substitution [Ogus et al. (2004)]3] Colorectal Cancer Boraska Jelavic et al. (2006) GT microsatellite repeat polymorphism in intron 2 of the TLR2 gene in sporadic colorectal cancer patients Frequency of TLR2 alleles with 20 and 21 GT repeats was decreased & that of 31 GT repeats was increased in patients versus controls. 23. TLR31] Herpes Simplex Encephalitis Zhang et al. (2007) detected a heterozygous P554S mutation in theTLR3 gene. The mutation occurred on different TLR3 haplotypes inthe children. TLR3 is vital for natural immunity to HSV-1 in the CNS andneurotropic viruses have contributed to the evolutionary maintenanceof TLR3.2] Age-related macular degeneration T allele of rs3775291 in the TLR3 gene, which results in a L412Fsubstitution, is protective against the development of geographicatrophy or advanced dry age-related macular degeneration 24. TLR4 (The human homolog of Drosophila Toll)1] ENDOTOXIN HYPORESPONSIVENESS Arbour et al. (2000) showed that 2 common cosegregating missense mutations (asp299 to gly and thr399 to ile) that affect the extracellular domain of the TLR4 receptor are associated with blunted response to inhaled lipopolysaccharide in humans. Susceptibility to Gm negative sepsis.2] D299G allele - Lower Levels Of Certain Proinflammatory Cytokines, More Susceptibility To Severe Bacterial Infections, Lower Risk Of Carotid Atherosclerosis, And A Smaller Intima-media Thickness In The Common Carotid Artery. [Kiechl et al. (2002)]3] gly299 allele of the TLR4 gene was more frequent in colorectal cancer patients than controls4] D299G and T399I variants of TLR4 as contributors to susceptibility to age-related macular degeneration5] D299G polymorphism metastasis after breast cancer surgery6] D299G polymorphism in TLR4 may influence the immunologic component of anthracycline-based chemotherapy in human cancer. 25. TLR51] Susceptibility to Legionnaire disease C-to-T transition at nucleotide 1174, changed arg392 to a stop codon (R392X) [ Hawn et al. 2003]2] Resistance to systemic lupus erythematosus allele 1174C of TLR5, but not allele 1174T, which encodes the premature stop codon, was preferentially transmitted to SLE-affected offspring. [Hawn et al. 2005] 26. TLR6 Enhancement of Lewis Lung Carcinoma (LLC) cellline growth. 27. TLR 7 Target of investigational agents with antitumor andantiviral properties 28. FUNCTIONS OF TOLL-LIKE RECEPTORS IN CNS TLR4 expression in the CNS is necessary to mount an appropriate cytokineresponse in the brain in response to systemic LPS exposure mice with peripheral TLR4-expressing cells, but lacking specific CNS TLR4, wereunable to mount a CNS cytokine response TLRs may play important roles in cerebral cell proliferation and braindevelopment. Inflammation has a strong effect on progenitor cells and reduces adult hippocampalneurogenesis. TLR2 deficiency in mice resulted in impaired hippocampal neurogenesis. absence of TLR4 enhanced proliferation and neuronal differentiation. Detrimental effects of TLR4 on progenitor cells was shown to be dependent onprostaglandin E2 receptors. protective effect on hippocampal neurogenesis by cyclooxygenase inhibitors TLR8 is also expressed at high levels during brain development, and in culturedcortical neurons, TLR8 stimulation inhibits neurite outgrowth 29. Immunostaining of cultured microglia and astrocytes for TLR3 and TLR4,revealed two opposite features. Both TLR3 and TLR4 were found exclusively localized in vesicular structuresinside microglia and not on the surface of the cells. With cultured astrocytes, TLR3 and TLR4 were found only on the cellularsurface Microglial TLRs are crucial as a first line of defence against bacterial or viralinfection. human astrocytes have been reported to express TLRs 15 and TLR 9 TLR signaling in astrocyte can activate the production of a wide range ofneuroprotective and anti-inflammatory mediators rather than merely stimulatingproinflammatory factors. The preference of astrocyte to express up to 200-fold elevated levels of TLR3upon activation is puzzling since the only currently known ligand for TLR3 isdsRNA, which is believed to emerge as an intermediate during viral replication. 30. TLR Signaling Link to Neurotoxicity TLR Signaling Link to Neurogenesis TLR Signaling in Neurodegenerative Diseases 31. Neurological diseases with possible link to TLRpathway1]Leprosy 9]Bacterial meningitis2]Herpes Simplex Encephalitis 10] Alzheimers Disease3]Entero and flaviviral 11] Prion Diseasesencephalitis12] Amyotrophic Lateral4]Malaria Sclerosis.5]Toxoplasmosis 13] Parkinsons Disease6]Trypanosomiasis 14]Perinatal brain injury7]Lyme disease15] Multiple sclerosis8]Neurocysticercosisand ????????????????? 32. Targeting TLR as Therapeutic Application in AD TLR activation may modulate glial cell activity in AD. Recent research suggests the involvement of TLRs 2, 4, 5, 7, and 9 in theproinflammatory response of microglia toward A, which may be linked toneurotoxicityActivation of TLRs 2, 4, and 9 were also linked to both phagocytosis ofthe neurotoxic A and to an anti-inflammatory response (TLR9), whichmay lead to neuroprotection (Figure 1).Therefore, elevated expression levels of TLRs 2, 4, and 9, through geneticmodification or toward specific agonists, may be a therapeutic applicationin AD.Use of TLRs 2 and 4 agonist as a specific macrophage activator to increasethe clearance of A in an AD mouse model. An alternative therapeutic approach may be the reduction of TLR5 and 7,by using shRNA or specific antagonists 33. Bisdemethoxycurcumin is a natural curcumin, a minorconstituent of turmeric , that enhances phagocytosis and theclearance of A in cells from most AD patients, and increasestranscription of the MGAT and TLR genes Administration of CpG, a TLR9 activator, in APP transgenicmice, resulted in clearance of A from microglial cells. 34. Targeting TLR as Therapeutic Application in Prion Diseases It has been suggested that TLR9 expression may be linked to the progression ofprion diseases. Treatment with synthetic oligodeoxynucleotides that contain cytosine phosphateguanosine (CpG-ODN) motifs, known to bind to TLR9, have been suggested aspossible treatment for prion diseases in a mouse model, by delaying the diseaseonset. Another explanation may be the effect of CpG-ODN on microglia activation thatmay lead to prion degradation . As the activation of TLRs in other amyloidogenic diseases, such as AD, has beenlinked to the clearance of neurotoxic amyloid, it may prove to be a potentialtherapeutic approach to the prion diseases. 35. Targeting TLR as a Therapeutic Application in Amyotrophic Lateral Sclerosis. A potential link between TLR signaling and an increase inneurotrophic factor secretion from glial cells may prove to be atherapeutic approach in ALS. 36. Targeting TLR as Therapeutic Application inParkinsons Disease. -synuclein immunization in a PD animal model mayameliorate disease progression. Targeting mechanisms in which -synuclein activates TLRsignaling, may open a new horizon for therapeutic applicationin PD. 37. Other therapeutic implications 38. Toll-Like Receptor 4: Agonism andAntagonism The best characterized ligand for the MD-2/TLR4 complex is lipid A (thebiologically active component of LPS) Different lipid A structures may be agonists or antagonists at the MD-2/TLR4 (Walsh et al., 2008). A synthetic compound CRX-527 is an agonist, but decreasing thesecondary acyl chain length below 6 or increasing it above 14 results in aloss of agonist activity Binding of lipid A to MD-2/TLR4 (Raetz et al., 2006) induces structuralrearrangements that trigger oligomerisation of TLR4 and initiate signaltransduction 39. In 1995, a synthetic form of Rhodobacter capsulatus lipid A was generatedthat antagonized E. coli lipid A and formed the basis for E5531 (Christ etal., 1995). Modification of E5531 generated the stable analog E5564 (eritoran), whichis currently undergoing clinical trials for use in treating Gram-negativeendotoxemia and sepsis Other antagonists at TLR4 include curcumin, auranofin (an antirheumaticgold compound), cinnamaldehyde, and acrolein, all of which preventhomodimerization of TLR4 Small molecules that inhibit MyD88 binding to TLR4 are also emerging TLR4 agonists are currently being developed as immunomodulators andadjuvants. TLR ligands have become a focus in therapeutic studies for their potentialuse as adjuvants in vaccine formulations 40. Toll-Like Receptor 2: Agonism and Antagonism Currently, the major use for compounds that activate TLR2 areas adjuvants.The synthetic compounds, such as Pam3CSK4 and MALP-2,could be developed for adjuvant usage. Another approach to blocking TLR2 is with a neutralizingantibody.One such antibody, T2.5, has been shown to prevent sepsisinduced by TLR2 ligands (Meng et al., 2004) Furthermore, when T2.5 is used in combination with an anti-TLR4/MD-2 antibody, it protects mice against sepsis inducedby Salmonella enterica or E. coli when given with antibiotics 41. Toll-Like Receptor 5: Agonism andAntagonism M2e is immunogenic component of influenza A M2e was recently fused with the TLR5 ligand S. typhimuriumflagellin (STF2). The resulting fusion protein can activate cells ina TLR5-dependent manner and elicits potent antibody responsesin mice. M2e sequence linked to the TLR5 ligand provides an effectiveapproach to developing vaccines against wide-spread epidemicand pandemic influenza In the case of IBD, TLRs can also amplify inappropriate immuneresponses that ultimately cause chronic inflammation. At low concentrations, flagellin can stimulate TLR5 on CD4 Tcells and enhance the expression of FOXP3, allowing for anincreased suppressive capacity 42. Toll-Like Receptors 7 and 8: Small-MoleculeTargets Activated by synthetic small-molecularweight compounds of theimidazoquinoline family, such as resiquimod and imiquimod. TLR7/TLR8 agonists act asantiviral agents. Imiquimod is the first approved topically active TLR7 agonist. It is prescribed for treatment of external virus induced skinlesions, such as the genital and perianal warts resulting frompapillomavirus infection Therapeutic interest in TLR7/TLR8 for cancer treatment cameabout because of the antitumoral activity of TLR7/TLR8 agonists 43. Toll-Like Receptor 9 Only TLR for which a systemically administered specific agonist has shownsubstantial evidence of antitumor activity in human clinical Trials TLR9 has evolved to recognize unmethylated CpG dinucleotides (CpGODN) that are prevalent in viral and bacterial DNA CpG ODNs seem to be the most promising of all adjuvants currently inpreclinical development ability to help vaccine hyporesponsive populations, such as persons positivefor HIV, to benefit from vaccination There are also promising results from studies into the adjuvant activity of CpGODN for tumor vaccination. Role of antagonists in SLE????? 44. Prophylactic and therapeutic targeting of TLRs in clinical trials of infectiousdiseases 45. TLR targets in different diseases 46. We have come a long way from the discovery of the first Tollin the fruit fly. The intense interest around TLRs, shared by immunologists,biomedical researchers, and pharmacologists, should surelyyield badly needed therapies for major pathologic conditions. 47. THANK YOU