Toll like receptors

1. TOLL LIKE RECEPTORS Dr. Tushar B. Patil, MD Senior Resident Departmet of Neurology King George’s Medical University , Lucknow, India

2. Introduction TLRs are germline -encoded pattern recognition receptors Sense conserved molecular structures produced by microorganisms Play essential role in host defence to microbial infection. Activate intracellular signalling pathways Induce genes involved in immune responses and inflammation. Act as a bridge between Innate and Adaptive immunity by mediating dendritic cell maturation and activation of pathogen-specific T lymphocytes.

3. Introduction TLRs recognize pathogens and generates an immediate defence response. Induce cytokines which destroy or limit invading pathogens. Activation of APCs & expression of MHC and co-stimulatory molecules 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 mediated immune responses.

4. Toll: Origin of the word (1985) The gene in question, when mutated, makes the Drosophila (fruit fly) embryo look unusual. The researchers were so surprised that they spontaneously shouted out in German "Das ist ja toll!" which translates as "That's great!". [The Nobel Prize in Physiology or Medicine 1995:Edward B. Lewis, Christiane Nüsslein-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 TLR TLR Location Phenotype Phenotype MIM no. TLR1 4p14 {Leprosy, protection against} 613223 {Leprosy, susceptibility to, 5} 613223 TLR2 4q31.3 {Colorectal cancer, susceptibility to} 114500 {Leprosy, susceptibility to} 246300 TLR3 4q35.1 Herpes simplex encephalitis, susceptibility to 613002 TLR4 9q33.1 Endotoxin hyporesponsiveness , {Colorectal cancer, susceptibility to} 114500 {Macular degeneration, age-related, 10} 611488

8. Genes for TLR TLR Location Phenotype Phenotype MIM no. TLR5 1q41 { Legionaire disease, susceptibility to} 608556 {Systemic lupus erythematosus , resistance to} 601744 {Systemic lupus erythematosus , susceptibility to, 1} 601744 TLR6 4p14   ?? ?? TLR7 Xp22.2 ?? ?? TLR8 Xp22.2   ?? ?? TLR9 3p21.2 ?? ?? TLR10 ?? ?? ??

9. Cellular Localization of TLRs

10. Cellular Localization of TLRs TLR1, TLR2, TLR4, TLR5, and TLR6 localized on the cell surface and recognize microbial membrane components. TLR3, TLR7, TLR8, and TLR9 expressed within intracellular vesicles and recognize nucleic acids. Intracellular vesicles with TLR3, TLR7, TLR8, and TLR9 are localized in endoplasmic reticulum (ER), endosomes , lysosomes , and endolysosomes . Intracellular localization important for avoiding contact with ‘‘self’’ nucleic acids and risk of autoimmunity. Regulated mechanism is present for TLR mobilization.

11. Structure of TLRs TLRs are type I membrane glycoproteins . Homology in the cytoplasmic region--- interleukin-1 receptors (IL-1Rs) superfamily Extracellular region of TLRs contains leucine -rich repeat (LRR) motifs, & IL 1Rs contains three immunoglobulin-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 the Toll/IL-1R (TIR) domain. The TIR domain is characterized by the presence of three highly homologous regions (known as boxes 1, 2 and 3).

13. Distribution of TLRs

14. Ligands for TLRs

15. Ligands for TLRs

16. TLR Signaling Pathway 1]MyD88 (myeloid differentiation primary-response protein 88) Forms homodimers through DD–DD and TIR-domain–TIR-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- κB (NF- κ 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 superfamily 4] NF- κ B (nuclear factor- κ B) Promote expression of genes and synthesis of cytokines

17. TLR-signalling pathways

18. TIR-domain-containing MyD88 mediates TLR pathway that activates IRAKs and TRAF6 Activation of the IKK complex (inhibitor of nuclear factor- κB ( IκB )- kinase complex), which consists of IKK-α, IKK-β and IKK-γ (also known as IKK1,IKK2 and NF- κB and releases NF- κB from its inhibitor so NF- κB translocates to the nucleus and induces expression of inflammatory cytokines. 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 factor 3 (IRF3) and the induction of IFN-β are observed in a MyD88-independent manner.

19. TLRs and susceptibility to diseases

20. TLR 1 1]PROTECTION AGAINST LEPROSY SNP in TLR1, 1805T-G, that results in an ile602-to-ser (I602S) substitution at 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. TLR2 1]Susceptibility to Leprosy Arg677-to-trp polymorphism (R677W; 603028.0001) in the intracellular domain of TLR2 in 10 (22%) of 45 Korean lepromatous leprosy patients [Kang and Chae 2001] R677W leads to poor cellular immune response associated with lepromatous leprosy.[ Bochud et al. 2003] R677W was undetectable in the Japanese patients [ Mikita et al. 2009], similar to the findings in Indian patients reported by [ Malhotra et al. 2005] Bochud et al. (2008 )analyzed 3 TLR2 polymorphisms in 441 patients and 187 controls 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 reversal reaction

22. TLR2 1]  Lyme Disease Monocytes and lymphocytes from healthy subjects produced more TNF and IFNG, respectively, in response to high concentrations of Borrelia lysate 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 due to 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. TLR3 1] Herpes Simplex Encephalitis Zhang et al. (2007) detected a heterozygous P554S mutation in the TLR3 gene. The mutation occurred on different TLR3 haplotypes in the children. TLR3 is vital for natural immunity to HSV-1 in the CNS and neurotropic viruses have contributed to the evolutionary maintenance of TLR3. 2] Age-related macular degeneration T allele of rs3775291 in the TLR3 gene, which results in a L412F substitution, is protective against the development of geographic atrophy 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 controls 4] D299G and T399I variants of TLR4 as contributors to susceptibility to age-related macular degeneration  5] D299G polymorphism– metastasis after breast cancer surgery 6] D299G polymorphism in TLR4 may influence the immunologic component of anthracycline -based chemotherapy in human cancer.

25. TLR5 1] 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) cell line growth.

27. TLR 7 Target of investigational agents with antitumor and antiviral properties

28. FUNCTIONS OF TOLL-LIKE RECEPTORS IN CNS TLR4 expression in the CNS is necessary to mount an appropriate cytokine response in the brain in response to systemic LPS exposure mice with peripheral TLR4-expressing cells, but lacking specific CNS TLR4, were unable to mount a CNS cytokine response TLRs may play important roles in cerebral cell proliferation and brain development. Inflammation has a strong effect on progenitor cells and reduces adult hippocampal neurogenesis . 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 on prostaglandin E2 receptors. protective effect on hippocampal neurogenesis by cyclooxygenase inhibitors TLR8 is also expressed at high levels during brain development, and in cultured cortical 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 structures inside microglia and not on the surface of the cells. With cultured astrocytes , TLR3 and TLR4 were found only on the cellular surface Microglial TLRs are crucial as a first line of defence against bacterial or viral infection. human astrocytes have been reported to express TLRs 1–5 and TLR 9 TLR signaling in astrocyte can activate the production of a wide range of neuroprotective and anti-inflammatory mediators rather than merely stimulating proinflammatory factors. The preference of astrocyte to express up to 200-fold elevated levels of TLR3 upon activation is puzzling since the only currently known ligand for TLR3 is dsRNA , 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 TLR pathway 1]Leprosy 2]Herpes Simplex Encephalitis 3] Entero and flaviviral encephalitis 4]Malaria 5]Toxoplasmosis 6] Trypanosomiasis 7]Lyme disease 8] Neurocysticercosis 9]Bacterial meningitis 10] Alzheimer’s Disease 11] Prion Diseases 12] Amyotrophic Lateral Sclerosis. 13] Parkinson’s Disease 14] Perinatal brain injury 15] Multiple sclerosis and ?????????????????

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 the proinflammatory response of microglia toward A β , which may be linked to neurotoxicity Activation of TLRs 2, 4, and 9 were also linked to both phagocytosis of the neurotoxic A β and to an anti-inflammatory response (TLR9), which may lead to neuroprotection (Figure 1). Therefore, elevated expression levels of TLRs 2, 4, and 9, through genetic modification or toward specific agonists, may be a therapeutic application in AD. Use of TLRs 2 and 4 agonist as a specific macrophage activator to increase the clearance of A β 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 minor constituent of turmeric , that enhances phagocytosis and the clearance of A β in cells from most AD patients, and increases transcription of the MGAT and TLR genes Administration of CpG , a TLR9 activator, in APP transgenic mice, resulted in clearance of A β 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 of prion diseases. Treatment with synthetic oligodeoxynucleotides that contain cytosine phosphate guanosine ( CpG -ODN) motifs, known to bind to TLR9, have been suggested as possible treatment for prion diseases in a mouse model, by delaying the disease onset. Another explanation may be the effect of CpG -ODN on microglia activation that may lead to prion degradation . As the activation of TLRs in other amyloidogenic diseases, such as AD, has been linked to the clearance of neurotoxic amyloid , it may prove to be a potential therapeutic 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 in neurotrophic factor secretion from glial cells may prove to be a therapeutic approach in ALS.

36. Targeting TLR as Therapeutic Application in Parkinson’s Disease. α- synuclein immunization in a PD animal model may ameliorate disease progression. Targeting mechanisms in which α- synuclein activates TLR signaling , may open a new horizon for therapeutic application in PD.

37. Other therapeutic implications

38. Toll-Like Receptor 4: Agonism and Antagonism The best characterized ligand for the MD-2/TLR4 complex is lipid A (the biologically 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 the secondary acyl chain length below 6 or increasing it above 14 results in a loss of agonist activity Binding of lipid A to MD-2/TLR4 ( Raetz et al., 2006) induces structural rearrangements that trigger oligomerisation of TLR4 and initiate signal transduction

39. In 1995, a synthetic form of Rhodobacter capsulatus lipid A was generated that antagonized E. coli lipid A and formed the basis for E5531 (Christ et al., 1995). Modification of E5531 generated the stable analog E5564 ( eritoran ), which is currently undergoing clinical trials for use in treating Gram-negative endotoxemia and sepsis Other antagonists at TLR4 include curcumin , auranofin (an antirheumatic gold compound), cinnamaldehyde , and acrolein , all of which prevent homodimerization of TLR4 Small molecules that inhibit MyD88 binding to TLR4 are also emerging TLR4 agonists are currently being developed as immunomodulators and adjuvants . TLR ligands have become a focus in therapeutic studies for their potential use as adjuvants in vaccine formulations

40. Toll-Like Receptor 2: Agonism and Antagonism Currently, the major use for compounds that activate TLR2 are as adjuvants . The synthetic compounds, such as Pam3CSK4 and MALP-2, could be developed for adjuvant usage. Another approach to blocking TLR2 is with a neutralizing antibody. One such antibody, T2.5, has been shown to prevent sepsis induced 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 induced by Salmonella enterica or E. coli when given with antibiotics

41. Toll-Like Receptor 5: Agonism and Antagonism M2e is immunogenic component of influenza A M2e was recently fused with the TLR5 ligand S. typhimurium flagellin (STF2). The resulting fusion protein can activate cells in a TLR5-dependent manner and elicits potent antibody responses in mice. M2e sequence linked to the TLR5 ligand provides an effective approach to developing vaccines against wide-spread epidemic and pandemic influenza In the case of IBD, TLRs can also amplify inappropriate immune responses that ultimately cause chronic inflammation. At low concentrations, flagellin can stimulate TLR5 on CD4 T cells and enhance the expression of FOXP3, allowing for an increased suppressive capacity

42. Toll-Like Receptors 7 and 8: Small-Molecule Targets Activated by synthetic small- molecularweight compounds of the imidazoquinoline family, such as resiquimod and imiquimod . TLR7/TLR8 agonists act as“antiviral agents.” Imiquimod is the first approved topically active TLR7 agonist. It is prescribed for treatment of external virus induced skin lesions, such as the genital and perianal warts resulting from papillomavirus infection Therapeutic interest in TLR7/TLR8 for cancer treatment came about because of the antitumoral activity of TLR7/TLR8 agonists

43. Toll-Like Receptor 9 Only TLR for which a systemically administered specific agonist has shown substantial evidence of antitumor activity in human clinical Trials TLR9 has evolved to recognize unmethylated CpG dinucleotides ( CpG ODN) that are prevalent in viral and bacterial DNA CpG ODNs seem to be the most promising of all adjuvants currently in preclinical development ability to help vaccine hyporesponsive populations, such as persons positive for HIV, to benefit from vaccination There are also promising results from studies into the adjuvant activity of CpG ODN for tumor vaccination. Role of antagonists in SLE?????

44. Prophylactic and therapeutic targeting of TLRs in clinical trials of infectious diseases

45. TLR targets in different diseases

46. We have come a long way from the discovery of the first Toll in the fruit fly. The intense interest around TLRs, shared by immunologists, biomedical researchers, and pharmacologists, should surely yield badly needed therapies for major pathologic conditions.

47. THANK YOU