Stanford Microbiology and Immunology 104 204 Innate Immunology Lecture 3 Vertebrate Toll The immune function of Toll was identified in the fly but how did this information affect vertebrate immunology There were several routes Identification of Toll family members in vertebrates There had been attempts over a decade to identify Toll homologs based on sequence similarity cross hybridization and antibody cross reactivity all failed Searches of EST databases revealed several Toll homologs in humans Medzhitov and Janeway first showed Toll could activate macrophages Made a Toll in which the extracellular domain had been removed and replaced with Cd4 2 reasons for doing this 1 could ligate with a monoclonal antibody and potentially induce activation of Toll 2 could produce a dominant mutation This chimeric protein was constitutively activated In monocytes activated Toll could Induce inflammatory cytokines Induce costimulatory molecule B7 1 CD80 In jurkat cells a T cell line activated Toll could activate NFkB This fit into their pattern recognition hypothesis by linking an innate immune response to infection with activation of the RAG dependent immune system The elicitor for this Toll remained a mystery Lipopolysaccharide is a potent endotoxin This compound if found in the outer leaflet of the outer membrane of Gram positive bacteria Lipopolysaccharide LPS is a potent immune inducer and can induce the symptoms of septic shock upon injection into the circulation Early on Tlr 2 implicated in LPS signaling but this doesn t hold true LPS added to cells expressing TLR2 caused the cells to be activated ELAM enhancer turned on in 293 cells 1 Stanford Microbiology and Immunology 104 204 Innate Immunology Lecture 3 Vertebrate Toll Demonstrate gross binding of labeled LPS to the cells and showed that LPS binding protein could increase the activity of the system Unfortunately this was likely one of the first casualties of impure LPS Note them methods section reveals that the LPS is from Sigma This is a very crude preparation Two years ago there was a spate of papers in which groups purified the real elicitors from LPS The Lps mutant in mice was identified in Tlr4 as was shown to be the receptor responsible for signaling LPS Mouse mutant lps which caused mice to be resistant to the toxic effects of LPS and sensitivity to gram negative bacterial infections was identified by positional cloning It seems unfair to summarize the amount of work that went into this in one sentence Lps is encoded by Tlr4 The original paper argues that Tlr2 cannot be the exclusive LPS receptor as Tlr4 has an immune phenotype related to TLR signaling Eventually it was shown that Tlr2 signals lipoprotein in addition to other PAMPS TLR4 is not a pattern recognition receptor rather a relay system extracts LPS from the bacteria and presents it to Tlr4 Tlr4 does not bind LPS directly rather the LPS is sandwiched inside of a protein called MD2 which binds to Toll The protein LPS binding protein helps remove LPS from the outer membrane of bacteria LPS is then transferred to the cell surface molecule CD14 CD14 then transfers the LPS to MD2 which binds Toll MD2 resembles the principal allergen in dust mites The dust mite allergen is a lipid transporter in the mite Why is dust mite allergen such a good antigen Is it because the antigen is also a Tlr elicitor Rather than show the analysis of each of 11 Tolls let s jump to the current understanding and look at some Tolls in detail 11 tolls in humans in mice 8 appears nonfunctional The chart is constantly expanding and changing 2 Stanford Microbiology and Immunology 104 204 Innate Immunology Lecture 3 Vertebrate Toll Contamination of elicitors can lead to misunderstanding about signaling pathways as shown above for Tlr2 Special care has to be taken to avoid contamination with LPS peptidoglycans and lipoproteins List of what each Toll interacts with In mice 6 2 interact with mycoplasmal lipoprotein 1 2 interact with peptidoglycan lipoprotein Tlr 3 interacts with dsRNA Tlr 4 LPS Tlr 5 Flagellin Tlr 7 imidazoquinolines antiviral guanine analogues Tlr 8mouse non functional human ssRNA Tlr 9 CpG DNA Tlr 11interaction with Uropathogenic E coli flagellin How does the only licensed human adjuvant alum signal Flagellin and Toll Monomeric flagellin is an elicitor Identified during purification of Tlr5 inducer Tlr 5 transduces the signal Clone flagellin into non flagellated E coli become elicitors of Tlr5 Remove flagellin from Salmonella no longer induces cells Conclude that Tlr5 is pattern recognition receptor for the PAMP flagellin Mutations in Toll show up as sensitivities to a variety of infectious agents Common polymorphism in Tlr5 associated with susceptibility to legionaires disease Legionaires disease is caused by Legionella pneumophila Intracellular pathogen Mutants produce a secreted extracellular protein due to a stop codon insertion This protein acts as a dominant negative This is a common trait 7 5 of population Is there a selective advantage Odds ratio of 1 8 for Legionaires disease when heterozygous for this trait With traits with this level in the population you should consider positive selection factors Tlr 2 signaling and leprosy Two forms of the leprosy Tubercular localized and lepromatous disseminated 3 Stanford Microbiology and Immunology 104 204 Innate Immunology Lecture 3 Vertebrate Toll Tubercular local rashes few bacteria Lepromatous large papules limited cellular immune response 10 45 lepromatous patients but no tuberculoid or healthy controls dominant mutation presumably reducing signaling TLR2 expression can mediate M leprae NFkB activation Shown assaying TNF induction in TLR2 vs TLR 4 mice New drugs based on Toll signaling are currently being tested Tlr7 8 signaling ligand is imiquimod and ssRNA Tlr7 in mice Tlr 8 in humans as far as I can tell Tlr8 in mice is inactive No direct binding data though this has been pursued heavily Drug target for genital warts FDA approved rub on cream and warts are removed Presumed function is to activate immune response in a low pathogenicity infection Imiquimod is also used for actinic keratosis Pre cancerous skin lesions 40 of 40 year old Australians have them Raises an immune reaction to these cells and they are removed Even destroys subclinical keratoses so that they don t come back Rub the cream all over the area and only lesions are destroyed Tlr9 agonists are being attached to antigens and being used as vaccine adjuvants This