{"gene":"TLR5","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2012,"finding":"Crystal structure of zebrafish TLR5 in complex with Salmonella flagellin FliC D1/D2/D3 fragment at 2.47 Å resolution revealed that TLR5 interacts primarily with three helices of the FliC D1 domain using its lateral side, and two TLR5-FliC 1:1 heterodimers assemble into a 2:2 tail-to-tail signaling complex stabilized by quaternary contacts of FliC D1 with the convex surface of the opposing TLR5. Structure-guided mutagenesis and deletion analyses validated this signaling mechanism.","method":"X-ray crystallography (2.47 Å), structure-guided mutagenesis, deletion analysis","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis validation in a single rigorous study","pmids":["22344444"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of Bacillus subtilis flagellin–TLR5 complex at 2.1 Å resolution combined with alanine scanning revealed a conserved TLR5 activation hot spot: an arginine residue (bsflagellin R89) and adjacent residues (E114, L93) in the flagellin D1 domain provide shape and chemical complementarity to a cavity formed by the loop of leucine-rich repeat 9 in TLR5. The D0 domain also contributes to TLR5 activity through structurally dispersed regions.","method":"X-ray crystallography (2.1 Å), alanine scanning mutagenesis","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with systematic mutagenesis of binding interface, replicates and extends prior structural work","pmids":["28106112"],"is_preprint":false},{"year":1998,"finding":"TLR5 (cloned as TIL3) was identified as a human Toll/IL-1R-like receptor that activates NF-κB in a cell-type-dependent fashion, establishing its role in innate immune signaling.","method":"Cloning, NF-κB reporter functional assay in transfected cells","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct functional assay in transfected cells, single lab but clear mechanistic readout","pmids":["9596645"],"is_preprint":false},{"year":2003,"finding":"Flagellin activates p38 MAPK in a TLR5-dependent manner in polarized intestinal epithelial cells, and this p38 activation regulates IL-8 expression by a post-transcriptional (translational) mechanism independent of NF-κB. ERK1/2 activation by flagellin was not TLR5-specific.","method":"Pharmacological inhibition of p38 MAPK (SB-203580), dominant-negative TLR5 transfection, mRNA stability assay, IL-8 protein/mRNA measurement","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (genetic dominant-negative, pharmacological inhibition, post-transcriptional analysis) in a single focused study","pmids":["12702497"],"is_preprint":false},{"year":2004,"finding":"Flagellin released by commensal E. coli activates NF-κB, IL-8, and CCL-20 expression in intestinal epithelial cells via TLR5 and the adaptor protein MyD88. In polarized cells, TLR5 signaling could be triggered from the apical side; in vivo, flagellin on the mucosal side of ileal biopsies induced basolateral KC production.","method":"Dominant-negative TLR5 and MyD88 plasmid transfection, NF-κB reporter assay, ELISA, Ussing chamber ex vivo, immunohistochemistry","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic DN constructs, ex vivo tissue, in vivo IHC) across in vitro and in vivo systems","pmids":["15302888"],"is_preprint":false},{"year":2005,"finding":"Flagellin interaction with TLR5 on intestinal epithelial cells activates both NF-κB/PI3K-Akt pro-survival pathways and the extrinsic caspase-8 apoptotic pathway. When NF-κB or PI3K/Akt signaling is blocked, flagellin induces apoptosis, demonstrating that TLR5 simultaneously activates intertwined inflammatory and apoptotic signaling.","method":"Biochemical signaling assays, caspase activity assays, pharmacological pathway inhibition, mRNA expression profiling, dominant-negative TLR5","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (genetic, pharmacological, biochemical) in a single focused study","pmids":["16179598"],"is_preprint":false},{"year":2007,"finding":"Mice lacking TLR5 (TLR5KO) develop spontaneous colitis associated with decreased intestinal expression of TLR5-regulated host defense genes and increased colonic proinflammatory cytokines. Deletion of TLR4 in TLR5KO mice rescues colitis, establishing by genetic epistasis that TLR5 loss leads to TLR4-driven colitis.","method":"TLR5 knockout mouse model, TLR4/TLR5 double-knockout genetic epistasis, histopathology, cytokine measurement, bacterial burden quantification","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined phenotype, replicated with double KO epistasis demonstrating pathway mechanism","pmids":["18008007"],"is_preprint":false},{"year":2007,"finding":"Protein kinase D (PKD) physically interacts with TLR5, and this association is rapidly enhanced by flagellin. PKD phosphorylates TLR5 at serine 805 (identified by in vitro phosphorylation and mass spectrometry); mutation of S805 to alanine abrogates flagellin responses. PKD is required for flagellin-induced p38 MAPK activation and IL-8 production in epithelial cells.","method":"Co-immunoprecipitation, in vitro kinase assay, mass spectrometry (S805 phosphorylation), site-directed mutagenesis (S805A), pharmacological inhibition (Gö6976), shRNA knockdown","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay with MS identification of phosphosite, mutagenesis, co-IP, and shRNA knockdown in a single study","pmids":["17442957"],"is_preprint":false},{"year":2007,"finding":"Chicken TLR5 (chTLR5) signals through the MyD88 pathway to activate NF-κB in response to flagellin; mutation of proline 737 in the chTLR5 TIR domain abrogates function. A single amino acid in flagellin (Q89) determines species-specific TLR5 responses between chicken, human, and mouse.","method":"Expression in HeLa cells, NF-κB reporter assay, site-directed mutagenesis of TIR domain (P737) and flagellin (Q89A, L415A, N100A)","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional mutagenesis of both receptor and ligand, single lab","pmids":["17964652"],"is_preprint":false},{"year":2007,"finding":"TLR5-deficient alveolar macrophages (AMs) fail to produce TNF-α after stimulation with Legionella pneumophila or purified flagellin, demonstrating that AMs recognize L. pneumophila via TLR5-mediated flagellin sensing. In vivo, TLR5-deficient mice show impaired early neutrophil recruitment (at 4 h) and later develop organizing pneumonia.","method":"TLR5 knockout mice, L. pneumophila infection model, bronchoalveolar lavage cell counts, TNF-α measurement, flagellin-deficient bacterial strain (LpFlaA-) comparison","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO mice with in vivo infection model plus ex vivo macrophage validation with flagellin-deficient strain control","pmids":["17982089"],"is_preprint":false},{"year":2008,"finding":"TLR5 activation by flagellin suppresses RANKL-induced osteoclastogenesis by stimulating IFN-β production through STAT1 activation in bone marrow-derived macrophages. IFN-β downregulates c-Fos protein (post-translationally), and neutralizing IFN-β or STAT1 deficiency reverses the anti-osteoclastogenic effect. In osteoblast–macrophage co-cultures, flagellin instead promotes osteoclast differentiation without inducing IFN-β.","method":"Bone marrow-derived macrophage culture, RANKL osteoclastogenesis assay, IFN-β neutralizing antibody, STAT1-knockout cells, JAK2 inhibitor (AG490), ectopic c-Fos and NFATc1 overexpression","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (KO cells, neutralizing Ab, pharmacological inhibition, OE rescue) in a single focused mechanistic study","pmids":["18209032"],"is_preprint":false},{"year":2006,"finding":"AsialoGM1 and TLR5 cooperate in flagellin signaling: TLR5 is required for NF-κB activation, while flagellin-induced ATP release (via Toll signaling) is required for Erk1/2 activation and mucin induction downstream of asialoGM1. TLR5 alone cannot activate Erk1/2 without extracellular ATP.","method":"Pharmacological inhibition, dominant-negative Toll signaling, ATP receptor signaling assays, Erk1/2 phosphorylation measurement in lung epithelial cells","journal":"American journal of respiratory cell and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection with dominant-negative approach and pharmacological inhibitors, single lab","pmids":["16439799"],"is_preprint":false},{"year":2010,"finding":"TLR5 functions as an endocytic receptor on dendritic cells to enhance MHC class II presentation of flagellin peptides to CD4+ T cells independently of the MyD88 adaptor. TLR5-deficient mice show poor flagellin-specific CD4+ T cell expansion even when other TLR agonists are provided, but robust responses occur when pre-processed flagellin peptide is used.","method":"TLR5-knockout mice, flagellin peptide vs. whole flagellin immunization, in vitro DC culture system, adoptive transfer, MyD88-knockout comparison","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple genetic models (TLR5-KO, MyD88-KO) with in vivo and in vitro validation, mechanistically dissects endocytic from signaling function","pmids":["21182074"],"is_preprint":false},{"year":2010,"finding":"TLR5 activation induces secretory IL-1 receptor antagonist (sIL-1Ra) in intestinal epithelia and macrophages in a TLR5-dependent manner (on non-hematopoietic cells), whereas IL-1β production from flagellin depends on IPAF (inflammasome). Loss of TLR5 increases the IL-1β/sIL-1Ra ratio and correlates with increased inflammatory pathology.","method":"TLR5-knockout mice, bone marrow chimera experiments to identify non-hematopoietic source of sIL-1Ra, Salmonella infection model, ELISA","journal":"Mucosal immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — bone marrow chimeras precisely localize TLR5-dependent sIL-1Ra to non-hematopoietic cells; multiple orthogonal readouts","pmids":["20844479"],"is_preprint":false},{"year":2010,"finding":"TLR5 or NLRC4 is necessary and sufficient for flagellin-mediated humoral immunity: TLR5-KO mice lack NF-κB-regulated cytokines (CXCL1) but retain IL-18, NLRC4-KO mice show the opposite pattern, and double-KO mice lack all cytokines and antibody responses to flagellin.","method":"TLR5-KO, NLRC4-KO, and TLR5/NLRC4 double-KO mice, prime/boost immunization, cytokine ELISA, antibody titer measurement","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with single, double KO mice dissecting pathway contributions to antibody responses","pmids":["21072873"],"is_preprint":false},{"year":2010,"finding":"TRIF induces proteolytic degradation of TLR5 protein through caspase activity (blocked by pan-caspase inhibitor but not by cathepsin B, ROS, or proteasome inhibitors), requiring the C-terminus of TRIF and the extracellular domain of TLR5. TRIF overexpression suppresses flagellin/TLR5-driven NF-κB activation without altering TLR5 mRNA levels.","method":"TRIF overexpression in HEK293 and NCM460 cells, caspase/cathepsin inhibitors, proteasome inhibitor, domain deletion constructs, Western blot, NF-κB reporter assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection of degradation pathway with domain mapping, single lab","pmids":["20452988"],"is_preprint":false},{"year":2012,"finding":"Cell surface expression of TLR5 on immune cells (macrophages, neutrophils, classical monocytes, specific DC subsets) is completely dependent on the TLR-specific chaperone PRAT4A. Silencing PRAT4A abolishes both surface TLR5 expression and flagellin-induced responses in the macrophage cell line J774.","method":"Anti-mouse TLR5 monoclonal antibody development, flow cytometry, PRAT4A siRNA silencing, cytokine ELISA, in vivo immune cell subset analysis","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — novel monoclonal antibody tool with siRNA knockdown and multiple cell types studied; single lab but two orthogonal methods","pmids":["22836022"],"is_preprint":false},{"year":2016,"finding":"HMGB1 binds TLR5 and activates NF-κB signaling in a MyD88-dependent manner, resulting in proinflammatory cytokine production and pain hypersensitivity in vivo. The C-terminal tail region of HMGB1 is essential for the interaction with TLR5.","method":"Biophysical binding assays, NF-κB reporter assay in TLR5-expressing cells, MyD88-dependence testing, in vivo allodynia model, domain mapping of HMGB1","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biophysical binding plus cell-based signaling assay plus in vivo pain model, single lab","pmids":["27760316"],"is_preprint":false},{"year":2019,"finding":"H. pylori T4SS component CagL contains a D1-like flagellin motif that mediates direct binding to TLR5, activating TLR5-dependent downstream signaling in gastric epithelial cells independently of flagellin. TLR5 is important for efficient control of H. pylori infection in vivo (TLR5-knockout vs. wild-type mice).","method":"TLR5 binding assays, NF-κB reporter assay, siRNA knockdown, TLR5-knockout mice, H. pylori infection model, human biopsy immunohistochemistry","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vitro binding/signaling mechanistic data combined with in vivo KO mouse validation, multiple orthogonal approaches","pmids":["31844047"],"is_preprint":false},{"year":2020,"finding":"TLR5 physically associates with TLR4 in primary murine macrophages (co-immunoprecipitation) and biases TLR4 signaling towards the MyD88 pathway. TLR5 impacts in vivo responses to LPS, hyaluronan, and ozone (TLR4-mediated stimuli), and human carriers of a dominant-negative TLR5 allele show decreased inflammatory responses to these stimuli.","method":"Co-immunoprecipitation, TLR5-knockout mice in vivo models (LPS, O3, hyaluronan), human dominant-negative TLR5 allele carrier studies","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal evidence from co-IP plus KO mouse models plus human genetic variant, but TLR4/TLR5 co-IP is a single-lab finding","pmids":["31989925"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of P. aeruginosa FliC flagellin (paFliC) at 2.1 Å, combined with gel filtration and native PAGE, demonstrated direct TLR5 binding. Structural modeling shows the paFliC D1 domain provides major TLR5-binding sites analogous to Salmonella FliC.","method":"X-ray crystallography (2.1 Å), gel filtration, native PAGE, structural modeling of TLR5 complex","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure with direct binding assays but TLR5 complex is modeled rather than solved directly","pmids":["24434155"],"is_preprint":false},{"year":2018,"finding":"Zebrafish TLR5 signals as a heterodimer composed of drTLR5b and drTLR5a (products of a duplicated gene), unlike mammalian TLR5 which signals as a homodimer. Flagellin-induced signaling requires both a heterodimeric ectodomain and cytoplasmic domain configuration; TLR5 trafficking chaperone UNC93B1 enhances signaling. Structure-guided substitution of the principal flagellin-binding site in human TLR5 with zebrafish TLR5 residues abrogated human TLR5 activation.","method":"Heterodimer co-expression and signaling assays, domain swap mutagenesis, UNC93B1 co-transfection, structure-guided site-directed mutagenesis of human TLR5","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal genetic approaches (domain swaps, structure-guided mutagenesis, chaperone co-expression) systematically dissecting heterodimeric mechanism","pmids":["29555749"],"is_preprint":false},{"year":2014,"finding":"TLR5-mediated sensing of gut microbiota flagellin is required for antibody responses to trivalent inactivated influenza vaccine (TIV): TLR5-KO mice have reduced antibody titers and fewer plasma cells. Mechanistically, TLR5 sensing promotes plasma cell differentiation directly and by stimulating lymph node macrophages to produce plasma cell growth factors.","method":"TLR5-KO mice, germ-free and antibiotic-treated mice, reconstitution with flagellated vs. aflagellated E. coli, plasma cell frequency measurement, antibody titer ELISA","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic and microbiota manipulation strategies converging on a defined mechanistic pathway","pmids":["25220212"],"is_preprint":false},{"year":2014,"finding":"Flagellin-induced protection against rotavirus requires both TLR5 (on dendritic cells) and NLRC4. TLR5 activation on DCs elicits IL-22 production which induces a protective gene expression program in intestinal epithelial cells; NLRC4 drives IL-18-dependent elimination of RV-infected cells. Administration of IL-22 and IL-18 together fully recapitulates flagellin protection.","method":"TLR5-KO and NLRC4-KO mice, flagellin treatment, rotavirus infection model, cytokine neutralization, IL-22 and IL-18 co-administration rescue experiment","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with cytokine rescue experiment precisely placing TLR5 in the pathway (TLR5→DC→IL-22→epithelial protection)","pmids":["25395539"],"is_preprint":false},{"year":2016,"finding":"TLR5 expression in intestinal epithelial cells is regulated at the transcriptional level by differential binding of Sp1 and Sp3 to GC-box sequences in the TLR5 promoter. Butyrate activates two PKC isoforms: one dephosphorylates/acetylates Sp1 (causing its displacement) and another phosphorylates Sp3 via ERK-MAPK, leading to Sp3 binding, p300 recruitment, histone acetylation, and TLR5 transcriptional activation.","method":"Promoter reporter assays, ChIP, siRNA knockdown of Sp1/Sp3, PKC isoform-specific inhibitors, ERK-MAPK inhibition, HDAC inhibition, mutagenesis of GC-box elements","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (ChIP, reporter assay, siRNA, pharmacological inhibitors) dissecting transcriptional mechanism in a single study","pmids":["27060138"],"is_preprint":false},{"year":2015,"finding":"TLR5 activation by flagellin induces RANKL expression in osteoblasts via a MyD88- and NF-κB-dependent mechanism, leading to robust osteoclast formation and bone loss in vitro and in vivo. These effects are absent in Tlr5-/- mice, establishing TLR5 as a direct activator of RANKL and osteoclastogenesis.","method":"TLR5-KO mice, neonatal calvarial bone culture, isolated osteoblast culture, local flagellin injection model, RANKL:OPG ratio measurement, osteoclast quantification, NF-κB inhibition","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — TLR5-KO mice confirm specificity in both in vitro and in vivo settings with defined molecular mechanism (MyD88/NF-κB/RANKL)","pmids":["26207027"],"is_preprint":false},{"year":2010,"finding":"TLR5 requires the trafficking chaperone PRAT4A for cell surface expression; without PRAT4A, TLR5 is not expressed on the cell surface and flagellin-induced cytokine responses are abolished. PRAT4A-dependent surface TLR5 is primarily found on neutrophils, CD11b(hi)Ly6C(hi) classical monocytes, and specific DC subsets in vivo.","method":"Anti-TLR5 monoclonal antibody, PRAT4A siRNA silencing, flow cytometry, IL-6/G-CSF ELISA","journal":"International immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — novel mAb tool plus siRNA knockdown with cytokine readout; single lab but mechanistically clean","pmids":["22836022"],"is_preprint":false},{"year":2020,"finding":"TLR5 activation by flagellin in hepatocytes stimulates ApoA1 production through NF-κB transcriptional activation at the Apoa1 promoter. Deletion of hepatic TLR5 suppresses HFD-stimulated HDL-C and ApoA1 levels; overexpression of TLR5 in the liver of TLR5-KO mice partially restores ApoA1 and HDL-C production.","method":"TLR5-KO mice, liver-specific TLR5 overexpression (AAV), primary hepatocyte stimulation, NF-κB ChIP on Apoa1 promoter, fecal microbiome transplantation, HDL-C/ApoA1 ELISA","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP of NF-κB on Apoa1 promoter with TLR5 KO and liver-specific rescue providing mechanistic pathway evidence","pmids":["32820707"],"is_preprint":false},{"year":2017,"finding":"TLR5 signaling in bovine cells requires PI3K activation for downstream responses; mutation of bTLR5 F798 (within a putative PI3K motif) to hTLR5 Y798 significantly reduces signaling. Species-specific TLR5 responses involve cognate MyD88 recognition differences between bovine and human TIR domains.","method":"Bovine vs. human TLR5 expression in cognate cell lines, siRNA knockdown, PI3K inhibitor, site-directed mutagenesis of TIR domain (F798Y), CXCL8 measurement","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis with functional readout, pharmacological inhibition; single lab","pmids":["29247203"],"is_preprint":false},{"year":2013,"finding":"TLR5 epithelial activation by flagellin results in decreased epithelial barrier resistance and altered tight junction protein (claudin-3, occludin, ZO-1) expression in ileal tissue of SAMP mice. The elevated TLR5 in this model is derived primarily from non-hematopoietic (epithelial) cells, as demonstrated by bone marrow chimera experiments.","method":"Bone marrow chimera experiments, TLR5-specific ex vivo activation of ileal tissue, transepithelial resistance measurement, tight junction protein expression (claudin-3, occludin, ZO-1)","journal":"Inflammatory bowel diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bone marrow chimera localizes TLR5 to non-hematopoietic cells; ex vivo barrier assay directly links TLR5 activation to functional consequence","pmids":["28146004"],"is_preprint":false},{"year":2014,"finding":"TLR5 signaling in myometrial and fetal membrane cells promotes pro-inflammatory cytokines (IL-6, IL-8), MMP-9, COX-2, and prostaglandin release through MyD88, TRAF6, and NF-κB. siRNA knockdown of TLR5, MyD88, TRAF6, or NF-κB inhibitor reduced flagellin-induced pro-labour mediator production.","method":"siRNA knockdown of TLR5, MyD88, TRAF6 in primary amnion and myometrium cells, NF-κB reporter assay, ELISA for cytokines, MMP-9 activity assay","journal":"American journal of reproductive immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA of multiple pathway components with defined functional readouts; single lab","pmids":["24635133"],"is_preprint":false},{"year":2016,"finding":"TLR5 mediates CD172α+ lamina propria DC (LPDC) induction of Th17 cells in the intestine in response to commensal flagellin. Wild-type CD172α+ LPDCs (but not TLR5-deficient LPDCs) induced Th17 cells when cultured with full-length flagellin; LPDCs expressed high levels of TLR5 and produced IL-23, IL-6, and TGFβ upon flagellin stimulation.","method":"TLR5-KO mice, microbiota antigen-specific T cell reporter mouse system, LPDC-T cell co-culture, flagellin vs. flagellin peptide comparison, cytokine measurement","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — TLR5-KO DC demonstrates requirement; multiple readouts (Th17 differentiation, cytokine production) in a mechanistically defined system","pmids":["26907705"],"is_preprint":false},{"year":2018,"finding":"MAP1S regulates the flagellin/TLR5 signaling pathway in breast cancer cells through enhancement of NF-κB activity and cytokine secretion. Knockdown of MAP1S abrogates flagellin-induced tumor suppression. MAP1S in later stages of TLR5 signaling degrades MyD88 via autophagy, providing a negative feedback mechanism.","method":"MAP1S knockdown (siRNA), NF-κB reporter assay, tumor growth assay, autophagy assay, MyD88 protein level measurement","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — siRNA knockdown with tumor growth and signaling readouts; MyD88 degradation via autophagy is shown but mechanism is single-lab, single method","pmids":["24466264"],"is_preprint":false},{"year":2018,"finding":"Nucleoside diphosphate kinase 3 (NME3) is a positive regulator of TLR5-mediated NF-κB signaling, acting downstream of MyD88. Knockdown of NME3 reduces flagellin-induced NF-κB activation; overexpression enhances it.","method":"High-throughput siRNA library screen (691 kinases), NFκB bioluminescent reporter, NME3 targeted knockdown and overexpression validation","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — screen confirmed by targeted knockdown and OE in the same signaling system; epistasis (downstream of MyD88) established by genetic order","pmids":["29523766"],"is_preprint":false},{"year":2021,"finding":"TLR5 ligation by α-synuclein monomers and oligomers (along with TLR2) activates the NLRP3 inflammasome in primary microglia, compromising α-syn degradation. TLR2 and TLR5 act on different signaling checkpoints of NLRP3 activation; NLRP3 inhibition improves overall clearance of α-syn oligomers.","method":"Primary microglia from wild-type mice, TLR2/TLR5 antibody blocking, NLRP3 inhibitor (CRID3), NLRP3-deficient cells, α-syn internalization and degradation assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor blocking and NLRP3 KO cells with defined functional readout; single lab","pmids":["34507948"],"is_preprint":false},{"year":2022,"finding":"Roseburia intestinalis stimulates TSLP production in intestinal epithelial cells specifically through TLR5 (not TLR2 or TLR4). TSLP from IECs induces IL-10 and TGFβ secretion from DCs, which drives Treg differentiation. TLR5 depletion or TSLP neutralization abrogates the protective effect of R. intestinalis on experimental colitis.","method":"TLR5-siRNA in Caco-2 cells, Tlr5-/- mice, bone marrow chimera mice, anti-TSLP/anti-TGFβ neutralizing antibodies, DC-T cell co-culture differentiation assays","journal":"EBioMedicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — TLR5 specificity established by siRNA and KO mice; TSLP pathway confirmed by neutralizing Ab; DC-T cell assay provides cellular mechanism","pmids":["36182776"],"is_preprint":false},{"year":2023,"finding":"Clostridia flagella (TLR5 ligand) signal through TLR5/MyD88 on CD11c+ antigen-presenting cells to induce IL-22 secretion from ileal explants, which contributes to barrier protection against food allergy. This TLR5/MyD88 pathway works together with AhR signaling in RORγt+ cells to maintain intestinal barrier integrity.","method":"TLR5-KO, MyD88-KO, CD11c-specific MyD88-KO, and AhR-KO mice; ileal explant IL-22 assay; intestinal permeability measurement; anaphylaxis model","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple conditional KO models precisely locating TLR5/MyD88 signaling to CD11c+ cells with functional IL-22 and barrier readouts","pmids":["37742185"],"is_preprint":false},{"year":2004,"finding":"TLR5 in the gastric epithelium undergoes dynamic relocalization from apical+basolateral to exclusively basolateral distribution during H. pylori infection, as determined by confocal microscopy in patient biopsies, suggesting infection-regulated polarized TLR5 localization modulates mucosal immune responses.","method":"Confocal immunofluorescence microscopy on human gastric biopsies from H. pylori gastritis patients vs. noninflamed controls","journal":"Clinical and experimental immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by confocal microscopy in human tissue; replicated across multiple patients but no functional consequence directly tested in this paper","pmids":["15147355"],"is_preprint":false}],"current_model":"TLR5 is a pattern recognition receptor that detects bacterial flagellin through its lateral surface contacting the flagellin D1 domain, assembling into a 2:2 tail-to-tail signaling complex (established by crystal structure); upon ligand binding, TLR5 recruits MyD88 (requiring PKD-mediated phosphorylation at S805 and PRAT4A chaperone for surface trafficking) to activate NF-κB and p38 MAPK (the latter via a post-transcriptional mechanism regulating IL-8 translation), while simultaneously activating caspase-8-dependent apoptotic pathways that are suppressed by NF-κB/PI3K-Akt survival signals; in the intestinal epithelium TLR5 induces sIL-1Ra (counterbalancing NLRC4/inflammasome-derived IL-1β), promotes barrier integrity through tight junction regulation, and on lamina propria DCs drives Th17 differentiation and IgA production; TLR5 additionally functions as an endocytic receptor on DCs to enhance MHC-II antigen presentation of flagellin in a MyD88-independent manner, physically associates with TLR4 to bias its signaling toward MyD88, and can be activated by non-flagellin ligands including HMGB1 and the H. pylori T4SS component CagL; TLR5 expression is transcriptionally regulated by Sp1/Sp3 balance through PKC-ERK signaling in response to butyrate, and negatively regulated post-translationally by TRIF-induced caspase-dependent degradation."},"narrative":{"mechanistic_narrative":"TLR5 is a Toll/IL-1R-family pattern recognition receptor that senses bacterial flagellin to drive innate immune signaling [PMID:9596645]. Crystal structures of the flagellin–TLR5 complex establish that TLR5 engages three helices of the flagellin D1 domain through its lateral surface, with a conserved activation hot-spot arginine fitting a cavity formed by the leucine-rich repeat 9 loop, and that two 1:1 heterodimers assemble into a 2:2 tail-to-tail signaling complex [PMID:22344444, PMID:28106112]. Surface display of TLR5 on immune cells requires the chaperone PRAT4A [PMID:22836022], and signaling competence depends on protein kinase D–mediated phosphorylation at serine 805 [PMID:17442957]. Ligand engagement recruits MyD88 to activate NF-κB and, via a parallel arm, p38 MAPK that controls IL-8 output post-transcriptionally [PMID:12702497, PMID:15302888]; TLR5 simultaneously engages a caspase-8 extrinsic apoptotic pathway held in check by NF-κB and PI3K-Akt survival signaling [PMID:16179598]. In the intestinal epithelium TLR5 induces secretory IL-1Ra to counterbalance NLRC4/inflammasome-derived IL-1β [PMID:20844479], regulates tight-junction proteins and barrier resistance [PMID:28146004], and on lamina propria and CD11c+ dendritic cells drives Th17 differentiation, IL-22 production, and antibody/plasma-cell responses to flagellin [PMID:25220212, PMID:26907705, PMID:37742185]. TLR5 also acts as an endocytic receptor that enhances MHC class II presentation of flagellin independently of MyD88 [PMID:21182074], physically associates with TLR4 to bias TLR4 signaling toward MyD88 [PMID:31989925], and is activated by non-flagellin ligands including HMGB1 and the H. pylori T4SS protein CagL [PMID:27760316, PMID:31844047]. Genetic loss of TLR5 causes spontaneous TLR4-driven colitis [PMID:18008007], and the receptor additionally couples flagellin sensing to osteoclastogenesis through RANKL induction [PMID:26207027] and to hepatic ApoA1/HDL production via NF-κB [PMID:32820707]. TLR5 transcription is set by the Sp1/Sp3 balance through PKC-ERK signaling in response to butyrate [PMID:27060138], and TLR5 protein is negatively regulated by TRIF-induced caspase-dependent degradation [PMID:20452988].","teleology":[{"year":1998,"claim":"Established TLR5 as a functional innate immune receptor by showing it activates NF-κB, placing it in the Toll/IL-1R signaling family before its ligand was known.","evidence":"Cloning and NF-κB reporter assay in transfected cells","pmids":["9596645"],"confidence":"Medium","gaps":["No ligand identified at this stage","Cell-type dependence of signaling unexplained","No downstream effectors mapped"]},{"year":2003,"claim":"Resolved how flagellin/TLR5 controls IL-8, showing a NF-κB-independent p38 MAPK arm that regulates IL-8 at the translational level.","evidence":"Dominant-negative TLR5, p38 inhibitor, mRNA stability assays in polarized intestinal epithelium","pmids":["12702497"],"confidence":"High","gaps":["Link between p38 and translational machinery not defined","ERK activation not TLR5-specific"]},{"year":2004,"claim":"Defined the core adaptor and output of TLR5 signaling and its apical/basolateral geometry in epithelium, establishing MyD88-dependent NF-κB induction of chemokines.","evidence":"Dominant-negative TLR5/MyD88, NF-κB reporter, Ussing chamber and IHC in tissue; confocal localization in gastric biopsies","pmids":["15302888","15147355"],"confidence":"High","gaps":["Functional consequence of polarized relocalization not directly tested","Mechanism of basolateral targeting unknown"]},{"year":2005,"claim":"Revealed that TLR5 simultaneously engages pro-survival and apoptotic programs, explaining how flagellin sensing is buffered against epithelial death.","evidence":"Caspase assays, pathway inhibitors, dominant-negative TLR5 in intestinal epithelial cells","pmids":["16179598"],"confidence":"High","gaps":["Molecular link from TLR5 to caspase-8 not defined","In vivo relevance of the apoptotic arm untested here"]},{"year":2007,"claim":"Identified PKD as a direct kinase phosphorylating TLR5 at S805 required for responses, and extended TLR5 flagellin sensing to macrophages and species-specific TIR-domain signaling.","evidence":"Co-IP, in vitro kinase assay/MS, S805A mutagenesis, shRNA; TLR5-KO macrophages with flagellin-deficient bacterial strains; TIR-domain mutagenesis","pmids":["17442957","17982089","17964652"],"confidence":"High","gaps":["How S805 phosphorylation gates MyD88 recruitment not resolved","Structural basis of species-specific flagellin recognition not solved"]},{"year":2008,"claim":"Showed TLR5 signaling can be anti-osteoclastogenic via an IFN-β/STAT1 axis, demonstrating context-dependent outputs beyond classical NF-κB inflammation.","evidence":"BMDM osteoclastogenesis assays, IFN-β neutralization, STAT1-KO cells, c-Fos rescue","pmids":["18209032"],"confidence":"High","gaps":["Opposing osteoclast-promoting effect in co-culture mechanistically unexplained","Connection between TLR5 and IFN-β induction not mapped"]},{"year":2007,"claim":"Demonstrated by genetic epistasis that TLR5 loss causes spontaneous colitis that is TLR4-dependent, defining TLR5 as a homeostatic regulator of intestinal inflammation.","evidence":"TLR5-KO and TLR4/TLR5 double-KO mice, histopathology and cytokine profiling","pmids":["18008007"],"confidence":"High","gaps":["Mechanism linking TLR5 loss to TLR4 hyperactivation not defined here","Microbiota contribution not dissected"]},{"year":2010,"claim":"Separated TLR5's signaling and antigen-presentation roles and defined surface-expression and negative-regulation requirements, showing endocytic MHC-II presentation, PRAT4A-dependent surface display, and TRIF/caspase-mediated TLR5 degradation.","evidence":"TLR5-KO/MyD88-KO mice with peptide vs whole flagellin; PRAT4A siRNA and anti-TLR5 mAb; TRIF overexpression with domain mapping and protease inhibitors","pmids":["21182074","22836022","20452988"],"confidence":"High","gaps":["Endocytic trafficking machinery for TLR5 not identified","Physiological trigger for TRIF-driven TLR5 degradation unknown"]},{"year":2010,"claim":"Defined the division of labor between TLR5 and the NLRC4 inflammasome in flagellin immunity, with TLR5 driving NF-κB cytokines and sIL-1Ra while NLRC4 produces IL-1β/IL-18.","evidence":"TLR5-KO, NLRC4-KO, double-KO mice; bone marrow chimeras; immunization and cytokine/antibody measurements","pmids":["20844479","21072873"],"confidence":"High","gaps":["Coordination between the two pathways at the cellular level not fully resolved","Source-specific contributions across tissues incompletely mapped"]},{"year":2012,"claim":"Provided the atomic mechanism of flagellin recognition, showing lateral-surface engagement of the flagellin D1 domain and assembly of a 2:2 tail-to-tail signaling complex.","evidence":"X-ray crystallography of zebrafish TLR5–FliC at 2.47 Å with structure-guided mutagenesis","pmids":["22344444"],"confidence":"High","gaps":["Cytoplasmic TIR-domain dimerization upon activation not captured","Human full-length receptor structure not solved"]},{"year":2014,"claim":"Connected TLR5 to systemic and mucosal adaptive immunity, showing microbiota flagellin sensing is required for vaccine antibody responses and that DC TLR5/IL-22 cooperates with NLRC4/IL-18 for antiviral epithelial protection; structural confirmation extended flagellin recognition across species.","evidence":"TLR5-KO, germ-free/antibiotic and reconstitution mice (influenza vaccine); TLR5-KO/NLRC4-KO mice with IL-22/IL-18 rescue (rotavirus); P. aeruginosa FliC crystal structure with binding assays","pmids":["25220212","25395539","24434155"],"confidence":"High","gaps":["How gut flagellin signal reaches systemic plasma cells not fully traced","P. aeruginosa TLR5 complex modeled, not solved"]},{"year":2015,"claim":"Extended TLR5 function to bone biology, showing flagellin/TLR5 induces RANKL in osteoblasts via MyD88/NF-κB to drive osteoclastogenesis and bone loss.","evidence":"TLR5-KO mice, calvarial and osteoblast cultures, in vivo flagellin injection, NF-κB inhibition","pmids":["26207027"],"confidence":"High","gaps":["Reconciliation with anti-osteoclastogenic IFN-β arm not addressed","Relevance to inflammatory bone disease not established"]},{"year":2016,"claim":"Broadened TLR5 ligand repertoire to the endogenous alarmin HMGB1 and defined transcriptional control of TLR5 by Sp1/Sp3, plus a DC-intrinsic Th17-inducing role.","evidence":"HMGB1 binding/signaling assays and in vivo pain model; promoter reporter, ChIP, Sp1/Sp3 siRNA and PKC/ERK inhibitors; TLR5-KO LPDC co-culture","pmids":["27760316","27060138","26907705"],"confidence":"High","gaps":["Structural basis of HMGB1–TLR5 interaction unknown","Whether endogenous ligand sensing uses the same activation hot-spot untested"]},{"year":2018,"claim":"Established the dimerization architecture in zebrafish (heterodimeric TLR5a/TLR5b) and identified intracellular regulators (MAP1S, NME3) of TLR5 signaling amplitude.","evidence":"Heterodimer co-expression, domain swaps, UNC93B1 co-transfection; siRNA kinase screen with NME3 validation; MAP1S knockdown with autophagy/MyD88 readouts; bovine PI3K-motif mutagenesis","pmids":["29555749","29523766","24466264","29247203"],"confidence":"Medium","gaps":["MAP1S/NME3 mechanisms are single-lab","Generalizability of zebrafish heterodimeric mode to other species limited"]},{"year":2019,"claim":"Showed TLR5 is activated by the non-flagellin H. pylori T4SS protein CagL through a D1-like motif, expanding pathogen recognition beyond flagellin.","evidence":"Binding/NF-κB assays, siRNA, TLR5-KO mice infection model, human biopsy IHC","pmids":["31844047"],"confidence":"High","gaps":["Structural detail of CagL–TLR5 engagement not solved","Whether CagL uses the canonical activation hot spot untested"]},{"year":2020,"claim":"Revealed a cross-receptor role in which TLR5 physically partners with TLR4 to bias signaling toward MyD88, and a metabolic role in hepatic ApoA1/HDL production.","evidence":"Co-IP in primary macrophages, TLR5-KO mouse LPS/hyaluronan/ozone models, human dominant-negative allele; TLR5-KO mice with liver-specific AAV rescue and Apoa1 promoter ChIP","pmids":["31989925","32820707"],"confidence":"Medium","gaps":["TLR4/TLR5 co-IP is single-lab without structural validation","Stoichiometry of the TLR4–TLR5 association unknown"]},{"year":2022,"claim":"Placed TLR5 in tolerogenic and barrier-protective circuits, showing commensal-driven TLR5 induces epithelial TSLP that promotes Treg differentiation and DC/IL-22-mediated barrier protection.","evidence":"TLR5-siRNA Caco-2, Tlr5-/- and BM chimera mice, anti-TSLP/TGFβ neutralization, DC-T cell co-culture; conditional CD11c-MyD88-KO and AhR-KO mice with IL-22 readouts","pmids":["36182776","37742185"],"confidence":"High","gaps":["Direct epithelial signaling steps from TLR5 to TSLP transcription not mapped","Interplay with AhR pathway mechanistically incomplete"]},{"year":2021,"claim":"Linked TLR5 to neuroinflammation, showing α-synuclein sensing by TLR5 (with TLR2) activates the NLRP3 inflammasome in microglia and impairs α-syn clearance.","evidence":"Primary microglia, antibody blocking, NLRP3 inhibitor and NLRP3-KO cells, α-syn degradation assays","pmids":["34507948"],"confidence":"Medium","gaps":["Direct α-syn–TLR5 binding not demonstrated","Distinct TLR2 vs TLR5 checkpoints not molecularly defined"]},{"year":null,"claim":"How activated human full-length TLR5 transduces signal across the membrane—linking the 2:2 ectodomain complex, S805 phosphorylation, and TIR-domain assembly to MyD88 recruitment, and how it discriminates flagellin from endogenous/non-flagellin ligands—remains unresolved.","evidence":"No discovery in the timeline resolves the activated cytoplasmic signaling architecture","pmids":[],"confidence":"Low","gaps":["No structure of the activated TIR-domain signaling module","Mechanism connecting ectodomain dimerization to MyD88 recruitment unknown","Whether non-flagellin ligands trigger the same conformational pathway untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[0,1,2,18]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,3,4,5]},{"term_id":"GO:0038024","term_label":"cargo receptor activity","supporting_discovery_ids":[12]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[16,26,37]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,4,12,22,31]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,7,30,33]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5]}],"complexes":[],"partners":["MYD88","TLR4","PRKD1","PRAT4A","HMGB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O60602","full_name":"Toll-like receptor 5","aliases":["Toll/interleukin-1 receptor-like protein 3"],"length_aa":858,"mass_kda":97.8,"function":"Pattern recognition receptor (PRR) located on the cell surface that participates in the activation of innate immunity and inflammatory response (PubMed:11323673, PubMed:18490781). Recognizes small molecular motifs named pathogen-associated molecular pattern (PAMPs) expressed by pathogens and microbe-associated molecular patterns (MAMPs) usually expressed by resident microbiota (PubMed:29934223). Upon ligand binding such as bacterial flagellins, recruits intracellular adapter proteins MYD88 and TRIF leading to NF-kappa-B activation, cytokine secretion and induction of the inflammatory response (PubMed:11489966, PubMed:20855887). Plays thereby an important role in the relationship between the intestinal epithelium and enteric microbes and contributes to the gut microbiota composition throughout life (By similarity)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/O60602/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TLR5","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TLR5","total_profiled":1310},"omim":[{"mim_id":"615592","title":"IMMUNODEFICIENCY 15B; IMD15B","url":"https://www.omim.org/entry/615592"},{"mim_id":"615557","title":"MELIOIDOSIS, SUSCEPTIBILITY TO","url":"https://www.omim.org/entry/615557"},{"mim_id":"609948","title":"RING FINGER PROTEIN 216; RNF216","url":"https://www.omim.org/entry/609948"},{"mim_id":"608556","title":"LEGIONNAIRE DISEASE, SUSCEPTIBILITY TO","url":"https://www.omim.org/entry/608556"},{"mim_id":"607955","title":"SAM- AND SH3 DOMAIN-CONTAINING PROTEIN 1; SASH1","url":"https://www.omim.org/entry/607955"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TLR5"},"hgnc":{"alias_symbol":["TIL3","FLJ10052","MGC126430","MGC126431"],"prev_symbol":["SLEB1"]},"alphafold":{"accession":"O60602","domains":[{"cath_id":"3.80.10.10","chopping":"20-173","consensus_level":"medium","plddt":95.1865,"start":20,"end":173},{"cath_id":"3.80.10.10","chopping":"183-339","consensus_level":"medium","plddt":94.3876,"start":183,"end":339},{"cath_id":"3.40.50.10140","chopping":"696-838","consensus_level":"high","plddt":87.7782,"start":696,"end":838}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60602","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60602-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60602-F1-predicted_aligned_error_v6.png","plddt_mean":88.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TLR5","jax_strain_url":"https://www.jax.org/strain/search?query=TLR5"},"sequence":{"accession":"O60602","fasta_url":"https://rest.uniprot.org/uniprotkb/O60602.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60602/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60602"}},"corpus_meta":[{"pmid":"22344444","id":"PMC_22344444","title":"Structural basis of TLR5-flagellin recognition and signaling.","date":"2012","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/22344444","citation_count":456,"is_preprint":false},{"pmid":"25220212","id":"PMC_25220212","title":"TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination.","date":"2014","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/25220212","citation_count":424,"is_preprint":false},{"pmid":"18008007","id":"PMC_18008007","title":"Deletion of TLR5 results in spontaneous colitis in mice.","date":"2007","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/18008007","citation_count":353,"is_preprint":false},{"pmid":"17690885","id":"PMC_17690885","title":"TLR5 and Ipaf: dual sensors of bacterial flagellin in the innate immune system.","date":"2007","source":"Seminars in immunopathology","url":"https://pubmed.ncbi.nlm.nih.gov/17690885","citation_count":228,"is_preprint":false},{"pmid":"15147355","id":"PMC_15147355","title":"Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection.","date":"2004","source":"Clinical and experimental immunology","url":"https://pubmed.ncbi.nlm.nih.gov/15147355","citation_count":192,"is_preprint":false},{"pmid":"9596645","id":"PMC_9596645","title":"Cloning and characterization of two Toll/Interleukin-1 receptor-like genes TIL3 and TIL4: evidence for a multi-gene receptor family in humans.","date":"1998","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/9596645","citation_count":182,"is_preprint":false},{"pmid":"25395539","id":"PMC_25395539","title":"Viral infection. 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surface of the opposing TLR5. Structure-guided mutagenesis and deletion analyses validated this signaling mechanism.\",\n      \"method\": \"X-ray crystallography (2.47 Å), structure-guided mutagenesis, deletion analysis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis validation in a single rigorous study\",\n      \"pmids\": [\"22344444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of Bacillus subtilis flagellin–TLR5 complex at 2.1 Å resolution combined with alanine scanning revealed a conserved TLR5 activation hot spot: an arginine residue (bsflagellin R89) and adjacent residues (E114, L93) in the flagellin D1 domain provide shape and chemical complementarity to a cavity formed by the loop of leucine-rich repeat 9 in TLR5. The D0 domain also contributes to TLR5 activity through structurally dispersed regions.\",\n      \"method\": \"X-ray crystallography (2.1 Å), alanine scanning mutagenesis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with systematic mutagenesis of binding interface, replicates and extends prior structural work\",\n      \"pmids\": [\"28106112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"TLR5 (cloned as TIL3) was identified as a human Toll/IL-1R-like receptor that activates NF-κB in a cell-type-dependent fashion, establishing its role in innate immune signaling.\",\n      \"method\": \"Cloning, NF-κB reporter functional assay in transfected cells\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct functional assay in transfected cells, single lab but clear mechanistic readout\",\n      \"pmids\": [\"9596645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Flagellin activates p38 MAPK in a TLR5-dependent manner in polarized intestinal epithelial cells, and this p38 activation regulates IL-8 expression by a post-transcriptional (translational) mechanism independent of NF-κB. ERK1/2 activation by flagellin was not TLR5-specific.\",\n      \"method\": \"Pharmacological inhibition of p38 MAPK (SB-203580), dominant-negative TLR5 transfection, mRNA stability assay, IL-8 protein/mRNA measurement\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (genetic dominant-negative, pharmacological inhibition, post-transcriptional analysis) in a single focused study\",\n      \"pmids\": [\"12702497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Flagellin released by commensal E. coli activates NF-κB, IL-8, and CCL-20 expression in intestinal epithelial cells via TLR5 and the adaptor protein MyD88. In polarized cells, TLR5 signaling could be triggered from the apical side; in vivo, flagellin on the mucosal side of ileal biopsies induced basolateral KC production.\",\n      \"method\": \"Dominant-negative TLR5 and MyD88 plasmid transfection, NF-κB reporter assay, ELISA, Ussing chamber ex vivo, immunohistochemistry\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic DN constructs, ex vivo tissue, in vivo IHC) across in vitro and in vivo systems\",\n      \"pmids\": [\"15302888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Flagellin interaction with TLR5 on intestinal epithelial cells activates both NF-κB/PI3K-Akt pro-survival pathways and the extrinsic caspase-8 apoptotic pathway. When NF-κB or PI3K/Akt signaling is blocked, flagellin induces apoptosis, demonstrating that TLR5 simultaneously activates intertwined inflammatory and apoptotic signaling.\",\n      \"method\": \"Biochemical signaling assays, caspase activity assays, pharmacological pathway inhibition, mRNA expression profiling, dominant-negative TLR5\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (genetic, pharmacological, biochemical) in a single focused study\",\n      \"pmids\": [\"16179598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Mice lacking TLR5 (TLR5KO) develop spontaneous colitis associated with decreased intestinal expression of TLR5-regulated host defense genes and increased colonic proinflammatory cytokines. Deletion of TLR4 in TLR5KO mice rescues colitis, establishing by genetic epistasis that TLR5 loss leads to TLR4-driven colitis.\",\n      \"method\": \"TLR5 knockout mouse model, TLR4/TLR5 double-knockout genetic epistasis, histopathology, cytokine measurement, bacterial burden quantification\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined phenotype, replicated with double KO epistasis demonstrating pathway mechanism\",\n      \"pmids\": [\"18008007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Protein kinase D (PKD) physically interacts with TLR5, and this association is rapidly enhanced by flagellin. PKD phosphorylates TLR5 at serine 805 (identified by in vitro phosphorylation and mass spectrometry); mutation of S805 to alanine abrogates flagellin responses. PKD is required for flagellin-induced p38 MAPK activation and IL-8 production in epithelial cells.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, mass spectrometry (S805 phosphorylation), site-directed mutagenesis (S805A), pharmacological inhibition (Gö6976), shRNA knockdown\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay with MS identification of phosphosite, mutagenesis, co-IP, and shRNA knockdown in a single study\",\n      \"pmids\": [\"17442957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Chicken TLR5 (chTLR5) signals through the MyD88 pathway to activate NF-κB in response to flagellin; mutation of proline 737 in the chTLR5 TIR domain abrogates function. A single amino acid in flagellin (Q89) determines species-specific TLR5 responses between chicken, human, and mouse.\",\n      \"method\": \"Expression in HeLa cells, NF-κB reporter assay, site-directed mutagenesis of TIR domain (P737) and flagellin (Q89A, L415A, N100A)\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional mutagenesis of both receptor and ligand, single lab\",\n      \"pmids\": [\"17964652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TLR5-deficient alveolar macrophages (AMs) fail to produce TNF-α after stimulation with Legionella pneumophila or purified flagellin, demonstrating that AMs recognize L. pneumophila via TLR5-mediated flagellin sensing. In vivo, TLR5-deficient mice show impaired early neutrophil recruitment (at 4 h) and later develop organizing pneumonia.\",\n      \"method\": \"TLR5 knockout mice, L. pneumophila infection model, bronchoalveolar lavage cell counts, TNF-α measurement, flagellin-deficient bacterial strain (LpFlaA-) comparison\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mice with in vivo infection model plus ex vivo macrophage validation with flagellin-deficient strain control\",\n      \"pmids\": [\"17982089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TLR5 activation by flagellin suppresses RANKL-induced osteoclastogenesis by stimulating IFN-β production through STAT1 activation in bone marrow-derived macrophages. IFN-β downregulates c-Fos protein (post-translationally), and neutralizing IFN-β or STAT1 deficiency reverses the anti-osteoclastogenic effect. In osteoblast–macrophage co-cultures, flagellin instead promotes osteoclast differentiation without inducing IFN-β.\",\n      \"method\": \"Bone marrow-derived macrophage culture, RANKL osteoclastogenesis assay, IFN-β neutralizing antibody, STAT1-knockout cells, JAK2 inhibitor (AG490), ectopic c-Fos and NFATc1 overexpression\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (KO cells, neutralizing Ab, pharmacological inhibition, OE rescue) in a single focused mechanistic study\",\n      \"pmids\": [\"18209032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"AsialoGM1 and TLR5 cooperate in flagellin signaling: TLR5 is required for NF-κB activation, while flagellin-induced ATP release (via Toll signaling) is required for Erk1/2 activation and mucin induction downstream of asialoGM1. TLR5 alone cannot activate Erk1/2 without extracellular ATP.\",\n      \"method\": \"Pharmacological inhibition, dominant-negative Toll signaling, ATP receptor signaling assays, Erk1/2 phosphorylation measurement in lung epithelial cells\",\n      \"journal\": \"American journal of respiratory cell and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection with dominant-negative approach and pharmacological inhibitors, single lab\",\n      \"pmids\": [\"16439799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TLR5 functions as an endocytic receptor on dendritic cells to enhance MHC class II presentation of flagellin peptides to CD4+ T cells independently of the MyD88 adaptor. TLR5-deficient mice show poor flagellin-specific CD4+ T cell expansion even when other TLR agonists are provided, but robust responses occur when pre-processed flagellin peptide is used.\",\n      \"method\": \"TLR5-knockout mice, flagellin peptide vs. whole flagellin immunization, in vitro DC culture system, adoptive transfer, MyD88-knockout comparison\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic models (TLR5-KO, MyD88-KO) with in vivo and in vitro validation, mechanistically dissects endocytic from signaling function\",\n      \"pmids\": [\"21182074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TLR5 activation induces secretory IL-1 receptor antagonist (sIL-1Ra) in intestinal epithelia and macrophages in a TLR5-dependent manner (on non-hematopoietic cells), whereas IL-1β production from flagellin depends on IPAF (inflammasome). Loss of TLR5 increases the IL-1β/sIL-1Ra ratio and correlates with increased inflammatory pathology.\",\n      \"method\": \"TLR5-knockout mice, bone marrow chimera experiments to identify non-hematopoietic source of sIL-1Ra, Salmonella infection model, ELISA\",\n      \"journal\": \"Mucosal immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bone marrow chimeras precisely localize TLR5-dependent sIL-1Ra to non-hematopoietic cells; multiple orthogonal readouts\",\n      \"pmids\": [\"20844479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TLR5 or NLRC4 is necessary and sufficient for flagellin-mediated humoral immunity: TLR5-KO mice lack NF-κB-regulated cytokines (CXCL1) but retain IL-18, NLRC4-KO mice show the opposite pattern, and double-KO mice lack all cytokines and antibody responses to flagellin.\",\n      \"method\": \"TLR5-KO, NLRC4-KO, and TLR5/NLRC4 double-KO mice, prime/boost immunization, cytokine ELISA, antibody titer measurement\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with single, double KO mice dissecting pathway contributions to antibody responses\",\n      \"pmids\": [\"21072873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TRIF induces proteolytic degradation of TLR5 protein through caspase activity (blocked by pan-caspase inhibitor but not by cathepsin B, ROS, or proteasome inhibitors), requiring the C-terminus of TRIF and the extracellular domain of TLR5. TRIF overexpression suppresses flagellin/TLR5-driven NF-κB activation without altering TLR5 mRNA levels.\",\n      \"method\": \"TRIF overexpression in HEK293 and NCM460 cells, caspase/cathepsin inhibitors, proteasome inhibitor, domain deletion constructs, Western blot, NF-κB reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection of degradation pathway with domain mapping, single lab\",\n      \"pmids\": [\"20452988\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cell surface expression of TLR5 on immune cells (macrophages, neutrophils, classical monocytes, specific DC subsets) is completely dependent on the TLR-specific chaperone PRAT4A. Silencing PRAT4A abolishes both surface TLR5 expression and flagellin-induced responses in the macrophage cell line J774.\",\n      \"method\": \"Anti-mouse TLR5 monoclonal antibody development, flow cytometry, PRAT4A siRNA silencing, cytokine ELISA, in vivo immune cell subset analysis\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel monoclonal antibody tool with siRNA knockdown and multiple cell types studied; single lab but two orthogonal methods\",\n      \"pmids\": [\"22836022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HMGB1 binds TLR5 and activates NF-κB signaling in a MyD88-dependent manner, resulting in proinflammatory cytokine production and pain hypersensitivity in vivo. The C-terminal tail region of HMGB1 is essential for the interaction with TLR5.\",\n      \"method\": \"Biophysical binding assays, NF-κB reporter assay in TLR5-expressing cells, MyD88-dependence testing, in vivo allodynia model, domain mapping of HMGB1\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biophysical binding plus cell-based signaling assay plus in vivo pain model, single lab\",\n      \"pmids\": [\"27760316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"H. pylori T4SS component CagL contains a D1-like flagellin motif that mediates direct binding to TLR5, activating TLR5-dependent downstream signaling in gastric epithelial cells independently of flagellin. TLR5 is important for efficient control of H. pylori infection in vivo (TLR5-knockout vs. wild-type mice).\",\n      \"method\": \"TLR5 binding assays, NF-κB reporter assay, siRNA knockdown, TLR5-knockout mice, H. pylori infection model, human biopsy immunohistochemistry\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding/signaling mechanistic data combined with in vivo KO mouse validation, multiple orthogonal approaches\",\n      \"pmids\": [\"31844047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TLR5 physically associates with TLR4 in primary murine macrophages (co-immunoprecipitation) and biases TLR4 signaling towards the MyD88 pathway. TLR5 impacts in vivo responses to LPS, hyaluronan, and ozone (TLR4-mediated stimuli), and human carriers of a dominant-negative TLR5 allele show decreased inflammatory responses to these stimuli.\",\n      \"method\": \"Co-immunoprecipitation, TLR5-knockout mice in vivo models (LPS, O3, hyaluronan), human dominant-negative TLR5 allele carrier studies\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal evidence from co-IP plus KO mouse models plus human genetic variant, but TLR4/TLR5 co-IP is a single-lab finding\",\n      \"pmids\": [\"31989925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of P. aeruginosa FliC flagellin (paFliC) at 2.1 Å, combined with gel filtration and native PAGE, demonstrated direct TLR5 binding. Structural modeling shows the paFliC D1 domain provides major TLR5-binding sites analogous to Salmonella FliC.\",\n      \"method\": \"X-ray crystallography (2.1 Å), gel filtration, native PAGE, structural modeling of TLR5 complex\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with direct binding assays but TLR5 complex is modeled rather than solved directly\",\n      \"pmids\": [\"24434155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Zebrafish TLR5 signals as a heterodimer composed of drTLR5b and drTLR5a (products of a duplicated gene), unlike mammalian TLR5 which signals as a homodimer. Flagellin-induced signaling requires both a heterodimeric ectodomain and cytoplasmic domain configuration; TLR5 trafficking chaperone UNC93B1 enhances signaling. Structure-guided substitution of the principal flagellin-binding site in human TLR5 with zebrafish TLR5 residues abrogated human TLR5 activation.\",\n      \"method\": \"Heterodimer co-expression and signaling assays, domain swap mutagenesis, UNC93B1 co-transfection, structure-guided site-directed mutagenesis of human TLR5\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal genetic approaches (domain swaps, structure-guided mutagenesis, chaperone co-expression) systematically dissecting heterodimeric mechanism\",\n      \"pmids\": [\"29555749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TLR5-mediated sensing of gut microbiota flagellin is required for antibody responses to trivalent inactivated influenza vaccine (TIV): TLR5-KO mice have reduced antibody titers and fewer plasma cells. Mechanistically, TLR5 sensing promotes plasma cell differentiation directly and by stimulating lymph node macrophages to produce plasma cell growth factors.\",\n      \"method\": \"TLR5-KO mice, germ-free and antibiotic-treated mice, reconstitution with flagellated vs. aflagellated E. coli, plasma cell frequency measurement, antibody titer ELISA\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic and microbiota manipulation strategies converging on a defined mechanistic pathway\",\n      \"pmids\": [\"25220212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Flagellin-induced protection against rotavirus requires both TLR5 (on dendritic cells) and NLRC4. TLR5 activation on DCs elicits IL-22 production which induces a protective gene expression program in intestinal epithelial cells; NLRC4 drives IL-18-dependent elimination of RV-infected cells. Administration of IL-22 and IL-18 together fully recapitulates flagellin protection.\",\n      \"method\": \"TLR5-KO and NLRC4-KO mice, flagellin treatment, rotavirus infection model, cytokine neutralization, IL-22 and IL-18 co-administration rescue experiment\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with cytokine rescue experiment precisely placing TLR5 in the pathway (TLR5→DC→IL-22→epithelial protection)\",\n      \"pmids\": [\"25395539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TLR5 expression in intestinal epithelial cells is regulated at the transcriptional level by differential binding of Sp1 and Sp3 to GC-box sequences in the TLR5 promoter. Butyrate activates two PKC isoforms: one dephosphorylates/acetylates Sp1 (causing its displacement) and another phosphorylates Sp3 via ERK-MAPK, leading to Sp3 binding, p300 recruitment, histone acetylation, and TLR5 transcriptional activation.\",\n      \"method\": \"Promoter reporter assays, ChIP, siRNA knockdown of Sp1/Sp3, PKC isoform-specific inhibitors, ERK-MAPK inhibition, HDAC inhibition, mutagenesis of GC-box elements\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (ChIP, reporter assay, siRNA, pharmacological inhibitors) dissecting transcriptional mechanism in a single study\",\n      \"pmids\": [\"27060138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TLR5 activation by flagellin induces RANKL expression in osteoblasts via a MyD88- and NF-κB-dependent mechanism, leading to robust osteoclast formation and bone loss in vitro and in vivo. These effects are absent in Tlr5-/- mice, establishing TLR5 as a direct activator of RANKL and osteoclastogenesis.\",\n      \"method\": \"TLR5-KO mice, neonatal calvarial bone culture, isolated osteoblast culture, local flagellin injection model, RANKL:OPG ratio measurement, osteoclast quantification, NF-κB inhibition\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TLR5-KO mice confirm specificity in both in vitro and in vivo settings with defined molecular mechanism (MyD88/NF-κB/RANKL)\",\n      \"pmids\": [\"26207027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TLR5 requires the trafficking chaperone PRAT4A for cell surface expression; without PRAT4A, TLR5 is not expressed on the cell surface and flagellin-induced cytokine responses are abolished. PRAT4A-dependent surface TLR5 is primarily found on neutrophils, CD11b(hi)Ly6C(hi) classical monocytes, and specific DC subsets in vivo.\",\n      \"method\": \"Anti-TLR5 monoclonal antibody, PRAT4A siRNA silencing, flow cytometry, IL-6/G-CSF ELISA\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel mAb tool plus siRNA knockdown with cytokine readout; single lab but mechanistically clean\",\n      \"pmids\": [\"22836022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TLR5 activation by flagellin in hepatocytes stimulates ApoA1 production through NF-κB transcriptional activation at the Apoa1 promoter. Deletion of hepatic TLR5 suppresses HFD-stimulated HDL-C and ApoA1 levels; overexpression of TLR5 in the liver of TLR5-KO mice partially restores ApoA1 and HDL-C production.\",\n      \"method\": \"TLR5-KO mice, liver-specific TLR5 overexpression (AAV), primary hepatocyte stimulation, NF-κB ChIP on Apoa1 promoter, fecal microbiome transplantation, HDL-C/ApoA1 ELISA\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP of NF-κB on Apoa1 promoter with TLR5 KO and liver-specific rescue providing mechanistic pathway evidence\",\n      \"pmids\": [\"32820707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TLR5 signaling in bovine cells requires PI3K activation for downstream responses; mutation of bTLR5 F798 (within a putative PI3K motif) to hTLR5 Y798 significantly reduces signaling. Species-specific TLR5 responses involve cognate MyD88 recognition differences between bovine and human TIR domains.\",\n      \"method\": \"Bovine vs. human TLR5 expression in cognate cell lines, siRNA knockdown, PI3K inhibitor, site-directed mutagenesis of TIR domain (F798Y), CXCL8 measurement\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis with functional readout, pharmacological inhibition; single lab\",\n      \"pmids\": [\"29247203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TLR5 epithelial activation by flagellin results in decreased epithelial barrier resistance and altered tight junction protein (claudin-3, occludin, ZO-1) expression in ileal tissue of SAMP mice. The elevated TLR5 in this model is derived primarily from non-hematopoietic (epithelial) cells, as demonstrated by bone marrow chimera experiments.\",\n      \"method\": \"Bone marrow chimera experiments, TLR5-specific ex vivo activation of ileal tissue, transepithelial resistance measurement, tight junction protein expression (claudin-3, occludin, ZO-1)\",\n      \"journal\": \"Inflammatory bowel diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bone marrow chimera localizes TLR5 to non-hematopoietic cells; ex vivo barrier assay directly links TLR5 activation to functional consequence\",\n      \"pmids\": [\"28146004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TLR5 signaling in myometrial and fetal membrane cells promotes pro-inflammatory cytokines (IL-6, IL-8), MMP-9, COX-2, and prostaglandin release through MyD88, TRAF6, and NF-κB. siRNA knockdown of TLR5, MyD88, TRAF6, or NF-κB inhibitor reduced flagellin-induced pro-labour mediator production.\",\n      \"method\": \"siRNA knockdown of TLR5, MyD88, TRAF6 in primary amnion and myometrium cells, NF-κB reporter assay, ELISA for cytokines, MMP-9 activity assay\",\n      \"journal\": \"American journal of reproductive immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA of multiple pathway components with defined functional readouts; single lab\",\n      \"pmids\": [\"24635133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TLR5 mediates CD172α+ lamina propria DC (LPDC) induction of Th17 cells in the intestine in response to commensal flagellin. Wild-type CD172α+ LPDCs (but not TLR5-deficient LPDCs) induced Th17 cells when cultured with full-length flagellin; LPDCs expressed high levels of TLR5 and produced IL-23, IL-6, and TGFβ upon flagellin stimulation.\",\n      \"method\": \"TLR5-KO mice, microbiota antigen-specific T cell reporter mouse system, LPDC-T cell co-culture, flagellin vs. flagellin peptide comparison, cytokine measurement\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TLR5-KO DC demonstrates requirement; multiple readouts (Th17 differentiation, cytokine production) in a mechanistically defined system\",\n      \"pmids\": [\"26907705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MAP1S regulates the flagellin/TLR5 signaling pathway in breast cancer cells through enhancement of NF-κB activity and cytokine secretion. Knockdown of MAP1S abrogates flagellin-induced tumor suppression. MAP1S in later stages of TLR5 signaling degrades MyD88 via autophagy, providing a negative feedback mechanism.\",\n      \"method\": \"MAP1S knockdown (siRNA), NF-κB reporter assay, tumor growth assay, autophagy assay, MyD88 protein level measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — siRNA knockdown with tumor growth and signaling readouts; MyD88 degradation via autophagy is shown but mechanism is single-lab, single method\",\n      \"pmids\": [\"24466264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Nucleoside diphosphate kinase 3 (NME3) is a positive regulator of TLR5-mediated NF-κB signaling, acting downstream of MyD88. Knockdown of NME3 reduces flagellin-induced NF-κB activation; overexpression enhances it.\",\n      \"method\": \"High-throughput siRNA library screen (691 kinases), NFκB bioluminescent reporter, NME3 targeted knockdown and overexpression validation\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — screen confirmed by targeted knockdown and OE in the same signaling system; epistasis (downstream of MyD88) established by genetic order\",\n      \"pmids\": [\"29523766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TLR5 ligation by α-synuclein monomers and oligomers (along with TLR2) activates the NLRP3 inflammasome in primary microglia, compromising α-syn degradation. TLR2 and TLR5 act on different signaling checkpoints of NLRP3 activation; NLRP3 inhibition improves overall clearance of α-syn oligomers.\",\n      \"method\": \"Primary microglia from wild-type mice, TLR2/TLR5 antibody blocking, NLRP3 inhibitor (CRID3), NLRP3-deficient cells, α-syn internalization and degradation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor blocking and NLRP3 KO cells with defined functional readout; single lab\",\n      \"pmids\": [\"34507948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Roseburia intestinalis stimulates TSLP production in intestinal epithelial cells specifically through TLR5 (not TLR2 or TLR4). TSLP from IECs induces IL-10 and TGFβ secretion from DCs, which drives Treg differentiation. TLR5 depletion or TSLP neutralization abrogates the protective effect of R. intestinalis on experimental colitis.\",\n      \"method\": \"TLR5-siRNA in Caco-2 cells, Tlr5-/- mice, bone marrow chimera mice, anti-TSLP/anti-TGFβ neutralizing antibodies, DC-T cell co-culture differentiation assays\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TLR5 specificity established by siRNA and KO mice; TSLP pathway confirmed by neutralizing Ab; DC-T cell assay provides cellular mechanism\",\n      \"pmids\": [\"36182776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Clostridia flagella (TLR5 ligand) signal through TLR5/MyD88 on CD11c+ antigen-presenting cells to induce IL-22 secretion from ileal explants, which contributes to barrier protection against food allergy. This TLR5/MyD88 pathway works together with AhR signaling in RORγt+ cells to maintain intestinal barrier integrity.\",\n      \"method\": \"TLR5-KO, MyD88-KO, CD11c-specific MyD88-KO, and AhR-KO mice; ileal explant IL-22 assay; intestinal permeability measurement; anaphylaxis model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple conditional KO models precisely locating TLR5/MyD88 signaling to CD11c+ cells with functional IL-22 and barrier readouts\",\n      \"pmids\": [\"37742185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TLR5 in the gastric epithelium undergoes dynamic relocalization from apical+basolateral to exclusively basolateral distribution during H. pylori infection, as determined by confocal microscopy in patient biopsies, suggesting infection-regulated polarized TLR5 localization modulates mucosal immune responses.\",\n      \"method\": \"Confocal immunofluorescence microscopy on human gastric biopsies from H. pylori gastritis patients vs. noninflamed controls\",\n      \"journal\": \"Clinical and experimental immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by confocal microscopy in human tissue; replicated across multiple patients but no functional consequence directly tested in this paper\",\n      \"pmids\": [\"15147355\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TLR5 is a pattern recognition receptor that detects bacterial flagellin through its lateral surface contacting the flagellin D1 domain, assembling into a 2:2 tail-to-tail signaling complex (established by crystal structure); upon ligand binding, TLR5 recruits MyD88 (requiring PKD-mediated phosphorylation at S805 and PRAT4A chaperone for surface trafficking) to activate NF-κB and p38 MAPK (the latter via a post-transcriptional mechanism regulating IL-8 translation), while simultaneously activating caspase-8-dependent apoptotic pathways that are suppressed by NF-κB/PI3K-Akt survival signals; in the intestinal epithelium TLR5 induces sIL-1Ra (counterbalancing NLRC4/inflammasome-derived IL-1β), promotes barrier integrity through tight junction regulation, and on lamina propria DCs drives Th17 differentiation and IgA production; TLR5 additionally functions as an endocytic receptor on DCs to enhance MHC-II antigen presentation of flagellin in a MyD88-independent manner, physically associates with TLR4 to bias its signaling toward MyD88, and can be activated by non-flagellin ligands including HMGB1 and the H. pylori T4SS component CagL; TLR5 expression is transcriptionally regulated by Sp1/Sp3 balance through PKC-ERK signaling in response to butyrate, and negatively regulated post-translationally by TRIF-induced caspase-dependent degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TLR5 is a Toll/IL-1R-family pattern recognition receptor that senses bacterial flagellin to drive innate immune signaling [#2]. Crystal structures of the flagellin–TLR5 complex establish that TLR5 engages three helices of the flagellin D1 domain through its lateral surface, with a conserved activation hot-spot arginine fitting a cavity formed by the leucine-rich repeat 9 loop, and that two 1:1 heterodimers assemble into a 2:2 tail-to-tail signaling complex [#0, #1]. Surface display of TLR5 on immune cells requires the chaperone PRAT4A [#16, #26], and signaling competence depends on protein kinase D–mediated phosphorylation at serine 805 [#7]. Ligand engagement recruits MyD88 to activate NF-κB and, via a parallel arm, p38 MAPK that controls IL-8 output post-transcriptionally [#3, #4]; TLR5 simultaneously engages a caspase-8 extrinsic apoptotic pathway held in check by NF-κB and PI3K-Akt survival signaling [#5]. In the intestinal epithelium TLR5 induces secretory IL-1Ra to counterbalance NLRC4/inflammasome-derived IL-1β [#13], regulates tight-junction proteins and barrier resistance [#29], and on lamina propria and CD11c+ dendritic cells drives Th17 differentiation, IL-22 production, and antibody/plasma-cell responses to flagellin [#22, #31, #36]. TLR5 also acts as an endocytic receptor that enhances MHC class II presentation of flagellin independently of MyD88 [#12], physically associates with TLR4 to bias TLR4 signaling toward MyD88 [#19], and is activated by non-flagellin ligands including HMGB1 and the H. pylori T4SS protein CagL [#17, #18]. Genetic loss of TLR5 causes spontaneous TLR4-driven colitis [#6], and the receptor additionally couples flagellin sensing to osteoclastogenesis through RANKL induction [#25] and to hepatic ApoA1/HDL production via NF-κB [#27]. TLR5 transcription is set by the Sp1/Sp3 balance through PKC-ERK signaling in response to butyrate [#24], and TLR5 protein is negatively regulated by TRIF-induced caspase-dependent degradation [#15].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established TLR5 as a functional innate immune receptor by showing it activates NF-κB, placing it in the Toll/IL-1R signaling family before its ligand was known.\",\n      \"evidence\": \"Cloning and NF-κB reporter assay in transfected cells\",\n      \"pmids\": [\"9596645\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ligand identified at this stage\", \"Cell-type dependence of signaling unexplained\", \"No downstream effectors mapped\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Resolved how flagellin/TLR5 controls IL-8, showing a NF-κB-independent p38 MAPK arm that regulates IL-8 at the translational level.\",\n      \"evidence\": \"Dominant-negative TLR5, p38 inhibitor, mRNA stability assays in polarized intestinal epithelium\",\n      \"pmids\": [\"12702497\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Link between p38 and translational machinery not defined\", \"ERK activation not TLR5-specific\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the core adaptor and output of TLR5 signaling and its apical/basolateral geometry in epithelium, establishing MyD88-dependent NF-κB induction of chemokines.\",\n      \"evidence\": \"Dominant-negative TLR5/MyD88, NF-κB reporter, Ussing chamber and IHC in tissue; confocal localization in gastric biopsies\",\n      \"pmids\": [\"15302888\", \"15147355\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of polarized relocalization not directly tested\", \"Mechanism of basolateral targeting unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Revealed that TLR5 simultaneously engages pro-survival and apoptotic programs, explaining how flagellin sensing is buffered against epithelial death.\",\n      \"evidence\": \"Caspase assays, pathway inhibitors, dominant-negative TLR5 in intestinal epithelial cells\",\n      \"pmids\": [\"16179598\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link from TLR5 to caspase-8 not defined\", \"In vivo relevance of the apoptotic arm untested here\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified PKD as a direct kinase phosphorylating TLR5 at S805 required for responses, and extended TLR5 flagellin sensing to macrophages and species-specific TIR-domain signaling.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay/MS, S805A mutagenesis, shRNA; TLR5-KO macrophages with flagellin-deficient bacterial strains; TIR-domain mutagenesis\",\n      \"pmids\": [\"17442957\", \"17982089\", \"17964652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How S805 phosphorylation gates MyD88 recruitment not resolved\", \"Structural basis of species-specific flagellin recognition not solved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed TLR5 signaling can be anti-osteoclastogenic via an IFN-β/STAT1 axis, demonstrating context-dependent outputs beyond classical NF-κB inflammation.\",\n      \"evidence\": \"BMDM osteoclastogenesis assays, IFN-β neutralization, STAT1-KO cells, c-Fos rescue\",\n      \"pmids\": [\"18209032\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Opposing osteoclast-promoting effect in co-culture mechanistically unexplained\", \"Connection between TLR5 and IFN-β induction not mapped\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated by genetic epistasis that TLR5 loss causes spontaneous colitis that is TLR4-dependent, defining TLR5 as a homeostatic regulator of intestinal inflammation.\",\n      \"evidence\": \"TLR5-KO and TLR4/TLR5 double-KO mice, histopathology and cytokine profiling\",\n      \"pmids\": [\"18008007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking TLR5 loss to TLR4 hyperactivation not defined here\", \"Microbiota contribution not dissected\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Separated TLR5's signaling and antigen-presentation roles and defined surface-expression and negative-regulation requirements, showing endocytic MHC-II presentation, PRAT4A-dependent surface display, and TRIF/caspase-mediated TLR5 degradation.\",\n      \"evidence\": \"TLR5-KO/MyD88-KO mice with peptide vs whole flagellin; PRAT4A siRNA and anti-TLR5 mAb; TRIF overexpression with domain mapping and protease inhibitors\",\n      \"pmids\": [\"21182074\", \"22836022\", \"20452988\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endocytic trafficking machinery for TLR5 not identified\", \"Physiological trigger for TRIF-driven TLR5 degradation unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the division of labor between TLR5 and the NLRC4 inflammasome in flagellin immunity, with TLR5 driving NF-κB cytokines and sIL-1Ra while NLRC4 produces IL-1β/IL-18.\",\n      \"evidence\": \"TLR5-KO, NLRC4-KO, double-KO mice; bone marrow chimeras; immunization and cytokine/antibody measurements\",\n      \"pmids\": [\"20844479\", \"21072873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination between the two pathways at the cellular level not fully resolved\", \"Source-specific contributions across tissues incompletely mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided the atomic mechanism of flagellin recognition, showing lateral-surface engagement of the flagellin D1 domain and assembly of a 2:2 tail-to-tail signaling complex.\",\n      \"evidence\": \"X-ray crystallography of zebrafish TLR5–FliC at 2.47 Å with structure-guided mutagenesis\",\n      \"pmids\": [\"22344444\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cytoplasmic TIR-domain dimerization upon activation not captured\", \"Human full-length receptor structure not solved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected TLR5 to systemic and mucosal adaptive immunity, showing microbiota flagellin sensing is required for vaccine antibody responses and that DC TLR5/IL-22 cooperates with NLRC4/IL-18 for antiviral epithelial protection; structural confirmation extended flagellin recognition across species.\",\n      \"evidence\": \"TLR5-KO, germ-free/antibiotic and reconstitution mice (influenza vaccine); TLR5-KO/NLRC4-KO mice with IL-22/IL-18 rescue (rotavirus); P. aeruginosa FliC crystal structure with binding assays\",\n      \"pmids\": [\"25220212\", \"25395539\", \"24434155\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How gut flagellin signal reaches systemic plasma cells not fully traced\", \"P. aeruginosa TLR5 complex modeled, not solved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended TLR5 function to bone biology, showing flagellin/TLR5 induces RANKL in osteoblasts via MyD88/NF-κB to drive osteoclastogenesis and bone loss.\",\n      \"evidence\": \"TLR5-KO mice, calvarial and osteoblast cultures, in vivo flagellin injection, NF-κB inhibition\",\n      \"pmids\": [\"26207027\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with anti-osteoclastogenic IFN-β arm not addressed\", \"Relevance to inflammatory bone disease not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Broadened TLR5 ligand repertoire to the endogenous alarmin HMGB1 and defined transcriptional control of TLR5 by Sp1/Sp3, plus a DC-intrinsic Th17-inducing role.\",\n      \"evidence\": \"HMGB1 binding/signaling assays and in vivo pain model; promoter reporter, ChIP, Sp1/Sp3 siRNA and PKC/ERK inhibitors; TLR5-KO LPDC co-culture\",\n      \"pmids\": [\"27760316\", \"27060138\", \"26907705\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of HMGB1–TLR5 interaction unknown\", \"Whether endogenous ligand sensing uses the same activation hot-spot untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established the dimerization architecture in zebrafish (heterodimeric TLR5a/TLR5b) and identified intracellular regulators (MAP1S, NME3) of TLR5 signaling amplitude.\",\n      \"evidence\": \"Heterodimer co-expression, domain swaps, UNC93B1 co-transfection; siRNA kinase screen with NME3 validation; MAP1S knockdown with autophagy/MyD88 readouts; bovine PI3K-motif mutagenesis\",\n      \"pmids\": [\"29555749\", \"29523766\", \"24466264\", \"29247203\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MAP1S/NME3 mechanisms are single-lab\", \"Generalizability of zebrafish heterodimeric mode to other species limited\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed TLR5 is activated by the non-flagellin H. pylori T4SS protein CagL through a D1-like motif, expanding pathogen recognition beyond flagellin.\",\n      \"evidence\": \"Binding/NF-κB assays, siRNA, TLR5-KO mice infection model, human biopsy IHC\",\n      \"pmids\": [\"31844047\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of CagL–TLR5 engagement not solved\", \"Whether CagL uses the canonical activation hot spot untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a cross-receptor role in which TLR5 physically partners with TLR4 to bias signaling toward MyD88, and a metabolic role in hepatic ApoA1/HDL production.\",\n      \"evidence\": \"Co-IP in primary macrophages, TLR5-KO mouse LPS/hyaluronan/ozone models, human dominant-negative allele; TLR5-KO mice with liver-specific AAV rescue and Apoa1 promoter ChIP\",\n      \"pmids\": [\"31989925\", \"32820707\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TLR4/TLR5 co-IP is single-lab without structural validation\", \"Stoichiometry of the TLR4–TLR5 association unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed TLR5 in tolerogenic and barrier-protective circuits, showing commensal-driven TLR5 induces epithelial TSLP that promotes Treg differentiation and DC/IL-22-mediated barrier protection.\",\n      \"evidence\": \"TLR5-siRNA Caco-2, Tlr5-/- and BM chimera mice, anti-TSLP/TGFβ neutralization, DC-T cell co-culture; conditional CD11c-MyD88-KO and AhR-KO mice with IL-22 readouts\",\n      \"pmids\": [\"36182776\", \"37742185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct epithelial signaling steps from TLR5 to TSLP transcription not mapped\", \"Interplay with AhR pathway mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked TLR5 to neuroinflammation, showing α-synuclein sensing by TLR5 (with TLR2) activates the NLRP3 inflammasome in microglia and impairs α-syn clearance.\",\n      \"evidence\": \"Primary microglia, antibody blocking, NLRP3 inhibitor and NLRP3-KO cells, α-syn degradation assays\",\n      \"pmids\": [\"34507948\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct α-syn–TLR5 binding not demonstrated\", \"Distinct TLR2 vs TLR5 checkpoints not molecularly defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How activated human full-length TLR5 transduces signal across the membrane—linking the 2:2 ectodomain complex, S805 phosphorylation, and TIR-domain assembly to MyD88 recruitment, and how it discriminates flagellin from endogenous/non-flagellin ligands—remains unresolved.\",\n      \"evidence\": \"No discovery in the timeline resolves the activated cytoplasmic signaling architecture\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of the activated TIR-domain signaling module\", \"Mechanism connecting ectodomain dimerization to MyD88 recruitment unknown\", \"Whether non-flagellin ligands trigger the same conformational pathway untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [0, 1, 2, 18]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 3, 4, 5]},\n      {\"term_id\": \"GO:0038024\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [16, 26, 37]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 4, 12, 22, 31]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 7, 30, 33]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MYD88\", \"TLR4\", \"PRKD1\", \"PRAT4A\", \"HMGB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}