TLR8 (Toll-like receptor 8, CD288) is an endosomal pattern recognition receptor of the innate immune system and the most highly expressed nucleic-acid-sensing Toll-like receptor of the human myeloid compartment, found in monocytes, macrophages and myeloid dendritic cells. It is a single-pass type I membrane glycoprotein whose horseshoe-shaped leucine-rich-repeat ectodomain faces the endosomal lumen and whose cytoplasmic TIR domain transmits the signal. TLR8 detects the degradation products of single-stranded RNA rather than intact RNA, using two distinct sites: uridine binds a pocket at the interface between the two protomers - the same pocket that accommodates synthetic imidazoquinolines such as R-848 - while a short oligonucleotide binds a second site on the concave LRR surface. Both sites must be occupied for activation. The ligands are generated in the same compartment by the lysosomal endoribonuclease RNase T2, which cleaves single-stranded RNA between purine and uridine residues, supplying catabolic uridine and purine-2',3'-cyclophosphate-terminated oligoribonucleotides. Natural agonists include GU-rich single-stranded RNA sequences from HIV-1, SARS-CoV-1 and SARS-CoV-2 and endogenous RNAs such as microRNAs. Activation has two structural prerequisites: the receptor exists as a pre-formed dimer whose Z-loop, the insertion between leucine-rich repeats 14 and 15, must first be cleaved by furin-like proprotein convertases and cathepsins in endosomes - the uncleaved loop sterically blocks dimerisation - after which ligand binding reorganises the dimer so that the two C-termini and hence the two TIR domains come together. The apposed TIR domains recruit MyD88 and assemble the IRAK4-IRAK1/IRAK2-TRAF6 myddosome, activating canonical NF-kappaB and IRF7 and driving IL-1beta, IL-6, IL-12, TNF and type I interferon. Endogenous TLR8 resides in the early endosome and the endoplasmic reticulum, distinguishing it from TLR7 and TLR9, which occupy endolysosomes; it associates with the chaperone UNC93B1, which is required for its trafficking and signalling. Beyond cytokine induction, TLR8 signalling in CD4-positive regulatory T cells relieves their suppressive activity. X-linked gain-of-function variants, frequently present as somatic mosaicism, cause an inborn error of immunity with refractory neutropenia, lymphoproliferation, autoinflammation, impaired B-cell maturation and in some cases bone marrow failure.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000139 Golgi membrane | TAS Reactome:R-HSA-1678927 | MARK AS OVER ANNOTATED | Summary: Reactome places endosomal TLRs in the Golgi membrane as a transit step of the UNC93B1-dependent route. Reason: Directly contradicted for TLR8. In human monocytes and in HeLa transfectants the Golgi marker p115 did not colocalise with TLR8, and the study concluded that TLR8's localisation differs from that of TLR7 and TLR9. The Reactome assertion is a generic modelling step for the whole TLR3/7/8/9 group rather than a TLR8 observation; over-annotation. Supporting Evidence: PMID:22164301 Late endosome (MPR), lysosome (LAMP-1), and Golgi (p115) markers did not colocalize with TLR8 PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:22164301 Thus, the localization site of TLR8 differed from TLR7 and TLR9, both of which reside in the endolysosome and the ER. |
| GO:0000139 Golgi membrane | TAS Reactome:R-HSA-1678998 | MARK AS OVER ANNOTATED | Summary: Reactome places endosomal TLRs in the Golgi membrane as a transit step of the UNC93B1-dependent route. Reason: Directly contradicted for TLR8. In human monocytes and in HeLa transfectants the Golgi marker p115 did not colocalise with TLR8, and the study concluded that TLR8's localisation differs from that of TLR7 and TLR9. The Reactome assertion is a generic modelling step for the whole TLR3/7/8/9 group rather than a TLR8 observation; over-annotation. Supporting Evidence: PMID:22164301 Late endosome (MPR), lysosome (LAMP-1), and Golgi (p115) markers did not colocalize with TLR8 PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:22164301 Thus, the localization site of TLR8 differed from TLR7 and TLR9, both of which reside in the endolysosome and the ER. |
| GO:0002224 toll-like receptor signaling pathway | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA placing toll-like receptor signaling pathway at the TLR7/8/9 clade node. Reason: The generic TLR signalling term is the right level for the node, and human TLR8 is inside the clade with direct evidence for ligand-induced dimer reorganisation and MyD88-IRAK4-dependent signalling. The specific GO:0034158 is also annotated three times. Supporting Evidence: PMID:23520111 Thus, ligand binding induces reorganization of the TLR8 dimer, which enables downstream signaling processes. PMID:16123302 This effect was independent of dendritic cells but required functional TLR8-MyD88-IRAK4 signaling in Treg cells. |
| GO:0003677 DNA binding | IDA PMID:16123302 Toll-like receptor 8-mediated reversal of CD4+ regulatory T ... | MARK AS OVER ANNOTATED | Summary: IDA for DNA binding from the study in which human TLR8 ligands reverse regulatory T cell suppression. Reason: DNA sensing is TLR9's function, not TLR8's: the two TLR8 ligand-binding sites resolved crystallographically bind uridine and a short RNA oligonucleotide, and RNase T2 cleavage of RNA is what supplies them. The cached record is abstract-only, so I do not assert that the curator misread the paper - the abstract does refer to 'synthetic and natural ligands', and G-rich oligodeoxynucleotides are known TLR8 stimulants - but an activation readout does not establish a distinct DNA-binding activity. Over-annotation of the receptor's nucleic-acid recognition. Supporting Evidence: PMID:16123302 We describe a mechanism linking Toll-like receptor (TLR) 8 signaling to the control of Treg cell function, in which synthetic and natural ligands for human TLR8 can reverse Treg cell function. PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. PMID:31778653 This is due to RNase T2's preferential cleavage of single-stranded RNA molecules between purine and uridine residues, which critically contributes to the supply of catabolic uridine and the generation of purine-2',3'-cyclophosphate-terminated oligoribonucleotides. |
| GO:0003723 RNA binding | TAS PMID:16123302 Toll-like receptor 8-mediated reversal of CD4+ regulatory T ... | MODIFY | Summary: Author-statement annotation of generic RNA binding. Reason: Correct but uninformative. TLR8's characterised activity is recognition of single-stranded RNA and its degradation products - uridine at the dimer interface and a short oligonucleotide on the concave LRR surface - so single-stranded RNA binding (GO:0003727) is the informative term and is already annotated by IDA. Proposed replacements: single-stranded RNA binding Supporting Evidence: PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. PMID:25599397 These results demonstrate that TLR8 is a sensor for both uridine and a short oligonucleotide derived from RNA. |
| GO:0003725 double-stranded RNA binding | IDA PMID:16111635 Suppression of RNA recognition by Toll-like receptors: the i... | MODIFY | Summary: IDA for double-stranded RNA binding from the nucleoside-modification study of TLR3/7/8 RNA sensing. Reason: The essence is right - TLR8 recognises RNA and nucleoside modification abolishes it - but the RNA species is wrong. Double-stranded RNA is the TLR3 ligand; TLR8's two sites bind uridine and a short single-stranded oligonucleotide, and its natural agonists are GU-rich single-stranded RNAs. Single-stranded RNA binding is the supported term. Proposed replacements: single-stranded RNA binding Supporting Evidence: PMID:16111635 We show that RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity. PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. PMID:14976262 By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. |
| GO:0003725 double-stranded RNA binding | IDA PMID:33718825 SARS-CoV-2, SARS-CoV-1, and HIV-1 derived ssRNA sequences ac... | MODIFY | Summary: IDA for double-stranded RNA binding from the SARS-CoV-2/SARS-CoV-1/HIV-1 GU-rich RNA study. Reason: The cited paper used GU-rich single-stranded RNA throughout, as its own abstract states, and UniProt summarises it the same way. No double-stranded RNA was tested. Single-stranded RNA binding is the correct term. Proposed replacements: single-stranded RNA binding Supporting Evidence: PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. |
| GO:0003727 single-stranded RNA binding | IDA PMID:14976262 Species-specific recognition of single-stranded RNA via toll... | ACCEPT | Summary: IDA: human TLR8 confers recognition of GU-rich single-stranded RNA oligonucleotides derived from HIV-1. Reason: Core molecular function, established by genetic complementation and species comparison. The structures later resolved the two sites that read such RNA - uridine at the dimer interface and a short oligonucleotide on the concave surface. Supporting Evidence: PMID:14976262 By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. PMID:14976262 We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. |
| GO:0003727 single-stranded RNA binding | IDA PMID:24942581 CD4 ligation on human blood monocytes triggers macrophage di... | ACCEPT | Summary: IDA from the CD4-ligation monocyte study, in which differentiated macrophages responded to the GU-rich HIV RNA ssRNA40 but not to the control ssRNA41. Reason: The cached record is abstract-only; I consulted the open full text (PMC4136363), which does assay TLR8 and shows a ssRNA40-specific, TLR8-attributed cytokine response. The readout is functional responsiveness rather than a binding measurement, but the activity itself is correct for TLR8 and is directly established elsewhere. Supporting Evidence: PMID:24942581 Altogether, our studies have identified a novel function for the CD4 molecule on peripheral monocytes and suggest that a unique set of events that lead to innate immune activation differ between humans and mice. PMID:14976262 By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. |
| GO:0005515 protein binding | IPI PMID:22164301 UNC93B1 physically associates with human TLR8 and regulates ... | REMOVE | Summary: IPI recording the TLR8-UNC93B1 interaction. Reason: Generic protein binding is uninformative. TLR8 is the client of UNC93B1 and the relevant biology - UNC93B1-dependent trafficking and signalling competence - is a localisation matter rather than a molecular function of TLR8. Removal does not dispute the interaction, which this paper establishes directly. Supporting Evidence: PMID:22164301 UNC93B1 physically associated with human TLR8, similar to TLRs 3, 7, and 9, and played a critical role in TLR8-mediated signaling. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | REMOVE | Summary: IPI from a proteome-scale affinity-purification interactome screen (BioPlex). Reason: Generic protein binding is uninformative, and a high-throughput interactome screen assigns no function. Removal does not dispute the interaction. Supporting Evidence: PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-1678921 | ACCEPT | Summary: Reactome reactions placing full-length TLR8 in the ER membrane during chaperone-assisted folding and UNC93B1 binding. Reason: Correct and independently confirmed: endogenous TLR8 colocalises with the ER marker calreticulin in human monocytes and HeLa cells, and UNC93B1 associates with it there. Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:22164301 UNC93B1 physically associated with human TLR8, similar to TLRs 3, 7, and 9, and played a critical role in TLR8-mediated signaling. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-1678944 | ACCEPT | Summary: Reactome reactions placing full-length TLR8 in the ER membrane during chaperone-assisted folding and UNC93B1 binding. Reason: Correct and independently confirmed: endogenous TLR8 colocalises with the ER marker calreticulin in human monocytes and HeLa cells, and UNC93B1 associates with it there. Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:22164301 UNC93B1 physically associated with human TLR8, similar to TLRs 3, 7, and 9, and played a critical role in TLR8-mediated signaling. |
| GO:0005789 endoplasmic reticulum membrane | TAS Reactome:R-HSA-1678998 | ACCEPT | Summary: Reactome reactions placing full-length TLR8 in the ER membrane during chaperone-assisted folding and UNC93B1 binding. Reason: Correct and independently confirmed: endogenous TLR8 colocalises with the ER marker calreticulin in human monocytes and HeLa cells, and UNC93B1 associates with it there. Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:22164301 UNC93B1 physically associated with human TLR8, similar to TLRs 3, 7, and 9, and played a critical role in TLR8-mediated signaling. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT IBA asserting that TLR8 is active in the plasma membrane, at the TLR7/8/9 clade node. Reason: Not where TLR8 acts. Endogenous TLR8 is found in the early endosome and the ER, and endosomal residence is a prerequisite rather than a detail: the Z-loop must be cleaved by endosomal furin-like convertases and cathepsins before the receptor can form the activation dimer at all. Retained as non-core because the node-level assertion covers three receptors, and the question of retiring it at the node is raised separately. Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:26929371 TLR8 with the uncleaved Z-loop is unable to form a dimer, which is essential for activation, irrespective of the presence of agonistic ligands. PMID:25297876 Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. |
| GO:0006952 defense response | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA assignment of defense response. Reason: True but very general; TLR8 initiates innate defence against RNA-bearing microbes. The specific pathway and antiviral terms carry the information. Supporting Evidence: PMID:31778653 TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment |
| GO:0006955 immune response | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA assignment of immune response. Reason: True but very general. The specific terms are preferred. Supporting Evidence: PMID:31778653 TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment |
| GO:0007165 signal transduction | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro TIR-domain mapping to signal transduction. Reason: Correct but uninformative: the TIR domain recruits MyD88. The TLR8 signalling pathway term is preferred. Supporting Evidence: PMID:16123302 This effect was independent of dendritic cells but required functional TLR8-MyD88-IRAK4 signaling in Treg cells. |
| GO:0007249 canonical NF-kappaB signal transduction | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for canonical NF-kappaB signal transduction at the TLR7/8/9 node. Reason: Correct; TLR8 expression alone confers R-848-induced NF-kappaB activation, and TLR8-specific antagonists suppress TLR8-driven proinflammatory signalling in human primary cells. Supporting Evidence: PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. PMID:29155428 we have demonstrated that these drug-like inhibitors are able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens |
| GO:0009615 response to virus | IDA PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to th... | MODIFY | Summary: IDA for response to virus, from the demonstration that TLR8 alone confers responsiveness to the antiviral imidazoquinoline R-848. Reason: The essence - TLR8 participates in the cellular response to viruses - is sound, but the term is a general parent and the experiment used a synthetic small molecule rather than a virus. TLR8's characterised role is defensive recognition of viral single-stranded RNA (GU-rich sequences from HIV-1, SARS-CoV-1 and SARS-CoV-2), which is the more specific child GO:0051607 that TLR8 already carries from four other lines of evidence. Proposed replacements: defense response to virus Supporting Evidence: PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis PMID:14976262 By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. |
| GO:0009897 external side of plasma membrane | IDA PMID:24942581 CD4 ligation on human blood monocytes triggers macrophage di... | REMOVE | Summary: IDA placing TLR8 on the external side of the plasma membrane, from the CD4-ligation monocyte differentiation study. Reason: Contradicted by the cited paper itself. The cached record is abstract-only, so I retrieved the open full text (PMC4136363): TLR8 is assayed there, but by intracellular staining after fixation and permeabilisation, and the discussion describes 'the increase in expression of pattern recognition receptors such as intracellular TLR8'. Nothing in the study places TLR8 on the outer face of the plasma membrane. Endogenous TLR8 is in the early endosome and the ER, and it cannot signal before endosomal proteases cleave its Z-loop. The paper's other two TLR8 annotations are accepted; this location row is removed. Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:26929371 TLR8 with the uncleaved Z-loop is unable to form a dimer, which is essential for activation, irrespective of the presence of agonistic ligands. PMID:25297876 Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. |
| GO:0010008 endosome membrane | EXP PMID:25297876 Endosomal localization of TLR8 confers distinctive proteolyt... | ACCEPT | Summary: Experimental localisation of TLR8 to the endosome membrane, in the study of its distinctive proteolytic processing in human myeloid cells. Reason: Core location. TLR8 is cleaved by furin-like convertases and cathepsins in early/late endosomes, and endosomal localisation is what confers that processing pattern. Supporting Evidence: PMID:25297876 Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. PMID:25297876 In this study, we show that proteolytic processing of TLR8 occurs in human monocytes and macrophages in a different manner compared with TLR7/9 cleavage. PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants |
| GO:0010008 endosome membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping to endosome membrane. Reason: Correct and matches the direct evidence: endogenous TLR8 is an endosome-membrane protein. Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants |
| GO:0010008 endosome membrane | TAS Reactome:R-HSA-9691204 | ACCEPT | Summary: Reactome reactions placing the TLR8 dimer in the endosome membrane, binding TLR7/TLR8 ligands and SARS-CoV-2 GU-rich ssRNA. Reason: Core location and the right compartment for TLR8, consistent with the early-endosome localisation of the endogenous receptor and with the GU-rich ssRNA agonists. Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis |
| GO:0010008 endosome membrane | TAS Reactome:R-HSA-9754830 | ACCEPT | Summary: Reactome reactions placing the TLR8 dimer in the endosome membrane, binding TLR7/TLR8 ligands and SARS-CoV-2 GU-rich ssRNA. Reason: Core location and the right compartment for TLR8, consistent with the early-endosome localisation of the endogenous receptor and with the GU-rich ssRNA agonists. Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis |
| GO:0016064 immunoglobulin mediated immune response | TAS PMID:16123302 Toll-like receptor 8-mediated reversal of CD4+ regulatory T ... | REMOVE | Summary: Author-statement annotation of immunoglobulin mediated immune response. Reason: The cited paper is about TLR8 signalling reversing CD4+ regulatory T cell suppression; neither immunoglobulin production nor antibody-mediated immunity is part of it, and TAS is not experimental evidence to defer to. No other evidence reviewed here supports TLR8 executing a step of an immunoglobulin-mediated response. Its effects on B cells in gain-of-function disease are indirect, appearing as impaired B-cell maturation in a broadly proinflammatory state. Supporting Evidence: PMID:16123302 We describe a mechanism linking Toll-like receptor (TLR) 8 signaling to the control of Treg cell function, in which synthetic and natural ligands for human TLR8 can reverse Treg cell function. PMID:33512449 All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. |
| GO:0032727 positive regulation of interferon-alpha production | IDA PMID:16286015 Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/be... | ACCEPT | Summary: IDA: IFN-alpha induction by TLR8 agonists in human blood cells, abolished in IRAK-4 deficiency. Reason: A genuine TLR8 output; GU-rich ssRNA from HIV-1 also drives interferon-alpha secretion from dendritic cells and macrophages, and IRAK-4 dependence places the response downstream of the myddosome. Supporting Evidence: PMID:16286015 We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. PMID:14976262 We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. |
| GO:0032727 positive regulation of interferon-alpha production | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA assignment of positive regulation of interferon-alpha production. Reason: Consistent with the direct human evidence. Supporting Evidence: PMID:16286015 We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. |
| GO:0032728 positive regulation of interferon-beta production | IDA PMID:16286015 Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/be... | ACCEPT | Summary: IDA: IFN-beta induction by TLR8 agonists in human blood cells, abolished in IRAK-4 deficiency. Reason: Type I interferon induction downstream of TLR8 is established by this study, with IRAK-4 dependence tying it to the receptor's own pathway. Supporting Evidence: PMID:16286015 We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. PMID:16286015 The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent |
| GO:0032728 positive regulation of interferon-beta production | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA assignment of positive regulation of interferon-beta production. Reason: Consistent with the direct human evidence. Supporting Evidence: PMID:16286015 We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. |
| GO:0032729 positive regulation of type II interferon production | IDA PMID:16286015 Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/be... | MODIFY | Summary: IDA annotated as positive regulation of type II interferon production, from the IRAK-4 deficiency study. Reason: The cited paper measured IFN-alpha/beta (type I) and IFN-lambda (type III) after TLR-7/8/9 stimulation, not IFN-gamma - its title says so. The finding supports positive regulation of type III interferon production (GO:0034346). Same decision as in the TLR9 review and in the TLR7 review of this batch. Proposed replacements: positive regulation of type III interferon production Supporting Evidence: PMID:16286015 We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. PMID:16286015 The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent |
| GO:0032729 positive regulation of type II interferon production | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA assignment of positive regulation of type II interferon production. Reason: No TLR8-specific IFN-gamma evidence appears in the literature reviewed here. Unlike TLR7, where loss-of-function patients show an IFN-gamma defect, TLR8 stimulation is characterised by IL-1beta, IL-6, IL-12, TNF and type I interferon; IFN-gamma after TLR8 ligation would arise indirectly from NK and T cells responding to those cytokines. Over-annotation, consistent with the TLR9 review. Supporting Evidence: PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis PMID:14976262 We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. |
| GO:0032731 positive regulation of interleukin-1 beta production | IDA PMID:33718825 SARS-CoV-2, SARS-CoV-1, and HIV-1 derived ssRNA sequences ac... | ACCEPT | Summary: IDA: GU-rich viral ssRNA drives TLR8-dependent mature IL-1beta production in human macrophages. Reason: A direct and mechanistically dissected output of TLR8 signalling - caspase-1/caspase-8 and NLRP3 dependent, without pyroptosis - and the ligand specificity is TLR8's own. Supporting Evidence: PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis PMID:33718825 we show that the induction of mature IL-1ฮฒ is through a non-classical pathway dependent on caspase-1, caspase-8, the NLRP3 inflammasome, potassium efflux, and autophagy |
| GO:0032755 positive regulation of interleukin-6 production | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for positive regulation of interleukin-6 production at the TLR7/8/9 node. Reason: Correct; TLR8 ligation of human macrophages and monocytes induces IL-6 among the proinflammatory cytokines, and TLR8 gain-of-function patients have elevated serum cytokines. Supporting Evidence: PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis PMID:33512449 All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. |
| GO:0032757 positive regulation of interleukin-8 production | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA assignment of positive regulation of interleukin-8 production. Reason: Consistent with the direct evidence that TLR8 expression raises virus-induced IL-8. Supporting Evidence: PMID:16188996 ectopic expression of TLR7 and TLR8 (but not TLR2, -3, -4, or -9) in HEK293 cells elevated SeV-induced activation of NF-ฮบB and expression of IL-8 and IP-10 |
| GO:0032757 positive regulation of interleukin-8 production | IMP PMID:16188996 Activation of innate defense against a paramyxovirus is medi... | ACCEPT | Summary: IMP: ectopic TLR8 expression raised Sendai-virus-induced IL-8 in HEK293 cells. Reason: Gain-of-function in a TLR-null background, with TLR2, -3, -4 and -9 as negative controls, makes this a clean attribution to TLR8. Supporting Evidence: PMID:16188996 ectopic expression of TLR7 and TLR8 (but not TLR2, -3, -4, or -9) in HEK293 cells elevated SeV-induced activation of NF-ฮบB and expression of IL-8 and IP-10 PMID:16188996 myeloid cells utilized both the single-stranded RNA sensing TLR7 and TLR8 and dsRNA-dependent mechanisms independent of RIG-I, TLR3, and dsRNA-activated protein kinase R to trigger this response |
| GO:0034158 toll-like receptor 8 signaling pathway | IDA PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to th... | ACCEPT | Summary: IDA: human TLR8 alone confers responsiveness to R-848. Reason: Core process and the correctly specific term. R-848 occupies the first (small-ligand) site at the TLR8 dimer interface, the same pocket that binds uridine. Supporting Evidence: PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. |
| GO:0034158 toll-like receptor 8 signaling pathway | IDA PMID:24942581 CD4 ligation on human blood monocytes triggers macrophage di... | ACCEPT | Summary: IDA: TLR8-upregulated macrophages responded to the GU-rich HIV RNA ssRNA40, but not to control ssRNA41, with IL-1beta and IL-6. Reason: Ligand-specific, TLR8-attributed signalling in primary human cells. The cached record is abstract-only; the open full text (PMC4136363) contains the ssRNA40/ssRNA41 experiment. Supporting Evidence: PMID:24942581 Altogether, our studies have identified a novel function for the CD4 molecule on peripheral monocytes and suggest that a unique set of events that lead to innate immune activation differ between humans and mice. PMID:14976262 By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. |
| GO:0034158 toll-like receptor 8 signaling pathway | IDA PMID:33718825 SARS-CoV-2, SARS-CoV-1, and HIV-1 derived ssRNA sequences ac... | ACCEPT | Summary: IDA: GU-rich ssRNA from SARS-CoV-2, SARS-CoV-1 and HIV-1 triggers a TLR8-dependent cytokine response in human macrophages. Reason: Core process, with genetic and pharmacological confirmation that the response runs through TLR8. Supporting Evidence: PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis PMID:33718825 we show that the induction of mature IL-1ฮฒ is through a non-classical pathway dependent on caspase-1, caspase-8, the NLRP3 inflammasome, potassium efflux, and autophagy |
| GO:0036020 endolysosome membrane | IEA GO_REF:0000117 | MODIFY | Summary: ARBA assignment of endolysosome membrane. Reason: The compartment is one step too far along the endocytic route for TLR8. Endogenous TLR8 in human monocytes and in HeLa transfectants colocalised with the early-endosome marker EEA1 and with an ER marker, but not with late-endosome or lysosome markers, and the authors drew the contrast with TLR7 and TLR9 explicitly. Early endosome membrane (GO:0031901) is the supported location; endosome membrane (GO:0010008), which TLR8 also carries, is correct as a parent. Proposed replacements: early endosome membrane Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:22164301 Thus, the localization site of TLR8 differed from TLR7 and TLR9, both of which reside in the endolysosome and the ER. PMID:22164301 Late endosome (MPR), lysosome (LAMP-1), and Golgi (p115) markers did not colocalize with TLR8 |
| GO:0036020 endolysosome membrane | TAS Reactome:R-HSA-1678927 | MODIFY | Summary: Reactome reaction placing TLR8 in the endolysosome membrane after UNC93B1 delivery. Reason: Same objection as the electronic row: the Reactome step is generic to TLR3/7/8/9, whereas endogenous TLR8 was found in the early endosome and not in the late endosome or lysosome. Early endosome membrane is the supported location. Proposed replacements: early endosome membrane Supporting Evidence: PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants PMID:22164301 Thus, the localization site of TLR8 differed from TLR7 and TLR9, both of which reside in the endolysosome and the ER. |
| GO:0038023 signaling receptor activity | NAS PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to th... | MODIFY | Summary: Author-statement annotation of generic signaling receptor activity. Reason: Correct but uninformative. TLR8 is specifically a pattern recognition receptor: it binds RNA degradation products at two defined sites and transduces the event through ligand-induced reorganisation of a pre-formed dimer. Pattern recognition receptor activity (GO:0038187) is the informative term and is already annotated by IBA; there is no TLR-specific MF term. Proposed replacements: pattern recognition receptor activity Supporting Evidence: PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. PMID:23520111 Thus, ligand binding induces reorganization of the TLR8 dimer, which enables downstream signaling processes. |
| GO:0038187 pattern recognition receptor activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for pattern recognition receptor activity at the TLR7/8/9 node. Reason: Core molecular function. TLR8 binds uridine and short RNA oligonucleotides directly at two crystallographically defined sites and signals through its TIR domain; human TLR8 is itself among the descendant evidences for the node, which marks experimental grounding on the target rather than circularity. There is no TLR-specific MF term, so this is the appropriate parent. Supporting Evidence: PMID:25599397 TLR8 recognized two degradation products of ssRNAโuridine and a short oligonucleotideโat two distinct sites: uridine bound the site on the dimerization interface where small chemical ligands are recognized, whereas short oligonucleotides bound a newly identified site on the concave surface of the TLR8 horseshoe structure. PMID:25599397 These results demonstrate that TLR8 is a sensor for both uridine and a short oligonucleotide derived from RNA. PMID:23520111 Thus, ligand binding induces reorganization of the TLR8 dimer, which enables downstream signaling processes. file:human/TLR8/TLR8-deep-research-falcon.md TLR8 serves as an endosomal pattern recognition receptor whose primary function is to detect viral and bacterial RNA degradation products and initiate downstream innate immune responses |
| GO:0043123 positive regulation of canonical NF-kappaB signal transduction | IDA PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to th... | ACCEPT | Summary: IDA: TLR8 expression confers R-848-induced NF-kappaB activation. Reason: Direct, receptor-attributable activation of canonical NF-kappaB. Supporting Evidence: PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. |
| GO:0043123 positive regulation of canonical NF-kappaB signal transduction | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA assignment of positive regulation of canonical NF-kappaB signal transduction. Reason: Consistent with the direct evidence and with the suppression of TLR8-driven proinflammatory signalling by TLR8-specific antagonists. Supporting Evidence: PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. PMID:29155428 we have demonstrated that these drug-like inhibitors are able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens |
| GO:0045087 innate immune response | NAS PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to th... | ACCEPT | Summary: Author-statement annotation of innate immune response. Reason: Correct though general: TLR8 is one of the most highly expressed innate sensors of human myeloid cells. The specific pathway and antiviral terms carry the detail. Supporting Evidence: PMID:31778653 TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment PMID:12032557 Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. |
| GO:0045089 positive regulation of innate immune response | IDA PMID:16123302 Toll-like receptor 8-mediated reversal of CD4+ regulatory T ... | ACCEPT | Summary: IDA: TLR8 ligands acting through TLR8-MyD88-IRAK4 in regulatory T cells relieve their suppression of immune responses. Reason: A positive-regulatory effect on immunity attributable to TLR8's own signalling pathway, shown to be dendritic-cell independent and to require the receptor's adaptors; transferred cells enhanced anti-tumour immunity in vivo. Supporting Evidence: PMID:16123302 We describe a mechanism linking Toll-like receptor (TLR) 8 signaling to the control of Treg cell function, in which synthetic and natural ligands for human TLR8 can reverse Treg cell function. PMID:16123302 This effect was independent of dendritic cells but required functional TLR8-MyD88-IRAK4 signaling in Treg cells. PMID:16123302 Adoptive transfer of TLR8 ligand-stimulated Treg cells into tumor-bearing mice enhanced anti-tumor immunity. |
| GO:0051607 defense response to virus | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for defense response to virus at the TLR7/8/9 node. Reason: Core process. TLR8 senses GU-rich single-stranded RNA from HIV-1, SARS-CoV-1 and SARS-CoV-2 and drives the antiviral cytokine response of human myeloid cells. Supporting Evidence: PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis PMID:14976262 By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. |
| GO:0051607 defense response to virus | IDA PMID:33718825 SARS-CoV-2, SARS-CoV-1, and HIV-1 derived ssRNA sequences ac... | ACCEPT | Summary: IDA: viral GU-rich ssRNA sequences trigger a TLR8-dependent proinflammatory response in human macrophages. Reason: Direct antiviral sensing function with genetic and pharmacological attribution to TLR8. Supporting Evidence: PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis PMID:33718825 we show that the induction of mature IL-1ฮฒ is through a non-classical pathway dependent on caspase-1, caspase-8, the NLRP3 inflammasome, potassium efflux, and autophagy |
| GO:0051607 defense response to virus | IEP PMID:32634389 Chikungunya virus infection induces differential inflammator... | ACCEPT | Summary: IEP: TLR transcript levels change in monocytes and macrophages on chikungunya virus infection. Reason: The IEP evidence is weak - an expression correlation, with the abstract not singling out TLR8 - but the term is correct for TLR8 on strong independent grounds. Accepted on that basis, consistent with the TLR3 and TLR7 reviews. Supporting Evidence: PMID:32634389 our data provides evidence that CHIKV infection activates the TLR pathways in primary monocytes and MDMs PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis |
| GO:0051607 defense response to virus | IMP PMID:16188996 Activation of innate defense against a paramyxovirus is medi... | ACCEPT | Summary: IMP: TLR8 mediates part of the myeloid-cell antiviral response to Sendai virus. Reason: Direct antiviral function attributable to TLR8 in a gain-of-function, controlled setting. Supporting Evidence: PMID:16188996 myeloid cells utilized both the single-stranded RNA sensing TLR7 and TLR8 and dsRNA-dependent mechanisms independent of RIG-I, TLR3, and dsRNA-activated protein kinase R to trigger this response PMID:16188996 ectopic expression of TLR7 and TLR8 (but not TLR2, -3, -4, or -9) in HEK293 cells elevated SeV-induced activation of NF-ฮบB and expression of IL-8 and IP-10 |
| GO:0051707 response to other organism | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA assignment of response to other organism. Reason: True but very general; the specific antiviral and pathway terms carry the content. Supporting Evidence: PMID:33718825 Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis |
| GO:0071260 cellular response to mechanical stimulus | IEP PMID:19593445 Expression of the Bcl-2 protein BAD promotes prostate cancer... | UNDECIDED | Summary: IEP for cellular response to mechanical stimulus, citing a paper on BAD and prostate cancer growth. Reason: The cached record for PMID:19593445 carries full text and contains no occurrence of 'TLR', 'toll' or any mechanical stimulation; it is 'Expression of the Bcl-2 protein BAD promotes prostate cancer growth'. The identifier is almost certainly wrong, but the intended reference cannot be recovered and no other evidence reviewed here links TLR8 to mechanical stimuli. Left undecided pending a corrected reference; the identical row exists on TLR7 and TLR3, where it was also left undecided. The reference is flagged in references[].reference_review. Supporting Evidence: PMID:19593445 Expression of the Bcl-2 protein BAD promotes prostate cancer growth. |
| GO:0140894 endolysosomal toll-like receptor signaling pathway | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA assignment of endolysosomal toll-like receptor signaling pathway. Reason: Correct at the level GO uses it: GO:0034158, which TLR8 carries three times by IDA, is a descendant of this term. The 'endolysosomal' label is a family-level designation for the intracellular nucleic-acid-sensing TLRs rather than a precise compartment claim, and for TLR8 specifically the receptor is processed in early/late endosomes and localises to the early endosome. Supporting Evidence: PMID:25297876 Both furin-like proprotein convertase and cathepsins contribute to TLR8 cleavage in the early/late endosomes. PMID:22164301 we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants |
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Download this section (compressed HTML)Q: Endogenous TLR8 colocalises with EEA1 and an ER marker but not with late-endosome, lysosome or Golgi markers (PMID:22164301). Should the endolysosome membrane (GO:0036020) and Golgi membrane rows be replaced by early endosome membrane (GO:0031901), and should the Reactome TLR3/7/8/9 trafficking model be split so that TLR8 does not inherit the endolysosomal steps?
Q: GO:0009897 external side of plasma membrane (IDA, PMID:24942581) rests on a paper that measured TLR8 by intracellular staining and calls it intracellular TLR8. Should this row be retired?
Q: The two structurally defined TLR8 sites bind uridine and a short oligonucleotide (PMID:25599397, PMID:31778653). GO has no term for uridine binding, and GO:0003727 single-stranded RNA binding does not describe site 1. Should a uridine binding term be created under GO:0001884 pyrimidine nucleoside binding, in parallel with GO:1905108 guanosine binding, which is what TLR7's equivalent site needs?
Q: GO:0003677 DNA binding (IDA, PMID:16123302) sits alongside TLR8's RNA-binding terms. Do the G-rich oligodeoxynucleotide ligands used in that study bind TLR8 directly, or do they act through another route? A direct binding measurement would settle whether the row should stand.
Q: GO:0016064 immunoglobulin mediated immune response (TAS) on TLR8 cites the regulatory T cell paper, which does not address antibody responses. Is there a source for it, given that the B-cell phenotype of TLR8 gain-of-function disease is impaired maturation rather than a positive contribution?
Q: GO:0071260 cellular response to mechanical stimulus (IEP) on TLR8, TLR7 and TLR3 all cite PMID:19593445, a paper on BAD and prostate cancer growth that never mentions TLRs. What was the intended reference?
Q: The TLR7/8/9 PAINT node carries is_active_in GO:0005886 plasma membrane, yet TLR8 cannot dimerise until endosomal proteases cleave its Z-loop. Should that assertion be retired or demoted at the node?
Q: Z-loop cleavage by furin-like convertases and cathepsins is a prerequisite for TLR8 activation. Should TLR8 carry a term for protein maturation by peptidase-dependent cleavage as a target of that process, or is this better modelled on the proteases with TLR8 as has input?
Experiment: Quantitative colocalisation of endogenous TLR8 with EEA1, Rab7, LAMP1, p115 and calreticulin in primary human monocytes and monocyte-derived macrophages before and after ssRNA40 stimulation, to establish whether the signalling-competent, Z-loop-cleaved receptor stays in early endosomes or moves onward.
Experiment: Direct binding assays (ITC or SPR) of the cleaved TLR8 ectodomain against uridine, cytidine, guanosine and the G-rich oligodeoxynucleotides used in PMID:16123302, to test whether TLR8 binds DNA at either site and to calibrate site-1 nucleoside specificity against TLR7's.
Experiment: Side-by-side stimulation of TLR7- and TLR8-expressing reporter cells with RNase T2 and PLD3/PLD4 digestion products of the same RNA, to test directly whether the guanosine/2',3'-cGMP versus uridine split between the two receptors' first sites holds for physiologically generated ligands.
Experiment: Cell-surface versus intracellular flow cytometry of TLR8 in primary human monocytes with and without permeabilisation, using a validated antibody and TLR8-knockout cells as a control, to determine whether any surface pool exists at all.
Experiment: Measurement of IFN-gamma and IFN-lambda, separately and with the producing cell identified, in human PBMC and purified monocyte cultures stimulated with a TLR8-selective agonist, to test whether the type II and type III interferon annotations belong to TLR8 and in which cells.
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