TLR3基因敲除HEK293细胞

Pattern recognition receptor (PRR)-induced interferon (IFN) is critical for effective immunity. The PRRs Toll-like receptor (TLR) 3, TLR4 and cyclic GMP-AMP synthase (cGAS), together with the stimulator of IFN genes (STING), signal through TANK-binding kinase 1 (TBK1), which activates the type-I/III IFN-inducing transcription factor interferon-response factor 3 (IRF3). The mechanism by which these PRRs activate TBK1 remains unresolved. Here we show that lysine-11 (K11)-linked ubiquitination drives TBK1 activation by these PRRs. The E3 ligase ANKIB1 attaches K11-linked ubiquitin chains to components of the TLR3- and cGAS-STING-induced signalosomes. This facilitates Optineurin recruitment to these complexes, in turn enabling recruitment and activation of TBK1 and IRF3, defining an uncharacterized signalling axis. In mice, ANKIB1 deficiency dampens IFN induction via TLR3 and cGAS-STING, reducing interferonopathy and compromising protection against HSV-1, respectively. Together, our results demonstrate an unanticipated and critical role for ANKIB1-generated K11-linked ubiquitination in the immune response activated by cGAS-STING, TLR3 and TLR4.

Toll-like receptor 3 (TLR3), an innate immune sensor for double-stranded RNA (dsRNA), traffics from the endoplasmic reticulum (ER) after synthesis to endolysosomes for proteolytic cleavage and activation. However, the molecular mechanisms governing TLR3 trafficking remain largely unclear. Here, we identify the ER-resident E3 ligase HMG-CoA reductase degradation protein 1 (HRD1), a core component of ER-associated degradation (ERAD), as a key regulator that promotes TLR3 trafficking and downstream signaling. HRD1 deficiency in macrophages significantly impairs poly(I:C)-induced TLR3 signaling and inflammatory responses in vitro and in vivo, caused by a marked reduction in TLR3 transport into endolysosomes and subsequent proteolytic processing. Mechanistically, HRD1 mediates ubiquitination of ER-localized TLR3 at lysine 813, which is required for its recognition and sorting by the endosomal sorting complex required for transport (ESCRT) machinery. This HRD1 function is decoupled from its canonical ERAD activity and the ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α). Hence, our study identifies a previously unrecognized mechanism controlling TLR3 signaling and links HRD1-mediated ubiquitination to immune sensor trafficking during innate immune responses.

BACKGROUND:Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. METHODS AND RESULTS:Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (<0.01 and <0.05), but not in TLR3 mice, by reinforcing neutrophil recruitment (<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (<0.05). Finally, eRNA triggered plasma clot formation in vitro (<0.01). CONCLUSIONS:We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.