Background: UNC93B1 sorts nucleic acid-sensing TLRs to endosomes. Results: Stimulation of HUVEC by TLR3, but not other TLR agonists, up-regulates the endogenous UNC93B1, which translocates TLR3 to plasma membrane. Poly(I:C) strongly potentiates activation of TLR9 via UNC93B1. Conclusion: Up-regulation of UNC93B1 through TLR3 activation forms a positive feedback loop that primes cells for the augmented response against infection. Significance: This work presents unique TLR3-UNC93B1 interrelationship.
Oxidative stress produced in response to infection or sterile injury activates the innate immune response. We found that extracellular vesicles (EVs) isolated from the plasma of patients with rheumatoid arthritis or secreted from cells subjected to oxidative stress contained oxidized phospholipids that stimulated cells expressing Toll-like receptor 4 (TLR4) in a manner dependent on its co-receptor MD-2. EVs from healthy subjects or reconstituted synthetic EVs subjected to limited oxidation gained the ability to stimulate TLR4-expressing cells, whereas prolonged oxidation abrogated this property. Furthermore, we found that 15-lipoxygenase generated hydro(pero)xylated phospholipids that stimulated TLR4-expressing cells. Molecular modeling suggested that the mechanism of activation of TLR4 by oxidized phospholipids in EVs was structurally similar to that of the TLR4 ligand lipopolysaccharide (LPS). This was supported by experiments showing that EV-mediated stimulation of cells required MD-2, that mutations that block LPS binding to TLR4 abrogated the stimulatory effect of EVs, and that EVs induced TLR4 dimerization. On the other hand, analysis of gene expression profiles showed that genes encoding factors that resolve inflammation were more abundantly expressed in responses to EVs than in response to LPS. Together, these data suggest that EVs act as an oxidative stress-induced endogenous danger signal that underlies the pervasive role of TLR4 in inflammatory diseases.
We show that substrate specificity of Toll-like receptor 3 (TLR3) is due to the presence of two binding sites in the ectodomain, separated by 50 A. This corresponds to two turns of a double-stranded RNA duplex, allowing differentiation between nucleic acids in the A- or B-type conformation. We propose that there are different arrangements of TLR3 ectodomains along the double-stranded RNA that could modulate the strength of the interferon response.
Toll-like receptor 3 (TLR3) is a dsRNA sensing receptor that is localized in the cellular compartments but also at the plasma membrane. Overexpression of UNC93B1 promoted localization of TLR3, but not other nucleic acid sensing TLRs, to the plasma membrane. Here we show that UNC93B1 itself is localized at the plasma membrane. We investigated the role of different domains of TLR3 on cell signaling by preparing chimeric receptors between TLR3 and TLR9 where each of the transmembrane segments or cytosolic domains has been exchanged. While the ectodomain completely governs ligand specificity and the cytosolic TIR domain determines the engagement of the signaling adapters as well as the potentiation of receptor activation by UNC93B1, the ectodomain but not transmembrane segment or cytosolic domain determines plasma membrane localization of TLR3. Nevertheless, TLR3 receptor and ligand endocytosis as well as endosomal acidification are important for the robust signaling of TLR3.
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