MABF2273-25UG Sigma-AldrichAnti-TLR7 Antibody, clone A94B10

TLR7 recognizes pathogen-derived and self-derived RNA, and thus a regulatory system for control of the TLR7 response is required to avoid excessive activation. Unc93 homolog B1 (Unc93B1) is a regulator of TLR7 that controls the TLR7 response by transporting TLR7 from the endoplasmic reticulum to endolysosomes. We have previously shown that a D34A mutation in Unc93B1 induces hyperactivation of TLR7, and that Unc93b1(D34A/D34A) mice (D34A mice) have systemic inflammation spontaneously. In this study, we examined the roles of inflammatory cytokines such as IFN-γ, IL-17A, and type I IFNs to understand the mechanism underlying the phenotype in D34A mice. mRNAs for IFN-γ and IL-I7A in CD4(+) T cells increased, but inflammatory phenotype manifesting as thrombocytopenia and splenomegaly was still observed in Ifng(-/-) or Il17a(-/-) D34A mice. In contrast to T cell-derived cytokines, Ifnar1(-/-) D34A mice showed an ameliorated phenotype with lower expression of TLR7 in B cells and conventional dendritic cells (cDCs). The amount of TLR7 decreased in B cells from Ifnar1(-/-) D34A mice, but the percentage of TLR7(+) cells decreased among CD8α(-) cDCs. In conclusion, type I IFNs maintain expression of TLR7 in B cells and cDCs in different ways; total amount of TLR7 is kept in B cells and TLR7(+) population is retained among cDCs. Our results suggested that these TLR7-expressing cells are activated initially and influence TLR7-dependent systemic inflammation.

Toll-like receptor 7 (TLR7) an innate immune sensor for microbial RNA, erroneously responds to self-derived RNA. To avoid autoimmune responses, TLR7 is suggested to be silenced until the N-terminal half of the TLR7 ectodomain (TLR7N) is cleaved off. Resultant truncated TLR7 (TLR7C) is thought to signal microbial RNA. We here show that TLR7N remains associated with TLR7C through a disulfide bond. By N-terminal amino acid sequencing, TLR7C was found to start at 461E or 462A. The newly established monoclonal anti-TLR7N showed that endogenous TLR7 in bone marrow-derived dendritic cells was almost all cleaved and cleaved TLR7N remained in endolysosomes. TLR7N in endolysosomes was linked with TLR7C by a disulfide bond. In contrast, TLR9 did not have a disulfide bond between TLR9N and TLR9C fragments. Among the cysteines unique to the ectodomain of TLR7 but not TLR9 (Cys98, Cys445, Cys475 and Cys722), Cys98 in TLR7N and Cys475 in TLR7C were required for an intramolecular disulfide bond. These cysteines were also needed for proteolytic cleavage of and RNA sensing by TLR7, but not for TLR7 trafficking from endoplasmic reticulum to endosomes. No response was seen in TLR7 mutants lacking the proteolytic cleavage site or TLR7C alone. These results demonstrate requirement for proteolytic cleavage and TLR7N in TLR7 responses and indicate RNA sensing by TLR7N + TLR7C.