Low-affinity IgG3 Abs to microbial membranes are important for primary immune defense against microbes, but little is known about the importance of TLRs in their production. IgG3 levels were extremely low in mice lacking radioprotective 105 (RP105), a B cell surface molecule structurally related to TLRs. RP105−/− B cells proliferated poorly in response to not only the TLR4 ligand LPS but also TLR2 ligand lipoproteins, both of which mediate the immunostimulatory activity of microbial membranes. RP105−/− mice were severely impaired in hapten-specific Ab production against LPS or lipoproteins. CD138 (syndecan-1)-positive plasma cells were detected after lipid A injection in wild-type spleen but much less in RP105−/− spleen. RP105 ligation in vivo induced plasma cell differentiation. RP105 expression was ∼3-fold higher on marginal zone B cells than on follicular and B1 cells and was down-regulated on germinal center cells. These results demonstrate that a signal via RP105 is uniquely important for regulating TLR-dependent Ab production to microbial membranes.
Natural immunoglobulin M (IgM) is reactive to autoantigens and is believed to be important for autoimmunity. Blood pentameric IgM loaded with antigens forms a large immune complex (IC) that contains various elements, including apoptosis inhibitor of macrophage (AIM). Here we demonstrate that this IgM-AIM association contributes to autoantibody production under obese conditions. In mice fed a high-fat diet, natural IgM increased through B cell TLR4 stimulation. AIM associated with IgM and protected AIM from renal excretion, increasing blood AIM levels along with the obesity-induced IgM augmentation. Meanwhile, the AIM association inhibited IgM binding to the Fcα/μ receptor on splenic follicular dendritic cells, thereby protecting the IgM IC from Fcα/μ receptor-mediated internalization. This supported IgM-dependent autoantigen presentation to B cells, stimulating IgG autoantibody production. Accordingly, in obese AIM-deficient (AIM(-/-)) mice, the increase of multiple IgG autoantibodies observed in obese wild-type mice was abrogated. Thus, the AIM-IgM association plays a critical role in the obesity-associated autoimmune process.
Toll-like receptor (TLR) 7and 8 were considered to recognize single-strand RNA (ssRNA) from viruses. Although these receptors also respond to synthetic small chemical ligands, such as CL075 and R848, it remains to be determined whether these receptors sense natural small molecules or not. In the structure of human TLR8 (huTLR8) with ssRNA, there are two ligand-binding sites: one binds a uridine and the other binds an oligoribonucleotide (ORN). This finding demonstrates that huTLR8 recognizes degradation products of ssRNA, suggesting the presence of natural small ligands. We here show that TLR7 works as the sensor for guanosine (G)/2'-deoxyguanosine (dG) in the presence of ORN where ORN strengthens TLR7 interaction with G/dG. In addition, modified nucleosides such as 7-methylguanosine, 8-hydroxyguanosine (8-OHG) and 8-hydroxydeoxyguanosine (8-OHdG) activated TLR7 with ORNs. Importantly, 8-OHdG-a well-known oxidative DNA damage marker with unknown function-induced strong cytokine production comparable to G and dG both in mouse and human immune cells. Although 8-OHdG bound TLR7/ORN with lower affinity than dG did in isothermal titration calorimetry, administered 8-OHdG was metabolically more stable than dG in the serum, indicating that 8-OHdG acts on TLR7 as an endogenous ligand in vivo To address a role of G analogs in the disease state, we also examined macrophages from Unc93b1 (D34A/D34A) mice, which suffer from TLR7-dependent systemic inflammation, and found that Unc93b1 (D34A/D34A) macrophages showed significantly enhanced response to G alone or 8-OHdG with ORN. In conclusion, our results provide evidence that G, dG, 8-OHG and 8-OHdG are novel endogenous ligands for TLR7.
Toll-like receptor 9 (TLR9) is an innate immune sensor for microbial DNA that erroneously responds to self DNA in autoimmune disease. To prevent autoimmune responses, Toll-like receptor 9 is excluded from the cell surface and silenced until the N-terminal half of the ectodomain (TLR9N) is cleaved off in the endolysosome. Truncated Toll-like receptor 9 (TLR9C) senses ingested microbial DNA, although the precise role of the truncation remains controversial. Here we show that TLR9 is expressed on the surface of splenic dendritic cells. Following the cleavage of TLR9 in the endolysosome, N-terminal half of the ectodomain remains associated with truncated TLR9, forming the complex TLR9N þ C. The TLR9-dependent cytokine production by Tlr9 À / À dendritic cells is rescued by a combination of TLR9N and TLR9C, but not by TLR9C alone. These results demonstrate that the TLR9N þ C complex is a bona fide DNA sensor.
Toll-like receptor 7 (TLR7) senses microbial-derived RNA but can also potentially respond to self-derived RNA. To prevent autoimmune responses, TLR7 is thought to localize in endolysosomes. Contrary to this view, we show here that TLR7 is present on the cell surface of immune cells and that TLR7 responses can be inhibited by an anti-TLR7 antibody. The anti-TLR7 antibody is internalized with TLR7 and accumulates in endolysosomes as an immune complex. TLR7 responses in dendritic cells, macrophages and B cells are all inhibited by the anti-TLR7 antibody. Furthermore, the anti-TLR7 antibody inhibits in vivo cytokine production induced by a TLR7 ligand. Spontaneous TLR7 activation in Unc93b1 D34A/D34A mice causes lethal inflammation. Progressive inflammation such as splenomegaly, thrombocytopenia and chronic active hepatitis are ameliorated by anti-TLR7 antibody treatment. These results demonstrate that cell surface TLR7 is a promising target for therapeutic intervention in autoimmune diseases.
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