402 Genetic dissection of TLR9 reveals complex regulatory and cryptic pro-inflammatory roles

BackgroundToll like receptors (TLR) 7 and 9, endosomal sensors for ssRNA and dsDNA, are key mediators of lupus autoreactivity. Although considered homologous, they have opposing effects on lupus severity: TLR7 exacerbates disease while TLR9 protects. One theory is that TLR9 induces protection by competing with TLR7 for endosomal trafficking, thus restraining TLR7 expression and signaling. However, since both TLR9 and TLR7 ligands are available in lupus, and both TLRs are supposed to signal via MyD88, this theory doesn’t explain the differential effects of the two TLRs on disease. To reconcile this, we hypothesized that TLR9 could induce counter-regulatory signaling and/or regulate TLR7 via a ligand- or signaling-independent mechanism.Methods and ResultsTo differentiate between these hypotheses, we created mutants of TLR9 that prevent ligand binding (TLR9K51E) or MyD88 signaling (TLR9P915H) in the TLR9 locus of MRL/lpr mice. Mutant mice were assessed by for dermatitis, nephritis, immune dysregulation, and auto-antibodies. TLR9K51E/K51E mice had increased survival and decreased kidney pathology compared to TLR9-/- mice, indicating that the restoration of TLR9 expression induces protection. To assess the effect of TLR9-MyD88 signaling, we compared TLR9P915H/P915H and TLR9-sufficient cohorts of MRL/lpr mice. Kidney disease, survival and immune activation were significantly more severe in TLR9± mice. Thus, there is a TLR9-MyD88 dependent pathway that promotes disease. Moreover, TLR9K51E/- mice had increased glomerulonephritis and immune activation compared to TLR9P915H/P915H mice, suggesting that TLR9 could regulate disease through a ligand binding-dependent but MyD88-independent mechanism (figure 1). Using a 3 way (TLR9WT, TLR9P915H and TLR9-/-)-mixed bone marrow chimera, we found that TLR9 inhibits B cell development and differentiation in a B cell-intrinsic fashion and that the absence of TLR9 (TLR9-/-) was very different from the inability of TLR9 to signal (TLR9P915H). RNA seq analysis of sorted age-associated B cells (ABC) revealed that TLR9WT, TLR9P915H and TLR9-/- ABC exhibit different transcriptional programs. Notably, the absence of TLR9 did not lead to an increase in genes that are induced by TLR7, arguing against the idea that TLR9 simply restrains TLR7 signaling.ConclusionThis in vivo genetic dissection of TLR9 reveals how it both promotes and regulates lupus. Inability of TLR9 to signal via MyD88 is different from absence of TLR9 and also is different from inability of TLR9 to bind ligand. These findings shed light on the basic biology of endosomal TLR signaling and are relevant to the design of TLR-targeted therapy.AcknowledgementFunded by R37-AI118841 (M. Shlomchik).