TLR8 on dendritic cells and TLR9 on B cells restrain TLR7-mediated spontaneous autoimmunity in C57BL/6 mice

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with diverse clinical presentations characterized by the presence of autoantibodies to nuclear components. Toll-like receptor (TLR)7, TLR8, and TLR9 sense microbial or endogenous nucleic acids and are implicated in the development of SLE. In mice TLR7-deficiency ameliorates SLE, but TLR8- or TLR9-deficiency exacerbates the disease because of increased TLR7 response. Thus, both TLR8 and TLR9 control TLR7 function, but whether TLR8 and TLR9 act in parallel or in series in the same or different cell types in controlling TLR7-mediated lupus remains unknown. Here, we reveal that double TLR8/9-deficient (TLR8/9^−/−) mice on the C57BL/6 background showed increased abnormalities characteristic of SLE, including splenomegaly, autoantibody production, frequencies of marginal zone and B1 B cells, and renal pathology compared with single TLR8^−/− or TLR9^−/− mice. On the cellular level, TLR8^−/− and TLR8/9^−/− dendritic cells were hyperesponsive to TLR7 ligand R848, but TLR9^−/− cells responded normally. Moreover, B cells from TLR9^−/− and TLR8/9^−/− mice were hyperesponsive to R848, but TLR8^−/− B cells were not. These results reveal that TLR8 and TLR9 have an additive effect on controlling TLR7 function and TLR7-mediated lupus; however, they act on different cell types. TLR8 controls TLR7 function on dendritic cells, and TLR9 restrains TLR7 response on B cells.Systemic lupus erythematosus (SLE) is a complex chronic autoimmune disease that arises spontaneously and is characterized by production of autoantibodies against self-nucleic acids and associated proteins (1). These autoantibodies bind self-nucleic acids released by dying cells and form immune complexes that accumulate in different parts of the body, leading to inflammation and tissue damage. The kidneys, skin, joints, lungs, serous membranes, as well as, the cardiovascular, nervous and musculoskeletal system become targets of inflammation at onset or during the course of the disease (2). The etiology of SLE is unknown, yet genetics, sex, infectious agents, environmental factors, and certain medications may play a role in the initiation of the disease by causing alterations in lymphoid signaling, antigen presentation, apoptosis, and clearance of immune complexes (3, 4).Toll-like receptors (TLRs) detect specific microbial components widely expressed by bacteria, fungi, protozoa, and viruses, and initiate signaling pathways critical for induction of immune responses to infection (5). In contrast to the cell surface TLRs that detect bacterial cell wall components and viral particles, nucleic acid-sensing TLRs are localized mainly within endosomal compartments (6). Human endosomal TLRs consist of TLR3, which senses viral double-stranded RNA (dsRNA) (7), TLR7 and TLR8, which recognize viral single-stranded RNA (8–10), and TLR9, which detects bacterial and viral unmethylated CpG-containing DNA motifs (11). Interestingly, these endosomal TLRs are also able to detect self-nucleic acids (12–14). Although the endosomal localization isolate TLR3, TLR7, TLR8, and TLR9 away from self-nucleic acids in the extracellular space, still self-RNA or -DNA can become a potent trigger of cell activation when transported into TLR-containing endosomes, and such recognition can result in sterile inflammation and autoimmunity, including SLE (4, 15, 16). The connection of the endosomal TLRs with SLE originates mainly from mouse models, where TLR7 signaling seems to play a central role. TLR7 gene duplication is the cause for the development of lupus in mice bearing the Y chromosome-linked autoimmune accelerating (Yaa) locus that harbors 17 genes, including TLR7 (17, 18). In TLR7 transgenic mouse lines, a modest increase in TLR7 expression promotes autoreactive lymphocytes with RNA specificities and myeloid cell proliferation, but a substantial increase in TLR7 expression causes fatal acute inflammatory pathology and profound dendritic cell (DC) dysregulation (17). In addition, studies in several lupus-prone mouse strains have revealed that TLR7-deficiency ameliorates disease, but TLR9-deficiency exacerbates it. Interestingly, this controversy can be explained by the enhanced TLR7 activity in the TLR9-deficient lupus mice (19, 20). Although murine TLR8 does not seem so far to be able to sense a ligand (21, 22), we have shown previously that it plays an important biological role in controlling TLR7-mediated lupus. Indeed, TLR8-deficiency in mice (on the C57BL/6 background that is not prone to lupus) leads to lupus development because of increased TLR7 expression and signaling in DCs (23). Thus, tight control and regulation of TLR7 is pivotal for avoiding SLE and inflammatory pathology in mice. Recent studies in humans have also revealed that increased expression of TLR7 is associated with increased risk for SLE (24–26).Nucleic acid TLRs are expressed in many cell types, including DCs, plasmacytoid DCs (pDCs) and B cells, all of which play a central role in SLE development. TLR7, TLR8, and TLR9 signal through the adaptor molecule myeloid differentiation primary response gene 88 (MyD88), whereas TLR3 signals via the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor inducing IFN-β) (5). MyD88-deficiency abrogates most attributes of lupus in several lupus-prone mouse strains (19, 27–29). Moreover, deficiency for Unc93B1, a multipass transmembrane protein that controls trafficking of TLRs from the endoplasmic reticulum to endolysosomes and is required for nucleic acid-sensing TLR function (30), also abrogates many clinical parameters of disease in mouse lupus strains, suggesting that endosomal TLRs are critical in this disease (31). Interestingly, TLR9 competes with TLR7 for Unc93B1-dependent trafficking and predominates over TLR7 (32). TLR9 predominance is reversed to TLR7 by a D34A mutation in Unc93B1 and mice that carry this mutation show TLR7-dependent, systemic lethal inflammation (32).Thus, in mice both TLR8 and TLR9 control TLR7-mediated lupus, but it is unknown if these TLRs act in parallel or in series in the same or different cell types and if they have an additive effect or not in controlling TLR7. To address these issues, we generated double TLR8/TLR9-deficient (TLR8/9^−/−) mice and analyzed and compared the lupus phenotype in TLR8^−/−, TLR9^−/−, and TLR8/9^−/− mice. Our data revealed that TLR8/9^−/− mice have increased abnormalities characteristic of SLE and that both TLR8 and TLR9 keep TLR7-mediated lupus under control, but they act in different cell types. On DCs TLR7 function is ruled by TLR8, whereas on B cells TLR7 is mastered by TLR9.