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Toll-like receptor 9 (TLR9) is a regulator of disease pathogenesis in systemic lupus erythematosus (SLE). Why TLR9 represses disease while TLR7 and MyD88 have the opposite effect remains undefined. To begin to address this question, we created 2 alleles to manipulate TLR9 expression, allowing for either selective deletion or overexpression. We used these to test cell type-specific effects of Tlr9 expression on the regulation of SLE pathogenesis. Notably, Tlr9 deficiency in B cells was sufficient to exacerbate nephritis while extinguishing anti-nucleosome antibodies, whereas Tlr9 deficiency in dendritic cells (DCs), plasmacytoid DCs, and neutrophils had no discernable effect on disease. Thus, B cell-specific Tlr9 deficiency unlinked disease from autoantibody production. Critically, B cell-specific Tlr9 overexpression resulted in ameliorated nephritis, opposite of the effect of deleting Tlr9. Our findings highlight the nonredundant role of B cell-expressed TLR9 in regulating lupus and suggest therapeutic potential in modulating and perhaps even enhancing TLR9 signals in B cells.

Objective: Toll-like receptors (TLRs) 7 and 9 are important innate signaling molecules with opposing roles in the development and progression of systemic lupus erythematosus (SLE). While multiple studies support the notion of a dependency on TLR-7 for disease development, genetic ablation of TLR-9 results in severe disease with glomerulonephritis (GN) by a largely unknown mechanism. This study was undertaken to examine the suppressive role of TLR-9 in the development of severe lupus in a mouse model., Methods: We crossed Sle1 lupus-prone mice with TLR-9-deficient mice to generate Sle1TLR-9 -/- mice. Mice ages 4.5-6.5 months were evaluated for severe autoimmunity by assessing splenomegaly, GN, immune cell populations, autoantibody and total Ig profiles, kidney dendritic cell (DC) function, and TLR-7 protein expression. Mice ages 8-10 weeks were used for functional B cell studies, Ig profiling, and determination of TLR-7 expression., Results: Sle1TLR-9 -/- mice developed severe disease similar to TLR-9-deficient MRL and Nba2 models. Sle1TLR-9 -/- mouse B cells produced more class-switched antibodies, and the autoantibody repertoire was skewed toward RNA-containing antigens. GN in these mice was associated with DC infiltration, and purified Sle1TLR-9 -/- mouse renal DCs were more efficient at TLR-7-dependent antigen presentation and expressed higher levels of TLR-7 protein. Importantly, this increase in TLR-7 expression occurred prior to disease development, indicating a role in the initiation stages of tissue destruction., Conclusion: The increase in TLR-7-reactive immune complexes, and the concomitant enhanced expression of their receptor, promotes inflammation and disease in Sle1TLR9 -/- mice., (© 2018 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)

Genetic deficiency in TLR9 accelerates pathogenesis in the spontaneous polygenic MRL.Faslpr murine model of systemic lupus erythematosus, despite the absence of anti-nucleosome autoantibodies. However, it could be argued that this result was dependent on Fas-deficiency rather than lupus-promoting genes in the MRL genetic background. Here we report the effects of TLR9 deficiency on autoimmune disease independent of the lpr mutation in Fas by characterizing Tlr9-/- and Tlr9+/+ mice on the Fas-intact MRL/+ genetic background. By 30 weeks of age, Tlr9-deficient MRL/+ had more severe renal disease, increased T cell activation, and higher titers of anti-Sm and anti-RNA autoantibodies than Tlr9-intact animals, as had been the case in the MRL.Faslpr model. In addition, Tlr9-deficient MRL/+ mice had increased numbers of germinal center phenotype B cells and an increase in splenic neutrophils and conventional dendritic cell populations. Thus, the disease accelerating effects of Tlr9 deficiency are separable from those mediated by the Fas mutation in the lupus-prone MRL genetic background. Nonetheless, disease acceleration in Tlr9-deficient MRL/+ mice was phenotypically distinct from that in Fas-deficient counterparts, which has important implications.

TLR9 suppresses TLR7-driven pathogenesis in the MRL.Fas(lpr) murine model of systemic lupus erythematosus, but the mechanisms by which TLR7 promotes and TLR9 prevents disease in this and other lupus models remain unclear. Type I IFNs (IFN-I) have also been implicated in the pathogenesis of lupus both in patients and in several murine models of disease, but their role in MRL.Fas(lpr) mice is controversial. Using MRL.Fas(lpr) mice genetically deficient in a subunit of the receptor for IFN-I, Ifnar1, we show that IFN-I contribute significantly to renal disease in this model. Ifnar1 had no effect on anti-nucleosome or anti-Sm autoantibody titers, but instead regulated anticytoplasmic and anti-RNA specificities. Moreover, Ifnar1 deficiency prevented the exacerbation of clinical disease observed in Tlr9-deficient animals in this lupus model. Thus, IFN-I signaling is an important mediator of lupus pathogenesis and anti-RNA Ab production that is dysregulated in the absence of Tlr9.

Circulating monocytes are divided into two major, phenotypically and functionally distinct subsets: Gr-1(hi) "inflammatory" and Gr-1(lo) "resting" monocytes. One of the unique cellular abnormalities in lupus-prone mice is monocytosis, which is characterized by a selective expansion of Gr-1(lo) monocytes and dependent on the expression of stimulatory IgG Fc receptors (FcγR). We speculated that IgG immune complexes containing nuclear antigens could stimulate Gr-1(hi) monocytes through interaction with FcγRs and then TLR7 and TLR9, thereby promoting the maturation towards Gr-1(lo) monocytes. In the present study, we assessed this hypothesis by analyzing effects of TLR9 or TLR7 agonist on monocytes in vivo. The analysis of various surface markers differentially expressed on both subsets of monocytes in combination with selective depletion of either subset revealed that within 48 h after injection of the TLR9 agonist CpG, approximately one third of Gr-1(hi) monocytes became phenotypically identical to Gr-1(lo) monocytes. In addition, we observed approximately two-fold increases in the total monocyte population 8-24 h after injection of CpG. Moreover, the activation of TLR9 resulted in an increased expression of stimulatory FcγRIV relative to inhibitory FcγRIIB on monocytes, thereby enhancing their responsiveness to IgG immune complexes. Essentially identical results were obtained after stimulation of TLR7 with a synthetic agonist (1V136). Our results indicate that the activation of TLR7 and TLR9 not only induced the maturation of a fraction of Gr-1(hi) monocytes towards Gr-1(lo) monocytes but also promoted the overall generation of monocytes, thereby supporting the critical role of TLR7 and TLR9 for the development of monocytosis in lupus-prone mice., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The nucleosome is a major autoantigen known to activate PMN in systemic lupus erythematosus (SLE). TLR9 recognizes bacterial and even mammalian DNA under certain circumstances. Nevertheless, the role of TLR9 in SLE development is still unclear. Since nucleosomes are composed of DNA, we investigated whether TLR9 is required for nucleosome-induced PMN activation. Isolated neutrophils were cultured with nucleosomes, plasma from lupus patients and other stimuli in the presence/absence of various inhibitors. Cells were analyzed by flow cytometry, ELISA and confocal microscopy. We found that nucleosomes circulating in lupus plasma induce the secretion of pro-inflammatory cytokines by PMN. Nucleosomes activate human PMN independently of unmethylated CpG sequences in nucleosomal DNA, leading to IL-8/IL-6/TNF secretion and CD11b up-regulation. Nucleosomes accumulate in the cytoplasm of PMN upon endocytosis, induce TLR9 up-regulation and act synergistically with TLR9 ligands. Nucleosome-induced activation was not inhibited by polymyxin B (PB), chloroquine (CQ), ammonium chloride (AC) or a TLR9 antagonist. Moreover, both PMN isolated from WT and TLR9-KO mice were activated by nucleosomes, as detected by MIP-2 secretion and CD11b up-regulation. Activation occurred therefore independently of endotoxins, endosomal acidification, TLR9 and CpG motifs. TLR9 may thus be differently required in the triggering of nucleosome-induced innate immunity and anti-nucleosome B-cell autoimmunity., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recognition of endogenous DNA and RNA by cells expressing TLR7 and TLR9 is an important contributor to the pathogenesis of systemic lupus erythematosus and has been suggested to contribute to cutaneous lupus and to a group of related inflammatory skin diseases termed interface dermatitis. We have developed a mouse model of TLR7- and TLR9-dependent skin inflammation using tape stripping. In normal mice, this resulted in a rapid but transient inflammatory cell infiltration accompanied by induction of type I IFN production by plasmacytoid dendritic cells (PDCs) and release of extracellular traps and proinflammatory cytokines by neutrophils. These responses were strongly reduced in MyD88-deficient mice and in mice treated with a bifunctional inhibitor of TLR7 and TLR9. In contrast, in lupus-prone (NZBxNZW)F(1) mice, tape stripping induced the development of chronic lesions characterized by a persistent type I IFN gene signature and many clinical and histological features of cutaneous lupus. Depletion of PDCs before injury prevented the development of skin lesions, whereas treatment with a bifunctional TLR7/9 inhibitor before tape stripping or after the initial lesion was established led to a significant reduction of the disease. These data suggest that inhibitors of TLR7 and TLR9 signaling have potential therapeutic application for the treatment of interface dermatitis.

Accumulating evidence supports the idea that TLR7 and TLR9 play pathogenic and protective roles, respectively, in the development of murine systemic lupus erythematosus (SLE). However, the molecular mechanism responsible for the accelerated development of SLE resulting from the deletion of TLR9 and the respective contributions of TLR7 and TLR9 to the development of different autoimmune responses against nuclear and non-nuclear autoantigens implicated in lupus nephritis have not been well defined. In the present study, we addressed these questions by assessing the effect of the TLR9 and/or TLR7 deletion on the production of various autoantibodies and the development of lupus nephritis in C57BL/6 mice congenic for the Nba2 (NZB autoimmunity 2) locus (B6.Nba2). TLR9-deficient B6.Nba2 mice displayed increased production of autoantibodies against nuclear antigens, serum retroviral gp70 and glomerular matrix antigens, and developed a markedly accelerated form of lupus nephritis. Enhanced disease was associated with functionally upregulated expression of TLR7, as documented by an increased TLR7-dependent activation of B cells and plasmacytoid dendritic cells. Notably, disease exacerbation in TLR9-deficient mice was completely suppressed by the deletion of TLR7. Our results indicate that TLR7 has a pivotal role in a wide variety of autoimmune responses against DNA- and RNA-containing nuclear antigens, retroviral gp70 and glomerular matrix antigens implicated in murine SLE, and that enhanced TLR7 activity is critical for the accelerated development of SLE in TLR9-deficient lupus-prone mice.

Systemic lupus erythematosus is characterized by the production of autoantibodies against nucleic acid-associated Ags. We previously found that Tlr7 was required for anti-Sm and Tlr9 for anti-chromatin autoantibodies. Yet, although Tlr7 deficiency ameliorated disease, Tlr9 deficiency exacerbated it. Despite the mechanistic and clinical implications of this finding, it has yet to be elucidated. In this study, we characterize MRL/lpr lupus-prone mice genetically deficient in Tlr7, Tlr9, both Tlr7 and Tlr9, or Myd88 to test whether Tlr7 and Tlr9 function independently or instead regulate each other. We find that disease that is regulated by Tlr9 (and hence is worse in its absence) depends on Tlr7 for its manifestation. In addition, although Tlr7 and Tlr9 act in parallel pathways on different subsets of autoantibodies, Tlr9 also suppresses the production of Tlr7-dependent RNA-associated autoantibodies, suggesting previously unrecognized cross-regulation of autoantibody production as well. By comparing disease in mice deficient for Tlr7 and/or Tlr9 to those lacking Myd88, we also identify aspects of disease that have Tlr- and Myd88-independent components. These results suggest new models for how Tlr9 regulates and Tlr7 enhances disease and provide insight into aspects of autoimmune disease that are, and are not, influenced by TLR signals.

Chronic graft-vs-host (cGVH) disease is induced in nonautoimmune mice by the transfer of alloreactive T cells that recognize foreign MHC class II. It closely resembles systemic lupus erythematosus, with antinuclear Abs and immune-mediated nephritis. Recent work has implicated TLRs, particularly TLR9, in the recognition of certain autoantigens in vitro and in vivo. To explore further the role of TLR9 in systemic autoimmunity, we induced cGVH disease in C57BL/6 (B6) mice lacking TLR9, including B6 mice expressing the anti-DNA-encoding IgH transgenes 3H9 or 56R (B6.3H9.TLR9(-/-), B6.56R.TLR9(-/-)). We found that cGVH disease caused breakdown of B cell tolerance to chromatin and DNA in TLR9(-/-) recipients of alloreactive cells, yet that nephritis was less severe and that some autoantibody titers were lower compared with B6-cGVH controls. Spleen lymphocyte analysis showed that cGVH disease strikingly depleted marginal zone B cells in B6 mice, but did not influence T cell subsets in either B6 or B6-TLR9(-/-) hosts. B6.56R.TLR9(-/-) mice had less spontaneous production of autoantibodies than B6.56R mice, but there were no significant differences between B6.56R and B6.56R.TLR9(-/-) postinduction of cGVH disease. Taken together, these results suggested that TLR9 may worsen some aspects of systemic autoimmunity while alleviating others.

Antibodies (Abs) to RNA- and DNA-containing autoantigens are characteristic of systemic lupus erythematosus (SLE). We showed previously that Toll-like receptor (TLR) 9, recognizing DNA, is required for the spontaneous generation of DNA autoantibodies, but not for the development of lupus nephritis in susceptible mice. We report that lupus-prone mice deficient in TLR7, a receptor for ssRNA, failed to generate Abs to RNA-containing antigens (Ags) such as Smith (Sm) Ag. TLR9 and TLR7 also had dramatic effects on clinical disease in lupus-prone mice. In the absence of TLR9, autoimmune disease was exacerbated, lymphocytes and plasmacytoid DCs were more activated, and serum IgG and IFN-alpha were increased. In contrast, TLR7-deficient mice had ameliorated disease, decreased lymphocyte activation, and decreased serum IgG. These findings reveal opposing inflammatory and regulatory roles for TLR7 and TLR9, despite similar tissue expression and signaling pathways. These results have important implications for TLR-directed therapy of autoimmune disease.

The generation of anti-DNA auto-antibodies is characteristic for the human autoimmune condition systemic lupus erythematosus (SLE) and its animal models. However, the contribution of the toll-like receptor (TLR) system of innate immunity receptors and, in particular, TLR9 to this B cell-mediated autoimmune process remains controversial. Here we report that in a novel murine model of SLE, based on hyper-reactive B cell activation mediated by mutant phospholipase Cg2, the genetic deficiency of TLR9 does not protect from spontaneous anti-DNA auto-antibody formation and glomerulonephritis. On the contrary, disease induction is aggravated and additional nucleolar antibody specificity develops in autoimmune TLR9-deficient mice. In vitro studies demonstrate that, in autoimmune-prone mice, dual signaling via the B cell receptor and non-CpG DNA results in synergistic B cell activation in a TLR9-independent manner. These results suggest that engagement of a TLR9-independent DNA activation pathway may promote autoimmunity, while TLR9 signaling can ameliorate SLE-like immune pathology in vivo.

Loss of tolerance in systemic lupus erythematosus (SLE) leads to the generation of autoantibodies, which accumulate in end-organs where they induce disease. Here we show that immunoglobulin (Ig)G2a and 2b autoantibodies are the pathogenic isotypes by recruiting FcgammaRIV expressing macrophages. Class switching, but not development, of IgM anti-self B cells to these pathogenic subclasses requires the innate immune receptor Toll-like receptor (TLR)9 and MyD88 signaling. In their absence, switching of autoreactive B cells to the IgG2a and 2b subclasses is blocked, resulting in reduced pathology and mortality. In contrast, switching of anti-self B cells to IgG1 is not perturbed and generation of nonautoreactive IgG2a and 2b antibodies is not impaired in TLR9-deficient mice. Thus, the TLR9 pathway is a potential target for therapeutic intervention in SLE.