Since the original clinical and serological description of MCTD by Sharp and colleagues in this journal (56
), many hypotheses have been put forward regarding the mechanisms by which high-titer IgG autoantibodies against the U1snRNP and other nucleic acid–containing autoantigens become immunogenic. Cross-reactivity with viral antigens such as U1-70K and the retroviral protein p30gag (57
), coassociation of RNPs with viral antigens such as EBER1 and EBER2 (58
), and apoptosis-specific modifications of many components of the U1snRNP (59
) have all been suggested to play important roles in the immunogenicity of the U1snRNP. Our present results do not refute these models, but identify a central, in vivo role for 2 molecules downstream of IFNAR in the production of IgG autoantibodies capable of immunoprecipitating RNP complexes targeted in MCTD and SLE, including the U1snRNP and RiboP.
In the current study, we describe the autoantibody response in mice deficient for IRF9 and STAT1, critical downstream mediators of IFN-I signaling. Our initial screen using autoantigen microarrays followed by rigorous statistical analysis using SAM revealed 4 significantly different antigens between pristane-treated WT and Irf9–/– mice, all of which are associated with RNPs. Further validation using conventional techniques demonstrated that IRF9 and STAT1 were required for the development of IgG autoantibodies to several different nucleic acid–associated complexes targeted in this model. In contrast, Irf9–/– mice mounted an effective IgG antibody response against a foreign antigen when immunized with the strong adjuvant CFA. Irf9–/– and Stat1–/– B cells had specific defects in expression of, and activation through, the nucleic acid–sensing TLRs, suggesting that defects in TLR7- and TLR9-dependent B cell responses may account for the lack of isotype-switched IgG autoantibodies.
The ISGF3 complex, composed of STAT1, STAT2, and IRF9, has also been shown to form upon activation by IFN-γ (10
). ISGF3 formation in response to IFN-γ treatment occurred at higher levels following pretreatment with IFN-α, which upregulates the expression of STAT1 and IRF9, or in the presence of highly overexpressed STAT1 and IRF9 (10
). The in vivo relevance of this pathway has not yet been established, and it is possible that IRF9 mediates signals through both the IFNAR and the IFN-γ receptor. To more directly address the role of the IFN-I pathway in the development of autoimmunity, we have initiated similar studies in pristane-treated mice deficient for the IFNAR2 chain of the IFNAR. Preliminary data suggest that pristane-treated Ifnar2–/–
mice have a phenotype similar to that seen in pristane-treated Irf9–/–
mice (D.L. Thibault and P.J. Utz, unpublished observations). Specifically, the production of IgG autoantibodies, and the expression of nucleic acid–sensing TLRs in B cells, was completely dependent on IFNAR2. Levels of total serum IgM were significantly increased in pristane-treated Ifnar2–/–
mice, and these mice developed high titers of IgM autoantibodies, suggesting a defect in isotype switching similar to that seen in Irf9–/–
mice. In addition, pristane-treated Ifnar1–/–
mice failed to develop anti-RNP autoantibodies; however, the expression of TLRs and the isotypes of the autoantibodies were not assessed in this study (60
). This differs from the autoantibody phenotype seen in pristane-treated IFN-γ–deficient mice, in which IFN-γ was not absolutely required for the development of high titers of IgG autoantibodies capable of precipitating the Sm/RNP complex (ref. 61
and D.L. Thibault, K.L. Graham, and P.J. Utz, unpublished observations). These data suggest that IRF9 mediates signals downstream of the IFNAR, but not downstream of the IFN-γ receptor, and that IFN-I drives the autoimmune response to RNA-associated autoantigens in vivo.
Molecules involved in the negative regulation of both TLR and IFN signaling pathways have previously been identified. In particular, the TAM receptors, which include Tyro3, Axl, and Mer, are pleiotropic inhibitors of TLR- and cytokine-induced signaling, and control the induction of SOCS-1 by IFN-α (62
). Both Socs1–/–
and TAM receptor triple-knockout mice develop spontaneous autoimmunity (63
), highlighting the critical role of negative regulation of the TLR and IFN pathways in the development of autoimmunity. The TAM receptors have previously been shown to be directly associated with IFNAR1, and Stat1–/–
mice display defects in TAM receptor function (62
). TAM-mediated induction of the molecule Twist results in the suppression of TNF-α production (65
), demonstrating a mechanism by which IFN-I mediates the suppression of TNF-α. The role of these molecules in the development of autoimmunity in the pristane model, as well as their function in Irf9–/–
mice, will need to be assessed in future studies.
The 9 known members of the IRF family play roles in the induction of IFN-regulated genes and of IFN-Is themselves (66
). Recent publications have highlighted important potential roles for members of the IRF family in the pathogenesis of SLE. A common haplotype of IRF5 is a genetic risk factor for human SLE (67
). Both IRF7 and IRF5 are critical for DC production of IFN-α, IFN-β, and IL-6 by anti-RNP immune complexes and by conventional TLR7 and TLR9 ligands (70
mice lacking the IRF1 gene develop less severe disease compared with WT mice (72
). Finally, female mice deficient for IRF4-binding protein develop a lupus-like disease characterized by hypergammaglobulinemia, autoantibody production, and immune complex–mediated glomerulonephritis (73
). To our knowledge, a role for IRF9 has not previously been addressed in the contexts of SLE or TLR signaling. Our present results suggest this molecule is a critical mediator of B cell responses, including autoantibody production, isotype switching, and the expression of and activation through nucleic acid–sensing TLRs.
The pristane model offers a unique opportunity to study the role of the IFN-I pathway in the development of murine SLE. Several recent studies have examined the contributions of TLRs and IFN-I to SLE disease in the MRL/lpr
). DNA microarray analysis of MRL/lpr
splenocyte subsets and kidneys clearly demonstrate an IFN-γ–regulated gene expression profile, whereas genes induced by IFN-I are not upregulated in MRL/lpr
). This observation may explain why MRL/lpr
mice deficient for the IFNAR1 chain of the IFNAR actually develop more severe disease: the lack of IFN-I signaling may further drive the IFN-γ response (75
). In contrast to the MRL/lpr
model, several IFN-I–inducible genes are upregulated in pristane-induced ectopic lymphoid tissue and peripheral blood mononuclear cells (18
), which mirrors the gene expression profile seen in human lupus patients (2
). In human SLE patients, high expression of these genes correlates with the production of anti-nucleoprotein and, in particular, anti-RNP autoantibodies (12
). (NZB × NZW)F1 mice do not develop autoantibodies that recognize RNA-containing autoantigens, although administration of pristane to these mice resulted in an accelerated disease course and induced the production of anti-RNP autoantibodies (77
). Therefore, disease pathogenesis in the pristane model, especially in regard to dysregulation of the IFN-I pathway, more closely resembles human SLE when compared with the spontaneous models described above. To more carefully assess the contribution of this pathway to lupus nephritis, further studies in these spontaneous models will be necessary.
Several recent studies have described the ability of nucleic acid–containing MCTD and SLE autoantigens to stimulate autoreactive B cells through nucleic acid–sensing TLRs. Here we demonstrated that IFN-I signaling components controlled the ability of B cells to upregulate the expression of TLR7 and TLR9 upon stimulation with IFN-α. The expression of TLR7 in particular was influenced by IFN-I, because its expression was enhanced more than 20-fold upon incubation with IFN-α in WT B cells. Interestingly, although B cell expression of TLR7 and secretion of IL-6 in response to a TLR7 agonist was dramatically reduced in Irf9–/– mice following treatment with IFN-α (Figure , B and G), TLR7 expression and activation were slightly enhanced by IFN-α in the absence of IRF9. Residual TLR7 expression following IFN-α treatment was sufficient for the secretion of lower levels of IL-6, suggesting that expression of TLR7 is partially IRF9 independent in B cells. We propose that residual expression of TLR7 may drive the partial activation of autoreactive B cells and the subsequent production of IgM-specific autoantibodies.
IRF9 is absolutely required for isotype switching to pathogenic, high-titer, high-affinity IgG autoantibodies. This is not caused by global defects in isotype switching in Irf9–/–
B cells, because there was no defect in adjuvant-enhanced isotype switching to all subtypes of IgG against a foreign antigen (Figure ). Unlike pristane-treated Irf9–/–
mice, pristane-treated Stat1–/–
mice did not develop high titers of total serum IgM or IgM-specific autoantibodies. Both PBS- and pristane-treated Stat1–/–
mice had very low serum titers of all antibody isotypes except for IgG1, and B cells from Stat1–/–
mice displayed a decreased ability to respond to multiple stimuli, including TLR7 and TLR9, and decreased crosslinking of the B cell receptor by anti-IgM (78
). These data suggest that there may be global defects in B cell activation in Stat1–/–
mice that are not limited to TLR7-specific responses; however, further studies to characterize potential defects are necessary. B cell stimulation by CpGs through TLR9 and MyD88 promotes T cell–independent isotype switching to IgG2a through direct induction of the transcription factor T-bet (32
). Because TLR7 also signals through MyD88, it is thought that signaling through TLR7 promotes a similar IgG2a response. In support of these studies, Irf9–/–
mice immunized with OVA using a TLR7 agonist as an adjuvant failed to produce IgG2a OVA-specific antibodies in a pilot study (D.L. Thibault and P.J. Utz, unpublished observations); however, a larger cohort of mice is necessary to confirm these findings. The lack of isotype switching to IgG in Irf9–/–
mice is not due to the inability of Irf9–/–
B cells to upregulate T-bet, as T-bet was highly induced in Irf9–/–
B cells following incubation with IFN-α together with TLR agonists (D.L. Thibault and P.J. Utz, unpublished observations). IFN-I together with IL-6 promotes B cell maturation to antibody-secreting plasma cells (43
), providing further support for a role for IRF9 in B cell isotype switching. Further studies are necessary to dissect the complex role for IFN-I signaling in TLR7- and TLR9-dependent and -independent B cell responses and to determine the role of this pathway in other cell types in SLE pathogenesis.
In summary, our data demonstrate what we believe to be a novel role for 2 downstream mediators of IFN-I signaling in the generation of IgG autoantibody responses against nucleic acid–associated autoantigens. We propose that IFN-I, which is produced by pDCs in vivo following pristane treatment, promotes the expression of TLR7 and TLR9 in B cells through the ISGF3, allowing for the activation of autoreactive B cells by MCTD- and SLE-associated autoantigens and the production of high-titer, high-affinity IgG autoantibodies. A schematic outlining this proposed model is depicted in Supplemental Figure 7. Pristane treatment has previously been shown to induce apoptosis both in vitro and in vivo, providing a potential source of lupus-associated autoantigens (17
). Our data suggest that IFN-I signaling is upstream of TLR responses in the activation of autoreactive B cells. Finally, specific inhibitors of IFN-I and TLRs are in preclinical or early-stage clinical trials for the treatment of SLE in human patients. Our results suggest that patients could be selected for clinical trials and monitored for response to therapy based on autoantibody profiles and provide a clear rationale for pursuing these therapeutic approaches.