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Keywords:

  • Autoantibody;
  • Interferon regulatory factor 5 (IRF5, IRF-5);
  • Systemic lupus erythematosus (SLE);
  • Type I interferon;
  • Th2

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information

Polymorphisms in the transcription factor interferon (IFN) regulatory factor 5 (IRF5) have been identified that show a strong association with an increased risk of developing the autoimmune disease systemic lupus erythematosus (SLE). A potential pathological role for IRF5 in SLE development is supported by the fact that increased IRF5 mRNA and protein are observed in primary blood cells of SLE patients and this correlates with an increased risk of developing the disease. Here, we demonstrate that IRF5 is required for pristane-induced SLE via its ability to control multiple facets of autoimmunity. We show that IRF5 is required for pathological hypergammaglobulinemia and, in the absence of IRF5, IgG class switching is reduced. Examination of in vivo cytokine expression (and autoantibody production) identified an increase in Irf5−/− mice of Th2 cytokines. In addition, we provide clear evidence that loss of Irf5 significantly weakens the in vivo type I IFN signature critical for disease pathogenesis in this model of murine lupus. Together, these findings demonstrate the importance of IRF5 for autoimmunity and provide a significant new insight into how overexpression of IRF5 in blood cells of SLE patients may contribute to disease pathogenesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder affecting multiple organs and is characterized by a type I interferon (IFN) gene signature, the production of auto-antibodies, and subsequent development of glomeruloneph-ritis [[1]]. Although the underlying etiology of SLE remains obscure, several lines of evidence document a complex interaction between environmental and genetic factors [[1-3]]. Results from genome-wide association studies (GWASs) have identified a number of SLE susceptibility genes [[2-5]]. With the availability of numerous models of experimental murine lupus, we can begin to elucidate their contribution(s) to SLE pathogenesis [[5]].

The transcription factor interferon regulatory factor 5 (IRF5) is one SLE susceptibility gene recently identified [[6]]. Multiple studies have confirmed the presence of IRF5 genetic variants that show strong association with increased risk of developing SLE [[6-8]]. Association has been convincingly replicated in SLE patients from multiple populations and distinct IRF5 haplotypes that confer either susceptibility to (risk), or protection from, SLE in persons of varying ethnic ancestry have been identified [[6-11]]. A potential biologic role for IRF5 in human SLE pathogenesis has been supported by the fact that elevated IRF5 mRNA levels are associated with specific IRF5 risk variants [[7, 8, 12, 13]]. Subsequently, we demonstrated that IRF5 mRNA and protein abundance were significantly elevated in primary blood cells of SLE patients, as compared to healthy donors, independent of IRF5 risk variants; however, a correlation between IRF5 expression and the IRF5 risk haplotype was obtained [[14]]. These data support a more global role for IRF5 in SLE pathogenesis that is both genotype dependent and genotype independent.

IRF5 regulates type I IFN expression in response to a variety of pathogenic stimuli and is a critical mediator of MyD88-dependent Toll-like receptor (TLR) signaling [[15-18]]. Proinflammatory cytokines elevated in the serum of lupus patients, that is IFN-α, interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-α, are regulated by IRF5 [[16]]. In mice, the production of IFN-α/β and IL-6 in response to sera or IgG–RNA immune complexes (IC) from lupus patients was shown to be Tlr7, Irf5, and Irf7 dependent [[19]]. These data support the conventional wisdom that elevated IRF5 expression in SLE patients may drive disease development by causing aberrant production of type I IFN through TLR7 and/or TLR9 signaling that is activated by IC [[20, 21]]. Correlative data supporting this has been obtained in SLE patients demonstrating association of an IRF5 risk haplotype with IFN-α activity that was dependent on autoantibodies [[22]].

Recently, it was demonstrated that FcRIIb−/− and FcRIIb−/−Yaa mice lacking Irf5 had significantly decreased autoantibody production, limited glomerular IgG deposition, and enhanced survival [[23]]. Little mechanistic insight was provided for the protective Irf5−/− phenotype. A subsequent study demonstrated that IRF5 regulates transcription of the γ2a locus resulting in decreased autoantibody production [[24]]. Surprisingly, neither study directly addressed whether loss of Irf5 affected type I IFN expression [[23, 24]]. We hypothesized that loss of Irf5 would alter multiple aspects of autoimmunity due to its regulation of the pleiotropic cytokine type I IFN and other proinflammatory cytokines [[15-18]]. The 2,6,10,14-tetramethylpentadecane (pristane) induced lupus model displays many key immunologic and clinical features of human SLE, including production of autoantibodies against dsDNA and small nuclear ribonucleoproteins (snRNPs), a type I IFN signature, and the development of IC-mediated glomerulonephritis [[25, 26]]. This murine model is at present the only one reported to recapitulate the IFN signature in peripheral blood (PB) and, similar to its proposed role in human SLE, IFN signaling is required for the production of pathogenic autoantibodies and glomerulonephritis [[25]]. As such, we assessed changes in immune status associated with Irf5 loss in this model.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information

Irf5 is required for production of IgG lupus-associated autoantibodies but not IgM

Autoantibodies directed against nuclear components, such as DNA/protein or RNA/protein macromolecular complexes, are a diagnostic feature of SLE and contribute to disease pathogenesis [[1]]. Pristane induces the production of lupus autoantibodies ∼4–6 month postperitoneal injection [[27]]. At 10 months postinjection, Savitsky et al. reported a decrease in antinuclear antibodies (ANAs) in the sera of pristane-injected Irf5−/− mice that was, in part, due to a decrease in anti-dsDNA and anti-Sm IgG2a and IgG2b lupus autoantibodies [[24]]. We observed a similar decrease in sera ANAs 6 months postinjection by HEp-2 immunostaining (Fig. 1A); ten of 12 Irf5−/− mice had no detectable ANA staining while the remaining two lacked cytoplasmic staining and gave weak positive homogenous nuclear staining (data not shown). To extend upon the repertoire of lupus autoantibodies that may be affected by loss of Irf5, we analyzed additional autoantibodies (anti-Ribosomal Phosphoprotein P0 (anti-RiboP0), anti-U1A, anti-sn RNP BB′ (RNP BB′), and anti-histone) that are present in pristane-induced SLE [[25, 28]]. This analysis confirmed a marked reduction in IgG autoantibody levels of Irf5−/− mice targeted against a variety of autoantigens (Fig. 1B). Furthermore, we show that IgM autoantibodies are unaffected by loss of Irf5.

image

Figure 1. IRF5 is required for pristane-induced IgG autoantibody production. (A) ANAs in sera obtained from 12 Irf5−/− and Irf5+/+ littermate mice 6 months postpristane injection were assayed by fluorescent immunostaining of Hep2 cells. A representative positive section from each genotype is shown; ten of 12 Irf5−/− mice were negative for ANA. Bar = 20 μM. (B) The relative amounts of IgG and IgMautoantibodies against ribo P0, U1A, RNP b/b, and histone(s), in the serum of pristane (PZ) injected Irf5+/+ (WT; black bar; n = 13) and Irf5−/− (KO; white bar; n = 10), and PBS (Ctl) injected WT(n = 6) and KO (n = 5) mice 6 months postinjection. Data are presented as mean + SEM and are representative of two experiments. *p < 0.05; **p < 0.01; ***p < 0.001, Mann–Whitney U test.

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Irf5−/− mice lack IgG2a production and drive IgG1 class switching

Pristane-induced lupus is associated with hypergammaglobulin-emia and marked polyclonal B-cell activation [[29]]. In mice, IgG2a/c autoantibodies are considered to be the most pathogenic, while IgG1 displays the poorest pathogenicity [[30]]. Of the total sera IgG produced in response to pristane, IgG2a/c predominates, with relatively smaller differences observed in IgG1 levels between pristane- and PBS-injected mice [[31]]. Examination of total serum IgG subclasses (IgG1, IgG2a/c, IgG2b, and IgG3) in wild-type and Irf5−/- mice revealed significant decreases in both IgG2a/c and IgG2b levels of Irf5-deficient mice; in addition, we observed a striking increase in IgG1 levels of Irf5−/− mice (Fig. 2A). The decrease in total IgG2a/c and IgG2b levels correlated with significant decreases in specific lupus autoantibodies (Supporting Information Fig. 1A).

image

Figure 2. IRF5 controls IgG1 in pristane-induced hypergammaglobulinemia. (A) The relative amount of total serum IgG isotypes from pristane-injected Irf5+/+ (WT; black bar; n = 12) and Irf5−/− (KO; white bar; n = 12), and PBS-injected WT (n = 5) and KO (n = 5) mice 6 months postinjection was quantified by ELISA at 1:100,000 serum dilution. Data are presented as mean + SD and are representative of three experiments. *p < 0.05, Student's t-test. (B) TLR7-associated serum IgG1 autoantibodies are decreased in Irf5−/− mice. Serum from pristane-injected Irf5+/+ (WT; black bar; n = 10) and Irf5−/– mice (KO; white bar; n = 10), and PBS-injected WT (n = 4) and KO (n = 5) mice 6 months postinjection were measured for IgG1 antibodies against P0, U1A, and dsDNA by ELISA. Data are presented as mean + SEM and are representative of three experiments. *p < 0.05, Mann–Whitney U test.

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Irf5 regulates antigen-specific IgG1 following pristane injection

T cells are required for IgG1 and IgG2a/c hypergammaglobulinemia in pristane-injected mice [[32]]. While data in Fig. 2A reveal that Irf5−/− mice produce robust levels of IgG1, indicating that Irf5−/− T cells are capable of supporting the generation of IgG1 hypergammaglobulinemia, the ability of Irf5−/− mice to generate antigen-specific IgG1 is inconclusive. Lien et al. recently showed that Irf5−/− mice were impaired in their generation of antigen-specific IgG1 by T cell-dependent or T cell-independent immunogens. [[33]]. A similar analysis revealed contrasting data demonstrating increased antigen-specific IgG1 production from Irf5−/− mice [[24]].

To clarify the role of IRF5 in generating antigen-specific IgG1 and to determine whether the observed increase in IgG1 hypergammaglobulinemia in Irf5−/− mice (Fig. 2A) results in elevated IgG1 autoantibody production, we analyzed autoantibody isotypes in Irf5+/+ and Irf5−/− mice 6 months postpristane injection. As expected, IgG2a/c and IgG2b autoantibodies against dsDNA, RiboP0, U1A, U1B′/B were absent or significantly reduced in pristane-injected Irf5−/− mice (Supporting Information Fig. 1A and data not shown). Similarly, serum anti-RiboP0 and anti-U1A IgG1 levels were significantly reduced in Irf5−/− mice, whereas anti-dsDNA IgG1 autoantibodies were similar between Irf5+/+ and Irf5−/− mice (Fig. 2B). Given that Irf5−/− mice have intact IgG1 class switching, the impaired production of certain IgG1 autoantibodies in this model of murine lupus supports the existence of a mechanism(s) other than class switching for the regulation of IgG1 autoantibodies by IRF5. Furthermore, the reduction in IgG1 anti-U1A, but not dsDNA, autoantibodies indicate that the TLR7-IRF5 axis is important for the development of pristane-induced autoantibodies and controls autoantibody specificity.

Pristane induces Th2 polarization in Irf5−/− mice

T helper (Th) 1 and 2 cells promote the production of IgG2a/c and IgG1, respectively; Th1-mediated autoimmune responses generate the more pathogenic autoantibodies and are thus associated with the progression of murine lupus [[34]]. Imbalance of Th1 and Th2 cytokine homeostasis is a prominent feature of both experimental and human SLE [[35, 36]]. Given that IRF5 has been linked to proinflammatory cytokine expression [[17]], and several cytokines, such as IL-4, IL-5, IL-6, and IL-10, are known to promote antibody production [[37-41]], serum cytokine levels in PBS- and pristane-injected Irf5+/+ and Irf5−/− littermates were measured using the MILLIPLEX mouse kit (Millipore, Billerica, MA, USA). As early as 2 weeks postinjection, serum levels of the Th2 cytokine IL-10 were significantly elevated in Irf5−/− mice compared with those of Irf5+/+ mice (Fig. 3A); 6 months postinjection, only Th2 cytokines IL-4 and IL-5 were upregulated (Fig. 3B). There was no difference in serum levels of the Th1 cytokine IL-12p40 (Fig. 3C). As expected, serum levels of IL-6 were decreased in Irf5−/− mice, whereas TNF-α levels remained unchanged between wild-type and Irf5−/− mice (Fig. 3C). The increase in serum Th2 cytokines may contribute to disease protection in Irf5−/− mice since Th2 cells promote production of the least pathogenic IgG1 isotype [[34]] observed in Irf5−/− mice (Fig. 2A).

image

Figure 3. Th2 polarization in Irf5-deficient mice. (A) Two weeks after injecting Irf5−/− (KO; n = 6) or Irf5+/+ (WT; n = 5) mice with pristane, serum was collected and IL-10 quantified using a Multiplex cytokine assay. Data are presented as mean + SEMand are representative of two experiments. *p < 0.05, Mann–Whitney U test. (B and C) WT (n = 16) or Irf5−/− (n = 15) mice were immunized with PBS or pristane. Six months later mice were culled and serum was tested for (B) IL-4 and IL-5 or (C) IL-12 p40, IL-6, and TNF-α. Data are presented as mean/median of six replicates and are representative of three independent experiments *p < 0.05; **p < 0.01, Mann–Whitney U test.

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To address whether T cells from pristane-injected Irf5−/− mice are capable of producing Th2 cytokines, PMA/ionomycin-stimulated splenocytes from Irf5+/+ and Irf5−/− littermate mice 6 months postinjection were surface-stained with CD4 antibodies followed by intracellular staining for IL-4 and IFN-γ. Our results demonstrate that CD4+ T cells from Irf5+/+ mice produce negligible amounts of IL-4; in contrast, a fraction of CD4+ T cells from Irf5−/− mice produced IL-4 (Fig. 4A). Both Th1 (IFN-γ+IL-4) and Th2 (IFN-γIL-4+) cells, but not Th0 (IFN-γ+IL-4+), exist in Irf5−/− mice, whereas only Th1 cells could be detected in Irf5+/+ mice. The frequency of IFN-γ producing CD4+ T cells from Irf5−/− mice was comparable with those from Irf5+/+ (Fig. 4A). Together, these data support a critical role for IRF5 in the regulation of Th1/Th2 polarization contributing to pristane-induced lupus pathogenesis. Since IFN-γ production was not impaired in T cells from Irf5−/− mice, the emergence of Th2 cells in Irf5−/− mice is not solely due to a lack of Th1 polarization in this model.

image

Figure 4. Intracellular IL-4 is upregulated in CD4+Tcells of pristane-injected Irf5−/− mice. (A) Flow cytometric dot plot of IL-4 and IFN-γ intracellular expression by splenic CD4+T cells from Irf5+/+ (WT) and Irf5−/− mice 6 months postpristane injection (left). Plot is representative of n = 5 replicates. Quantification of the percentage of CD4+T cells from WT(n = 5) and KO (n = 5) mice expressing IFN-γ or IL-4 (right). (B) IRF5 controls early activation of T cells. Flow cytometric dot plot of CD4 and CD69 expression by splenic T cells from Irf5+/+ (WT) and Irf5−/− mice 6 months postpristane injection (left). Quantification of CD4+CD69+T cells from n = 5 mice per group (right). Data are presented as mean + SD and are representative of two experiments. **p < 0.01, Student's t-test.

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Irf5 is required for early T-cell activation

In SLE, activated T and B cells can infiltrate tissues to cause organ damage. Recent data in the Yaa murine lupus model [[23]] indicated that IRF5 was critical for T-cell activation. In a similar manner, we investigated whether loss of Irf5 affects lymphocyte activation. At 6 months postpristane, we found that expression of the early activation marker CD69, in splenic CD4+ T cells of Irf5−/− mice, was significantly reduced (Fig. 4B).

Loss of type I IFN signature in pristane-induced Irf5−/− mice

Because IRF5 regulates type I IFN production [[15, 42]] and type I IFN signaling is central to the pathogenesis of pristane-induced SLE [[25]], we examined the contribution of IRF5 to pristane-induced type I IFN production. Levels of serum IFN were determined by the type I IFN reporter cell assay that measures the ability of sera to cause IFN-induced gene expression [[43]]. A significant decrease in mRNA levels of the IFN stimulated gene (ISG) IRF7 was observed in L929 cells stimulated with sera from pristane-injected Irf5−/− mice as compared with Irf5+/+ (Fig. 5A). No increase in surface expression of the ISG Sca-1 [[44, 45]] was observed on CD19+ B cells from the PB of Irf5−/− mice 2 weeks postpristane injection (Fig. 5B). Similar results were found at 6 months postinjection in different cellular compartments of Irf5−/− mice (Fig. 5C) and decreased mRNA expression of the ISGs — MCP-1 (ccl2) and MX1 (myxoma response protein) — was also observed in the bone marrow (BM) of Irf5−/− mice (Supporting Information Fig. 2).

image

Figure 5. Type I IFN gene signature induced by pristane is IRF5 dependent. (A) IRF7 mRNA expression in L929 reporter cells was determined after stimulation in vitro with sera from Irf5+/+ (WT; n = 5) and Irf5−/− (KO; n = 6) mice 2 weeks postpristane injection. (B) Representative flow cytometric analysis of Sca-1 surface expression on CD19+PBMCs 2 weeks postpristane injection. Mean fluorescence intensity (MFI) of Sca-1 on CD19+ cells is shown. (C) Quantification of Sca-1 on B220+B cells from PB (n = 5 per group), BM, and spleen (n = 9 per group) of Irf5+/+ (WT) and Irf5−/− (KO) mice 6 months postpristane injection. Data are presented as mean + SEM and are representative of two experiments. **p < 0.01; *p = 0.05, Mann–Whitney U test. (D) qPCR analysis of IRF7 and MX1 expression in purified peritoneal Ly6GCD11B+ monocytes (normalized to peritoneal cells from untreated mice) of Irf5+/+ (WT; n = 3) and Irf5−/− (KO; n = 3) mice 4 weeks after pristane injection. Data are presented as mean + SD and are representative of two experiments. *p < 0.05, Student's t-test.

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Ly6Chi monocytes, which are recruited rapidly to the peritoneal cavity (PC) in response to pristane, are thought to be the major source of type I IFN in this model [[44]]. As such, Ly6Chi monocytes were isolated from the PC [[45]] and quantitative PCR (qPCR) performed to determine IRF7 and MX1 mRNA levels. Expression of IRF7 and MX1 were significantly decreased in Irf5−/− mice (Fig. 5D). Thus, we provide evidence to show that the IFN signature in pristane-injected Irf5−/− mice is greatly diminished, but not completely absent, at both acute (2 weeks) and chronic (6 months) stages of the disease suggesting yet another mechanism of protection for Irf5−/− mice.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information

The findings presented here strongly support a multifaceted role for IRF5 in the regulation of autoimmunity. Consistent with recent reports [[23]], we show that IRF5 is required for the development of ANAs in response to pristane. We replicate the lack of IgG2a/c autoantibodies in pristane-injected Irf5−/− mice [[24]]; however, in addition, we found that a defect in IgG2a/c class switch recombination (CSR) is already present in naive Irf5−/− mice (Supporting Information Fig. 1B). A similar defect in the generation of IgG2a and 2b was shown in Tlr9- and Myd88-deficient FcγRIIB−/−.B6 lupus mice and T-bet-deficient MRL/lpr mice [[46, 47]]. These results implicate IRF5 as a critical factor regulating both basal and stimuli-induced IgG2a/c class switching. The finding that Irf5 is required for pristane-induced IgG2a/c and IgG2b hypergammaglobulinemia and not IgG1 hypergammaglobulinemia led us to examine whether the skewing of IgG isotypes was an intrinsic or extrinsic effect. Data from in vitro stimulations examining IgG1 class switching support a B-cell intrinsic defect in Irf5−/− mice (Supporting Information Fig. 3). Whether T-cell intrinsic/extrinsic defects in Irf5−/− mice as well contribute to the overall skewing of IgG isotypes is not currently clear. Data from Savitsky et al. [[24]] suggest that in vitro T-cell polarization is unaffected in Irf5−/−mice, while in vivo data presented here indicate impaired production of IL-4 in CD4+ T cells from pristane-injected Irf5−/− mice (Fig. 4A). Together, these data suggest a T-cell extrinsic defect in Irf5−/− mice. Similar to findings by Richez et al. [[23]], we observed a defect in T-cell activation in Irf5−/− mice (Fig. 4B). Additional studies are required to clarify the role of IRF5 in T-cell polarization, activation, and function. Nevertheless, results from the present study suggest that dysregulation of IRF5 expression in human SLE is likely to affect both B- and T-cell function(s) ultimately contributing to pathogenic autoantibody production.

In animal models, TLR9 contributes to the development of anti-chromatin autoantibodies and TLR7 to the development of anti-RNP autoantibodies; MyD88 and a number of transcription factors including IRF5 mediate the effects of TLR7/9 engagement [[48]]. Our data indeed support a downstream role for IRF5 in both TLR pathways since Irf5-deficient mice are unable to generate TLR7- or TLR9-associated IgG2a autoantibodies (Supporting Information Fig. 1A); however, they do not preclude a TLR-independent role for IRF5 in autoantibody production since antigen specificity could not be addressed by studying the IgG2a isotype. Indeed, a thorough analysis of nonisotype-specific autoantibodies in Irf5-deficient RII.Yaa mice led Richez et al. to suggest that IRF5 plays a nonredundant role in TLR7 and TLR9 signaling in SLE and/or contributes to autoantibody production independent of these two pathways [[23]]. In Irf5−/− and Irf5+/− RII.Yaa mice, all four IgG isotypes were dramatically decreased, whereas sera IgG1 levels in Irf5+/− RII mice were comparable with Irf5+/+ RII mice [[23]]. In the pristane-induced model of murine lupus, we found that Irf5−/− mice had only striking reductions in IgG2a/c and IgG2b antibody levels whereas IgG1 levels were elevated. These data suggest that a lack of Irf5 does not reduce long-lived IgG1 expressing plasma cells. After class switching, autoreactive B cells may undergo further selection and expansion. In order to address the role of IRF5 in selecting or expanding B-cell clones with autoreactive specificity, we examined the production of antigen-specific IgG1. We found that Irf5−/− mice are deficient in their production of lupus IgG1 autoantibodies, suggesting that a mechanism other than class switching regulates antigen specificity in these mice. Instead, IRF5 may be critical for selection or expansion of autoreactive clones from the B-cell repertoire. The selective impairment of TLR7- and not TLR9-associated IgG1 autoantibody production indicates a distinct and likely more critical role for IRF5 in mediating TLR7 signaling in pristane-induced lupus. Whether this proves true in human SLE is not currently known.

CSR of B cells from IgM to IgG is dependent on the cognate interaction of B cells with Th cells [[49]]. Although CD40L–CD40 interaction is necessary to initiate Ab isotype switching [[50]], it is assumed that Th cell-derived cytokines determine whether B cells are switched to IgG1 or IgG2a [[51]]. IFN-γ and IL-4 are key cytokines of Th1 and Th2 cells, respectively, although IL-5, IL-10, and IL-13 are also produced by Th2 cells. To determine whether the cytokine milieu in Irf5−/− mice contribute to their production (or inhibition) of IgG isotypes, we measured serum cytokine levels in response to pristane. The Th2 cytokines IL-4 and IL-5 were significantly upregulated in the serum of pristane-injected Irf5−/− mice; intracellular IL-4 was also elevated in CD4+ T cells from pristane-injected Irf5−/− mice (Fig. 4A). IL-4 and IL-5 have been shown to be protective against SLE in certain murine models [[35, 52]]. These data support a Th2 polarization in Irf5−/− mice that would be expected to drive IgG1 class switching. However, Th2 polarization does not necessarily entail inhibition of Th1 as Th1/Th2 coexist and tipping the balance one way or the other is all that may be required to affect a systemic autoimmune disease such as lupus [[53, 54]]. Indeed, we did not observe downregulation of the key Th1 cytokine IFN-γ in T cells. Given that IgG2a/c CSR is induced by IFN-γ, and Irf5−/− mice make sufficient levels to induce IgG2a CSR (Fig. 4A), the inability of Irf5−/− mice to produce IgG2a/c autoantibodies in the presence of IFN-γ provides further support for an intrinsic defect in IgG2a/c CSR.

Other factors capable of regulating IgG class switching include IFN-α (potently induces IgG2a and IgG3 [[55]]). The significant decrease in the type I IFN signature of pristane-injected Irf5−/− mice may also contribute to the loss of IgG2a class switching, although recent data suggest that exogenous type I IFN does not rescue the defect in IgG2a secretion in Irf5−/− B cells [[24]]. Previous studies on IFNAR−/− mice [[23, 31]] provide further support of differences in lupus development between Irf5−/− and IFNAR−/− mice. Pristane-injected IFNAR−/− mice retained positive ANA staining with a mean titer value lower than wild-type controls and equivalent IgG2a autoantibodies [[31]]. In the FcRIIb−/− murine lupus model, mice lacking Irf5 were completely protected from disease development while mice lacking IFNAR maintained a substantial level of residual disease [[23]]. These data support distinct phenotypic differences between Irf5−/− and IFNAR−/− mice suggesting that the role of IRF5 in lupus pathogenesis exceeds beyond its regulation of type I IFN production.

Interestingly, we also detected significantly elevated levels of IL-10 in the sera of Irf5−/− mice 2 weeks postpristane injection (Fig. 3A). Given that IL-10 is a Th2 cytokine and downregulates IFN-α production [[56, 57]], early expression in Irf5−/− mice may indirectly contribute to reduction of the type I IFN signature. Recent data in human macrophages reveal that IL-10 is a direct target of IRF5 and overexpression of IRF5 represses IL-10 expression while M1 murine macrophages lacking Irf5 express elevated levels [[58]]. Although IRF5 has been shown to directly regulate type I IFN expression [[15, 42]], other indirect mechanisms via IRF5 may contribute to the downregulation of a type I IFN signature in pristane-induced lupus. With respect to serum IL-10 levels, our data suggest that two mechanisms exist that control the acute (2 weeks) and chronic (6 months) expression of type I IFNs in this model.

In summary, our study highlights the regulatory role of IRF5 in the onset of pathological hypergammaglobulinemia in pristane-induced lupus. We reveal that Irf5 is indispensable for the maintenance and production of IgG2a/c autoantibodies. In addition, we demonstrate that IRF5 regulates not only CSR, but also antigen specificity. We show that loss of Irf5 significantly alters cyto-kine production in response to pristane, ultimately skewing the cytokine (and autoantibody) profile toward a Th2-like response, and inhibits the type I IFN signature that is critical for disease pathogenesis in this model of lupus. Given the current data in human SLE and murine models of lupus [[35, 36, 39]], it would be expected that factors capable of regulating the Th1/Th2 balance would potentially alter lupus development. To this extent, we also provide evidence that T-cell polarization is altered in Irf5−/− mice and that IRF5 has a critical role in T-cell activation. These findings greatly extend our current, but limited understanding of the global effect that IRF5 has on autoimmunity and provides significant new insight into how overexpression of IRF5 in blood cells of SLE patients may contribute to disease pathogenesis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information

Mice

Irf5−/− mice backcrossed eight generations to C57Bl/6 were obtained from T. Taniguchi (University of Tokyo, Tokyo, Japan) and T. Mak (University of Toronto, Toronto, Ontario, Canada) [[17]]. Wild-type C57Bl/6 were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Backcrossed heterozygotes were intercrossed to obtain a cohort of Irf5+/+ and Irf5−/− littermates, by standard breeding techniques. Littermate Irf5+/+ mice were used as controls. Presence of the recently described Dock2 mutation in Irf5−/− mice was analyzed by PCR genotyping [60]. 80% of the Irf5−/− mice used in this study had wild-type Dock2 and 20% were heterozygous for the Dock2 mutation; none of the mice used in this study were homozygous for the Dock2 mutation. Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry of New Jersey, New Jersey Medical School. Eight-week-old mice received a single intraperitoneal (i.p.) injection of 0.5-mL pristane (Sigma-Aldrich, St. Louis, MO, USA) or PBS. Mice were sacrificed for tissue and blood at 6 months postinjection unless otherwise indicated.

Ig isotype detection

Nunc Maxisorp plates (VWR, West Chester, PA, USA) were coated with 5 μg/mL goat anti-mouse Ig (heavy and light chain) antibody (Southern Biotechnology, Birmingham, AL, USA) overnight at 4°C. Coated wells were blocked with 3% BSA for 1 h and then diluted sera samples (1:100,000 for serum from pristane-injected mice; 1:1 for in vitro supernatants) in 3% FBS and 0.05% Tween-20 were added. After washing, 2 μg/mL of biotinylated rat anti-mouse isotype-specific antibodies (Biolegend, CA, USA) were added and incubated for 1 h then 0.5 μg/mL avidin-conjugated HRP (Biolegend) was added for 30 min at room temperature (RT). After additional washing, 1-Step Ultra TMB-ELISA (Thermo Scientific, Waltham, MA, USA) was used for color development. All dilution buffers contained 3% BSA.

Autoantibody detection

For dsDNA, plates were coated with 0.01% (w/v) poly-L-lysine (Sigma-Aldrich) for 45 min at RT followed by addition of 5 μg/mL double-stranded (ds) plasmid DNA and incubated overnight at 4°C. For other types of ELISA, 1 μg/mL of antigen including U1A and RiboP (Diarect, Freiburg, Germany) were used to coat the plate overnight at 4°C. Sera were diluted 1:100 and incubated in the plate for 1 h at RT. Biotinylated rat anti-mouse isotype-specific antibodies (Biolegend) were then incubated for 1 h. As described previously, avidin-conjugated HRP (Biolegend) was added for 30 min at RT, followed by 1-Step Ultra TMB-ELISA. The method of Thibault et al. was used to detect IgG and IgM autoantibody levels [59].

Cell purification and class switching

Naive B cells were negatively isolated from splenocytes using Miltenyi's B-cell isolation kit containing biotin-conjugated monoclonal antibodies (mAbs) to CD43, CD4, and Ter-119 beads. B cells were cultured in RPMI 1640 medium supplemented with 1% glutamine, 1% penicillin/streptomycin, 10% FBS, and 50 μM β-ME. 2 × 105 B cells per well were seeded in 96-well plates and stimulated with 1 μg/mL Gardiquimod (Invivogen, San Diego, CA, USA), 10 μg/mL anti-CD40 mAb (Biolegend), or in combination with 20 ng/mL IL4 (R&D Systems, Minneapolis, MN, USA). Supernatants were collected after 7 days and Ig isotype was assayed.

Cytokine detection

Bead-based sandwich immunoassay for cytokines using MILLIPLEX MAP multiplex mouse cytokine/chemokine kit (Millipore, Billerica, MA, USA) was performed according to the manufacturer's instruction. Samples were analyzed with a Luminex 100 Multi-Analyte Profiling System (Luminex Corp, Austin, TX, USA). Cytokine concentrations were determined by standard curve, which were generated using the mixed standard provided with the kit.

Flow cytometry

Single-cell suspensions of spleen cells, BM, or PB cells were stained with fluorochrome-labeled mAb (Biolegend) against CD4 and CD8 for T cells, B220 or CD19 for B cells, Sca-1 for B-cell activation, and CD69 for T-cell activation. For intracellular cytokine detection, 106 splenocytes or isolated cells were stimulated with phorbol myristate acetate (PMA) (Sigma, St Louis, MO, USA) (0.02 μg/mL) and Ionomycin (3 μM) for 4 h in the presence of Brefeldin A (10 μg/mL; Sigma). After incubation, cells were fixed using 2% PFA and then permeabilized in 0.5% saponin buffer, followed by addition of cytokine detection antibodies. Samples were acquired on a FACS Calibur and data analyzed using FlowJo (Tree Star, Inc., Ashland, OR, USA) software.

Real-time qPCR

BM cells were collected from femurs of pristane-injected mice. Peritoneal lavage was collected from pristane-injected mice. Peritoneal cells were harvested by centrifugation and enriched for monocytes by negative selection using biotinylated mAb (Biolegend) against Ly6G+, Ter119+, CD3+, CD19+, and anti-biotin MACS MicroBeads (Miltenyi Biotec, Cambridge, MA, USA). qPCR was performed as previously described [[14]]. Briefly, total RNA was extracted from cells using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA), cDNA was prepared using qScript cDNA supermix kit (Quanta Biosciences, Gaithersburg, MD, USA), and qPCR was performed using iTaq SYBR Green Supermix (Bio-rad, Hercules, CA, USA). Primer sequences used were as follows: MCP1 F: 5-TTAA AAAC CTGGA TCGGAA CCAA-3 and R: 5-GCATTAG CTT CAGAT TTACG GGT-3; MX1 F: 5-GATC CGA CTTC ACTTC CAG ATGG-3 and R: 5-CATCTC AGTGG TAGT CAAC CC-3; b-actin F: 5-AT GCTCT CCCT CACG CCATC-3 and R: 5-CACGC ACGAT TTCCC TCTCA-3. All reactions were performed in the 7300 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) under the following conditions: 1 cycle of 45°C (3 min) and 95°C (10 min), followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The delta Ct method was used to calculate relative expression.

IFN-α reporter cell assay

A modified IFN-α assay [[45]] was used to measure the ability of murine lupus sera to cause IFN-induced gene expression. A total of 1 × 105 reporter cells (L929, no. CCL-1; American Type Tissue Collection, Manassas, VA, USA) were cultured with 15% wild-type or Irf5-deficient serum for 6 h and total cellular mRNA isolated. Reverse-transcription and qPCR were performed as described above with primers for murine IRF7 (F: 5-GACCTTGGATCTACTGTGG-3 and R: 5-TAGAAAGCAGAGGGCTTG-3) and b-actin. The delta Ct method was used to calculate relative IRF7 expression.

Statistical analysis

For normally distributed variables, differences between groups were analyzed by the Student's t-test. For variables not normally distributed, the Mann–Whitney U test was used. Normality was assessed by the Shapiro–Wilk test. Data are presented as mean ± SD (normal distribution) or mean ± SEM (nonnormal distribution). p value < 0.05 was considered significant. Statistical analyses were performed using Prism 4.0 (GraphPad Software, San Diego, CA, USA).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank I. Rifkin for providing the Irf5-deficient mice by approval from Tadatsugu Tanaguchi and Tak Mak. We thank R. Donnelly for help with the Luminex assays. This work was supported by grants from the National Institute of Health (NIH)/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS; 5R03AR054070) and the Arthritis Foundation (to B.J.B.).

The authors declare no financial or commercial conflict of interest.

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information
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Abbreviations
ANA

antinuclear antibodies

CSR

class switch recombination

IC

immune complex

IRF

interferon regulatory factor

ISG

IFN-stimulated gene

PB

peripheral blood

PC

peritoneal cavity

RNP

ribonucleoprotein

SLE

systemic lupus erythematosus

snRNP

small nuclear RNP

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. References
  9. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
eji2258-sup-0001-s1.pdf303KFigure 1. Serum IgG2a/c and IgG2b autoantibodies are decreased in Irf5–/– mice.
eji2258-sup-0001-s1.pdf303KFigure 2. Decreased MCP-1 and MX-1 mRNA expression in bone marrow of Irf5–/– mice.
eji2258-sup-0001-s1.pdf303KFigure 3. Loss of Irf5 drives production of the IgG1 isotype in a TLR-dependent and TLR-independent manner.

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