Anti–IFN-α/β Receptor Antibody Treatment Ameliorates Disease in Lupus-Predisposed Mice

Roberto Baccala, Rosana Gonzalez-Quintial, Robert D. Schreiber, Brian R. Lawson, Dwight H. Kono and Argyrios N. Theofilopoulos
J Immunol December 15, 2012, 189 (12) 5976-5984; DOI: https://doi.org/10.4049/jimmunol.1201477

Abstract

The demonstration in humans and mice that nucleic acid-sensing TLRs and type I IFNs are essential disease mediators is a milestone in delineating the mechanisms of lupus pathogenesis. In this study, we show that Ifnb gene deletion does not modify disease progression in NZB mice, thereby strongly implicating IFN-α subtypes as the principal pathogenic effectors. We further document that long-term treatment of male BXSB mice with an anti–IFN-α/β receptor Ab of mouse origin reduced serologic, cellular, and histologic disease manifestations and extended survival, suggesting that disease acceleration by the Tlr7 gene duplication in this model is mediated by type I IFN signaling. The efficacy of this treatment in BXSB mice was clearly evident when applied early in the disease process, but only partial reductions in some disease characteristics were observed when treatment was initiated at later stages. A transient therapeutic effect was also noted in the MRL-Faslpr model, although overall mortality was unaffected. The combined findings suggest that IFN-α/β receptor blockade, particularly when started at early disease stages, may be a useful treatment approach for human systemic lupus erythematosus and other autoimmune syndromes.

Introduction

Type I IFNs, particularly IFN-αs and IFN-β, have received prominent attention for their role in the pathogenesis of systemic lupus erythematosus (SLE) and other autoimmune and inflammatory syndromes (1, 2). By signaling through a common receptor (IFN-α/β receptor [IFNAR]), these pleiotropic cytokines affect almost every aspect of innate and adaptive immune responses, including upregulation of MHC and costimulatory molecules and production of B cell survival factors (BAFF, a proliferation-inducing ligand) by APCs, culminating in the engagement and expansion of autoreactive T and B cells (1, 2). Of particular relevance to lupus pathogenesis is the induction of type I IFNs under sterile conditions through the engagement of endosomal TLRs by self-nucleic acids (3–6). This systemic autoimmunity-inducing pathway has been well documented by studies showing reduced disease in predisposed mice lacking expression of endosomal TLRs (7), IFNAR (8, 9), or Unc93b1 (10), a molecule that acts as a transporter of TLR3, -7, and -9 from endoplasmic reticulum to endolysosomes.

These findings have stimulated considerable interest in creating treatments based on blocking reagents against either the multiple IFN-αs and the single IFN-β or their common receptor. The potential utility of these approaches would be considerably advanced by further defining the role of type I IFNs in lupus mice with diverse genetic abnormalities, the potential difference in pathogenicity between the IFN-α subtypes and IFN-β, and the clinical stage at which blockade of signaling by these cytokines is effective. In this study, we address some of these issues and demonstrate that the disease-promoting effect of type I IFNs in lupus is primarily mediated by the IFN-αs, type I IFN signaling significantly contributes to disease in BXSB mice but minimally in MRL-Faslpr mice, treatment with an anti–IFNAR Ab has therapeutic efficacy even with partial IFNAR blockade, and effectiveness is most evident when treatment is initiated at early disease stages. These findings provide support for the potential utility of IFNAR blockade for the treatment of human SLE, but suggest that the type of patient and timing of treatment may be crucial factors in determining the outcome.

Materials and Methods

Mice

BXSB.Yaa, MRL-Faslpr, and C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or The Scripps Research Institute Animal Facility. NZB mice deficient for IFNAR1 (Ifnar1−/−) have been reported (8), and marker-assisted congenic NZB mice deficient for IFN-β (Ifnb−/−) were generated as described (8). Mice were housed under specific pathogen-free conditions, and all experimental protocols were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by The Scripps Research Institute Animal Care and Use Committee.

Treatment with a monoclonal anti–IFNAR Ab

Male BXSB mice were treated with a monoclonal anti–IFNAR Ab of mouse origin (clone MAR1-5A3; Leinco Technologies) (11). Injections (i.p., 500 μg for three consecutive days, followed by 250 μg three times per week until experiment termination) were started either before (12 wk of age) or after (17 wk of age) appearance of disease manifestations, as suggested by detectable autoantibody titers and proteinuria. MRL-Faslpr mice were similarly treated, but starting at 7 wk of age due to the expedited disease course in this strain.

Cell preparations

Single-cell suspensions were prepared from bone marrow (BM), blood, peritoneal cavity, spleen, and lymph nodes (inguinal, axillary, brachial, and cervical), as described (12). B cells were purified from spleen or peritoneal cavity using magnetic beads (MACS; Miltenyi Biotec), whereas conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) were prepared by stimulating BM cells with recombinant mouse GM-CSF or Flt3 ligand (R&D Systems), respectively (13).

Flow cytometry

mAbs to mouse CD4, CD8, B220, CD11b, CD11c, pDC Ag-1 (PDCA-1), IFNAR1, CD69, CD86, CD25, CD21, CD23, AA4.1, CD138, I-Ab, H2-Kb, and GR-1 were obtained from BD Pharmingen, BioLegend, or eBioscience. For surface staining, cells were sequentially incubated with various combinations of Abs or streptavidin (BD Pharmingen). Cell events were acquired on four-color FACSCalibur and data analyzed using FlowJo software (Tree Star).

In vitro studies

Purified splenic B cells and BM-derived cDCs and pDCs were cultured in complete medium and stimulated or not with mouse IFN-α11 (1000 U/ml; Miltenyi Biotec), the TLR7 ligand R848 (30 ng/ml; InvivoGen), or both, in the presence or absence of the anti–IFNAR Ab (10 μg/ml). Splenic T cells were stimulated with plate-bound anti-CD3 and plate-bound or soluble anti-CD28 Abs in the presence or absence of anti–IFNAR Ab (10 μg/ml). At the indicated time points, cells were harvested, counted, and analyzed by flow cytometry, whereas supernatants were assayed for cytokines or IgM titers by ELISA.

ELISA

ELISA for polyclonal IgM and IgG was performed using 96-well plates coated with goat anti-mouse Ig (Jackson ImmunoResearch Laboratories) and for anti-chromatin and anti-ribonucleoprotein (RNP) autoantibodies using plates coated with chromatin or RNP (Inova Diagnostics), respectively. Bound Abs were detected using alkaline phosphatase–conjugated goat Abs specific for mouse IgM, IgG, and IgG isotypes (Southern Biotechnology Associates), and standard curves were generated using calibrated mouse serum (Nordic Immunology). Commercial ELISA kits were used to examine B cell and DC culture supernatants for the presence of IL-6, IL-10 (BioLegend), or IFN-α (PBL InterferonSource).

Anti-nuclear and anti-erythrocyte autoantibodies

Anti-nuclear autoantibodies (ANA) were detected using HEp-2 slides (Bion Enterprises), serial serum dilutions (1/10 to 1/3000), and Alexa Fluor 488–conjugated goat anti-mouse IgG (Invitrogen), and the results were expressed as the inverse of the maximal serum dilution giving positive staining. Erythrocyte-bound autoantibodies were quantified by flow cytometry after sequential incubation of washed RBCs with biotinylated goat anti-mouse IgG (MP Biomedicals) and streptavidin-PE (BioLegend).

Kidney pathology and immunohistology

Proteinuria was determined using Albustix strips (Bayer Corporation) and graded semiquantitatively (0, negative to traces; 1, 30 mg/ml; 2, 100 mg/ml; 3, 300 mg/ml; and 4, 2000 mg/ml). Zinc formalin–fixed and periodic acid–Schiff/hematoxylin stained tissue sections were scored blindly on a 0–4 scale for degree of glomerulonephritis (GN) (14). Kidney sections were examined by immunofluorescence for the presence of immune deposits and cellular infiltrates using Abs to mouse IgG2a (Invitrogen), C3 (Nordic Immunology), CD11c, or CD11b (BD Biosciences), whereas spleen sections were stained with Abs to CD19 (B cells), PDCA-1 (pDC) (BD Biosciences), or TLR7 (Imgenex). Glomerular IgG2a deposits and CD11b and CD11c staining were scored blindly on a 0–4 scale based on the intensity of the immunofluorescence signal.

Statistical analysis

Group comparisons were analyzed by unpaired two-tailed Student t test. Survival was analyzed by Kaplan–Meier plot and log-rank test. The p values <0.05 were considered significant.

Results

Differential roles of IFN-α and IFN-β in systemic autoimmunity

We have reported that lupus-predisposed NZB mice lacking the IFNAR1 subunit of the common receptor for type I IFNs showed significant reductions of disease manifestations (8). We generated Ifnb−/− NZB mice to determine whether the disease-inhibiting effect of Ifnar1 gene deletion results from absence of signaling by the multiple IFN-α or the single IFN-β. We found that, unlike Ifnar1−/− NZB mice, disease progression and severity were unaltered in Ifnb−/− NZB mice compared with wild-type (WT) controls, including autoantibody production, hemolytic anemia, kidney disease, and mortality (Fig. 1). The results indicated that the autoimmunity-promoting effects of type I IFNs in this model are primarily mediated by IFN-αs. Therefore, because of the multiplicity of IFN-αs, therapeutic inhibition of signaling by these cytokines may be best accomplished using an anti–IFNAR Ab.

FIGURE 1.
FIGURE 1.

IFN-β is not required for lupus-like disease in NZB mice. Groups of Ifnb−/− and WT NZB mice (n = 8–9/group) were followed for disease manifestations. (A) Anti-RBCs and anti-chromatin autoantibody levels at 9 mo of age. (B) Proteinuria and GN scores at 10 mo of age. (C) Survival. p > 0.05.

In vitro effects of IFNAR blockade

Using the anti–IFNAR mAb MAR1-5A3 (11), we assessed surface IFNAR expression in immunocytes from male BXSB mice, which carry the Yaa-associated Tlr7 gene duplication (15, 16). IFNAR levels were comparable in CD4 and CD8 T cells, B cells, and monocytes (CD11b+CD11c−), but lower in cDCs (CD11b+CD11c+ and CD11b−CD11c+) and higher in pDCs (PDCA-1+CD11clow) (Fig. 2A), and similar findings were obtained with cells from control C57BL/6 mice (not shown). IFNAR expression remained unchanged in B cells cultured for 24 h in the absence of stimuli, modestly decreased following incubation with IFN-α, and increased ∼2-fold upon TLR7 stimulation, whereas the combination of IFN-α and a TLR7 ligand had a null effect (Fig. 2B). Thus, ligand-induced IFNAR downmodulation, a mechanism critical for signal termination (17–19), is either inhibited or compensated upon TLR7 signaling, likely to sustain an efficient response.

FIGURE 2.
FIGURE 2.

IFNAR expression in BXSB mice. (A) Ex vivo analysis. Spleen cells from BXSB mice (16 wk old, n = 3) were examined by flow cytometry to assess cell-surface IFNAR expression after gating on T cells (CD4+ and CD8+), B cells (B220+), monocytes (CD11b+CD11c−), cDCs (CD11c+CD11b+ and CD11c+CD11b−), and pDCs (PDCA-1+CD11clow). Gray-filled histograms indicate background staining. (B) IFNAR modulation. Splenic B cells from BXSB mice (n = 2) were cultured in vitro for 24 h with medium alone (control) or medium containing IFN-α, the TLR7 ligand R848, or both. IFNAR expression on gated B220+ cells (black-lined histograms) was examined as in (A). Background staining (gray-lined histograms) and IFNAR levels in noncultured ex vivo B cells (gray-filled histograms) are also depicted. Numbers in histograms represent GMFI ± SD. Asterisks indicate statistical significance (*p < 0.05).

We next examined the effect of anti–IFNAR Ab on in vitro responses by B cells, T cells, and DCs. Complete IFNAR blockade (Fig. 3A) significantly inhibited the synergistic effect of IFN-α and TLR7 stimulation on B cells, including upregulation of CD69 and CD86, and production of IL-6, IL-10, and IgM (Fig. 3B, 3C). The Ab treatment also partially inhibited TLR7 ligand–induced production of IL-6 by cDCs and pDCs and IFN-α by pDCs (Fig. 3D), but T cell activation through CD3 and CD28 ligation was unaffected, as suggested by equal upregulation of CD25 and CD69 (not shown). These results indicate that type I IFNs and TLR7 act synergistically, and IFNAR blockade partially inhibits the effects of TLR7 engagement in B cells and DC subsets.

FIGURE 3.
FIGURE 3.

Anti–IFNAR Ab inhibits in vitro activation of B cells, DCs, and pDCs from BXSB mice. (A–C) Effects of IFNAR blockade in B cells. (A) B cells from BXSB mice were incubated for 120 h in medium supplemented or not with anti–IFNAR Ab. Efficiency of IFNAR blockade on BXSB B cells (compared with similarly cultured B cells from WT and Ifnr1−/− C57BL/6 mice) was assessed by flow cytometry and expressed as GMFI ± SD (n = 2–3/group). (B and C) B cells from BXSB mice were stimulated for 120 h with medium alone (control) or medium containing IFN-α, the TLR7 ligand R848, or both in the presence or absence of anti–IFNAR Ab. The effect of IFNAR blockade on B cell activation was evaluated by measuring expression of CD69 and CD86 by flow cytometry (B) and production of IL-6, IL-10, and IgM by ELISA (C). (D) Effects of IFNAR blockade in DC subsets. BM-derived cDCs and pDCs from BXSB mice were stimulated for 24 h with the TLR7 ligand R848 in the presence or absence of anti–IFNAR Ab. Production of IL-6 and IFN-α was determined by ELISA. Bars represent average (± SD) of individual mice (n = 3). Asterisks indicate statistical significance (*p < 0.05).

Early anti–IFNAR treatment of BXSB mice

To examine the effect of IFNAR blockade on systemic autoimmunity, male BXSB mice were treated with anti–IFNAR Ab starting at the preclinical stage (12 wk of age). Treatment consisted of three consecutive daily injections of 500 μg of Ab, followed by 250 μg three times weekly until termination. Assessment of IFNAR expression 4 wk after treatment commencement showed significant, although incomplete, receptor blockade (Fig. 4A). Nonetheless, considerable reductions in most disease parameters were observed. Thus, at 20 wk of age, anti–IFNAR Ab–treated mice showed decreases in polyclonal IgG isotypes (IgG2a, 1.5 ± 0.9 versus 2.7 ± 1.0 mg/ml; IgG2b, 1.2 ± 0.7 versus 2.1 ± 1.0 mg/ml; p < 0.05) and IgG2a anti-chromatin autoantibodies (Fig. 4B). Moreover, whereas ANA patterns of nontreated mice were diverse (homogeneous, nucleolar, and, in some mice, speckled and peripheral), those of treated mice were primarily homogeneous and with reduced titers (Fig. 4B and not shown). In addition, at the termination of the experiment (38 wk of age), GN scores, kidney deposits (IgG2a, C3), and cellular infiltrates (monocytes, DCs) were significantly reduced, and survival was extended compared with controls (Fig. 4C–E). Because glomerular CD11b staining (Fig. 4C) in untreated BXSB mice could be a reflection of the high frequency of blood monocytes in this strain, we performed control staining comparing perfused and unperfused kidneys and found minimal differences (Supplemental Fig. 1). Notably, the glomerular CD11b staining of perfused kidneys remained markedly more intense than that of unperfused kidneys from anti–IFNAR–treated mice. Thus, the reduction of renal mononuclear cell infiltration following IFNAR blockade could not be accounted for by the degree of reduction of these cells in the circulation, but rather suggests that type I IFN signaling mediates attraction of inflammatory cells in the afflicted organs, either directly by inducing specific sets of chemokines or indirectly by promoting autoantibody production and immune complex deposition.

FIGURE 4.
FIGURE 4.

Early anti–IFNAR Ab treatment of BXSB mice. Mice (n = 8–10/group) were treated with anti–IFNAR Ab (or PBS) starting at 12 wk of age. (A) Efficiency of IFNAR blockade as defined 4 wk posttreatment initiation by flow cytometry (average GMFI ± SD) in T cells (CD4+ and CD8+), B cells (B220+), monocytes (MC, CD11b+CD11c−), DCs (DC1, CD11c+CD11−; DC2, CD11c+CD11b+), and pDCs (PDCA-1+, CD11clow). Ifnar1−/− mice were used as negative staining controls (shown for B cells). (B) Serum autoantibody titers (anti-chromatin and ANA) at 20 wk of age. (C) Kidney histology showing glomerular size (periodic acid–Schiff), immune deposits (IgG2a and complement C3), and immunocyte infiltration (CD11b+ and CD11c+ cells) at 38 wk of age (n = 3–4/group). Original magnification ×20 (PAS), ×40 (immunofluorescence). (D) GN scores at 38 wk of age (n = 3–4/group). (E) Survival. Arrow indicates start of Ab treatment. At the termination of the experiment (38 wk of age), three PBS-treated and five anti–IFNAR–treated mice were still alive. Asterisks indicate statistical significance (*p < 0.05).

Lymphoid organ examinations of 38-wk-old anti–IFNAR Ab–treated mice showed reduced splenomegaly and lymphadenopathy (Fig. 5A), mostly resulting from numerical decreases in B cells (Fig. 5B), which are typically expanded in this strain (20). These decreases primarily affected CD21−CD23− B cells, which include the AA4.1low population phenotypically similar to a subset that accumulates in aged normal mice (21, 22) and found in this study to expand in untreated male BXSB mice (Fig. 5C). T2-follicular B cells (CD21lowCD23+) were also reduced in several of the treated mice, whereas the low number of marginal zone B cells (CD21+CD23−) characteristic of male BXSB and other Yaa+ mice (23–25) was retained (Fig. 5B). The percent of CD4+ and CD8+ T cells expressing the CD69 activation marker was also decreased (19 ± 5 versus 32 ± 0.3% for CD4+ cells, and 2 ± 1 versus 12 ± 3% for CD8+ cells; p < 0.05), as were the number of monocytes (CD11b+CD11c−) and the level of MHC class I expression by cDCs (geometric mean fluorescence intensity [GMFI] 68.3 ± 0.2 versus 142.5 ± 37.5; p < 0.05). Other changes in the spleen included reductions in follicle sizes, number of pDCs clustering at the marginal sinus, and TLR7 expression within follicles (Fig. 5E). Overall, the results indicate that early anti–IFNAR Ab treatment significantly reduces disease in male BXSB mice likely by inhibiting activation and expansion of B cells, monocytes, and DC subsets.

FIGURE 5.
FIGURE 5.

Anti–IFNAR Ab treatment inhibits expansion of B cells, monocytes, and DCs in BXSB mice. Mice (n = 8–10/group) were treated with anti–IFNAR Ab (or PBS) from 12–38 wk of age. (A) Weights (± SD) of spleen and lymph nodes (LN; inguinal, axillary, and cervical). (B) B cell subsets. Spleen cells were examined for CD21 and CD23 expression after gating on B220+IgM+ B cells to identify CD21−CD23+ T2/follicular (T2-FO), CD21+CD23− marginal zone (MZ), and CD21−CD23− T1 immature and ABCs. Numbers within plots correspond to average frequency of the indicated subsets. Bar graph indicates cell numbers ± SD. (C) Expansion of CD21−CD23−AA4.1low B cells in untreated BXSB mice. Spleen cells were obtained from male BXSB mice (n = 5; age 21–26 wk) with varying degrees of splenomegaly. The numbers of CD21−CD23−AA4.1low (ABCs) and CD21−CD23−AA4.1+ (T1) B cells were determined by flow cytometry and plotted as a function of the frequency of CD21−CD23− B cells for each individual mouse. Linear regression was calculated using Prism 4 software (GraphPad). (D) Spleen monocytes (CD11b+CD11c−) and DC subsets (CD11c+CD11b− and CD11c+CD11b+). Frequencies and cell numbers (± SD) were defined by flow cytometry. (E) pDCs and TLR7 expression in spleen B cell follicles. Spleen sections were stained with fluorescent anti–PDCA-1 (pDC, green) and anti-TLR7 (red) or with anti-CD19 (B cells, green) Abs. Original magnification ×40. Asterisks indicate statistical significance (*p < 0.05).

Late anti–IFNAR treatment of BXSB mice

A group of male BXSB mice was treated starting at 17 wk of age, when anti-chromatin autoantibodies and proteinuria had begun to emerge. This treatment had no significant effects on autoantibody titers, lymphadenopathy, splenomegaly, or mortality (not shown). However, at 26–33 wk of age, there were significant reductions of blood Gr-1−CD11b+ resident monocytes (Fig. 6A), the expansion of which is a characteristic of this lupus strain (26), and splenic CD21−CD23− B cells (Fig. 6B). Although GN scores were not modified by this late treatment (2.83 ± 1.15 versus 2.95 ± 0.87 in controls), and proteinuria was only marginally affected (Fig. 6C), there were significant reductions in IgG2a deposits and mononuclear cells infiltrates (Fig. 6D). Interestingly, in contrast to the untreated mice in which the CD11b+ and CD11c+ cells were within the glomeruli, in treated animals, these cells were confined to the interstitium, suggesting that signaling by type I IFNs contributes to the entry of inflammatory cells in the glomerulus. Thus, therapeutic anti–IFNAR treatment of male BXSB mice initiated after appearance of disease manifestations has measurable effects on monocytosis, B cell expansion, and kidney disease, but is significantly less efficient than treatment initiated at an earlier disease phase.

FIGURE 6.
FIGURE 6.

Late anti–IFNAR Ab treatment of BXSB mice. Mice (n = 7–8/group) were treated with anti–IFNAR Ab starting at 17 wk of age. (A) Blood monocyte subsets (CD11b+Gr-1− resident, CD11b+Gr-1+ inflammatory) at 26 wk of age. Numbers indicate average frequencies (± SD) of the gated cell subsets (*p < 0.05). (B) Spleen B cell subsets at 33 wk of age. Numbers indicate average frequencies (± SD) of the gated cell subsets (*p < 0.05). (C) Proteinuria at 15 and 28 wk of age. (D) Kidney glomerular IgG2a deposits and immunocyte infiltration (CD11b+ and CD11c+) at 33 wk. Shown are representative immunofluorescence images of kidneys from treated and control mice (n = 4/group). Original magnification ×40. Scoring of individual mice (0–4, based on fluorescence intensity) indicated significant treatment-associated reductions in IgG2a deposits (2.25 ± 1.26 versus 3.75 ± 0.5, p < 0.05), CD11b staining (1.13 ± 0.63 versus 3.13 ± 1.75, p < 0.05) and CD11c staining (1.00 ± 0.82 versus 2.5 ± 1.29, p < 0.05).

Anti–IFNAR treatment of Faslpr mice

Prophylactic anti–IFNAR Ab treatment was also initiated in 7-wk-old MRL-Faslpr mice. At 12 wk of age, both anti-RNP autoantibody titers and proteinuria were significantly reduced. However, with advancing age, these parameters and mortality converged with those of control mice (Fig. 7). Yet, there was no disease enhancement in the treated mice, in contrast to a previous report with Ifnar1−/− MRL-Faslpr mice (27).

FIGURE 7.
FIGURE 7.

Prophylactic anti–IFNAR Ab treatment of MRL-Faslpr mice. Mice (n = 10/group) were treated with anti–IFNAR Ab starting at 7 wk of age. (A) Serum anti-chromatin and anti-RNP autoantibody titers. (B) Proteinuria. (C) Survival. Asterisks indicate statistical significance (*p < 0.05).

Discussion

In this study, we further examined the pathogenic role of type I IFNs in mouse models of lupus. We demonstrated that IFN-β does not contribute to disease, pointing to the multiple IFN-αs as the most critical type I IFNs in lupus. Moreover, we showed that Ab-mediated IFNAR blockade significantly reduced disease in male BXSB mice as well as, transiently, in MRL-Faslpr mice. Of clinical relevance, this treatment was considerably less effective in BXSB mice when initiated late in the disease process.

The contribution of type I IFNs in lupus pathogenesis is supported by several findings, including the predominant representation of IFN-inducible genes correlating with disease flares in PBMCs of SLE patients (2, 28–33). Moreover, in lupus-predisposed mice, administration of rIFN-α enhanced autoimmunity (34–37), whereas Ifnar1 gene deletion reduced disease manifestations (8, 9). These findings have raised the possibility of blocking type I IFN signaling as a means to treat lupus and other syndromes mediated by these cytokines (1). Classically, interference with pathogenic cytokines entails neutralization with Abs or recombinant receptors or direct blockade of the receptors with Abs. The first approach is limited by the multiplicity of IFN-α subtypes, demonstrated in this study to be the main pathogenic mediators of mouse lupus. Therefore, soluble recombinant receptors or direct receptor blockade with Abs appear to be the methods of choice.

The availability of an anti-mouse IFNAR Ab of mouse origin allowed us to examine the effects of long-term treatment without interference by a host immune response. This anti–IFNAR Ab has been shown to potently inhibit type I IFN–mediated Stat1 phosphorylation, MHC class I upregulation, and inducible NO synthase induction in cell lines (11). In this study, we showed that this Ab also significantly inhibited in vitro TLR7- and type I IFN–mediated B cell, DC, and pDC activation and production of proinflammatory cytokines.

Through Ifnar1 gene deletion, we previously established the central role of type I IFNs in NZB mice (8). In this study, we extend these observations to BXSB male mice with the Tlr7 gene duplication (15, 16) and MRL-Faslpr mice with a mutation in the major apoptosis-controlling Fas gene (38). It has been demonstrated that the Tlr7 gene duplication is responsible for the accelerated disease phenotype in male BXSB mice and that transgenic overexpression of TLR7 can cause systemic autoimmunity even in normal background mice (24, 25, 39). In the current study, we found that IFNAR blockade significantly reduced most autoimmune manifestations in male BXSB mice, including autoantibody production, splenomegaly, lymphadenopathy, kidney disease, and mortality. These results clearly indicate that the disease-enhancing effect of the Tlr7 gene duplication is primarily mediated by hyperproduction of type I IFNs.

IFNAR blockade in BXSB mice was optimally effective when initiated at an early stage of disease. This is consistent with our proposed two-phase model of lupus pathogenesis (5), in which the initiation phase mediated by the innate immune system is required for the subsequent amplification phase mediated by the adaptive immune system. Nonetheless, even when treatment was initiated at a relatively advanced disease stage, some reductions in glomerular IgG2a deposits and mononuclear cell infiltrates were noted, although most disease parameters were unaffected. These findings are compatible with previous studies showing that local engagement of TLR7 and type I IFN signaling are involved in renal pathology of humans and mice with lupus (40–42). The limited effect of late anti–IFNAR Ab treatment suggests that IFNAR blockade is less efficient once autoantibody production has exceeded a certain threshold, or inflammatory molecules produced by the adaptive immune system may sustain end organ damage in a type I IFN–independent manner. These molecules may include IFN-γ, which has also been shown to promote disease in lupus-predisposed models (43). Overall, if the results can be translated to human SLE, they suggest that IFNAR blockade should be applied at relatively early disease stages, and, as disease progresses, this treatment may require supplementation with additional interventions aimed at inhibiting downstream injurious adaptive responses and associated inflammatory factors.

It is notable that although the dose and schedule of anti–IFNAR Ab injections led to incomplete occupation of the receptor, the disease reducing effects were still remarkable. This is consistent with our previous study, in which not only homozygous but also heterozygous Ifnar1 gene–deleted NZB mice showed significantly reduced disease and increased survival (8). Together, these findings strongly suggest that effectiveness of this treatment does not require complete receptor blockade, but rather reduction of receptor availability below a threshold level. Therefore, it could be surmised that translation of this treatment to humans may, upon proper titration, reduce autoimmunity and yet permit relatively normal responses to microbial pathogens.

Considerable evidence indicates that, in most cell types, production of IFN-α requires a preceding induction of IFN-β that, upon binding to the common receptor, induces the transcription factor IFN regulatory factor 7 necessary for IFN-α expression (44–46). In contrast, evidence has been presented that this IFN-β feedback loop is not required for IFN-α production by pDCs, presumably due to constitutive expression of IFN regulatory factor 7 by these cells (46–48). Our finding that IFN-β is not involved in lupus progression provides indirect evidence that pDCs, likely acting by hyperproduction of type I IFNs, are intimately involved in disease pathogenesis. Interestingly, we also found that IFNAR blockade inhibited in vitro production of type I IFNs by pDCs, consistent with reports in both humans and mice showing that signaling by these cytokines promotes their own expression via autocrine stimulation (49–51). Accordingly, in vivo anti–IFNAR treatment of BXSB mice reduced both follicle size and number of pDCs clustering at the marginal sinus. The latter finding may also relate to the observation that nucleic acid–sensing TLRs and type I IFNs affect expression of chemokine receptors and thus the migration patterns of pDCs (49).

IFNAR blockade also inhibited accumulation of monocytes, cDCs, and B cells. Monocytosis has been described as a unique characteristic of male BXSB mice (26), but what drives this expansion is incompletely defined. It appears that the Tlr7 gene duplication, although in itself insufficient, is required for severe monocytosis (25). Our results showing reduction of monocytosis by IFNAR blockade strongly suggest that type I IFNs are key mediators of this feature. The role of monocytosis in lupus pathogenesis remains unresolved, but possible contributions may include production of proinflammatory cytokines or conversion to mature Ag-presenting DCs (52).

B cell responses in vitro were potentiated by the combined IFN-α and TLR7 stimulation, an effect abolished by IFNAR blockade. Accordingly, in vivo treatment with anti–IFNAR Ab reduced B cell expansion, particularly CD21−CD23−AA4.1low B cells, a subset recently shown to derive from overstimulated follicular B cells, to expand with aging and autoimmunity, and to be enriched in autoreactive clonotypes (21, 22). Expansion of these age-associated B cells (ABCs) in normal mice was reported to depend on signaling by TLR7, but not IFNAR (22). In contrast, our results clearly showed that, in male BXSB mice, expansion of these cells is partly dependent on type I IFNs, perhaps due to engagement of the overexpressed TLR7.

At variance with the strong evidence in both humans and mice that type I IFNs are critical effectors of lupus pathogenesis, one study reported disease acceleration in Ifnar1−/− MRL-Faslpr mice, suggesting that these cytokines may exert a protective effect in this model (27). We observed that both anti-RNP autoantibody levels and proteinuria were reduced when assessed 5 wk after initiation of anti–IFNAR Ab treatment, but rebounded 4 wk later to levels similar to those in untreated controls. Other parameters, such as anti-chromatin autoantibodies, lymphadenopathy, and mortality, were not modified by this treatment. However, no disease acceleration or enhanced severity was observed in anti–IFNAR Ab–treated mice compared with controls. These results suggest that early autoimmune events in this model are also influenced by type I IFNs, particularly anti-RNP autoantibody responses known to be dependent on TLR7 engagement (7, 10, 15, 16, 25) and to correlate with type I IFN signaling in SLE (29–31) and mouse models of lupus (36, 53–55). However, the beneficial effect of IFNAR blockade in MRL-Faslpr mice was eventually overcome, likely due to apoptosis defects that may favor the engagement of type I IFN–independent pathogenic pathways. In fact, genome-wide mRNA expression analysis in this strain indicated a predominant IFN-γ–induced gene expression signature, whereas type I IFN genes were minimally modulated (42). The differential disease expression in anti–IFNAR Ab–treated versus Ifnar1 gene–deleted MRL-Faslpr mice might be explained by partial versus complete inhibition of IFNAR signaling, respectively. Thus, it may be hypothesized that, depending on the genetic predisposition, initiation and/or continuance of the pathogenic process in lupus might require high IFNAR availability and signaling, whereas a lower degree of type I IFN signaling is sufficient for the beneficial antiproliferative effects of these cytokines (56).

To summarize, the results of this and previous studies (8, 9, 55) clearly establish the pathogenic role of IFN-α in several models of lupus and strongly support the notion that anti–IFNAR Abs should be contemplated as a potential treatment for human SLE. In addition, because pDCs, TLRs, and type I IFNs have been implicated in the pathogenesis of several other autoimmune diseases, including rheumatoid arthritis, Sjogren’s syndrome, and type I diabetes (1, 5), this approach may also be applicable to these diseases.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Anthony Nguyen for excellent technical assistance and Kat Occhipinti-Bender for expert manuscript editing.

Footnotes

  • This work was supported by grants from the National Institutes of Health (AR53228 and AR31203).

  • This is article number 21807 from The Scripps Research Institute, Department of Immunology and Microbial Science.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ABC
    age-associated B cell
    ANA
    anti-nuclear autoantibody
    BM
    bone marrow
    cDC
    conventional dendritic cell
    GMFI
    geometric mean fluorescence intensity
    GN
    glomerulonephritis
    IFNAR
    IFN-α/β receptor
    pDC
    plasmacytoid dendritic cell
    PDCA-1
    plasmacytoid dendritic cell Ag-1
    RNP
    ribonucleoprotein
    SLE
    systemic lupus erythematosus
    WT
    wild-type.

  • Received May 29, 2012.
  • Accepted October 5, 2012.

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