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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: J Immunol. 2011 Nov 23;188(1):270–278. doi: 10.4049/jimmunol.1102311

Distinct regulation of murine lupus susceptibility genes by the IRF5-Blimp-1 axis

Ravichandran Panchanathan 1,2, Hongzhu Liu 1, Hongqi Liu 1, Chee-Mun Fang 3, Loren D Erickson 4, Paula M Pitha 3, Divaker Choubey 1,2
PMCID: PMC3244553  NIHMSID: NIHMS337531  PMID: 22116829

Abstract

Genome-wide association studies have identified lupus susceptibility genes such as IRF5 and PRDM1 (encoding for the IRF5 and Blimp-1) in the human genome. Accordingly, the murine Irf5 and Prdm1 genes have been shown to play a role in lupus susceptibility. However, it remains unclear how IRF5 and Blimp-1 (a transcriptional target of IRF5) contribute to lupus susceptibility. Given that the murine lupus susceptibility locus Nba2 includes the interferon-regulated genes Ifi202 (encoding for the p202 protein), Aim2 (encoding for the Aim2 protein), and Fcgr2b (encoding for the FcγRIIB receptor), we investigated whether the IRF5-Blimp-1 axis could regulate the expression of these genes. We found that an Irf5-deficiency in mice decreased the expression of Blimp-1 and reduced the expression of the Ifi202. However, the deficiency increased the expression of Aim2 and Fcgr2b. Correspondingly, increased expression of IRF5 in cells increased levels of Blimp-1 and p202 protein. Moreover, Blimp-1 expression increased the expression of Ifi202, whereas it reduced the expression of Aim2. Interestingly, an Aim2-deficiency in female mice increased the expression of IRF5. Similarly, the Fcgr2b-deficient mice expressed increased levels of IRF5. Moreover, increased expression of IRF5 and Blimp-1 in lupus-prone B6.Nba2, NZB, and B6.Sle123 female mice (as compared to age-matched C57BL/6 female mice) was associated with increased levels of the p202 protein. Together, our observations demonstrate that the IRF5-Blimp-1 axis differentially regulates the expression of Nba2 lupus susceptibility genes, and suggest an important role for the IRF5-Blimp-1-p202 axis in murine lupus susceptibility.

Keywords: IRF5, Blimp-1, Nba2 locus, p202, interferon, autoimmunity, SLE

Introduction

Genetic studies involving systemic lupus erythematosus (SLE) patients have identified several lupus susceptibility genes, including the IRF5 and PRDM1 (encoding for the IRF5 and Blimp-1 transcriptional regulators) (1, 2). Correspondingly, the murine Irf5 (3–7) and Prdm1 (4) genes have been shown to play an important role in the development of lupus disease. However, it remains unclear how IRF5 and Blimp-1 transcription factors contribute to lupus susceptibility.

The interferon (IFN)-regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors (8, 9). The murine IRF5 is primarily expressed as a full-length transcript in the B220+ mature B cells and levels of the IRF5 decrease in CD138+ plasma cells (10). Moreover, the female hormone estrogen up-regulates the expression of the murine Irf5 gene (11). IRF5 could be activated by both TBK1 and MyD88 to form homodimers and the activated IRF5 induces the transcription of type I interferon genes and the Prdm1 gene (4, 8). The Prdm1 gene encodes Blimp-1 protein, a master regulator of the B cell differentiation (12). The Irf5−/− mice, which were generated using embryonic stem (ES) cells from 129sv strain on the mixed (B6 × 129) genetic background, show reduced serum levels of type I IFNs and develop an aging-dependent splenomegaly that is associated with an accumulation of CD19+B220− B cells (4). Moreover, splenic cells from the Irf5−/− mice exhibit a decrease in the number of plasma cells and down-regulation of Blimp-1 expression (4). Notably, the murine IRF5 is required for the development of lupus-like disease in the FcγRIIB−/− Yaa and FcγRIIB−/− mouse models (3). Additionally, IRF5 is critical for the development of lupus in MRL/lpr mice (7). Interestingly, type I IFN receptor subunit 1-deficient FcγRIIB−/− Yaa mice maintained a substantial level of residual disease (3), thus, raising the possibility of the IFN-signaling independent role for the IRF5 in the development of murine lupus disease. Accordingly, a recent study (5) has noted that IRF5 contributes to murine SLE-like disease through its direct control of class switch recombination of the γ2a locus in B cells.

The transcriptional activation of the Prdm1 gene by murine IRF5 is associated with the terminal differentiation of B cells to CD138+ plasma cells (12). Blimp-1-mediated transcriptional repression of certain target genes, such as Pax5 and c-Myc, is required for the terminal differentiation of B cells. However, Blimp-1 induces the expression of XBP-1 in plasma cells (12). The optimal core DNA consensus sequence (GAAAG) that is bound by human and murine Blimp-1 is essentially identical to that bound by the IRF family members (13). Interestingly, the Blimp-1 represses the transcription of the Aim2 gene (14).

IRF5 transcription factor participates in cell type-dependent key signal transduction pathways, such as toll-like receptor (TLR)-signaling and type I IFN production (8, 9). These signaling pathways are implicated in the development of SLE (9, 15). The IFN-family of cytokines includes type-I (IFN-α and β) and type II (IFN-γ) IFNs (16). The IFNs exert multiple biological effects on the immune system by affecting differentiation, proliferation, and survival of immune cells (17, 18). The IFN-inducible genes encode “effector” proteins that mediate the immunomodulatory functions of IFNs (19). Increased serum levels of IFN-α and the “IFN-signature” have been reported in SLE patients (20, 21). Accordingly, lupus-prone NZB mice that are deficient in the type-I receptor do not develop disease (22). Interestingly, the SLE-associated variant of IRF5 has been linked to higher IFN-α levels in sera of human SLE patients (23).

The telomeric chromosome 1 in the mouse (and its syntenic equivalent 1q21-44 region in humans) has shown a strong linkage to systemic autoimmunity (24). In the mouse, three loci have been identified in the autoimmunity susceptibility region: New Zealand White (NZW)-derived Sle1 in NZM2410 strain (25), and New Zealand Black (NZB)-derived Lbw7 (26) and Nba2 (24, 27–32) in (NZB × NZW)F1 mice. Interestingly, both Sle1 and Nba2 intervals contain candidate lupus susceptibility genes, which include members of the Fcgr family, members of the SLAM family, and members of the Ifi200-family (28, 30). The Nba2 locus (~90–97 cM) has been shown to be a major genetic contributor from the NZB strain to lupus susceptibility in the (NZB × NZW)F1 spontaneous mouse model of SLE (28–32). Accordingly, C57BL/6 (B6) mice congenic for the Nba2 interval (congenic mice indicated as B6.Nba2-ABC) produce detectable levels of antinuclear Abs ~7-month of age, but do not develop a kidney disease (28), thus, suggesting interactions of the Nba2 locus with other loci for the development of the kidney disease. Based on the sequence polymorphisms that are identified in the Nba2 interval genes, it has been proposed that the interval may contain several candidate lupus susceptibility genes (28–32). The candidate genes include the Slam-family genes (31, 32), the Fcgr2b gene (encoding for the inhibitory FcγRIIB receptor) (29), and the interferon (IFN)-inducible Ifi202 gene (28, 33, 34). Interestingly, the B6.Nba2 congenic mice that are deficient in the type-I receptor do not develop a lupus-like disease and express reduced levels of the Ifi202 mRNA (35).

Generation of B6.Nba2-ABC sub-congenic lines (B6.Nba2-A, -A’B, -B, and -C) and their characterization reveal that the B6.Nba2-C sub-congenic mice, which harbor the Ifi200-gene cluster, do not develop ANAs and do not produce type I IFN (32). Consistent with these observations, we were able to detect the expression of the p202 protein in the B6.Nba2-ABC female mice (age ~4-months), but not the parental B6 or B6.Nba2-C congenic mice (36). Moreover, genes within the sub-interval C (comprising the Ifi200-family genes) negatively regulate the expression of the Fcgr2b gene and inhibit the FcγRIIB-induced apoptosis (32).

The IFN-inducible Ifi200-genes family includes structurally-related mouse genes, including the Ifi202a, Ifi202b, and Aim2 (33, 34). The 200-gene family region of the mouse chromosome 1 (~ 6,000 kb) is syntenic to a region at the 1q23 on human chromosome 1, which contains four genes (IFI16, IFIX, MNDA, and AIM2) (33, 34). Proteins in the p200-family share at least one HIN200 domain. The domain participates in protein-protein interactions as well as detection of cytosolic DNA (37). The Aim2 protein, which contains a pyrin domain (PYD), recruits ASC adapter protein to form an Aim2 inflammasome (38, 39). Generation of the Aim2-deficient mice revealed that the Aim2 protein negatively regulates type I IFN responses and the expression of the Ifi202 (possibly both Ifi202a and Ifi202b genes) lupus susceptibility gene (36, 40). Given that the expression of Ifi202 gene is regulated by the female sex hormone estrogen (41) and p202 protein suppresses the expression of both Aim2 and Fcgr2b genes located within the Nba2 interval (42), we investigated whether the IRF5-Blimp-1 axis could regulate the expression of the Nba2 lupus susceptibility genes. We report that the axis differentially regulates the expression of Nba2 lupus susceptibility genes. These observations suggest that the IRF5-Blimp-1-p202 axis contributes to lupus susceptibility in mice.

Materials and Methods

Mice

Generations of an Irf5 (4) and Aim2-deficient mice (40) on the mixed (129 × B6) genetic background have been described. Wild type and Irf5-deficient mice either 76% (KO-P) or 98% (KO-C) on the C57BL/6 (B6) genetic background were housed in specific pathogen-free animal facilities of the Johns Hopkins University, Baltimore, Maryland. The B6.Nba2 (or B6-Nba2-ABC) and B6.Nba2-C mice were housed in specific pathogen-free animal facilities at the University of Virginia, Charlottesville, VA. The Fcgr2b-deficient mice on the C57BL/6 genetic background and the corresponding wild type mice were purchased from Taconic Farms (Germantown, NY). C57BL/6 and NZB male and female mice were purchased from The Jackson Laboratory (Bar Harbor, Maine). The B6.Sle123 (B6.NZMSle1/Sle2/Sle3) female mice (43, 44) (age, ~10 wk) were purchased from The Jackson Laboratory. Mice were housed in specific pathogen-free animal facilities at the University of Cincinnati, Cincinnati. The Institutional Animal Care and Use Committee (IACUC) at the institution, where the mice were housed, approved the protocol for studies using mice that are described here.

Splenocytes isolation, cell culture, and treatments

Total splenocytes were prepared from age and strain-matched male or female mice as described previously (36). Cells were re-suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum. When indicated, splenic B cells (B220+) or plasma cells (CD138+) were purified using magnetic beads (purification kit purchased from Miltenyi Biotech) by the positive selection of cells. The purified (90–95% pure) cells were used immediately for the experiments. In most cases, unless indicated otherwise, cells from two or more age and gender-matched mice were pooled to prepare total RNA or protein extracts.

RAW264.7 murine macrophage cell line was purchased from the American Type Culture Collection and cells were maintained as suggested by the supplier.

Plasmids and nucleofections

Plasmids to express murine IRF5 (pCMV-mIRF5; ref. 10), Blimp-1 (pCMV-mPrdm1; ref. 13), and p202 (pCMV-202; ref. 41) have been described. RAW264.7 cells (2 × 106) from sub-confluent cultures were nucleofected with highly purified (endotoxin-free) plasmid DNA encoding for the indicated protein. As a control, we used pCMV plasmid. For nucleofections, we used the Nucleofector-II device (Amaxa Biosystems, Germany), the kit-V, and the program D-032. After nucleofections, cells were incubated for 24–30 h before harvesting for total RNA or proteins. Nucleofections resulted in ~60% cell survival after 24 h.

Reporter assays

Promoter reporter assays were performed essentially as described previously (41). In brief, sub-confluent cultures of RAW264.7 cells (in a 6-well plate) were transfected with the reporter plasmids 202-Luc (1.0 µg) and pRL-TK (0.2 µg) along with either an empty vector pCMV (0.8 µg) or equal amounts of a plasmid that allowed the expression of murine IRF5 (pCMV-mIRF5) or Blimp-1 (pCMV-mBlimp1) using the FuGENE 6 (Roche Applied, Indianapolis, IN) transfection reagent as suggested by the supplier. Cells were harvested between 40–45 h after transfections and the firefly and Renilla dual luciferase activities were determined.

Isolation of RNA from splenocytes and RT-PCR

Splenocytes (5–8 × 106 cells) were used to prepare total RNA using TRIzol (Invitrogen, Carlsbad, CA) method (36). RNA (0.5–2 µg) was used for RT-PCR reaction using the Superscript one-step RT-PCR system (from Invitrogen). Semi-quantitative regular PCR reaction was performed using a pair of primers specific to the Irf5 (primers: forward: 5’-aataccccaccaccttttga-3’; reverse primer: 5’-ttgagatccgggtttgagat-3’), Prdm1 (primers: forward: 5’-ttcttgtgtggtattgtcgggactt-3’; reverse primer: 5’-ttggggacactctttgggtagagtt-3’), Ifi202 (primers: forward: 5'-ggtcatctaccaact cagaat-3'; reverse primer: 5'-ctctaggatg ccactgctgttg-3'), Aim2 (primers: forward: 5’-acagtgg ccacggaga- 3’; reverse: 5’-aggtgacttcactccaca-3’), Fcgr2b (primers: forward: 5’-aagtctagga aggacactgc-3’; reverse: 5’- atcctggcctttctggcttgc-3’) or the murine Ifnb (primers: forward: 5’-ctgcgttcctgctgtgcttctcca- 3’; reverse: 5’- ttctccgtcatctccatagggatc-3’) genes. The conditions for the regular PCR have been described (36).

To perform quantitative real-time TaqMan PCRs, we used the 7300 Real-Time PCR System (from Applied Biosystems, Foster City, CA, USA) and the commercially available real-time TaqMan gene expression assays. The PCR cycling program has been described previously (36). The TaqMan assays for the murine Irf5 (Assay Id# Mm00496478_g1), Prdm1 (Assay Id# Mm0118 7285_ml), Ifi202 (Assay Id# Mm0304 8198_m1; the assay allows the detection of both the Ifi202a and Ifi202b mRNA levels), Aim2 (Assay Id# Mm01295719_m1), Ifnb (Assay Id# Mm00439552_s1), Fcgr2b (Assay Id# Mm00438875_m1; the assay allows the detection of mRNA encoding both the B1 and B2 isoforms of the FcγRIIB receptor), the endogenous Actb control (cat # 4352933E), and β2-microglobulin (Assay Id# Mm00437762_m1) were purchased from the Applied Biosystems (Foster City, CA) and used as suggested by the supplier.

Immunoblotting

Total cell extracts containing equal amounts of proteins were subjected to immunoblotting as described previously (36). The p202 antiserum, which allows the detection of both p202a and p202b proteins in immunoblotting, has been described (45). Additionally, we also used the polyclonal (cat # IMG-6670A from Imgenex, San Diego, CA) or monoclonal (sc-166253; from Santa Cruz Biotech, Santa Cruz, CA) anti-p202 antibodies to detect the p202 protein. We have described polyclonal antibodies against the murine Aim2 protein (36). Antibodies to murine IRF5 (cat # 4950), Blimp-1 (cat # 9115), STAT1 (# 9172), and β-actin (# 4967) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to detect the murine FcγRIIB receptor (sc-28842) were purchased from Santa Cruz Biotech.

Statistical analyses

The statistical significance of differences in the measured mean frequencies between the two groups of observations was calculated using the Student’s two-tailed t test. A p value <0.05 was considered significant.

Results

An Irf5-deficiency in mice decreases expression of Blimp-1 and p202

Expression of IRF5 is critical for the development of lupus in certain mouse models, including in FcγRIIB−/− Yaa and FcγRIIB−/− (3), and MRL/lpr (7). Because development of lupus-like disease in B6.Nba2 congenic female mice depends on type I IFN-signaling (35) and alterations in the expression of Ifi202, Aim2, and Fcgr2b genes that are located within the Nba2 autoimmunity locus are associated with the development of lupus-like disease in mice (28, 32, 36, 42), we first investigated whether an Irf5-deficiency in non autoimmune mice could regulate the expression of these genes. As shown in Fig. 1, an Irf5-deficiency in splenic cells isolated from the female mice (age ~ 12-weeks) on the C57BL/6 (B6) genetic background, decreased levels of both Blimp-1 (a transcriptional target of IRF5) and p202 proteins as compared to age-matched B6 females. Given that the increased levels of the p202 protein in immune cells negatively regulates the expression of Aim2 and Fcgr2b genes (36, 42), we also compared the steady-state levels of Aim2 and FcγRIIB proteins. Notably, levels of the Aim2 and FcγRIIB proteins were higher in IRF5-null cells than the wild-type cells. Consistent with our above observations (Fig. 1A), we also noted decreases in steady-state levels of Prdm1 and Ifi202 mRNA levels in Irf5-deficient splenocytes from female mice as compared to age-matched B6 females in regular PCR (Fig. 1B) and quantitative real-time PCR (Fig. 1C). Moreover, levels of the Aim2 and Fcgr2b mRNA were higher in Irf5-deficient cells than wild-type cells. Interestingly, we also detected reduced levels of Ifnb mRNA in Irf5-deficient cells than wild-type cells. Together, these observations indicated that an Irf5 gene deficiency in mice decreases the expression of Blimp-1, IFN-β, and p202 proteins, whereas the deficiency increases the levels of Aim2 and FcγRIIB proteins.

FIGURE 1. An Irf5-deficiency in mice decreases expression of Blimp-1 and p202 proteins.

FIGURE 1

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(a) Total cell lysates prepared from splenic cells isolated from wild-type (lane 1) or Irf5-deficient female mice (age ~12-weeks) either 76% (lane 2) or 98% (lane 3) on the C57BL/6 background were analyzed by immunoblotting using antibodies specific to the indicated proteins. (b) Total RNA prepared from splenic cells isolated from wild-type (lane 1) or an Irf5-deficient female mice (98% on C57BL/6 background; lane 2) was analyzed by semi-quantitative PCR using a pair of primer specific to the indicated genes. (c) The RNA samples in panel (b) were also subjected to quantitative real-time PCR using TaqMan assays specific to the indicated genes. The ratio of the mRNA levels for a test gene to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the test gene to β2-microglobulin mRNA). The ratio of mRNA levels in the wild-type mice is indicated as 1. The error bars represent the standard deviation.

IRF5 up-regulates the expression of Blimp-1, IFN-β, and p202

To investigate further whether the IRF5 stimulates the expression of the Prdm1, Ifnb, and Ifi202 genes, we chose to overexpress the murine IRF5 in murine macrophage RAW264.7 cells. We chose these cells because they do not express adaptor protein ASC (46), thus, allowing expression of a desired gene after nucleofection of a plasmid DNA into cells without the activation of the Aim2 inflammasomes, which can induce cell death upon sensing cytosolic DNA (38, 39). Additionally, these cells express low levels of TLR9, which allows the activation of IRF5 upon sensing DNA that is taken up by cells that survive nucleofection. As shown in Fig. 2A and B, nucleofection of the pCMV-mIRF5 plasmid allowing the expression of IRF5 in cells, but not an empty vector (V), increased steady-state levels of IRF5 and Blimp-1 mRNA.

FIGURE 2. IRF5 up-regulates the expression of Blimp-1, IFN-β, and p202 proteins.

FIGURE 2

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(a) RAW264.7 murine macrophage cells (2 × 106) were either nucleofected with an empty vector pCMV (2 µg) or equal amounts of pCMV-mIRF5 plasmid. 24 h after nucleofections, cells were harvested and total RNA was analyzed by quantitative real-time PCR to assess the steady-state levels of IRF5 mRNA. The ratio of the IRF5 mRNA levels to actin mRNA was calculated in units (one unit being the ratio of the test gene to actin mRNA). The ratio of mRNA levels in the vector nucleofected cells is indicated as 1. The error bars represent the standard deviation. (b) The RNA samples described in the panel (a) were analyzed by regular PCR using a pair of primer specific to the Prdm1 gene or actin. (c and d) The RNA samples described in the panel (a) were analyzed by quantitative real-time PCR to assess steady-state levels of Ifnb (c) or Ifi202 (d) mRNA levels. The ratio of mRNA levels in the vector nucleofected cells is indicated as 1. The error bars represent the standard deviation. (e) The RNA samples described in the panel (a) were also analyzed by regular PCR using a pair of primer specific to the indicated genes. (f) Actively proliferating RAW264.7 murine macrophage cells (2 × 106) were either nucleofected with an empty vector pCMV (2 µg) or equal amounts of pCMV-mIRF5 plasmid. 24 h after nucleofections, cells were harvested and total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins. (g) Sub-confluent cultures of RAW264.7 cells in a 6-well plate were transfected with the reporter plasmids 202-Luc (1.0 µg) and pRL-TK (0.2 µg) along with either an empty vector pCMV (0.8 µg) or equal amounts of a plasmid that allowed the expression of murine IRF5 (pCMV-mIRF5). Cells were harvested between 40–45 h after transfections and the firefly and Renilla dual luciferase activities were determined. The normalized relative luciferase activity in the vector transfected cells is indicated as 1.0.

Expression of IRF5 and its activation induces the expression of type I IFNs (8, 9). Therefore, our above observation that nucleofection of the pCMV-mIRF5 plasmid into RAW264.7 cells induced the expression of Blimp-1 expression prompted us to test whether IRF5 expression could induce the IFN-β expression and IFN-inducible Ifi202 gene. As shown in Fig. 2C and D, nucleofection of the plasmid DNA also increased steady-state levels of Ifnb and Ifi202 mRNAs. Moreover, consistent with our earlier observation (36), levels of the Aim2 mRNA were inversely correlated with Ifi202 mRNA (Fig. 2E). Correspondingly, levels of STAT1, p202, and Blimp-1 proteins were higher in cells that were nucleofected with the pCMV-mIRF5 plasmid as compared to an empty vector (Fig. 2F). Furthermore, increased expression of IRF5 in promoter-reporter assays using RAW264.7 cells stimulated the activity of the 202-luc-reporter plasmid ~6-fold (Fig. 2G). Together, these observations indicated that overexpression of the murine IRF5 in RAW264.7 cells after nucleofection of plasmid DNA stimulates the expression of Blimp-1, IFN-β, and p202.

Blimp-1 induces expression of Ifi202

Blimp-1 suppresses expression of c-Myc and E2F1 transcription factors (12). Interestingly, both c-Myc and E2F-1 negatively regulate the Ifi202 expression (47, 48). Moreover, the Blimp-1 can bind to the “GA” sequence (bound by the IRFs) that is present in the 5’-regulatory region of the Ifi202 gene (13, 49). Therefore, our above observations that IRF5 expression can stimulate the expression of Blimp-1 and p202 proteins raised the possibility that Blimp-1 could regulate the expression of Ifi202 gene. As shown in Fig. 3, nucleofection of pCMV-Prdm1 plasmid (allowing the expression of the murine Blimp-1 protein), but not an empty vector (V), into RAW264.7 cells increased levels of p202 mRNA (Fig. 3A) and protein (Fig. 3B). Furthermore, increased expression of Blimp-1 in promoter-reporter assays using RAW264.7 cells stimulated the activity of the 202-luc-reporter plasmid ~14-fold (Fig. 3C). Given that the p202 protein suppresses the expression of the Aim2 gene (33, 39) and Blimp-1 suppresses the expression of the Aim2 gene (14), we also explored whether p202 protein could regulate the Blimp-1 expression. Interestingly, p202 overexpression in RAW264.7 cells increased levels of Blimp-1 protein (Fig. 3D). Together, these observations revealed that the Blimp-1 and p202 proteins are part of a positive feedback loop.

FIGURE 3. Blimp-1 induces expression of Ifi202 gene.

FIGURE 3

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(a) RAW264.7 cells (2 × 106) that were proliferating actively were nucleofected with an empty pCMV plasmid (2 µg) or a plasmid (pCMV-mPrdm1) allowing expression of murine Blimp-1 protein. Nucleofected cells were harvested after 24 h and total RNA was prepared. Samples were analyzed for steady-state levels of mRNA for the indicated genes by regular PCR. (b) Cells were nucleofected as described in section (a) and 24 h after nucleofections total cells lysates were prepared. Lysates containing equal amounts of proteins were analyzed for levels of indicated protein by immunoblotting. (c) Sub-confluent cultures of RAW264.7 cells in a 6-well plate were transfected with the reporter plasmids 202-Luc (1.0 µg) and pRL-TK (0.2 µg) along with either an empty vector pCMV (0.8 µg) or equal amounts of a plasmid that allowed the expression of murine Blimp-1 (pCMV-mBlimp-1). Cells were harvested between 40–45 h after transfections and the firefly and Renilla dual luciferase activities were determined. The normalized relative luciferase activity in the vector transfected cells is indicated as 1.0. (d) RAW264.7 cells were nucleofected as described in the section (a) with either an empty vector or a plasmid (pCMV-202) allowing the expression of p202 protein. 24 h after nucleofections cells lysates were analyzed by immunoblotting.

Gender-dependent up-regulation of IRF5 levels in Aim2-deficient mice

Expression levels of IRF5 and its nuclear localization are regulated by the female sex hormone estrogen (11). Given that an Irf5-deficiency in mice increased expression levels of Aim2 protein (Fig. 1), we sought to investigate whether an Aim2-deficiency could regulate levels of IRF5 protein. As shown in Fig. 4A, levels of IRF5 protein were higher in splenic B220+ B cells from wild type female mice as compared to age-matched males. Interestingly, levels of the IRF5 protein were higher in the Aim2-deficient females than the age-matched wild-type females (compare lane 4 with 3) and IRF5 expression was not detectable in Aim2-deficient males. Consistent with these observations, we detected increased levels of IRF5 mRNA in splenic B220+ B cells from Aim2-deficient females as compared to age and gender-matched wild-type mice (Fig. 4B). Correspondingly, we also detected increased levels of mRNA encoding for Blimp-1, IFN-β, and p202 proteins in Aim2-deficient B cells (B220+) than wild-type cells. Together, these observations indicated that expression of Aim2 and IRF5 proteins is inversely correlated and the gender-dependent factors regulate the expression of IRF5 in Aim2-deficient immune cells.

FIGURE 4. Gender-dependent up-regulation of IRF5 levels in Aim2-deficient mice.

FIGURE 4

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(a) Total cell lysates prepared from purified splenic B cells (B220+) that were isolated from wild-type (lanes 1 and 3) and age-matched Aim2-deficient (lanes 2 and 4) male or female mice (age 6–8 weeks) were analyzed by immunoblotting using antibodies specific to the indicated proteins. (b) Total RNA prepared from purified splenic B cells (B220+), which were isolated from wild-type (lanes 1 and 3) and age-matched Aim2-deficient (lanes 2 and 4) male or female mice (age 6–8 weeks), were analyzed by regular PCR for mRNA levels.

Fcgr2b-deficiency increases IRF5 levels

Deficiency of the inhibitory FcγRIIB receptor in immune cells induces type I IFN response and also induces expression of the Ifi202 gene (42). Therefore, increased levels of FcγRIIB receptor in IRF5-deficient cells (Fig. 1) prompted us to test whether the deficiency of the FcγRIIB receptor could regulate expression of IRF5. As shown in Fig. 5A, levels of IRF5 protein were higher in Fcgr2b-deficient cells from male and female mice as compared to age and gender matched B6 mice. Correspondingly, we also detected higher steady-state levels of IRF5 mRNA in Fcgr2b-deficient cells as compared to age and gender-matched B6 mice (Fig. 5B). Together, these observations indicated that the lack of FcγRIIB receptor expression in immune cells increases levels of IRF5 mRNA and protein.

FIGURE 5. Fcgr2b-deficiency increases IRF5 levels.

FIGURE 5

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(a) Total cell lysates prepared from splenic cells that were isolated from wild-type (lanes 1 and 3) and age-matched Fcgr2b-deficient (lanes 2 and 4) male or female mice (age ~9-weeks) were analyzed by immunoblotting using antibodies specific to the indicated proteins. (b) Total RNA prepared from splenic cells, which were isolated from wild-type (lanes 1 and 3) and age-matched Fcgr2b-deficient (lanes 2 and 4) male or female mice (age 6–8 weeks), were analyzed by quantitative real-time PCR for steady-state levels of IRF5 mRNA. The ratio of the IRF5 mRNA levels to actin mRNA was calculated in units (one unit being the ratio of the test gene to actin mRNA). The ratio of the mRNA levels in the wild-type male splenic cells is indicated as 1. The error bars represent the standard deviation (* p <0.01).

Increased expression of IRF5 and Blimp-1 in lupus susceptible mice is associated with increased expression levels of p202 protein

Lupus-prone preautoimmune (much before the detection of autoantibodies; age ~4-months) B6.Nba2-ABC (same as B6.Nba2) female mice express increased levels of IFN-β, exhibit activation of the IFN-signaling, and express increased levels of p202 protein as compared with age-matched B6.Nba2-C and B6 non lupus-prone female mice (36). Therefore, our above observations that both IRF5 and Blimp-1 stimulated the expression of Ifi202 gene in immune cells and an Irf5-deficiency decreased p202 protein levels (Fig. 1) encouraged us to investigate whether levels of IRF5 and/or Blimp-1 proteins are higher in the lupus-prone B6.Nba2-ABC mice as compared to non lupus-prone B6.Nba2-C and B6 mice. As shown in Fig. 6A and B, we detected higher levels of IRF5 and Blimp-1 mRNA in splenic cells from B6.Nba2-ABC female mice as compared to the age-matched B6.Nba2-C or B6 female mice. Accordingly, we also detected the increased levels of both IRF5 and Blimp-1 proteins in splenic cells from B6.Nba2-ABC female mice as compared to the age-matched B6.Nba2-C and B6 mice (Fig. 6C). Interestingly, B6.Nba2-C splenic cells isolated from the female mice had higher levels of IRF5, but not Blimp-1. Because IRF5 expression decreases in plasma cells (4) and Blimp-1 promotes B cell differentiation (12), we also compared levels of IRF5, Blimp-1, and p202 proteins in splenic CD138+ plasma cells. As shown in Fig. 6D, we found that levels of IRF5 and p202 proteins were higher in lupus-prone B6.Nba2-ABC females than age-matched non lupus-prone B6 females. However, levels of the Blimp-1 were comparable between the B6 and B6.Nba2 plasma cells and levels of Aim2 protein were lower in the B6.Nba2-ABC females than B6.

FIGURE 6. Increased expression of IRF5 and Blimp-1 proteins in lupus susceptible mice is associated with increased expression levels of the p202 protein.

FIGURE 6

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(a and b) Total RNA was prepared from splenic cells, which were isolated from B6, B6.Nba2-C (C), or B6.Nba2-ABC (ABC) females (age ~4-months). Steady state levels of mRNA for IRF5 (a) or Blimp-1 (b) were analyzed by quantitative real-time PCR. The ratio of the IRF5 mRNA levels to β-microglubulin mRNA was calculated in units (one unit being the ratio of the test gene to β-microglubulin mRNA). The ratio of the mRNA levels in the B6 splenic cells is indicated as 1. The error bars represent the standard deviation (NS, not significant; *** p <0.001). (c) Total cell extracts were prepared from splenic cells, which were isolated from B6, B6.Nba2-C (C), or B6.Nba2-ABC (ABC) females (age ~4-months). Steady-state levels of the indicated proteins were analyzed by immunoblotting. (d) Total cell lysates prepared from purified splenic plasma cells (CD138+) that were isolated from B6 (lanes 1) or age-matched B6.Nba2-ABC (Nba2) female mice (age ~9-weeks) were analyzed by immunoblotting using antibodies specific to the indicated proteins. (e) Total cell lysates prepared from the purified splenic plasma cells (CD138+) that were isolated from B6.Nba2-ABC male (M) or female (F) mice (age ~9-weeks) were analyzed by immunoblotting using antibodies specific to the indicated proteins.

We have noted earlier that expression levels of IRF5, Blimp-1, and p202 are higher in splenic cells from females than males and the female sex hormone estrogen through estrogen receptor-α (ERα) up-regulates their expression (11, 41). Therefore, we also compared levels of these proteins in splenic CD138+ plasma cells isolated from B6.Nba2-ABC males and age-matched females. As shown in Fig. 6E, we could detect the expression of ERα in CD138+ plasma cells and the levels were higher in females than males. Consistent with our previous observations (11, 41), we detected much higher levels of IRF5, Blimp-1, and p202 in cells from females than males. Interestingly, we also detected much higher levels of STAT1 in females than males. Together, these observations indicated that the gender-dependent increased levels of IRF5 and Blimp-1 proteins in the B6.Nba2-ABC female CD138+ plasma cells, as compared to age-matched males, are associated with increased expression levels of the p202 protein.

B6.Sle123 congenic (congenic for the NZM2410 lupus-prone strain-derived Sle1, Sle2, and Sle3 loci) mice spontaneously develop lupus-like disease, which is characterized by autoantibody production, lymphosplenomegaly, and glomerulonephritis (44). As noted above, the Sle1 locus (derived from chromosome 1 from NZW strain) contains the genomic region corresponding to the Nba2 interval (43, 44). Because splenic cells from NZW females express higher levels of Ifi202 mRNA than the age-matched B6 females (28) and the B6.Sle123 congenic females develop detectable levels of autoantibodies beginning ~6-month of age (44), we decided to compare levels of IRF5, Blimp-1, p202, Aim2, and FcγRIIB proteins in splenic cells from age-matched (age 8–10 weeks; much before detection of autoantibodies) B6, B6.Nba2, NZB, and B6.Sle123 females. As shown in Fig. 7A, we detected increased levels of IRF5 and Blimp-1 in B6.Nba2, NZB, and B6.Sle123 cells as compared to B6 cells. Similarly, levels of p202 protein were higher in B6.Nba2, NZB, and B6.Sle123 cells than B6 cells. As expected from our experiments above (Fig. 1), steady-state levels of Aim2 and FcγRIIB proteins in these four strains of female mice were inversely correlated with the levels of IRF5 and p202 proteins. Furthermore, we detected significantly higher steady-state levels of IRF5 mRNA in all lupus-prone strains of female mice (Fig. 7B). Accordingly, we also detected increased levels of Ifnb and Ifi202 mRNAs (Fig. 7C and D). Together, these observations indicated that increased expression levels of IRF5 and Blimp-1 in lupus susceptible strains of mice are associated with increased levels of the p202 protein.

FIGURE 7. Increased expression of IRF5 in lupus susceptible female mice is associated with increased expression levels of the p202 protein and decreased levels of Aim2 and FcγRIIB.

FIGURE 7

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(a) Total cell lysates prepared from splenic cells that were isolated from B6 (lanes 1) or age-matched B6.Nba2, NZB, and B6.Sle123 females were analyzed by immunoblotting using antibodies specific to the indicated proteins. (b–d) Total RNA prepared from splenic cells that were isolated from B6 or age-matched B6.Nba2, NZB, and B6.Sle123 female mice was analyzed by quantitative real-time PCR for steady-state levels of Irf5 (b), Ifnb (c), and Ifi202 (d) mRNAs. The ratio of the indicated mRNA levels to β-microglubulin mRNA was calculated in units. The ratio of the mRNA levels in the B6 splenic cells is indicated as 1. The error bars represent the standard deviation (** p <0.005; *** p <0.001).

Discussion

Genetic studies involving SLE patients have identified IRF5 and PRDM1 genes as candidate lupus susceptibility genes (1, 2). Correspondingly, the murine Irf5 and Prdm1 genes have been shown to play a role in lupus disease in FcγRIIB−/− Yaa and FcγRIIB−/− (3), and MRL/lpr mice (7). These studies suggest that the IRF5 transcription factor plays an important role in the development of lupus disease. Because the “IFN-signature” in SLE patients is associated with the disease activity (20, 21) and a variant of human IRF5 is linked to increased serum levels of IFN-α in SLE patients (23), we tested whether the IRF5-Blimp-1 axis could regulate the expression of IFN-regulated lupus susceptibility genes within the Nba2 autoimmunity locus. Our observations revealed that: (i) an Irf5 gene deficiency in mice reduced the expression of Blimp-1 and p202 proteins, whereas increased the expression of Aim2 and FcγRIIB proteins (Fig. 1); (ii) overexpression of murine IRF5 in RAW264.7 cells up-regulated expression of Blimp-1, IFN-β, and p202 proteins and also stimulated the activity of the 202-luc-reporter (Fig. 2); (iii) overexpression of Blimp-1 protein in RAW264.7 cells induced expression of Ifi202 gene (Fig. 3); (iv) increased levels of the p202 protein induced expression of Blimp-1 (Fig. 3); (v) gender-dependent factors increase steady-state levels IRF5 mRNA and protein in Aim2-deficient mice (Fig. 4); (vi) deficiency of the inhibitory receptor FcγRIIB in immune cells increases IRF5 mRNA and protein levels (Fig. 5); (vii) increased expression of IRF5 and Blimp-1 proteins in lupus susceptible B6.Nba2 (Fig. 6) and B6.Sle123 (Fig. 7) congenic female mice, as compared to age-matched B6 female mice. is associated with increased expression of p202 protein. These observations revealed that the IRF5-Blimp-1 axis differentially regulates the expression of lupus susceptibility genes within the Nba2 interval and suggest that the axis contributes to lupus susceptibility in part by down-regulating the expression of Aim2 and Fcgr2b genes (Fig. 8).

FIGURE 8. Proposed roles of IRF5 and Blimp-1 proteins in differential regulation of the p202, Aim2, and FcγRIIB proteins encoded by the Nba2 lupus susceptibility genes.

FIGURE 8

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Notably, mice that are deficient in the Irf5 gene do not exhibit any significant change in the number of CD4+ and CD8+ cells (4). However, in these mice, some increase in the number of B cells (CD19+) was evident with the age: the old mice (~14 month) exhibited an expansion of CD19+B220− cells presumably due to plasma blasts. Given that we used cells from ~12-week old Irf5-deficient female mice (Fig. 1), it is unlikely that changes in the expression of Blimp-1, IFN-β, p202, Aim2, and FcγRIIB proteins that are detected in splenic cells from the Irf5-deficient mice (as compared to age-matched wild type mice) are due to changes in the composition of lymphoid compartment.

Type I IFN receptor subunit 1-deficient FcγRIIB−/− Yaa mice maintain a substantial level of residual disease (3), thus, raising the possibility that IRF5 contributes to lupus susceptibility independent of its role in type I IFN expression and activation of the IFN-signaling. Accordingly, a recent study (5) has noted that the IRF5 contributes to murine SLE-like disease through its direct control of class switch recombination of the γ2a locus in B cells. Given that increased levels of p202 protein in B6.Nba2-ABC lupus-prone female mice are associated with inhibition of the transcriptional activity of p53 (50) and that p53 represses class switch recombination (51), further work is needed to test whether the IRF5-induced p202 protein levels promote the class switch recombination by inhibiting the p53-mediated transcription.

Expression of FcγRIIB receptor is detectable in B cells and plasma cells (both splenic and bone marrow) and the receptor controls persistence and apoptosis of plasma cells in the bone marrow (52, 53). Consequently, mice that are deficient in the FcγRIIB receptor expression exhibit B-cell hyperactivity and develop SLE disease spontaneously on certain genetic backgrounds (53). Interestingly, reduced expression of the Fcgr2b gene in the B6.Nba2-ABC congenic mice is associated with defects in apoptosis of germinal center B cells and plasma cells (32). Moreover, increased levels of p202 protein suppress the expression of the FcγRIIB receptor (42). Therefore, our observations that an IRF5-deficiency in mice, which decreased levels of both Blimp-1 and p202 proteins, whereas increased levels of the FcγRIIB receptor, are consistent with the negative regulation of the Fcgr2b gene by the IRF5-Blimp-1-p202 axis.

Previous studies indicate that Aim2 protein is not needed for type I IFN expression after sensing cytosolic double-stranded DNA (40). Moreover, Aim2-deficiency in female mice increased steady-state levels of mRNA encoding for Ifnb and Ifi202 and also stimulated the expression of IFN-inducible genes (36). These observations supported the idea that Aim2 protein suppresses type I IFN responses. Therefore, our observation that the Aim-deficiency increased levels of IRF5 in female mice, as compared to age-matched wild-type female mice (Fig. 4), provides a potential molecular mechanisms by which the Aim2 protein suppresses the type I IFN responses in immune cells.

Expression levels of p202 and Aim2 mRNA and proteins are inversely correlated in immune cells derived from certain strains of male and female mice (36). Moreover, increased levels of p202 protein in RAW264.7 cells reduced levels of Aim2 protein (42). Given that Blimp-1 is reported to suppress transcription of the Aim2 gene (14), our observation that increased levels of the p202 protein induce expression of Blimp-1 (Fig. 3) support the possibility that p202-mediated increased levels of Blimp-1 negatively regulate transcription of the Aim2 gene. Further work is in progress to test this exciting possibility.

The 5’-regulatory region of the Ifi202 gene contains a “GA” box (49), which contains a potential DNA-binding consensus sequence (GAAAG) for the IRF5 and Blimp-1 transcription regulators (14). Therefore, our observations that increased expression of IRF5 or Blimp-1 protein in RAW264.7 cells stimulated the expression of Ifi202 gene and increased the activity of the 202-luc-reporter in promoter-reporter assays (Figs 2 and 3 respectively) are consistent with the transcriptional regulation of the Ifi202 gene by these two regulators. Because Blimp-1 negatively regulates expression of c-Myc and E2F1 transcription factors (12), which negatively regulate the expression of Ifi202 gene (47, 48), our observations do not rule out the possibility that Blimp-1 stimulates the expression of Ifi202 gene through suppression of c-Myc and E2F1 activity. Therefore, further studies are in progress to investigate the molecular mechanisms.

Expression levels of p202 protein in immune cells are regulated by sex hormones (41): levels are higher in B cells, which express increased levels of estrogen receptor-α (ERα), whereas levels are lower in T cells, which express increased levels of androgen receptor (AR). Therefore, our observations that increased levels of ERα in CD138+ plasma cells from female B6.Nba2 than age-matched males are associated with increased levels of IRF5, Blimp-1, and p202 proteins are consistent with our previous observations that levels of IRF5, Blimp-1, and p202 proteins are up-regulated by the female sex hormone estrogen through ERα (11, 41). Moreover, these observations suggest that the IRF5-Blimp-1-p202 axis contributes to sex bias in lupus disease in mice in part through down-regulation of Aim2 and FcγRIIB expression.

In summary, our observations support our model (Fig. 8), which predicts that gender-dependent increased levels of IRF5 in mature B cells and Blimp-1 in plasma cells contribute to lupus susceptibility in part by differentially regulating the expression of Nba2 lupus susceptibility genes, such as Ifi202 and Fcgr2b. Our observations will serve basis to understand the role of the IRF5-Blimp-1-p202 axis in lupus susceptibility in mice.

Acknowledgments

We would like to thank Dr. K. Calame (Columbia University, New York) for generously providing the murine Blimp-1 expression plasmid. We also thank Drs. K. Fitzgerald and V. Rathinam (University of Massachusetts Medical School, Worcester) for providing spleens from the Aim2-deficient mice.

This work is supported by a grant (AI066261 and AI089775) from the National Institutes of Health (NIH) to D.C. Research in the laboratory of P. M. P. is supported by a NIH grant AI067632. A grant from the Lupus Research Institute supported work in the laboratory of L. D. E.

Footnotes

Publisher's Disclaimer: This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.”

Disclosures

The authors have no financial conflicts of interest.

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