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IntervalNba2Gene within the an Interferon-Inducible Lupus Susceptibility

,Ifi202Differentially Regulate Expression of Female and Male Sex Hormones

Bupp, Karen A. Gould and Divaker ChoubeyRavichandran Panchanathan, Hui Shen, Melanie Gubbels

http://www.jimmunol.org/content/183/11/7031doi: 10.4049/jimmunol.0802665November 2009;

2009; 183:7031-7038; Prepublished online 4J Immunol 

Referenceshttp://www.jimmunol.org/content/183/11/7031.full#ref-list-1

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Female and Male Sex Hormones Differentially RegulateExpression of Ifi202, an Interferon-Inducible LupusSusceptibility Gene within the Nba2 Interval1

Ravichandran Panchanathan,* Hui Shen,* Melanie Gubbels Bupp,2† Karen A. Gould,‡

and Divaker Choubey3*

Increased expression of IFN-inducible Ifi202 gene in certain strains of female mice is associated with susceptibility to systemiclupus erythematosus (SLE). Although, the development of SLE is known to have a strong sex bias, the molecular mechanismsremain unknown. Here we report that in vivo treatment of orchiectomized (NZB � NZW)F1 male mice with the female sexhormone 17�-estradiol significantly increased steady-state levels of Ifi202 mRNA in splenic cells, whereas treatment with the malehormone dihydrotestosterone decreased the levels. Moreover, increased expression of Ifi202 in B6.Nba2 B cells and reducedexpression in T cells were associated with increased levels of estrogen receptor-� (ER�) and androgen receptor, respectively.Furthermore, the steady-state levels of Ifi202 mRNA were higher in splenic cells from C57BL/6, B6.Nba2, NZB, and (NZB �

NZW)F1 female mice as compared with males. 17�-estradiol treatment of B cells and WT276 cells increased Ifi202 mRNA levels,whereas treatment with dihydrotestosterone decreased the levels. Interestingly, overexpression of ER� in WT276 cells increasedthe expression of Ifi202 and stimulated the activity of the 202-luc-reporter through the c-Jun/AP-1 DNA-binding site. Accordingly,ER� preferentially associated with the regulatory region of the Ifi202 gene in female B6.Nba2 B cells than in males. Furthermore,Ifi202 mRNA levels were detectable in splenic cells of wild-type (Esr1�/�), but not null (Esr1�/�), (NZB � NZW)F1 female mice.Collectively, our observations demonstrate that the female and male sex hormones differentially regulate the expression of Ifi202,thus providing support for the role of Ifi202 in sex bias in SLE. The Journal of Immunology, 2009, 183: 7031–7038.

S tudies have demonstrated gender bias in the developmentof systemic lupus erythematosus (SLE),4 which occurs ata female-to-male ratio of 10:1 (1–4). The disease, which

predominantly affects women of childbearing age, is characterizedby the production of pathogenic autoantibodies to nuclear Ags anddevelopment of lupus nephritis (5–7). Studies in human SLE pa-tients and in mouse models of SLE have provided evidence thatSLE is a polygenic disease (5, 6, 8–11), which involves defects ina number of cell signaling pathways, resulting in increased sur-vival of autoreactive cells (5, 12).

Clinical studies suggest that the gender bias in SLE is influencedby sex hormones, such as estrogen and androgen (2–4). It is welldocumented that immune reactivity is more enhanced in female

SLE patients than in males, and lymphocytes and monocytes fromfemale patients show higher Ag-presenting activity (2, 3). In gen-eral, female SLE patients exhibit higher levels of serum IgG thando males and mount more robust humoral immune response.Therefore, it seems likely that enhanced activation of B cells infemales contributes to lupus susceptibility. Moreover, female hor-mone estrogen is known to have immunostimulatory effects,whereas male hormone androgen is known to have immunosup-pressive effects (2–4).

As for SLE patients, in a (NZB � NZW)F1 spontaneous mousemodel of SLE disease, female mice develop the disease earlier andhave shorter lifespans than do males (13, 14). Moreover, castratedmale (NZB � NZW)F1 mice have earlier onset of lupus and shorterlifespan than do their intact littermates (14). Additionally, treatment ofthese mice with estrogen exacerbates disease activity and causes earlymortality (13, 14). In contrast, administration of exogenous testoster-one, when begun between at 2 and 6 mo of age, extends the lifespanof ovariectomized (NZB � NZW)F1 females (13, 14). These obser-vations suggest that sex hormones, such as estrogen and testosterone,influence the pathogenesis of murine lupus.

Sex hormone estrogen classically functions by activating one ofits two nuclear receptors, estrogen receptor-� (ER�) and estrogenreceptor-� (ER�) (15–17). Although both estrogen receptors areexpressed in most immune cells, ER� is shown to be predomi-nantly expressed (17). Several recent studies involving variousmouse models of SLE have suggested a prominent role for ER� inthe development of lupus disease (18–20). Interestingly, the ER�

deficiency in (NZB � NZW)F1 female mice attenuated glomeru-lonephritis and increased survival of mice (20). Of note, the in-creased survival of ER�-deficient female mice was associated withreduced development of anti-chromatin and anti-dsDNA Abs aswell as reduced serum levels of IFN-� (20).

*Department of Environmental Health, University of Cincinnati, Cincinnati, OH45267; †Division of Clinical Immunology and Human Medical Genetics, Universityof Colorado Health Sciences Center, Denver, CO 80262; and ‡Department of Genet-ics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha,NE 68198

Received for publication August 13, 2008. Accepted for publication October 1, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grant R01 AI066261 toD.C. A Predoctoral Award (no. 0315287Z) from the American Heart Associationsupported M.G.B.2 Current address: Roche Palo Alto, Palo Alto, CA 94304.3 Address correspondence and reprint requests to Dr. Divaker Choubey, Departmentof Environmental Health, University of Cincinnati, 3223 Eden Avenue, P.O. Box670056, Cincinnati, OH 45267. E-mail address: Divaker.choubey@uc.edu4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; AR, androgenreceptor; ChIP, chromatin immunoprecipitation; DHT, dihydrotestosterone; E2, 17�-estradiol; ER, estrogen receptor; ERE, estrogen responsive core element.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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Binding of 17�-estradiol (E2) to ERs results in activation ofERs and transcriptional activation of ER target genes (15–17).Many ER target genes contain a minimal estrogen responsive coreelement (ERE) sequence (GGTCANNNTGACC) in the 5� regu-latory or promoter region. The ERE sequence functions in an ori-entation- and distance-independent manner, both of which arecharacteristics of an enhancer (21). Moreover, ER is also known tobind DNA through half ERE sites (GGTCAN) (15, 21). Becausemolecular mechanisms of the recruitment of ER to the promoterregion of its target genes remain relatively complex, it remains anactively investigated research area. Importantly, proteins that areencoded by the ER target genes mediate many of the biologicalactivities of female sex hormone estrogen (15–17).

Male sex hormone androgen signals via the intracellular andro-gen receptor (AR), a member of the superfamily of nuclear hor-mone receptors (22). Androgen-dependent activation and nucleartranslocation of the AR is followed by its binding to specific re-sponse elements in the promoter regions of target genes to mod-ulate gene expression either positively or negatively (22). Inter-estingly, expression of AR mRNA has been reported in enrichedpopulations of CD4� T lymphocytes, CD8� T lymphocytes, andmacrophages (23). However, the enriched populations of B lym-phocytes expressed only low levels of AR mRNA (23).

IFN-inducible Ifi200 gene family includes structurally and func-tionally related murine (E.G., Ifi202a, Ifi202b, Ifi203, Ifi204,Ifi205, And AimII) and human (e.g., IFI16, MNDA, AIM2, andIFIX) genes (24–30). The Ifi202a and Ifi202b are highly homolo-gous murine genes that encode p202a and p202b proteins, respec-tively (26, 27). These two proteins differ in only 7 amino acids (outof 445 amino acids) (27). Because Abs, which have been raisedagainst the p202a protein (31), also detect p202b protein (26, 27),in this study, we have referred both p202a and p202b proteins asp202 protein.

Generation of B6.Nba2 congenic (congenic for NZB-derivedNba2 interval on C57BL/6 genetic background) mice and geneexpression analyses identified Ifi202 (probably both Ifi202a andIfi202b genes) gene as a lupus susceptibility gene (26, 32). Impor-tantly, consistent with promoter polymorphisms contributing todifferential expression of Ifi202a gene between C57BL/6 and NZBmice (26, 32, 33), increased steady-state levels of Ifi202a andIfi202b mRNAs (as compared with C57BL/6 mice) are detectablein splenic cells from NZB and B6.Nba2 mice (33). Moreover, lev-els of Ifi202a mRNA are relatively higher than the Ifi202b mRNA(33). Interestingly, increased expression of p202 protein (probablyboth p202a and p202b proteins) in B6.Nba2 splenic B and T cells(more in B cells than in T cells) is associated with defects inapoptosis of B cells and increased susceptibility to develop lupus-like disease (26, 32). Furthermore, the B6.Nba2 congenic femalemice produce higher levels of antinuclear autoantibodies than didthe age-matched male mice, and (B6.Nba2 � NZW)F1 femalemice develop severe proteinuria with much higher frequency (34).These observations prompted us to investigate whether sex hor-mones could regulate expression of the Ifi202 gene. Here, we re-port that female and male sex hormones differentially regulate theexpression of Ifi202.

Materials and MethodsMice, orchiectomy, and sex hormone treatment

Spleens were isolated from wild-type (Esr1�/�) or null (Esr1�/�) (NZB �NZW)F1 female mice (20) (age �10 wk) that were housed in animal fa-cilities of University of Nebraska Medical Center (Omaha, NE). Age-matched (6–8 wk old) male and female nonautoimmune (C57BL/6J) andpreautoimmune (B6.Nba2, NZB, and (NZB � NZW)F1) mice were pur-chased from The Jackson Laboratory and housed in the animal facilities of

the University of Cincinnati. All animal experimental protocols were ap-proved by the Institutional Animal Care and Use Committee at the insti-tution where animals were housed.

Male (NZB � NZW)F1 mice were orchiectomized at 3 mo of age asdescribed (35). After orchiectomy, pellets (Innovative Research of Amer-ica) releasing E2, dihydrotestosterone (DHT), or placebo for up to 3 wkwere inserted s.c. with a 10-gauge needle. Serum was collected on day 7,8, or 9 and then analyzed by the Endocrine Laboratory at Colorado StateUniversity (Fort Collins, CO) for luteinizing hormone, estradiol, and tes-tosterone by radioimmunoassay as described (35). Orchiectomized malemice that were treated with E2 pellets exhibited serum E2 levels of 162pg/ml, while levels in placebo-treated mice were not detectable (data notshown). Intact and orchiectomized mice treated with DHT had similarlylow luteinizing hormone levels (�1 ng/ml), while orchiectomized micetreated with placebo had �20 ng/ml luteinizing hormones (data notshown). Orchiectomized male mice treated with placebo demonstrated tes-tosterone levels �2 ng/ml, while intact (NZB � NZW)F1 male mice ex-hibited 12 ng/ml testosterone (35).

Splenocyte isolation, purification of B or T cells, cell culture,and treatments

Total single-cell splenocytes were prepared from age-matched male or fe-male mice as described previously (36). After lysis of RBC, splenocyteswere resuspended in RPMI 1640 medium supplemented with 10% FBS,1% penicillin/streptomycin/glutamate, and 1� MEM, nonessential aminoacids/sodium pyruvate. Unless otherwise indicated, splenic cells from twoor more age-matched male or female mice were pooled to purify B or Tcells and to prepare total RNA or protein extracts. B or T cells were pu-rified from splenic cells using magnetic beads (purification kit purchasedfrom Miltenyi Biotec), allowing the positive selection of either B or T cells.The purified (�90–95% pure) T and B cells were used immediately foradditional experiments.

Estrogen-responsive mouse breast cancer cell line WT276 (37) was pro-vided by Dr. J. Welsh (University of Notre Dame, Notre Dame, IN). Cellswere maintained in DMEM medium supplemented with 10% FBS and 1�antibiotic-antimycotic solution (Invitrogen). For treatment of WT276 cellswith E2 or DHT, cells were cultured in phenol red-free RPMI 1640 me-dium (Invitrogen) and the medium was supplemented with 10% charcoal-stripped FBS (Invitrogen). Cells were treated in vitro with either DHT (23)or E2 (38) at the concentration used previously.

Plasmids and expression vectors

Dr. P. Chambon (Centre National de la Recherche Scientifique, France)provided ER� expression plasmid that allowed expression of ER� (39).The 202-luc-reporter (36) and the mutant 202AP-1mutCS1-luc reporter(40) plasmids have been described.

Reporter assays

For reporter assays, subconfluent cultures of WT276 cells (in 6-well plates)were transfected with the 202-luc (2.5 �g) or the 202AP-1mutCS1-luc(with mutated AP-1CS1 site; see Ref. 40) reporter plasmid along withpRL-TK reporter plasmid (0.5 �g), using calcium phosphate transfectionkit (Invitrogen), as suggested by the supplier. When indicated, cells wereeither treated with ethanol (vehicle) or the indicated concentration of E2 orDHT for 16–24 h. Unless otherwise indicated, cells were harvested be-tween 40 and 45 h after transfections. Cells were lysed, and the firefly andRenilla dual luciferase activities were determined as described previously(36). Student’s t test for paired samples was used to determine statisticalsignificance of the reporter activity data. Differences were considered sta-tistically significant at p � 0.05.

Isolation of RNA from splenocytes and RT-PCR

Splenocytes (5–8 � 106 cells) were used to isolate total RNA using TRIzol(Invitrogen). Total RNA was digested with DNase I (to remove any con-taminating genomic DNA in the preparation), and 0.5–2 �g of RNA wasused for RT-PCR reaction using a pair of the Ifi202 primer (forward, 5�-ggtcatctaccaactcagaat-3�; reverse, 5�-ctctaggatg ccactgctgttg-3�). For RT-PCR, we used the SuperScript One-Step RT-PCR system from Invitrogen.Primers for Esr1 gene (forward, 5�-aattctgacaatcgacgccag-3�; reverse,5�-ctctaggatgccactgctgttg-3�).

Quantitative real-time TaqMan PCR technology (7300 Real-Time PCRSystem; Applied Biosystems) and commercially available real-time Taq-Man gene expression assays were used to compare expression of genesbetween male and female mice. The PCR cycling program consisted ofdenaturation at 95oC for 10 min, 40 cycles at 95oC for 15 s, followed byannealing and elongation at 60oC for 1 min. The TaqMan assays for Ifi202

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(assay Mm0304 8198_m1; the assay allowing the detection of both Ifi202aand Ifi202b mRNA levels), Esr1 (assay Mm00433149_m1), and the en-dogenous control �2-microglobulin (assay Mm00437762_m1) were pur-chased from Applied Biosystems and used as suggested by the supplier.

Chromatin immunoprecipitation (ChIP) assays

Splenic B cells were purified using magnetic beads, and the purified splenicB cells (�2–4 � 106) were processed for ChIP assays using Champion-ChIP One-Day Kit (SABioscience) as suggested by supplier. In brief, celllysates containing equal amounts of protein were immunoprecipitated witheither an isotope Ab or anti-ER� Abs (sc-542X; Santa Cruz Biotechnol-ogy). The immunoprecipitates were collected using protein A beads. Im-mune complexes were eluted from beads, proteins were digested, and DNAwas collected. The isolated DNA was purified, precipitated, washed, anddissolved in water. Semiquantitative PCR was performed with DNA sam-ples for 28 cycles. PCR products were resolved in an agarose gel andvisualized. The PCR primers that were used have been described previ-ously (41). Quantitative PCR was performed using commercially availableprimer set (GPM041367; SABioscience) corresponding to the genomic 5�regulatory region of the Ifi202 gene and real-time PCR conditions as de-scribed above.

Immunoblotting

Total splenocytes, WT276, or NIH 3T3 cells were collected in PBS andresuspended in a modified radioimmunoprecipitation assay (RIPA) lysisbuffer (50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 1% Nonidet P-40, 0.5%sodium deoxycholate, 0.1% SDS), supplemented with 1� protease inhib-itor (Roche Diagnostics), and incubated at 4°C for 30 min. Cell lysateswere sonicated briefly before centrifugation at 14,000 rpm in a microcen-trifuge for 10 min at 4°C. The supernatants were collected, and the proteinconcentration was measured by Bio-Rad protein assay kit. Equal amountsof protein were processed for immunoblotting. Antiserum to p202 proteinhas been described previously (31). The p202 antiserum detects both p202aand p202b proteins in immunoblotting (27, 31). Abs to detect mouse ER�(sc-542; MC-20), AR (sc-816), and �2-mcroglobulin (sc-13565) were pur-chased from Santa Cruz Biotechnology. Abs to detect �-actin (no. 4967)were purchased from Cell Signaling Technology.

Statistical analyses

Data are presented as mean � SEM. A p value of �0.05 was consideredstatistically significant. These methods were performed using GraphPadPrism 5.02 software for Windows (GraphPad Software).

ResultsIn vivo treatment of orchiectomized male mice with estrogen in-creased steady-state levels of Ifi202a mRNA, whereas treatmentwith DHT reduced the mRNA levels.

To investigate the role of sex hormones in the regulation of theIfi202 gene, we chose to compare expression levels of Ifi202amRNA (because splenic cells express both Ifi202a and Ifi202bgenes and steady-state levels Ifi202a mRNA are more than Ifi202b;see Ref. 33) in (NZB � NZW)F1 male mice (age 12 wk) that wereorchiectomized and reconstituted with slow-releasing pellets re-leasing E2, DHT, or placebo. After 3 wk of the reconstitution,splenic cells were analyzed for steady-state levels of Ifi202amRNA by real-time PCR. As shown in Fig. 1, treatment of malemice with the E2-releasing pellet, under our experimental condi-tions (35), measurably increased levels of Ifi202a mRNA in mostof the E2-treated mice as compared with control mice. Interest-ingly, treatment of mice with DHT decreased the Ifi202 mRNAlevels to a measurable extent in most mice as compared with thecontrol mice. These observations are consistent with the possibilitythat increased in vivo levels of female sex hormone E2 in orchi-ectomized (NZB � NZW)F1 male mice contribute to increasedsteady-state levels of Ifi202a mRNA. Moreover, our observationsalso indicated that increased levels of male sex hormone androgen inmice contribute to decreased steady-state levels of Ifi202a mRNA.

Increased expression of Ifi202 in splenic B cells in female miceis associated with increased levels of ER� and reduced levelsof AR

Our earlier studies had revealed that splenic B cells from preau-toimmune (4-mo-old) B6.Nba2 female mice express higher levelsof Ifi202 mRNA as compared with T cells (32). Therefore, ourabove observations (Fig. 1) that in vivo treatment of orchiecto-mized (NZB � NZW)F1 male mice with estrogen increasedIfi202a mRNA levels, whereas treatment with DHT reduced themRNA levels, prompted us to compare steady-state levels of Ifi202mRNA in splenic B and T cells from male or female preautoim-mune (age �10 wk) B6.Nba2 mice (32). We noted that steady-state levels of Ifi202 mRNA were significantly (�7-fold) higher insplenic B cells than in T cells in the female mice (Fig. 2A). Inter-estingly, we found that levels of Ifi202 mRNA were consistentlyhigher in B and T cells from female mice than the age-matchedmale mice (Fig. 2A). Because sex hormones, such as E2 and DHT,regulate gene expression through binding to their respective re-ceptors (15, 22), we also compared expression levels of ER�, AR,and p202 proteins in B and T cells from male and female B6.Nba2mice. As shown in Fig. 2B, increased levels of p202 protein insplenic B cells from B6.Nba2 female mice as compared with age-matched male mice (compare lane 4 with lane 3) were associatedwith increased levels of ER� protein and reduced levels of ARprotein. In contrast, reduced levels of p202 protein in T cells frommale mice as compared with age-matched female mice (comparelane 1 with lane 2) were associated with increased levels of ARand reduced levels of ER� protein. Furthermore, consistent withour above observations (Fig. 2, A and B), basal steady-state levelsof Ifi202 mRNA were relatively high in B cells from C57BL/6 or(NZB � NZW)F1 female mice than from the age-matched malemice, and treatment of B cells with estrogen (10 nM for 24 h)resulted in further increases in the steady-state levels of the mRNA(Fig. 2C). Taken together, these observations revealed that in-creased expression of the Ifi202 in splenic B cells from B6.Nba2female mice is associated with increased levels of ER� and re-duced levels of AR. Additionally, our observations revealed thatthe basal steady-state levels of the Ifi202 mRNA are relativelyhigher in B cells from nonautoimmune (C57BL/6) and preautoim-mune (B6.Nba2 and (NZB � NZW)F1) female mice than fromage-matched males, and the treatment with E2 can increase themRNA levels further.

FIGURE 1. Ifi202 mRNA levels are regulated by sex hormones insplenocytes. Male (NZB � NZW)F1 mice (age 3 mo) were orchiectomizedand reconstituted with slow-releasing pellets releasing E2 (n 6), DHT(n 6), or placebo (n 6). Three weeks later, splenic total RNA wassubjected to quantitative real-time PCR using primers specific for theIfi202a gene. The ratio of Ifi202a mRNA to �2-microglobulin mRNA wascalculated in units, with 1 unit being the ratio of Ifi202a mRNA to �2-microglobulin mRNA in (NZB � NZW)F1 splenocytes). Units are indi-cated for an individual mouse in each group of mice.

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Sex-dependent regulation of Ifi202 expression

Our earlier studies had revealed that increased expression of Ifi202in preautoimmune B6.Nba2 female mice (as compared with non-autoimmune C57BL/6 mice) is associated with increased lupussusceptibility (26, 32). Because steady-state levels of Ifi202mRNA are very low (as compared with age-matched B6.Nba2mice), but detectable in C57BL/6 mice (33), we decided to com-pare the steady-state levels of Ifi202 mRNA between male andfemale C57BL/6 mice. We noted that basal levels of Ifi202 mRNAwere detectable in splenic cells from C57BL/6 male and femalemice (age �10 wk), and IFN treatment of cells increased the levelsfurther (Fig. 3A). Interestingly, the basal levels of Ifi202 mRNAwere �5-fold higher in C57BL/6 females than age-matched males.This observation indicated that steady-state levels of Ifi202 mRNAare regulated in sex-dependent manner in C57BL/6 splenic cells.

Encouraged by the above observations, we also compared thesteady-state levels of Ifi202 mRNA between young male and fe-

male lupus-prone mice (B6.Nba2, NZB, and (NZB � NZW)F1).As shown in Fig. 3B, basal steady-sate levels of the Ifi202 mRNAwere consistently higher in splenic cells from the females thanfrom the age-matched males. Taken together, these observationssuggested that the steady-state levels of Ifi202 mRNA in spleniccells are regulated in a sex-dependent manner.

Treatment of WT276 cells with female or male sex hormoneregulates Ifi202 expression

The mouse mammary tumor cell line WT276 has been reported to beestrogen-responsive (37). Therefore, to identify molecular mecha-nisms by which E2 treatment up-regulates expression of Ifi202 gene,we explored whether treatment of WT276 cells with sex hormonescould regulate the expression of Ifi202 gene. For this purpose, wetreated cells with increasing concentrations (1, 5, or 10 nM) of femalesex hormone E2 (these concentrations were chosen based on an earlierstudies; see Ref. 38) for 16 h. As shown in Fig. 4A, the treatment withE2 resulted in an increase in steady-state levels of Ifi202 mRNA asdetermined by semiquantitative RT-PCR. Moreover, consistent withE2-mediated up-regulation of Ifi202 expression by ER�, treatment ofcells with tamoxifen (100 nM), a selective estrogen receptor modu-lator (42), which resulted in increases in ER� mRNA and proteinlevels (data not shown), abrogated the E2-mediated increases in Ifi202mRNA levels (data not shown). Consistent with the above observa-tions, we also noted increases in p202 protein levels after treatment ofcells with increasing concentrations of E2 (Fig. 4B). Interestingly,

FIGURE 2. Increased steady-state levels of Ifi202 mRNA and proteinin splenic B cells from B6.Nba2 female mice are associated with in-creased levels of ER� and reduced levels of AR. Total splenocytesisolated from preautoimmune (age �9 wk) male or female mice (spleencells pooled from three age-matched male or female mice) were sub-jected to purification of B or T cells using a kit (from Miltenyi Biotec)that allowed positive selection of either B or T cells. Total RNA andproteins were prepared from the purified (cells �90% pure) T or Bcells. A, Steady-state levels of Ifi202 mRNA were analyzed by quanti-tative TaqMan real-time PCR. The ratio of Ifi202 mRNA to �2-micro-globulin mRNA was calculated in units (one unit being the ratio ofIfi202 mRNA to �2-microglobulin mRNA in B6.Nba2 splenocytes). Therelative levels of Ifi202 mRNA in T cells from male mice are indicatedas in Fig. 1. A representative experiment is shown. Results are meanvalues of triplicate experiments and error bars represent SD (��, p �0.005; ���, p � 0.0005). B, Total cell extracts prepared from T (lanes1 and 2) or B (lanes 3 and 4) cells were analyzed by immunoblottingusing Abs specific to the indicated proteins. A representative experi-ment is shown. M, male; F, female. The numbers below the figureindicate relative fold change (FC) in the p202 protein levels as com-pared with levels detected in T cells (indicated as 1.0) from male mice.C, Steady-state levels of Ifi202 mRNA were analyzed in splenic B cells(isolated from male or female mice) without any or after E2 (10 nM,24 h) treatment by quantitative TaqMan real-time PCR. The ratio ofIfi202 mRNA to �2-microglobulin mRNA was calculated in units, with1 unit being the ratio of Ifi202 mRNA to �2-microglobulin mRNA. Therelative levels of Ifi202 mRNA in B cells from C57BL/6 male mice areindicated as in Fig. 1. A representative experiment is shown. Results aremean values of triplicate experiments and error bars represent SD (�,p � 0.03; ���, p � 0.005).

FIGURE 3. Sex-dependent regulation of Ifi202 expression. A,Splenic cells were prepared from age-matched C57BL/6J male or fe-male mice (age �10 wk; cells pooled from two age-matched male orfemale mice) and cells were either left untreated or treated with IFN-�(1000 U/ml, for 14 h). Total RNA was isolated from control and IFN-treated cells, and steady-state levels of Ifi202 mRNA were analyzed byquantitative TaqMan real-time PCR. The ratio of Ifi202 mRNA to �2-microglobulin mRNA was calculated in units, with 1 unit being the ratioof Ifi202 mRNA to �2-microglobulin mRNA in C57BL/6J splenocytes.The relative levels of Ifi202 mRNA in the male mice are indicated as inFig. 1. A representative experiment is shown. Results are mean valuesof triplicate experiments, and error bars represent SD (�, p � 0.05).B, Splenic cells were prepared from age-matched nonautoimmune(C57BL/6J) or preautoimmune (B6Nba2, NZB, and (NZB/W)F1) maleor female mice (age �10 wk; cells pooled from two age-matched maleor female mice). Total RNA was isolated and steady-state levels ofthe Ifi202 mRNA were analyzed by quantitative TaqMan real-timePCR. The ratio of Ifi202 mRNA to �2-microglobulin mRNA was cal-culated in units, with 1 unit being the ratio of Ifi202 mRNA to �2-microglobulin mRNA in C57BL/6J. A representative experiment isshown. Results are mean values of triplicate experiments, and error barsrepresent SD (��, p � 0.005; ���, p � 0.0005).

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treatment of cells with 5 nM concentration of E2 resulted in increasesin p202 protein levels (compare lane 3 with lane 2). However, treat-ment of cells with 10 nM E2 resulted in moderate decreases in p202protein levels (compare lane 4 with lane 3). Furthermore, treatment ofWT276 cells with the male sex hormone DHT decreased basal levelsof p202 protein in a dose-dependent manner. Taken together, theabove observations demonstrated that in vitro treatment of WT276cells with female sex hormone E2 or male sex hormone DHT differ-entially regulated the levels of the p202 protein.

Estrogen through ER� up-regulates expression of the Ifi202gene

To further investigate whether estrogen treatment of cells acti-vates transcription of the Ifi202 gene through ER�, we firsttransfected estrogen-responsive WT276 cells with 202-luc-re-porter plasmid and treated cells with increasing concentrationsof E2. As shown in Fig. 5A, treatment of cells with increasingconcentrations of E2 stimulated the activity of the 202-luc-re-porter in a concentration-dependent manner. Next, we trans-fected WT276 cells with 202-luc-reporter along with either anempty vector or a plasmid encoding the ER� receptor. Aftertransfections, cells were either treated with ethanol alone (ve-hicle) or 10 nM E2. As shown in Fig. 5B, transfection of cellswith the plasmid encoding ER� protein stimulated the activityof the 202-luc-reporter. Interestingly, transfection of cells withthe plasmid encoding the ER� protein and subsequent treatmentof the transfected cells with E2 strongly stimulated the activityof the reporter. To further test whether ER� regulates expres-sion of the Ifi202 gene, we transfected NIH 3T3 mouse fibro-blasts (we chose these cells because basal levels of p202 proteinare detectable and these cells are not known to express ER�)with either an empty vector or the plasmid encoding ER� (cellstreated with 5 nM E2 in phenol red-free medium) and analyzedthe expression of p202 protein. As shown in Fig. 5C, ectopicexpression of ER� protein in NIH 3T3 cells resulted in in-creases in p202 protein levels. Taken together, these observa-

tions suggested that activation of ER� by E2 in WT276 andNIH 3T3 cells up-regulates the expression of the Ifi202 gene.

Transcriptional activation of Ifi202 by ER�

The 5�-regulatory region (�800 bp) of Ifi202 gene contains atleast two potential ERE half-sites (Fig. 6A). Moreover, one ofthe potential ERE half-sites is located next to an AP-1 DNA-binding site (AP-1CS1) that can bind to c-Jun/AP-1 in gel-mo-bility shift assays (40). Molecular mechanisms through whichthe ER� regulates the transcription of its target genes throughthe half ERE sites are relatively complex (15, 16) and known toinvolve collaborations with other transcription factors, such asc-Jun/AP-1 (15, 16). Therefore, to investigate the role of ER�in the regulation of Ifi202 expression, we compared the activityof the wild-type (202-luc) and the mutant (202-AP-1CS1-luc, inwhich the AP-1CS1 site is mutated) reporters without or afterE2 treatment of WT276 cells. As shown in Fig. 6B, the activityof the wild-type reporter was stimulated �2.5-fold by the treat-ment of cells with E2. However, the mutation in the AP-1CS1site in the 5� regulatory region of the Ifi202 gene abrogated thestimulation of the activity of the reporter after E2 treatment.This suggested that E2 treatment of WT276 cells stimulatestranscription of the Ifi202 gene through the AP-1CS1 site. Tofurther examine the role of ER� in the transcriptional activationof the Ifi202 gene by E2, we also compared in vivo associationof ER� with the 5� regulatory region of the Ifi202 gene insplenic B6.Nba2 B cells between female and age-matchedmales by ChIP assays. As shown in Fig. 6C, some binding ofER� to Ifi202 regulatory region was detected in male B cells(lane 5). Interestingly, relatively more ER� bound to the Ifi202regulatory region in female B cells (compare lane 6 with lane5). Moreover, a quantitative real-time pPCR revealed (Fig. 6D)that there was �4-fold more binding of ER� to the Ifi202 reg-ulatory region in the female B cells than in males. These ob-servations indicated that relatively higher levels of ER� asso-ciated with the regulatory region of the Ifi202 gene in femaleB6.Nba2 B cells than in males.

FIGURE 4. Treatment of WT276 cells with sex hormones regulates theIfi202 expression. A, Subconfluent cultures of WT276 cells were eithertreated with ethanol (vehicle) or increasing concentration of estrogen (1, 5,or 10 nM) for 16 h in phenol red-free medium. Total RNA isolated fromcells was analyzed by semiquantitative PCR for steady-state levels ofIfi202 or actin mRNA levels. B, Subconfluent cultures of WT276 cells weretreated with ethanol (vehicle; lane 2), 5 (lane 3), or 10 nM (lane 4) con-centration of E2, or 5 (lane 5) or 10 nM (lane 6) concentration of DHT for16 h in phenol red-free medium. Total cell lysates from control and treatedcells were analyzed by immunoblotting using Abs specific to the indicatedproteins. We also included a positive control for p202 protein in our ex-periment (lane 1). The numbers below the figure indicate the fold differ-ence (FD) in p202 protein levels as compared with control (lane 2).

FIGURE 5. Treatment of estrogen-responsive WT276 cells with E2 oroverexpression of ER� stimulated the activity of 202-luc-reporter. A, Sub-confluent cultures of WT276 cells in a 6-well plate were transfected with202-luc-reporter plasmid (2.5 �g) along with pRL-TK (0.5 �g) plasmidusing calcium phosphate precipitation method. Twenty-four hours aftertransfections, cells were either treated with ethanol (vehicle) or E2 (5 or 10nM). Forty to 45 h after transfections, cells were processed for dual lucif-erase activity. B, Subconfluent cultures of WT276 cells in a 6-well platewere transfected with 202-luc-reporter plasmid (2.5 �g), pRL-TK (0.5 �g)plasmid along with either empty vector, or a plasmid encoding ER� usingcalcium phosphate precipitation method. Twenty-four hours after transfec-tions, cells were either treated with ethanol (vehicle) or E2 (5 or 10 nM).Forty to 45 h after transfections, cells were processed for dual luciferaseactivity. C, Subconfluent cultures of NIH 3T3 cells in a 60-mm plate weretransfected either empty vector or a plasmid encoding ER� using calciumphosphate precipitation method. Forty to 45 h after transfections, cells wereprocessed for immunoblotting using Abs specific to the indicated proteins.

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Basal steady-state levels of Ifi202 mRNA are not detectable inER�-deficient mice

Because our above observations demonstrated that activation ofER� by E2 up-regulates the expression of the Ifi202 gene inWT276 cells, we compared steady-state levels of Ifi202 mRNAbetween ER�-expressing wild-type (Esr1�/�) and ER�-deficient(Esr1�/�) age-matched (NZB � NZW)F1 female mice (20). Asshown in Fig. 7, we found that basal levels of Ifi202 mRNA weredetectable in splenic cells from the wild-type mice. However,Ifi202 mRNA levels were not detectable in splenic cells from theEsr1-null mice. These observations indicated that the basal steady-state levels of the Ifi202 mRNA in (NZB � NZW)F1 splenic cellsare regulated by expression levels of ER�.

DiscussionIncreased levels of estrogen in certain lupus-prone strains offemale mice are known to activate and increase survival of au-toreactive cells in a naive repertoire (3, 4, 43– 45). However,molecular mechanisms that contribute to increased cell survivalin female mice remain to be elucidated. Therefore, identifica-tion of lupus-susceptibility genes whose expression is regulatedby genetic factors and sex hormones, as well as elucidation ofthe role of encoded proteins in cell survival, is expected toprovide insights into the molecular basis of sex bias in lupussusceptibility. Interestingly, estrogen treatment of femaleBALB/c transgenic (transgenic for R4A-�2b H chain) mice re-sults in up-regulation of Bcl-2 expression in splenic B cells(43). Furthermore, a recent study has identified a number ofgenes whose expression is regulated by sex hormones in spleniccells of (NZB � NZW)F1 lupus-prone mice (35). The study alsoidentified the Trp53 gene (encoding the p53 protein), whoseexpression is up-regulated in male mice (as compared with fe-male mice). p53 represses transcription of the Ifi202 gene (46).Therefore, our observation that expression of the Ifi202 gene isdown-regulated by male sex hormone (DHT) in orchiectomized(NZB � NZW)F1 male mice (Fig. 1) makes it likely thatincreased levels of male sex hormone negatively regulate Ifi202expression, in part by up-regulating the p53 expression. More-over, the study by Xin et al. (35) also identified other genes thatare known to encode proteins with immunomodulatory func-tions. However, none of the identified estrogen-responsivegenes mapped within the NZB-derived Nba2 lupus susceptibil-ity interval, which is syntenic to the human lupus susceptibilitylocus (26, 32).

Promoter polymorphisms-dependent increased expression ofIfi202 gene in female mice of certain strains is associated withdefects in apoptosis of B cells and the development of lupus-like disease (26, 32, 33). Because the development of lupus-likedisease in B6.Nba2 mice has sex bias (34), we investigatedwhether sex hormones could regulate the expression of theIfi202, an IFN-inducible lupus susceptibility gene within theNba2 interval. Our experiments revealed that: (1) in vivo treat-ment of orchiectomized (NZB � NZW)F1 male mice with fe-male sex hormone E2 increased steady-state levels of Ifi202mRNA, whereas treatment with male sex hormone DHT de-creased the mRNA levels (Fig. 1); (2) increased steady-statelevels of Ifi202 mRNA and protein in splenic B cells fromB6.Nba2 male and female mice were associated with increasedlevels of ER� and reduced levels of AR (Fig. 2); (3) steady-state levels of Ifi202 mRNA were relatively higher in C57BL/6,B6.Nba2, NZB, and (NZB � NZW)F1 female mice than in age-matched male mice (Fig. 3); (4) treatment of E2-responsiveWT276 cells with increasing concentrations of E2 increased thesteady-state levels of Ifi202 mRNA and protein, whereas treat-ment of cells with DHT decreased the p202 protein levels (Fig.4); (5) treatment of WT276 cells with E2 or overexpression ofER� stimulated the activity of 202-luc-reporter plasmid (Fig. 5,A and B), and overexpression of ER� in NIH 3T3 cells up-regulated the p202 protein levels (Fig. 5C); (6) E2 treatment ofWT276 cells activated transcription from 202-luc-reporterthrough the AP-1CS1 site and increased levels of ER� associ-ated with the 5� regulatory region of the Ifi202 gene in B6.NBa2female B cells than in age-matched males (Fig. 6); and (7)steady-state levels of Ifi202 mRNA were detectable in (NZB �NZW)F1 splenic cells of wild-type (Esr1�/�), but not ER�-deficient(Esr1�/�), age-matched female mice (Fig. 7). Taken together, theseobservations demonstrated that female sex hormone and male sex

FIGURE 6. ER� associates with the potential DNA-binding site in the5� regulatory region of Ifi202 gene in ChIP assays. A, Schematic represen-tation of the 5� regulatory region of the Ifi202 gene containing two potentialER� half DNA-binding sites, which are next to an AP-1 DNA-binding site.Relative locations of PCR primers that were used to amplify the immuno-precipitated chromatin are shown. B, Subconfluent cultures of WT276 cellswere either transfected with 202-luc reporter or 202AP1CS1-luc reporterplasmid along with pRL-TK plasmid. Twenty-four hours after transfec-tions, cells were left untreated or treated with E2 (10 nM). Forty hours aftertransfections, cells were processed for dual luciferase activity as describedin Materials and Methods. Normalized reporter activity is indicated. C,Soluble chromatin was prepared from B6.Nba2 male (lanes 1, 3, and 5) orfemale (lanes 2, 4, and 6) B cells. Chromatin was incubated with Abs toER� (lanes 5 and 6) or, as a negative control, with isotype IgG1 Abs (lanes3 and 4). DNA was extracted from immunoprecipitates and PCR amplified(30 cycles) using a pair of primers that covered ER� DNA-binding site inthe 5� regulatory region of the Ifi202 gene. As a positive control for PCR,we also amplified the input chromatin DNA from male (lane 1) and female(lane 2) B cells. D, Soluble DNA precipitated in B was also subjected toquantitative real-time PCR using PCR primers flanking the 5� regulatoryregion of the Ifi202 gene as described in Materials and Methods.

FIGURE 7. Levels of Ifi202 mRNA in splenic cells depend on the ER�status. Total RNA isolated from spleens of wild-type (Esr1�/�; lanes 1–3)or ER-�-deficient (Esr1�/�; lanes 4–6) age-matched (NZB � NZW)F1

female mice was analyzed by semiquantitative RT-PCR for expressionlevels of Esr1, Ifi202, and actin mRNA.

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hormone differentially regulate the expression of the Ifi202 gene inimmune cells.

Of note, our observations revealed that basal and IFN-in-duced steady-state levels of Ifi202 mRNA were significantlyhigher in nonautoimmune C57BL/6 females than in age-matched males (Fig. 3A). Moreover, steady-state levels ofIfi202 mRNA were also significantly higher in preautoimmuneB6.Nba2, NZB, and (NZB � NZW)F1 female splenic cells thanin age-matched males (Fig. 3B). Similarly, steady-state levels ofIfi202 mRNA and protein were significantly higher in preauto-immune B6.Nba2 female splenic B or T cells than age-matchedmales (Fig. 2). Taken together, these observations are consistentwith sex hormone-dependent (and disease-independent) regula-tion of Ifi202 expression.

ER�-deficient mice are reported to have elevated levels of es-trogen and testosterone (47). Therefore, our observations that invivo treatment of orchiectomized (NZB � NZW)F1 male micewith DHT reduced the mRNA levels (Fig. 1) and the lack of de-tection of Ifi202 mRNA in Esr1-null mice (Fig. 7) make it likelythat increased levels of testosterone in ER�-deficient female micethrough AR down-regulate the expression of Ifi202 gene. Furtherwork will be needed to test this possibility.

A study (48) has revealed that treatment of BALB/c mice withER subtype-selective agonists that results in activation of ER�,but not ER�, plays a major role in estrogen-induced thymicatrophy and thymic T cell and splenic B cell phenotype alter-ations. Moreover, the study also revealed that ER�, but notER�, mediates the estrogen-induced up-regulation of IFN-�.Consistent with a role for ER� in IFN-� production, our study(20) involving generation of ER� knockout (NZB �NZW)F1

mice and their characterization revealed that estrogen throughER� promotes lupus disease, at least in part, by inducing IFN-�production. Moreover, estrogen is known to enhance IFN-� pro-duction by CD11c� cells (49). Taken together, these observa-tions raise the possibility that activation of ER� by E2 in im-mune cells of certain strains of female mice up-regulates Ifi202expression in part by increasing IFN-� production. Therefore,further work will be needed to test this hypothesis.

Previous studies (43, 44, 50) have suggested that estrogentreatment of R4A-�2b BALB/c mice (transgenic for the H chainof an anti-DNA Ab) with E2 leads to the survival and activationof autoreactive cells in a naive repertoire. Moreover, studies(43) also revealed that estrogen treatment of B cells also up-regulates the expression of several genes, such as cd22, shp-1,and bcl-2, which are involved in B cell activation and survival.Interestingly, treatment of mice with tamoxifen, a selective ERmodulator (42), blocked estrogen-induced B cell maturation butnot survival (50). Because increased expression of p202 proteinin splenic B cells from B6.Nba2 congenic mice is associatedwith defects of apoptosis of cells (26, 32, 33) and down-regu-lation of expression of p53 and E2F-responsive proapoptoticgenes (36, 41), we compared basal transcriptional activity ofNF-�B between B6.Nba2 female B cells and age-matchedmales. These experiments indicated that increased levels ofp202 in female B cells (as compared with males) are associatedwith increased transcriptional activity of NF-�B (data notshown). Collectively, our observations support the idea that fe-male sex hormone-dependent increased levels of p202 proteinin B cells by increasing cell survival contribute to sex bias inlupus susceptibility (Fig. 8).

The 5� regulatory region (�800 bp) of Ifi202 gene contains atleast two ERE half-sites (Fig. 6A). Interestingly, one of the EREhalf-sites is located next to an AP-1 consensus DNA-bindingsite (the AP-1CS1), which can bind to AP-1 and can stimulate

the transcription of the Ifi202 gene (40). Because the ER� canregulate transcription of its target genes through the c-Jun/AP-1DNA-binding sites (15, 16), we tested whether ER� regulatestranscription of the Ifi202 gene through the AP-1CS1 site-de-pendent manner. Our experiments demonstrated that E2-medi-ated stimulation of the activity of 202-luc-reporter was abro-gated due to a mutation in the AP-1CS1 site. Further work willbe needed to investigate how ER� and c-Jun/AP-1 collaboratewith each other to up-regulate the expression of the Ifi202 genein B cells.

A search for an AR-responsive element (ARE) in the 5�-regulatory region of the Ifi202 gene did not result in identifi-cation of an ARE. Therefore, further work will be needed todetermine whether 5� regulatory region of the Ifi202 gene,which is upstream to an �800-bp region, contains an ARE. Inthis regard, note that bone marrow stromal cells mediateandrogenic suppression of B lymphocytes development throughup-regulation of TGF-� expression in response to DHT treat-ment (51). Consistent with these observations, we have notedthat TGF-� treatment of splenic cells reduced steady-state lev-els of Ifi202 mRNA (data not shown). Therefore, further workwill be needed to determine how androgens negatively regulatethe expression of Ifi202 in T cells.

Interestingly, a recent study (29) has provided evidence thatthe p202 protein can recognize double-stranded DNA in cyto-plasm (29). Moreover, the study proposed that increased levelsof p202 protein in immune cells could inhibit the ability of theAIM2 protein, a pyrin domain containing member of the p200-family (28, 30), which can also sense DNA in cytoplasm andcan form a caspase-1-activating inflammasome (30). In light ofthese observations it will be important to investigate whetherthe expression of the murine AimII is also differentially regu-lated by the female and male sex hormones in immune cells.

In summary, our observations provide support for our model(Fig. 8). The model predicts that increased levels of male hor-mone androgen through activation of AR down-regulate the ex-pression of the Ifi202 gene. In contrast, increased levels of fe-male hormone estrogen through activation of ER� up-regulatethe expression of Ifi202. Consequently, increased levels of p202protein in immune cells of certain strains of female mice con-tribute to increased survival of autoreactive cells, resulting inincreased susceptibility to lupus disease. Our observations willserve as a molecular basis to identify signaling pathways andmolecules that contribute to sex bias in the development of SLEin human patients.

FIGURE 8. Differential regulation of Ifi202 expression by sex hor-mones in male and female mice and the role of p202 protein in sex bias inlupus susceptibility.

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AcknowledgmentsWe thank Dr. JoEllen Welsh for providing the WT276 cell line. We alsothank Dr. Shuk-mei Ho for thoughtful suggestions concerning ourmanuscript.

DisclosuresThe authors have no financial conflicts of interest.

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7038 ROLE OF Ifi202 IN SEX BIAS IN LUPUS SUSCEPTIBILITY

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