http://informahealthcare.com/aut ISSN: 0891-6934 (print), 1607-842X (electronic) Autoimmunity, 2014; 47(1): 57–66 ! 2014 Informa UK Ltd. DOI: 10.3109/08916934.2013.832220

RESEARCH ARTICLE

Consequences of perinatal bisphenol A exposure in a mouse model of multiple sclerosis Candice Brinkmeyer-Langford1, Aline Rodrigues2, Kelli J. Kochan3, Rachel Haney4, Fenan Rassu4, Andrew J. Steelman1, Colin Young1, Penny Riggs3, Ralph Storts2, Mary W. Meagher4, and C. Jane Welsh1 Department of Veterinary Integrative Biosciences, 2Department of Veterinary Pathobiology, 3Department of Animal Science, and 4Department of Psychology, Texas A&M University, College Station, TX, USA Abstract

Keywords

Multiple sclerosis (MS) is a complex disease influenced by genetic and environmental contributing factors. Endocrine disrupting compounds (EDCs) such as bisphenol A (BPA) affect gene expression and hormone-regulated systems throughout the body. We investigated the effects of BPA on Theiler’s-virus induced demyelination (TVID), a mouse model of MS. Perinatal BPA exposure, combined with viral infection, resulted in a decreased level of viral antibodies, accelerated the onset of TVID symptoms, increased inflammation in both the spinal cord and digestive tract, and amplified immune-related gene expression changes induced by viral infection. These results demonstrate the effect of BPA on the trajectory of TVID, and illustrate how multiple factors collectively influence autoimmune disease.

Demyelination, endocrine disruptor, gene expression, immune response, inflammation

Introduction Autoimmune diseases such as multiple sclerosis (MS) have become alarmingly more prevalent in the last century [1,2]. MS is the most common inflammatory disease of the central nervous system (CNS) in humans and is characterized by acute, focal demyelination and neurodegeneration. Diagnosis rates have steadily increased for decades [1–4]. Both genetic and environmental components [5,6], including estrogen levels in the body [7], have been implicated in the etiology of MS. The correlation between increased presence of estrogenic endocrine disrupting compounds (EDCs) in the environment and the increased rates of autoimmune disease demands attention. EDCs are abundant in the environments of industrialized nations and are commonly found in everyday products – for example, polycarbonate plastic bottles. Vulnerability of the developing fetus to EDCs is of paramount concern, as relatively high levels of certain EDCs have been detected in the blood of pregnant women and their fetuses as well as the umbilical cord [8,9]. An increased incidence of MS has been observed in patients exposed prenatally to the EDC diethylstilbestrol [10,11] and the effects of prenatal exposure to the EDC bisphenol A (BPA) have been documented extensively (for review, see [12]). Exposure to BPA in the nanograms per kilograms body weight range

Correspondence: Candice Brinkmeyer-Langford, PhD, Department of Veterinary Integrative Biosciences, Texas A&M University, MS4458, College Station, TX 77845, USA. E-mail: [email protected]

History Received 19 April 2013 Revised 29 July 2013 Accepted 31 July 2013 Published online 5 November 2013

during the perinatal period can result in changes within the fetus that are manifested later in life [13,14]. Pre/perinatal EDC exposure may shift the normal immune balance towards a disproportionately robust proinflammatory response. Such an effect has been observed in rodent models of autoimmune diseases (reviewed in [15]). Prenatal exposure to BPA alters the normal balance of the immune cells – for example, by reducing the number of regulatory T cells [16]. Adult mice exposed to BPA via their drinking water showed an increase in Th1 response and suppression of the Th2 response [17], consistent with a shift toward a pro-inflammatory (pro-autoimmune) versus anti-inflammatory response. Yoshino et al. [18] found that the prenatal exposure to BPA over the first 18 d of gestation resulted in the up-regulation of Th1 and Th2 immune responses in both male and female mice, with a much greater increase in Th1 response, suggestive of a developmental impact on the immune system. Furthermore, perinatal BPA exposure resulted in increased inflammation of the gut in female adult rats [19]. To study the effects of BPA on autoimmune disease, we have used the Theiler’s murine encephalomyelitis virus (TMEV)induced demyelination (TVID) model of MS, in the genetically-susceptible SJL strain of mice. In this model, mice infected with TMEV develop a biphasic disease comprised of a polioencephalitic phase followed by a chronic demyelinating phase propagated by the autoimmune responses of T and B cells [20]. The phenotypes resulting from TMEV infection parallel human MS in a number of ways, including chronic central nervous system inflammation involving CD4þ and CD8þ T cells, B cells and macrophages [21,22], gender

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influenced disease phenotypes [23] and clinical symptoms such as incontinence and gait disorders [24,25]. As with human MS, the mouse model includes an exogenous contributing factor (virus) that interacts with genetic factors. The present study was designed to test the hypothesis that perinatal BPA exposure impacts disease manifestation and gene expression in the TVID model of MS. We demonstrate that BPA, in combination with TMEV infection, renders mice more vulnerable to TMEV infection and accelerates the onset of clinical symptoms. Furthermore, we report unexpected observations of colitis in mice affected by both BPA exposure and TMEV infection. These data indicate that BPA, a common environmental agent, can influence the presentation of TVID in mice.

Materials and methods Mice Four-week-old male (n ¼ 12) and female (n ¼ 24) SJL mice were purchased from Harlan (Indianapolis, IL). To habituate mice to human contact, each mouse was handled for several minutes each day for 2 weeks prior to the initiation of experimentation. Once habituated, females were administered either 10 mg/kg body weight (BW)/day BPA (dissolved in a charcoal-stripped corn oil vehicle; n ¼ 12) or corn oil only (n ¼ 12), delivered orally via gastric lavage. This concentration is considered an environmentally relevant dose [8,26,27] and oral administration provided for better control of dosage in a manner that mimics human exposure, where BPA is typically ingested and subsequently metabolized in the liver. During BPA administration, mice were housed for breeding with 2 females and 1 male per cage and fed a diet of 9% fat and 20.5% protein, and a regular light/dark cycle (0700– 1900 h). Food and water were provided ad libitum. Dams continued to receive BPA or corn oil at the same time of day throughout pregnancy and lactation. Pups were weaned at 21 d of age and separated by sex. Based on the treatment, each pup was assigned to one of the following groups: infected with TMEV/perinatally-exposed to BPA (inf/BPA), not infected/BPA-exposed (no inf/BPA), infected/ not BPA-exposed (inf/no BPA), or not infected/not BPAexposed (no inf/no BPA). Numbers of mice assigned to each group were: inf/BPA – 34 (20 female, 14 male), no inf/BPA – 7 (1 female, 6 male), inf/no BPA – 21 (9 female, 12 male), no inf/no BPA – 7 (2 female, 5 male). Mice in this study tested negative for the presence of Helicobacter species. All offspring were sacrificed 20 weeks post-infection by intra peritoneal (i.p.) injection of a lethal dose of Beuthanasia special 150 mg/kg (Schering-Plough Animal Health) as described [28]. Mice were perfused through the left ventricle with phosphate-buffered saline (PBS) followed by 10% formalin in phosphate buffered at pH 7.2. Blood and serum were collected as described and stored at 80  C [28]. Perfused mice were necropsied, organs were evaluated macroscopically and selected tissues were harvested for testing. The spinal cord was removed and a set of spinal cord samples from 25 mice was snap-frozen on dry ice, then stored at 80  C for RNA extraction. Spinal cord samples from an additional 26 mice and selected organs including the heart, spleen, liver, kidneys, lymph nodes, lungs and

Autoimmunity, 2014; 47(1): 57–66

gastrointestinal tract from 45 mice were fixed overnight in 4% paraformaldehyde for histopathology. After fixation, tissues were paraffin-embedded and sectioned for routine light microscopy using hematoxylin and eosin staining (H&E) and evaluated histologically. All animal care protocols were in accordance with NIH Guidelines for Care and Use of Laboratory Animals and were approved by the Texas A&M University Laboratory Animal Care and Use Committee. TMEV infection At 4 weeks of age, male (n ¼ 26) and female (n ¼ 29) offspring were anesthetized with isoflurane (MWI, Meridian, ID) and injected with 5.0  104 plaque-forming units (PFU) of the BeAn strain of TMEV in 20 ml of PBS placed into the right mid-parietal cortex at a depth of approximately 1.5 mm [29,30]. Sham-infected mice (n ¼ 11 males and 3 females) were similarly anesthetized, and injected with PBS only. Experiments to assess effects of perinatal BPA exposure on TMEV infection Clinical scoring All mice were assessed weekly for clinical signs of inflammatory disease, using an established scoring system known to correlate with histological scores [31]. Scoring was based on established markers of the progression of TVID, including ruffled fur, grooming, arched back, limb weakness, wobbly gait, and righting reflex. The severity of these markers was scored on a scale of 0 to 4, with 0 being normal and 4 being moribund [29,31]. Scoring was performed at the same time of day on the same day each week, by the same person, without knowledge of treatment to the animals. Histological evaluation of spinal cords TMEV-induced disease is characterized by inflammation of the spinal cord [32], particularly affecting the thoracic section. Spinal cords were sectioned and processed for H&E staining as described [29]. We evaluated thoracic sections of the spinal cord for perivascular cuffing and meningitis. Histological assessments were made without prior knowledge of experimental condition. Sections were evaluated based on severity (layers of inflammatory cells observed in perivascular cuffs or regions of meningitis) and percentage of total area of inflammation [31,33]. Image J software (US National Institutes of Health, Bethesda, MD; [34]) was used to measure area semi-quantitatively. Serum antibody and protein evaluation by radioimmunoassays (RIA) and ELISA Serum was prepared from blood collected from the left ventricles of 40 mice at the time of sacrifice, and stored at 80  C. RIAs were used to test the serum for antibodies against TMEV, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein peptide (MOG35-55), and proteolipid protein peptide (PLP139-151) as described previously [31,35,36]. Serum concentrations of TNFa were determined using a mouse TNFa-specific ELISA kit (eBioscience, San Diego, CA) according to the manufacturer’s instructions.

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DOI: 10.3109/08916934.2013.832220

Statistical analysis Clinical scores were analyzed with a mixed two-way ANOVA entering TMEV infection and BPA exposure as betweengroup variables and day post infection as a within subject repeated measure variable. Spinal cord inflammation scores were analyzed using a two-way ANOVA with TMEV infection and BPA exposure as between group variables. RIA values were also analyzed using a two-way ANOVA, with BPA exposure and sex as between group variables. Where appropriate, means comparisons were used for post hoc analyses. In all cases, p  0.05 was considered significant.

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same gender. Dye-normalized, quality-processed signal information was obtained using Agilent’s Feature Extraction Software for downstream analysis. Quality control and statistical evaluation of microarray data was performed using Agilent GeneSpring 12.0 GX software (Santa Clara, CA). Probes were filtered and eliminated on expression level; probes with expression values 520% were excluded. Genes with 5.0-fold change in expression were considered for further analysis. Gene ontology (GO) analysis was performed using a false discovery rate (FDR)-adjusted p value of 50.05 as a threshold for significance. Real-time PCR (RT-PCR)

RNA extraction Total RNA from 25 spinal cords was extracted in TRI Reagent (Life Technologies, Carlsbad, CA) and purified using Qiagen RNeasy Mini kit columns, according to manufacturer’s instructions (Qiagen, Valencia, CA). RNA concentration, quality, and integrity were assessed using the NanoDrop 1000 (NanoDrop, Wilmington, DE) and RNA 6000 Pico Chip Kit (Agilent, Santa Clara, CA) on an Agilent 2100 Bioanalyzer. Samples deemed of sufficient quality and concentration for microarray analysis included 3 inf/BPA (all female), 2 inf/no BPA (1 female, 1 male), 1 no inf/BPA (male), and 2 no inf/no BPA (1 female, 1 male). Microarray analysis About 150–300 ng of total RNA for each sample was labeled with the Agilent LowInput QuickAmp two-color labeling kit and hybridized to the Agilent SurePrint G3 Unrestricted GE 8x60K mouse genome expression array (G4858A) according to the manufacturer’s protocol. Each experimental sample (inf/BPA, inf/no BPA, and no inf/BPA) was co-hybridized with a control sample (no inf/no BPA) from a mouse of the

Synthesis of cDNA was carried out in a 50 ml reaction with the TaqManÕ Reverse Transcription Reagents (Applied Biosystems, Carlsbad, CA), utilizing the provided random hexamer primer and the manufacturer’s protocol. Up to 1 mg of RNA was reverse transcribed; however, due to very little RNA remaining for some of the samples, as little as 20 ng were used. One reaction containing no reverse transcriptase was also performed as a control against DNA contamination. Quantitative polymerase chain reactions (qPCR) were performed in triplicate 10 ml reactions containing 1X PowerSYBRÕ PCR master mix (Applied Biosystems) and 300 nM primers, with 1 mL cDNA as template. Primer sequences were either taken from literature or designed using Primer3 [37]; information for each primer is listed in Table 1. Amplification was carried out in a 7900 HT real-time thermal cycler (Applied Biosystems). Cycling parameters were 95  C for 10 min for initial denaturation, followed by 40 cycles of 95  C for 15 s and 60  C for 1 min. Amplification and dissociation data were analyzed with SDS software v.2.2.2 (Applied Biosystems). Relative expression data were calculated using the DDCt method of Livak and Schmittgen [38], and normalized to beta-actin (Actb) and matrix metallopeptidase 9 (Mmp9) expression using GeNorm [39].

Table 1. RT-PCR primers. Gene ID

Gene name

Accession/Reference

Actb

b-actin

[68]

Mmp9

matrix metallopeptidase 9

[69]

B2m

b-2-microglobulin

ENSMUS-T00000102476

Dnmt1

DNA methyltransferase(cytosine-5) 1

NM_010066

Fgf2

fibroblast growth factor 2

NM_008006

Gapdh

Glyceraldehyde-3-phosphate dehydrogenase

[70]

Gusb

glucuronidase beta

ENSMUS-G00000025534

Pten

phosphate and tensin homolog

ENSMUS-G00000013663

Vegfa

vascular endothelial growth factor A

[71]

Primers F: 50 -GACAGGATGCAGAAGGAGATTACT R: 50 -TGATCCACATCTGCTGGAAGGT F: 50 -CGAACTTCGACACTGACAAGAAGT R: 50 -GCACGCTGGAATGATCTAAGC F: 50 -CCTGGTCTTTCTGGTGCTTG R: 50 -TATGTTCGGCTTCCCATTCT F: 50 -CGGCTCAAAGACTTGGAAAG R: 50 -TAGCCAGGTAGCCTTCCTCA F: 50 -AGCGGCTCTACTGCAAGAAC R: 50 -GCCGTCCATCTTCCTTCATA F: 50 -TGTGTCCGTCGTGGATCTGA R: 50 -GCATCGAAGGTGGAAGAGTGG F: 50 -CCAGCCACTATCCCTACTCA R: 50 -GCCACAGACCACATCACAAC F: 50 -TTGAAGACCATAACCCACCA R: 50 -TACACCAGTCCGTCCCTTTC F: 50 -GCACTGGACCCTGGCTTTACT R: 50 -ACTTGATCACTTCATGGGACTTCTG

Primers used for RT-PCR are listed along with gene IDs and names. Accession numbers from which these primers were designed are provided; publications are cited when applicable. Primer sequences are listed 50 to 30 in the rightmost column.

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Figure 1. Comparison of clinical scores. Mice exposed perinatally to BPA (n ¼ 43) showed an accelerated onset of clinical symptoms of demyelination compared with unexposed mice (n ¼ 26), as shown via comparison of mean clinical scores (y-axis) over the first 9 weeks postinfection. Data are shown as mean þ/ SEM; asterisks (*) denote significance.

Results Effects of dams’ BPA treatment on fecundity and litter characteristics No differences were observed in litter sizes, gender ratio, or weaning weights in offspring of dams receiving daily BPA treatment compared with offspring of non-exposed dams. However, dams given BPA lost, ate, or abandoned more litters than dams being given corn oil alone (7 versus 3). This observation is supported by previous studies which have reported that exposure to estrogenic EDCs during pregnancy can reduce maternal behaviors such as nursing and spending time in the nest with the pups [40,41]. BPA treatment accelerates onset of clinical scores for TVID Mice perinatally exposed to BPA and infected with TMEV showed an earlier onset of TVID symptoms. Figure 1 depicts the effects of BPA exposure on mean clinical score in TMEV infected mice during the acute phase of the infection, the first nine weeks. A mixed two-way ANCOVA entering infection and BPA exposure as a between group variables and time post infection as a repeated measures variable, whereas sex, experimenter and infection date were entered as covariates. There was a significant main effect of infection [F(1,57) ¼ 48.11, p50.001], as well as significant interactions between infection and time [F(8,456) ¼ 6.52, p50.001], BPA exposure and time [F(8,456) ¼ 3.30, p50.01]. These findings confirmed that the infected mice had higher clinical scores, which became worse over time, and that BPA also worsened clinical scores over time. No other main effects or interactions were significant. To focus on the effects of BPA on infection, a one-way ANCOVA with time as a repeated measures variable was conducted on the TMEV infected mice alone. This analysis revealed a significant main effect of BPA exposure [F(1,46) ¼ 9.38, p50.01]. Bonferroni post hoc mean comparisons indicated that clinical scores were significantly elevated in the BPA exposed infected group on weeks 4, 5 and 6 relative to the non-exposed infected group (Figure 1).

Figure 2. Antibody levels for TMEV and myelin proteins. Panel A shows average Ab levels for TMEV, PLP, MOG and MBP for each treatment group. Panels B and C illustrate the effects of BPA (B) and sex (C) on TMEV antibody levels. Y-axes represent average Ab levels in counts per minute (CPM). Data are shown as mean þ SEM; asterisks (*) denote significance. Number of mice per group: no inf/no BPA ¼ 5; inf/no BPA ¼ 8; no inf/BPA ¼ 6; inf/BPA ¼ 21.

Antibody responses to myelin and Theiler’s virus proteins To evaluate the effect of BPA exposure on virus and myelinspecific adaptive immunity, plasma antibody (Ab) levels against TMEV and myelin proteins were determined by RIA. Figure 2 depicts the effects of BPA exposure and infection on averaged dilutions of Ab levels to TMEV, PLP, MOG and MBP (panel A). A series of ANOVAs were conducted to examine the effects of BPA exposure and infection on mean dilutions of Ab levels to TMEV, PLP, MOG and MBP. A twoway ANOVA was conducted on Ab to TMEV, using BPA exposure and sex as a between group variables. This analysis revealed a significant main effect of BPA exposure [F[1,25)58.28, p ¼ 0.008] indicating that BPA exposure led

DOI: 10.3109/08916934.2013.832220

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to lower levels of Ab to TMEV (Figure 2 panel B). Although the interaction was not significant, the main effect of sex approached significance [F(1,25)53.90, p ¼ 0.06]. Post hoc mean comparisons indicated that the infected females exposed to BPA had higher levels of Ab to TMEV than males (p ¼ 0.048; panel C). Moreover, BPA exposed males had lower levels of Abs to TMEV (p ¼ 0.050) when compared to non-exposed males. A two-way ANOVA conducted on Ab to PLP revealed a marginal main effect of infection [F(1,36)53.89, p ¼ 0.056], with the infected mice having slightly higher levels of Ab to PLP yet this failed to reach significance. The main effect and interaction were not significant [F(1,36)53.89, p40.05]. No significant differences were found for Ab to MOG and MBP [both F’s (1,36)50.983, p40.05]. Other clinical observations In addition to clinical evaluation of neurological signs, all mice were evaluated daily for presentation of other clinical signs. We particularly noted those signs not explainable by aggressive behavior typical of the SJL strain of mice, such as bites or fighting injuries [42,43]. Non-neurological clinical signs observed included diarrhea (3 inf/BPA females and 1 inf/BPA male), inflamed anal area and anal prolapse (6 inf/ BPA females and 2 inf/BPA males), prolapsed penis (4 males from all groups except no inf/no BPA), testicular swelling (3 no inf/BPA males), and ocular anomalies (4 inf/BPA and 2 inf/no BPA, all female). Only one male mouse had both diarrhea and inflammation of the anal area and another male presented with both penile and testicular inflammation. Ocular abnormalities were characterized by moderate to complete ptosis of the eyelid, in particular, presumably of neurogenic etiology [44]. Anal inflammation, diarrhea, and ocular symptoms were first noticed 12 weeks p.i.; penile/ testicular abnormalities were observed as early as 7 weeks p.i. Gross pathology and histological examination of tissues Necropsies and gross evaluation were performed on 45 mice: 25 inf/BPA (16 females, 9 males), 9 no inf/BPA (1 female, 8 males), and 11 inf/no BPA (5 females, 6 males).

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Thirteen mice presented with a markedly thickened intestinal wall, most often affecting the colon. This change was observed in 10 inf/BPA mice (9 females, 1 male), 2 female inf/no BPA mice, and 1 female no inf/BPA mouse. Six of the ten inf/BPA female mice and one inf/BPA male mouse had presented with inflammation in the anal area or diarrhea prior to death. Of the 45 mice evaluated, 16 showed splenomegaly: 12 inf/BPA (10 females, 2 males), 2 no inf/BPA, and 2 inf/no BPA (1 female and 1 male each for no inf/BPA and inf/no BPA groups). Of these, 10 also had thickened intestinal walls. Histological examination of the intestines of all mice with thickened intestinal wall revealed a multifocal, chronic, proliferative, lymphoplasmacytic and histiocytic colitis (Figure 3). Evidence of marked to severe colitis was seen in 10 inf/BPA mice (9 females, 1 male) and 1 female no inf/BPA mouse. The male inf/BPA mouse had the most severe, fulminant colitis. Mild to marked colitis was also observed in 2 females inf/no BPA mice, which were clinically asymptomatic. Histological evaluation of the spinal cord sections revealed perivascular cuffing and meningitis in all TMEV-infected mice, irrespective of BPA exposure. Figure 4 depicts the effect of BPA exposure on mean spinal cord area of inflammation in TMEV infected mice. A one-way ANOVA revealed a significant main effect of BPA exposure [F(1,22) ¼ 4.48, p ¼ 0.046], indicating that BPA lead to increased spinal cord inflammation (panel C). However, a two-way ANOVA entering BPA exposure and sex as between group variables did not show a main effect of sex or a sex by BPA interaction effect [both Fs50.09, p40.05]. Microarray results Gene expression profiles were compared between the four treatment groups, with all results normalized and filtered as described in the section ‘‘Materials and methods’’. Foldchange values for each probe and sample are listed in Supplementary Table 1. Following normalization, a total of 9555 probes were found to be expressed differently among groups (p value 0.05, fold change 5). TMEV infection had the strongest effects on gene expression, with 3105 probe sets differentially expressed in samples from infected mice

Figure 3. Comparison of colon inflammation severity. Panel A shows the colon of a mouse not exposed to BPA, with normal goblet cells and few inflammatory cells (dark spots). Panel B shows the colon of a mouse prenatally exposed to BPA. The intestinal mucosa is markedly thickened by a proliferative colitis, characterized by increased numbers of epithelial cells in colonic crypts with loss of goblet cells and by numerous inflammatory infiltrates. Inflammatory cells also infiltrate the submucosa and wall of the intestine. Inset shows inflammatory infiltrates composed of numerous macrophages, scattered multinucleated cells, lymphocytes and plasma cells. H&E, 100X; histopathology data collected from the colons of 13 mice with colitis (10 inf/BPA; 2 inf/no BPA; 1 not inf/BPA).

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Figure 4. Comparison of spinal cord inflammation severity. In panels A and B, thoracic spinal cord sections illustrate the degree of inflammation in TMEV-infected mice. Panel A shows the spinal cord of a female mouse not exposed to BPA; Panel B shows the spinal cord of a female mouse perinatally exposed to BPA. In panel B, examples of more severe inflammation in the BPA-exposed mouse are indicated as follows: meningitis is shown by an arrow and perivascular cuffing by an oval (both are shown in greater detail in insets). H&E, 40X. Panel C shows the effect of BPA exposure and sex on spinal cord (S.C.) inflammation in infected mice. The percent area of inflammation in the spinal cord is shown for BPA-exposed and unexposed mice whose histopathology images were analyzed (females: no BPA ¼ 4, BPA ¼ 10; males: no BPA ¼ 4; BPA ¼ 8). The amount of inflammation was significantly more severe in mice exposed to BPA, regardless of gender. Results are shown as mean þ SEM; asterisks (*) denote significance.

(inf/BPA and inf/no BPA) compared with no inf/no BPA. Exposure to BPA altered expression profiles to a lesser degree: compared to no inf/no BPA, 1374 probes showed differential expression in the no inf/BPA sample (p value 0.05, fold change 5). The effects of BPA exposure in combination with TMEV infection were more difficult to determine: for many genes, expression levels varied widely between mice of the same group. However, trends in the data (vis-a`-vis up- or downregulation of genes) provided insight into the effects of infection plus exposure. Of the 9555 probes differentially expressed between any of the groups relative to the control (no inf/no BPA), regulation patterns (up or down) were in concurrence for 8461 probes in all 3 inf/BPA samples and in 2 out of 3 samples for the remaining 1094 probes. The directions of regulation were also in agreement for 8411 probes in both inf/no BPA samples. When compared with gene expression in no inf/no BPA (control), TMEV-induced expression changes were correlated with 3 gene ontology (GO) pathways (p value cutoff of 0.05): biological process (GO:0008150jGO:0000004jGO:0007582), cellular component (GO:0005575jGO:0008372), and molecular function (GO:0003674jGO:0005554). Included in these 3 clusters were 257 GO terms (Supplementary Table 2). The most significant of these terms was immune system process (GO:0002376), and many other GO terms represented were connected to the immune system in some way. The GO term with the highest count number was response to stimulus (GO:0050896jGO:0051869), which included over 26% of the

total count. The ontology of the differentially-expressed genes was consistent with our expectations: viral infection induces effector populations of immune cells to secrete specific cytokines, and the demyelination associated with TMEV infection has been previously shown to be immune-mediated [45,46]. No GO terms were connected explicitly to BPA exposure, regardless of TMEV infection status. However, expression changes were observed for several genes relevant to adaptive immunity and MS. Forkhead box P3 (Foxp3); transforming growth factor, beta receptor 1 (Tgfbr1); transforming growth factor, beta receptor associated protein 1 (Tgfbrap1); interleukin 7 receptor (Il7r); interleukin 10 receptor, alpha (Il10ra); interleukin 18 binding protein (Il18bp); and interleukin 21 receptor (Il21r) were down-regulated substantially in no inf/BPA mice compared with other treatment groups. Interferon gamma (Ifng) was more strongly up-regulated in inf/BPA mice than in inf/no BPA mice, suggesting that BPA exacerbates the effects of TMEV infection on the increased expression of Ifng. Independent of TMEV infection, BPA changed the expression levels of numerous genes not directly related to MS. We compared the gene expression fold-change differences caused strictly by TMEV infection against those caused strictly by BPA exposure, by comparing log fold-change counts for inf/no BPA mice against the same values for no inf/ BPA mice. Relevant examples include fukutin (Fktn) and tubulin, gamma complex associated protein 6 (Tubgcp6), both which play a role in brain development; and sialic acid

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DOI: 10.3109/08916934.2013.832220

binding Ig-like lectin 5 (Siglec5), which inhibits the activation of monocytes, macrophages and neutrophils. Other BPAsensitive transcripts identified in this study included olfactory receptor genes (e.g. Olfr890 and Olfr93) and regulatory proteins such as long non-coding RNAs (lincRNAs). TMEV infection played the strongest role in the differential expression of genes related to MS/TVID. The expression of genes specific to macrophage/microglia function changed substantially in response to TMEV infection. Tumor necrosis factor (Tnf), interleukin 1 beta (Il1b), interleukin 23 receptor (Il23r), and interleukin 10 receptor, alpha (Il10ra) were all strongly up-regulated. Interleukin 17 receptor B (Il17rb), which plays a role in adaptive immunity and the pathogenesis of MS/TVID, was up-regulated as well. CD68 antigen (CD68) was also up-regulated, especially in inf/BPA mice - suggesting that the effects of TMEV infection on macrophages and microglia were augmented by perinatal BPA exposure. Microarray results were confirmed by RT-PCR for beta-2 microglobulin (B2m), fibroblast growth factor 2 (Fgf2), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), DNA methyltransferase (cytosine-5) 1 (Dnmt1), glucuronidase beta (Gusb), phosphatase and tensin homolog (Pten), and vascular endothelial growth factor A (Vegfa). Several of these genes (B2m, Gapdh, Gusb) are often considered ‘‘housekeeping genes’’, typically used for normalization; however, they showed variable expression in the microarray results and so were included in our experimental cohort for RT-PCR.

Discussion Autoimmune diseases are thought to result from the interplay of multiple factors, including genetic susceptibility and environmental triggers. The present study elucidates the influences of a common environmental agent, BPA, on a genetically-susceptible mouse model in the context of inflammatory demyelinating disease. Viral antibody titers against TMEV were reduced in inf/BPA mice versus inf/non-BPA mice, suggesting that BPA reduced the immune response to TMEV infection. The lower immune response likely resulted in a stronger TMEV infection, which augmented TMEV-induced inflammation and gene expression changes in inf/BPA mice relative to inf/non-BPA mice. This may also explain the earlier onset of demyelination symptoms. TMEV infection was the primary factor determining clinical severity, but BPA exposure accelerated the worsening of symptoms in the first several weeks post-infection. In all infected mice (inf/BPA and inf/no BPA groups) symptoms of demyelination ultimately reached the same level of clinical severity about 7 weeks post-infection. This is consistent with the onset of demyelination, observed at 42 d p.i. by Ulrich et al. [47]. The earlier onset of severe clinical symptoms seen exclusively in the inf/BPA mice was both statistically and biologically significant, implying that TMEV infection and BPA exposure induced demyelination earlier when combined than when encountered separately. These observations are supported by previous studies [48,49] as well as our own oligodendrocyte cell culture experiments demonstrating the influence of BPA on the fate of myelin progenitors in cell culture (C. Brinkmeyer-Langford, A. Steelman, S. Kim and J. Li, unpublished results).

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A higher degree of inflammation resulted from the combination of BPA exposure and TMEV infection than either treatment alone. The spinal cords of inf/BPA mice showed more meningitis and perivascular cuffing, both signs of inflammation and correlative to TVID clinical severity and demyelination [31]. Interestingly, we did not observe significantly raised levels of antibodies for myelin epitopes (MBP, MOG35-55, and PLP) in any group of mice, which would have indicated an autoimmune attack against myelin. This may be attributed to the fact that all serum samples used in these assays were collected at the time of sacrifice: 20 weeks p.i., long after the initial autoimmune response would have occurred. Colitis was typically more severe in mice in the inf/BPA group; these findings are particularly intriguing in light of human studies that describe the co-occurrence of inflammatory bowel diseases (IBDs), such as ulcerative colitis and Crohn’s disease, with MS [50–55]. The prenatal period is critical to the development of the intestinal barrier [56] and perinatal exposure to BPA influences gut barrier permeability and may contribute to colitis in females in adulthood [19]. Estrogenic compounds play a key role in permeability and inflammation in epithelial barriers, such as those in the gut, brain, and reproductive organs, through the regulation of tight junction integrity [57]. The increase in barrier permeability also disrupts normal immune homeostasis [58] and contributes to a loss of tolerance that can initiate autoimmune responses in the colon and in the brain [55,59]. Comorbidity of autoimmune diseases is not unusual [60] and in the case of MS and IBDs, ‘‘leaky’’ endothelial barriers in both brain and mucosa are associated with the increased production of antibodies against non-pathogenic proteins. Our observations are consistent with a role for estrogens in maintenance of epithelial barriers as the increase in inflammation and colitis in inf/BPA mice occurred almost exclusively in females. The lower immune response against TMEV in the inf/BPA mice likely acted in combination with the estrogenic effects of BPA to produce a more severe colitis than either treatment alone. The combined effects of BPA plus TMEV infection on changes in gene expression provide important evidence of the complexity of combined environmental influences. Our TMEV findings expand on those of previous expressionarray analyses in the TVID model, which investigated the effects of TMEV infection alone [47,61]. For many genes upor down-regulated in response to TMEV infection, the addition of BPA augmented the degree of expression change. However, other genes known to be key players in autoimmunity or demyelination were affected very little or not at all. Of the two treatment variables TMEV infection had the strongest effect on gene expression: no inf/BPA mice showed only modest expression level changes relative to controls. Our results imply that BPA alone does not cause autoimmunity but may deregulate the immune response such that, for some genes, the normal response to viral infection is enhanced. In light of our TMEV viral titer results, this may explain why the immune response to TMEV was lower in mice that were also exposed to BPA. The complex effects of BPA on gene expression are a testimony to the multiple ways BPA exerts its influence. First, BPA can modify gene expression through its activity as an

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estrogenic compound able to weakly bind to estrogen receptors a and b [62]. Receptor-bound endogenous estrogen regulates many processes throughout the body by recruiting and binding to various co-factors; the resulting complex modulates gene transcription [12,63]. Receptor-bound BPA, on the other hand, interferes with the proper recruitment/ binding of these co-factors and can thereby disrupt normal estrogen-regulated gene expression. Another avenue through which BPA can alter gene expression is through epigenetic modification. BPA causes methylation changes, for example, which modulate gene expression (e.g. [64–67]). In this project, BPA affected the expression levels of genes normally governed by both endocrine and epigenetic factors. These included regulatory genes and sequences (such as lncRNAs) involved in modulating the immune response, along with the primary (non-regulatory) genes involved. Moreover, these effects are pleiotropic, affecting multiple genes/pathways in a domino effect. Therefore, the mechanism of BPA in this project is complicated and multi-factorial.

Conclusions Our findings support the hypothesis that BPA can affect TVID presentation in mice. Because this is a powerful mouse analog of human MS, our results suggest that EDCs likely influence the presentation of MS – for example, by causing an earlier disease onset. These compounds should also be considered in the co-morbidity of MS and IBD.

Acknowledgements The authors gratefully acknowledge the assistance of Francisco Gomez, Collin Mulcahy, Deren Koseoglu, Kristin Deason and Christina Dudash in the care and daily treatment of the mice. Dr Jianrong Li and Dr Sunja Kim provided assistance with oligodendrocyte progenitor cultures in support of this project (unpublished data) and generously supplied space and materials for these experiments. Histology of the spinal cord samples was performed by Lin S. Bustamante and Chaitali Mukherjee. Dr Steve Safe provided the BPA for this project. The Texas A&M Whole Systems Genomics Initiative, Dr Loren Skow, Dr Scott Dindot and Ryan Doan, all provided lab space, training and/or equipment in support of the RT-PCR and microarray experiments.

Declaration of interest The authors declare no competing interests. Funds for purchasing materials and reagents for this work were provided by a Texas A&M College of Veterinary Medicine and Biomedical Sciences postdoctoral trainee research grant (awarded to C. B.-L.).

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Supplementary material available online

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Supplementary Tables 1–2