Gut microbiome-host interactions in driving environmental pollutant trichloroethene-mediated autoimmunity.

Trichloroethene (TCE), a widely used industrial solvent, is associated with the development of autoimmune diseases (ADs), including systemic lupus erythematosus and autoimmune hepatitis. Increasing evidence support a linkage between altered gut microbiome composition and the onset of ADs. However, it is not clear how gut microbiome contributes to TCE-mediated autoimmunity, and initial triggers for microbiome-host interactions leading to systemic autoimmune responses remain unknown. To achieve this, female MRL+/+ mice were treated with 0.5 mg/ml TCE for 52 weeks and fecal samples were subjected to 16S rRNA sequencing to determine the microbiome composition. TCE exposure resulted in distinct bacterial community revealed by ß-diversity analysis. Notably, we observed reduction in Lactobacillaceae, Rikenellaceae and Bifidobacteriaceae families, and enrichment of Akkermansiaceae and Lachnospiraceae families after TCE exposure. We also observed significantly increased colonic oxidative stress and inflammatory markers (CD14 and IL-1ß), and decreased tight junction proteins (ZO-2, occludin and claudin-3). These changes were associated with increases in serum antinuclear and anti-smooth muscle antibodies and cytokines (IL-6 and IL-12), together with increased PD1 + CD4+ T cells in TCE-exposed spleen and liver tissues. Importantly, fecal microbiota transplantation (FMT) using feces from TCE-treated mice to antibiotics-treated mice induced increased anti-dsDNA antibodies and hepatic CD4+ T cell infiltration in the recipient mice. Our studies thus delineate how imbalance in gut microbiome and mucosal redox status together with gut inflammatory response and permeability changes could be the key factors in contributing to TCE-mediated ADs. Furthermore, FMT studies provide a solid support to a causal role of microbiome in TCE-mediated autoimmunity.

Interplay and roles of oxidative stress, toll-like receptor 4 and Nrf2 in trichloroethene-mediated autoimmunity.

Previous studies in MRL+/+ mice suggest involvement of oxidative stress (OS) in trichloroethene (TCE)-mediated autoimmunity. However, molecular mechanisms underlying the autoimmunity remain to be fully elucidated. Even though toll-like receptors (TLRs) and Nuclear factor (erythroid-derived 2)-like2 (Nrf2) pathways are implicated in autoimmune diseases (ADs), interplay of OS, TLR and Nrf2 in TCE-mediated autoimmune response remains unexplored. This study was, therefore, undertaken to clearly establish a link among OS, TLR4 and Nrf2 pathways in TCE-induced autoimmunity. Groups of female MRL+/+ mice were treated with TCE, sulforaphane (SFN, an antioxidant) or TCE + SFN (TCE, 10 mmol/kg, i.p., every 4th day; SFN, 8 mg/kg, i.p., every other day) for 6 weeks. TCE exposure led to greater formation of serum 4-hydroxynonenal (HNE)-protein adducts, HNE-specific circulating immune complexes (CICs) and protein carbonyls which were associated with significant increases in serum antinuclear antibodies (ANAs). Moreover, incubation of splenocytes from TCE-treated mice with HNE-modified proteins resulted in enhanced splenocyte proliferation and cytokine release evidenced by increased expression of cyclin D3, Cyclin-dependent kinase 6 (CDK6) and phospho-pRb as well as increased release of IL-6, TNF-α and INF-γ. More importantly, TCE exposure resulted in increased expression of TLR4, MyD88, IRAK4, NF-kB and reduced expression of Nrf2 and HO-1 in the spleen. Remarkably, SFN supplementation not only attenuated TCE-induced OS, upregulation in TLR4 and NF-kB signaling and downregulation of Nrf2, but also ANA levels. These results, in addition to providing further support to a role of OS, also suggest that an interplay among OS, TLR4 and Nrf2 pathways contributes to TCE-mediated autoimmune response. Attenuation of TCE-mediated autoimmunity by SFN provides an avenue for preventive and/or therapeutic strategies for ADs involving OS.

Contribution of poly(ADP-ribose)polymerase-1 activation and apoptosis in trichloroethene-mediated autoimmunity.

Trichloroethene (TCE), a common environmental toxicant and widely used industrial solvent, has been implicated in the development of various autoimmune diseases (ADs). Although oxidative stress has been involved in TCE-mediated autoimmunity, the molecular mechanisms remain to be fully elucidated. These studies were, therefore, aimed to further explore the contribution of oxidative stress to TCE-mediated autoimmune response by specifically assessing the role of oxidative DNA damage, its repair enzyme poly(ADP-ribose)polymerase-1 (PARP-1) and apoptosis. To achieve this, groups of female MRL +/+ mice were treated with TCE, TCE plus N-acetylcysteine (NAC) or NAC alone (TCE, 10 mmol/kg, i.p., every 4th day; NAC, 250 mg/kg/day in drinking water) for 6 weeks. TCE treatment led to significantly higher levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the livers compared to controls, suggesting increased oxidative DNA damage. TCE-induced DNA damage was associated with significant activation of PARP-1 and increases in caspase-3, cleaved caspase-8 and -9, and alterations in Bcl-2 and Bax in the livers. Moreover, the TCE-mediated alterations corresponded with remarkable increases in the serum anti-ssDNA antibodies. Interestingly, NAC supplementation not only attenuated elevated 8-OHdG, PARP-1, caspase-3, cleaved caspase-9, and Bax, but also the TCE-mediated autoimmune response supported by significantly reduced serum anti-ssDNA antibodies. These results suggest that TCE-induced activation of PARP-1 followed by increased apoptosis presents a novel mechanism in TCE-associated autoimmune response and could potentially lead to development of targeted preventive and/or therapeutic strategies.

Autoimmune potential of perchloroethylene: Role of lipid-derived aldehydes.

Tetrachloroethene (perchloroethylene, PCE), an ubiquitous environmental contaminant, has been implicated in inducing autoimmunity/autoimmune diseases (ADs), including systemic lupus erythematosus (SLE) and scleroderma in humans. However, experimental evidence suggesting the potential of PCE in mediating autoimmunity is lacking. This study was, therefore, undertaken to explore PCE's potential in inducing/exacerbating an autoimmune response. Six-week old female MRL+/+ mice, in groups of 6 each, were treated with PCE (0.5mg/ml) via drinking water for 12, 18 and 24weeks and markers of autoimmunity and oxidative stress were evaluated. PCE exposure led to significant increases in serum anti-nuclear antibodies (ANA), anti-dsDNA and anti-scleroderma-70 (anti-Scl-70) antibodies at 18weeks and, to a greater extent at 24weeks, suggesting that PCE exposure exacerbated autoimmunity in our animal model. The increases in autoantibodies were associated with time-dependent increases in malondialdehyde (MDA)-protein adducts and their antibodies, as well as significantly decreased levels of antioxidants GSH and SOD. The splenocytes isolated from mice treated with PCE for 18 and 24weeks showed greater Th17 cell proliferation and increased release of IL-17 in culture supernatants following stimulation with MDA-mouse serum albumin adducts, suggesting that MDA-modified proteins may act as an immunologic trigger by activating Th17 cells and contribute to PCE-mediated autoimmunity. Our studies thus provide an experimental evidence that PCE induces/exacerbates an autoimmune response and lipid-derived aldehydes (such as MDA) contribute to this response.

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen.

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.

N-Acetylcysteine protects against trichloroethene-mediated autoimmunity by attenuating oxidative stress.

Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, is known to induce autoimmunity both in humans and animal models. However, mechanisms underlying TCE-mediated autoimmunity remain largely unknown. Previous studies from our laboratory in MRL+/+ mice suggest that oxidative stress may contribute to TCE-induced autoimmune response. The current study was undertaken to further assess the role of oxidative stress in TCE-induced autoimmunity by supplementing with an antioxidant N-acetylcysteine (NAC). Groups of female MRL+/+ mice were given TCE, NAC or TCE+NAC for 6 weeks (TCE, 10mmol/kg, i.p., every 4th day; NAC, 250mg/kg/day through drinking water). TCE exposure led to significant increases in serum levels of anti-nuclear, anti-dsDNA and anti-Sm antibodies. TCE exposure also led to significant induction of anti-malondiadelhyde (MDA)- and anti-hydroxynonenal (HNE)-protein adduct antibodies which were associated with increased ANA in the sera along with increased MDA-/HNE-protein adducts in the livers and kidneys, and increases in protein oxidation (carbonylation) in the sera, livers and kidneys, suggesting an overall increase in oxidative stress. Moreover, TCE exposure also resulted in increased release of IL-17 from splenocytes and increases in IL-17 mRNA expression. Remarkably, NAC supplementation attenuated not only the TCE-induced oxidative stress, IL-17 release and mRNA expression, but also the markers of autoimmunity, as evident from decreased levels of ANA, anti-dsDNA and anti-Sm antibodies in the sera. These results provide further support to a role of oxidative stress in TCE-induced autoimmune response. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for preventive and/or therapeutic strategies.

Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.

Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of oxidative DNA damage in aniline-induced splenic toxicity.

Liver proteomics in progressive alcoholic steatosis.

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber-DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (-1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (-1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified d-dopachrome tautomerase (-1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum.

Differential Expression of miRNAs in Trichloroethene-Mediated Inflammatory/Autoimmune Response and Its Modulation by Sulforaphane: Delineating the Role of miRNA-21 and miRNA-690.

Trichloroethene (TCE), an occupational and ubiquitous environmental contaminant, is associated with the induction of autoimmune diseases (ADs). Although oxidative stress plays a major role in TCE-mediated autoimmunity, the underlying molecular mechanisms still need to be delineated. Altered non-coding RNAs, including the expression of microRNAs (miRNAs), can influence target genes, especially related to apoptosis and inflammation, and contribute to ADs. Therefore, the objective of this study was to delineate the contribution of miRNAs in TCE-mediated inflammatory and autoimmune response. To achieve this, we treated female MRL+/+ mice with TCE (10 mmol/kg in corn oil, i.p., every fourth day) with/without antioxidant sulforaphane (SFN; 8 mg/kg in corn oil, i.p., every other day) for 6 weeks. With the use of miRNA microarray, 293 miRNAs were analyzed, which included 35 miRNAs that were relevant to inflammation and ADs. Among those 35 miRNAs, 8 were modulated by TCE and/or TCE+SFN exposure. TCE treatment led to increased expression of 3 miRNAs and also decreased expression of 3 miRNAs. Interestingly, among the 35 differentially expressed miRNAs, antioxidant SFN modulated the expression of 6 miRNAs. Based on the microarray findings, we subsequently focused on two miRNAs (miRNA-21 and miRNA-690), which are known to be involved in inflammation and autoimmune response. The increases in miRNA-21 and miR-690 (observed using miRNA microarray) were further validated by RT-PCR, and the TCE-mediated increases in miR-21 and miR-690 were ameliorated by SFN treatment. Modulating miR-21 and miR-690 by respective inhibitors or mimics suppressed the expression of NF-κB (p65) and IL-12 in RAW 264.7 cells. Our findings suggest a contributory role of miR-21 and miR-690 in TCE-mediated and its metabolite dichloroacetyl chloride (DCAC)-mediated inflammation and autoimmune response and support that antioxidant SFN could be a potential therapeutic candidate for inflammatory responses and ADs.

Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen.

Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in the spleen.

Aniline-induced nitrosative stress in rat spleen: proteomic identification of nitrated proteins.

Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity.

Induction of NEIL1 and NEIL2 DNA glycosylases in aniline-induced splenic toxicity.

The mechanisms by which aniline exposure elicits splenotoxic response, especially the tumorigenic response, are not well-understood. Earlier, we have shown that aniline-induced oxidative stress is associated with increased oxidative DNA damage in rat spleen. The base excision repair (BER) pathway is the major mechanism for the repair of oxidative DNA base lesions, and we have shown an up-regulation of 8-oxoguanine glycosylase 1 (OGG1), a specific DNA glycosylase involved in the removal of 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts, following aniline exposure. Nei-like DNA glycosylases (NEIL1/2) belong to a family of BER proteins that are distinct from other DNA glycosylases, including OGG1. However, contribution of NEIL1/2 in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on evaluating if NEILs also contribute to the repair of oxidative DNA lesions in the spleen following aniline exposure. To achieve that, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. The BER activity of NEIL1/2 was assayed using a bubble structure substrate containing 5-OHU (preferred substrates for NEIL1 and NEIL2) and by quantitating the cleavage products. Aniline treatment led to a 1.25-fold increase in the NEIL1/2-associated BER activity in the nuclear extracts of spleen compared to the controls. Real-time PCR analysis for NEIL1 and NEIL2 mRNA expression in the spleen revealed 2.7- and 3.9-fold increases, respectively, in aniline-treated rats compared to controls. Likewise, Western blot analysis showed that protein expression of NEIL1 and NEIL2 in the nuclear extract of spleens from aniline-treated rats was 2.0- and 3.8-fold higher than controls, respectively. Aniline treatment also led to stronger immunoreactivity for NEIL1 and NEIL2 in the spleens, confined to the red pulp areas. These studies, thus, show that aniline-induced oxidative stress is associated with an induction of NEIL1/2. The increased NIEL-mediated BER activity is another indication of aniline-induced oxidative damage in the spleen and could constitute another important mechanism of removal of oxidative DNA lesions, especially in transcribed DNA following aniline insult.

Markers of oxidative and nitrosative stress in systemic lupus erythematosus: correlation with disease activity.

OBJECTIVE: Free radical-mediated reactions have been implicated as contributors in a number of autoimmune diseases, including systemic lupus erythematosus (SLE). However, the potential for oxidative/nitrosative stress to elicit an autoimmune response or to contribute to disease pathogenesis, and thus be useful when determining a prognosis, remains largely unexplored in humans. This study was undertaken to investigate the status and contribution of oxidative/nitrosative stress in patients with SLE. METHODS: Sera from 72 SLE patients with varying levels of disease activity according to the SLE Disease Activity Index (SLEDAI) and 36 age- and sex-matched healthy controls were evaluated for serum levels of oxidative/nitrosative stress markers, including antibodies to malondialdehyde (anti-MDA) protein adducts and to 4-hydroxynonenal (anti-HNE) protein adducts, MDA/HNE protein adducts, superoxide dismutase (SOD), nitrotyrosine (NT), and inducible nitric oxide synthase (iNOS). RESULTS: Serum analysis showed significantly higher levels of both anti-MDA/anti-HNE protein adduct antibodies and MDA/HNE protein adducts in SLE patients compared with healthy controls. Interestingly, not only was there an increased number of subjects positive for anti-MDA or anti-HNE antibodies, but also the levels of both of these antibodies were statistically significantly higher among SLE patients whose SLEDAI scores were > or = 6 as compared with SLE patients with lower SLEDAI scores (SLEDAI score <6). In addition, a significant correlation was observed between the levels of anti-MDA or anti-HNE antibodies and the SLEDAI score (r = 0.734 and r = 0.647, respectively), suggesting a possible causal relationship between these antibodies and SLE. Furthermore, sera from SLE patients had lower levels of SOD and higher levels of iNOS and NT compared with healthy control sera. CONCLUSION: These findings support an association between oxidative/nitrosative stress and SLE. The stronger response observed in serum samples from patients with higher SLEDAI scores suggests that markers of oxidative/nitrosative stress may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.

Redox-sensitive Nrf2 and MAPK signaling pathways contribute to trichloroethene-mediated autoimmune disease progression.

Trichloroethene (TCE) exposure is associated with the induction of autoimmune diseases (ADs). Although oxidative stress plays a major role in TCE-mediated autoimmunity, the underlying molecular mechanisms still need to be delineated. Dysregulation of redox-sensitive nuclear factor (erythroid-derived 2)-like2 (Nrf2), resulting in uncontrolled antioxidant and cytoprotective genes, and pro-inflammatory MAPK signaling pathways could be critical in TCE-mediated disease progression. This study was, therefore, focused on establishing status and contribution of Nrf2 and MAPK signaling in TCE-mediated inflammatory and autoimmune responses, especially during disease progression. To achieve these objectives, time-response studies were conducted by treating female MRL+/+ mice with TCE (0.5 mg/mL, a dose relevant to human exposure) for 24, 36 and 52 wks. TCE exposure led to reduction in Nrf2 expression, but increased phos-NF-κB (p65) and iNOS along with increased phosphorylation of MAPKs (p38, ERK and JNK) and downstream pro-inflammatory cytokines IL-12, TNF-α and RANTES in the livers in a time-dependent manner. These changes were also associated with time-dependent increases in liver protein carbonyls and induction of serum anti-dsDNA antibodies (marker of systemic lupus erythematosus disease), further supporting the role of oxidative stress and Nrf2/MAPK signaling in TCE-mediated autoimmune response progression. The mechanistic role of MAPK in TCE-mediated autoimmunity was further established by treating MRL+/+ mice with sulforaphane (SFN; 8 mg/kg, i.p., every other day) along with TCE (10 mmol/kg, i.p., every 4th day) for 6 wks using an established protocol, and by in vitro treatment of T cells with dichloroacetyl chloride (a TCE metabolite) with/without p38 MAPK inhibitor. SFN treatment attenuated the TCE-mediated phosphorylation of p38 MAPK. More importantly, treatment with SFN or p38 inhibitor led to suppression of downstream pro-inflammatory cytokines IL-12 and TNF-α. These findings thus support the contribution of Nrf2 and MAPK signaling pathways and help in delineating novel potential therapeutic targets against TCE-mediated autoimmunity.

Aberrant Gut Microbiome Contributes to Intestinal Oxidative Stress, Barrier Dysfunction, Inflammation and Systemic Autoimmune Responses in MRL/lpr Mice.

Microbiome composition and function have been implicated as contributing factors in the pathogenesis of autoimmune diseases (ADs), including systemic lupus erythematosus (SLE), rheumatoid arthritis and autoimmune hepatitis (AIH). Furthermore, dysbiosis of gut microbiome is associated with impaired barrier function and mucosal immune dysregulation. However, mechanisms by which gut microbiome contributes to the ADs and whether antioxidant treatment can restore gut homeostasis and ameliorate the disease outcome are not known. This study was, therefore, focused on examining the involvement of gut microbiome and host responses in the pathogenesis of SLE using unique female mouse models (C57BL/6, MRL+/+ and MRL/lpr) of 6 and 18 weeks with varying degrees of disease progression. Fecal microbiome diversity and composition, gut oxidative stress (OS), barrier function and inflammation, as well as systemic autoimmunity were determined. Interestingly, each mouse strain had distinct bacterial community as revealed by ß-diversity. A lower Firmicutes/Bacteroidetes ratio in 6-week-old MRL/lpr mice was observed, evidenced by decrease in Peptostreptococcaceae under Firmicutes phylum along with enrichment of Rikenellaceae under Bacteroidetes phylum. Additionally, we observed increases in colonic OS [4-hydroxynonenal (HNE)-adducts and HNE-specific immune complexes], permeability changes (lower tight junction protein ZO-2; increased fecal albumin and IgA levels) and inflammatory responses (increased phos-NF-κB, IL-6 and IgG levels) in 18-week-old MRL/lpr mice. These changes were associated with markedly elevated AD markers (antinuclear and anti-smooth muscle antibodies) along with hepatic portal inflammation and severe glomerulonephritis. Notably, antioxidant N-acetylcysteine treatment influenced the microbial composition (decreased Rikenellaceae; increased Akkeransiaceae, Erysipelotrichaceae and Muribaculaceae) and attenuated the systemic autoimmunity in MRL/lpr mice. Our data thus show that gut microbiome dysbiosis is associated with increased colonic OS, barrier dysfunction, inflammatory responses and systemic autoimmunity markers. These findings apart from delineating a role for gut microbiome dysbiosis, also support the contribution of gut OS, permeability changes and inflammatory responses in the pathogenesis of ADs.

Enhancing the Nrf2 Antioxidant Signaling Provides Protection Against Trichloroethene-mediated Inflammation and Autoimmune Response.

Trichloroethene (trichloroethylene, TCE) and one of its reactive metabolites dichloroacetyl chloride (DCAC) are associated with the induction of autoimmunity in MRL+/+ mice. Although oxidative stress plays a major role in TCE-/DCAC-mediated autoimmunity, the underlying molecular mechanisms still need to be delineated. Nuclear factor (erythroid-derived 2)-like2 (Nrf2) is an oxidative stress-responsive transcription factor that binds to antioxidant responsive element (ARE) and provides protection by regulating cytoprotective and antioxidant gene expression. However, the potential of Nrf2 in the regulation of TCE-/DCAC-mediated autoimmunity is not known. This study thus focused on establishing the role of Nrf2 and consequent inflammatory responses in TCE-/DCAC-mediated autoimmunity. To achieve this, we pretreated Kupffer cells (KCs) or T cells with/without tert-butylhydroquinone (tBHQ) followed by treatment with DCAC. In both KCs and T cells, DCAC treatment significantly downregulated Nrf2 and HO-1 expression along with induction of Keap-1 and caspase-3, NF-κB (p65), TNF-α, and iNOS, whereas pretreatment of these cells with tBHQ attenuated these responses. The in vitro findings were further verified in vivo by treating female MRL+/+ mice with TCE along with/without sulforaphane. TCE exposure in mice also led to reduction in Nrf2 and HO-1 but increased phospho-NF-κB (p-p65) and iNOS along with increased anti-dsDNA antibodies. Interestingly, sulforaphane treatment led to amelioration of TCE-mediated effects, resulting in Nrf2 activation and reduction in inflammatory and autoimmune responses. Our results show that TCE/DCAC mediates an impairment in Nrf2 regulation. Attenuation of TCE-mediated autoimmunity via activation of Nrf2 supports that antioxidants sulforaphane/tBHQ could be potential therapeutic agents for autoimmune diseases.

Increased nitration and carbonylation of proteins in MRL+/+ mice exposed to trichloroethene: potential role of protein oxidation in autoimmunity.

Even though reactive oxygen and nitrogen species (RONS) are implicated as mediators of autoimmune diseases (ADs), little is known about contribution of protein oxidation (carbonylation and nitration) in the pathogenesis of such diseases. The focus of this study was, therefore, to establish a link between protein oxidation and induction and/or exacerbation of autoimmunity. To achieve this, female MRL +/+ mice were treated with trichloroethene (TCE), an environmental contaminant known to induce autoimmune response, for 6 or 12 weeks (10 mmol/kg, i.p., every 4(th) day). TCE treatment resulted in significantly increased formation of nitrotyrosine (NT) and induction of iNOS in the serum at both 6 and 12 weeks of treatment, but the response was greater at 12 weeks. Likewise, TCE treatment led to greater NT formation, and iNOS protein and mRNA expression in the livers and kidneys. Moreover, TCE treatment also caused significant increases ( approximately 3 fold) in serum protein carbonyls (a marker of protein oxidation) at both 6 and 12 weeks. Significantly increased protein carbonyls were also observed in the livers and kidneys (2.1 and 1.3 fold, respectively) at 6 weeks, and to a greater extent at 12 weeks (3.5 and 2.1 fold, respectively) following TCE treatment. The increases in TCE-induced protein oxidation (carbonylation and nitration) were associated with significant increases in Th1 specific cytokine (IL-2, IFN-gamma) release into splenocyte cultures. These results suggest an association between protein oxidation and induction/exacerbation of autoimmune response. The results present a potential mechanism by which oxidatively modified proteins could contribute to TCE-induced autoimmune response and necessitates further investigations for clearly establishing the role of protein oxidation in the pathogenesis of ADs.

Environmental Exposures and Autoimmune Diseases: Contribution of Gut Microbiome.

Environmental agents have been gaining more attention in recent years for their role in the pathogenesis of autoimmune diseases (ADs). Increasing evidence has linked environmental exposures, including trichloroethene (TCE), silica, mercury, pristane, pesticides, and smoking to higher risk for ADs. However, potential mechanisms by which these environmental agents contribute to the disease pathogenesis remains largely unknown. Dysbiosis of the gut microbiome is another important environmental factor that has been linked to the onset of different ADs. Altered microbiota composition is associated with impaired intestinal barrier function and dysregulation of mucosal immune system, but it is unclear if gut dysbiosis is a causal factor or an outcome of ADs. In this review article, we first describe the recent epidemiological and mechanistic evidences linking environmental/occupational exposures with various ADs (especially SLE). Secondly, we discuss how changes in the gut microbiome composition (dysbiosis) could contribute to the disease pathogenesis, especially in response to exposure to environmental chemicals.

Cytochrome P450 2E1-deficient MRL+/+ mice are less susceptible to trichloroethene-mediated autoimmunity: Involvement of oxidative stress-responsive signaling pathways.

Reactive trichloroethene (TCE) metabolites and oxidative stress are involved in TCE-mediated autoimmunity, as evident from our earlier studies in MRL+/+ mice. However, molecular mechanisms underlying the autoimmunity remain largely unknown. Cytochrome P450 2E1 (CYP2E1), the major enzyme responsible for TCE metabolism, could contribute to TCE-induced toxic response through free radical generation. The current study was, therefore, aimed to further evaluate the significance of TCE metabolism leading to oxidative stress and autoimmune response by using MRL+/+ mice that lack CYP2E1. The Cyp2e1-null MRL+/+ mice were generated by backcrossing Cyp2e1-null mice (B6N; 129S4-Cyp2e1) to MRL +/+ mice. Female MRL+/+ and Cyp2e1-null MRL+/+ mice were given TCE (10 mmol/kg, i.p., every 4th day) for 6 weeks; their respective controls received corn oil only. TCE treatment in MRL+/+ mice induced oxidative stress, evident from significantly increased serum malondiadelhyde (MDA)-protein adducts, their antibodies and reduced liver GSH levels. TCE treatment also modulated Nrf2 pathway with decreased Nrf2 and HO-1, and elevated NF-κB (p65) expression in the liver. TCE exposure also led to increases in serum antinuclear antibodies (ANA) and anti-double stranded DNA antibodies (anti-dsDNA). Although TCE treatment in Cyp2e1-null MRL+/+ mice also led to increases in serum MDA-protein adducts and their antibodies, changes in liver GSH, Nrf2, HO-1 and NF-κB along with increases in serum ANA, anti-dsDNA, the alterations in the oxidative stress and autoimmunity markers in these mice were less pronounced compared to those in MRL+/+ mice. These findings support the contribution of CYP2E1-mediated TCE metabolism in autoimmune response and an important role of Nrf2 pathway in TCE-mediated autoimmunity.

Redox regulation of hepatic NLRP3 inflammasome activation and immune dysregulation in trichloroethene-mediated autoimmunity.

Trichloroethene (TCE) exposure is associated with the development of various autoimmune diseases (ADs), including autoimmune hepatitis (AIH) and systemic lupus erythematosus (SLE), potentially through the generation of excessive reactive oxygen and nitrogen species (RONS; oxidative stress). However, the mechanisms by which oxidative stress contributes to these TCE-mediated ADs are not fully understood, and are the focus of current investigation. Female MRL+/+ mice were treated with TCE along with or without antioxidant N-acetylcysteine (NAC) for 6 weeks (TCE, 10 mmol/kg, i. p., every 4th day; NAC, 250 mg/kg/day via drinking water). TCE-treated mice had elevated antinuclear antibodies (ANA) and 4-hydroxynonenal (HNE)-specific circulating immune complexes, suggesting the association of TCE-induced oxidative stress with autoimmune response. In addition, TCE exposure led to prominent lobular inflammation with sinusoid dilation, increased sinusoidal cellularity and increased staining for proliferating cell nuclear antigen (PCNA), confirming inflammatory and hepatocellular cell proliferation. Importantly, TCE exposure resulted in the activation of hepatic inflammasome (NLRP3 and caspase-1) and up-regulation of pro-inflammatory cytokine IL-1ß, and these changes were attenuated by NAC supplementation. TCE treatment also led to dysregulation of hepatic immune response as evident from markedly increased hepatic lymphocyte infiltration (especially B cells) and imbalance between Tregs (decreased) and Th17 cells (increased). Interestingly, TCE-mediated dysregulation of various hepatic and splenic immune cells was also effectively attenuated by NAC. Taken together, our findings provide evidence for TCE-mediated inflammasome activation, infiltration of various immune cells, and skewed balance of Treg and Th17 cells in the liver. The attenuation of TCE-mediated hepatic inflammasome activation and immune responses by NAC further supports a critical role of oxidative stress in TCE-mediated inflammation and autoimmunity. These novel findings could help in designing therapeutic strategies for such ADs.