Immortalized mouse RAW 264.7 cell line was purchased from the American Type Tissue Collection (ATCC; Manassas, VA, USA). The cells were cultured in complete Dulbecco’s modified Eagle medium (DMEM) media, with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin and streptomycin according to the supplier’s guidelines. RAW 264.7 cells were incubated at 37°C and treated with either dichloroacetyl chloride (DCAC; a TCE metabolite, 5 mM) (Sigma-Aldrich, St. Louis, MO, USA) alone or treated first with SFN (10 µM) (Sigma-Aldrich) 1 h prior to incubation with DCAC for 24 h (7).

The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Medical Branch (UTMB), and all the experiments were conducted as per the National Institutes of Health (NIH) guidelines. Female MRL+/+ (Murphy Roths Large; MRL/MpJ) mice (5 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and kept at the UTMB animal facility under controlled conditions of temperature and humidity with a 12-h light/dark cycle. Mice had free access to food and water ad libitum and were acclimatized for a week before starting the treatments. Mice were divided randomly into 4 groups of 6 mice each and designated as control, TCE, SFN, or TCE+SFN treatment groups [TCE (Sigma-Aldrich), 10 mmol/kg in corn oil, i.p., every 4th day; SFN 8 mg/kg in corn oil, i.p., every other day] (7–10, 26, 27). The control animals received an equal volume of corn oil only. The selection of the dose and duration of TCE or SFN exposure was based on previous studies (8–10, 26, 27). MRL+/+ mice were chosen for the study, as these mice are autoimmune-prone where most autoantibodies appear several months after their birth and SLE disease appears late in the second year of their lives (8, 9). This slow development of the disease allows us to evaluate the autoimmune potential of specific agents in relatively young animals. After 6 weeks of treatment, the mice were euthanized under ketamine/xylazine anesthesia, and blood was withdrawn. Major organs were then removed and weighed. Liver portions were snap-frozen in liquid nitrogen and stored at −80°C for future use.

Microarray assay was performed by a service provider (LC Sciences, Houston, TX, USA). Briefly, total RNA was isolated from the liver samples using the mirVana miRNA Isolation Kit (Applied Biosystems, Foster City, CA, USA). A strand-labeling reaction was carried out with total RNA (1 µg), control oligonucleotides, App-Cap3 (5′-adenylated and 3′-dideoxy oligo-adaptor, Integrated DNA Technologies, Coralville, IA, USA), and T4 RNA ligase (Rnl2tr-K227Q, New England BioLabs, Ipswich, MA, USA) in T4 RNA ligase buffer with PEG 8000 and RNase inhibitors (Promega, Madison, WI, USA). Following the manufacturer’s protocol, the reaction mixture was incubated at 16°C for 16 h and stopped by adding an equal volume of hybridization buffer (28). Finally, fluorescence images were monitored using a laser scanner (GenePix 4000B, Molecular Device, San Jose, CA, USA) and digitized using Array-Pro image analysis software (Media Cybernetics, Rockville, MD, USA). Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (locally-weighted regression) (29).

Total RNA from cells and tissues was extracted using the RiboPure™ RNA Purification Kit (Invitrogen, Carlsbad, CA, USA). The design of primer sequencing was done using the Primer3Plus program. The primers were obtained from Integrated DNA Technologies (Coralville, IA, USA), and mRNA expressions of NF-kB (p65), cytokine IL-12, and miR-21 and miR-690 were determined. cDNA was synthesized using iScript™ cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as the endogenous loading controls for mRNA and miRNA, respectively (13, 14). Primer sequences are provided in Supplementary Table S1 .

For the extraction of proteins from cells or liver tissues, radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Danvers, MA, USA) with protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) was used. Thirty micrograms of protein lysates was used for Western blotting analyses. The immunoblots were probed with primary antibodies against, pNF-κB (p-p65) and β-Actin (Cell Signaling Technology, Danvers, MA, USA) (10, 30). The immunoblot images were quantified by densitometry and normalized using loading control protein β-actin.

Mouse splenocytes were isolated from the spleens of control or TCE-treated mice following an earlier method (17). The spleens were gently crushed in RPMI-1640 medium through a cell strainer. Red blood cells were removed by using Red Cell Lysis buffer (Sigma). Splenocytes were collected by centrifugation and re-suspended in complete RPMI-1640 with 10% FBS.

Mouse cytokine antibody pair-based array spotted on a membrane kit (Abcam, Cambridge, MA, USA) was used to measure the cytokines in the splenocyte protein samples according to the manufacturer’s instructions. Briefly, proteins were extracted from spleen cells of MRL+/+ mice using RIPA buffer with protease inhibitor. Proteins (300 μg) were incubated at 4°C overnight with previously blocked membranes (provided). The membranes were then washed with the washing buffer followed by incubation with biotin-conjugated anti-cytokines at 4°C overnight. After being washed, the membranes were then incubated with streptavidin–horseradish peroxidase (HRP) at 4°C overnight. After being washed, the cytokines on membranes were detected using chemiluminescence detection solution (31).

RAW 264.7 cells were plated separately into 6-well flat-bottom plates at 3 × 10⁶/well in a total volume of 2 ml and incubated at 37°C. RAW 264.7 cells were transfected with miR-21 and miR-690 inhibitors or mimics (20 nm) and negative controls (NCs) (20 nm) by using Lipofectamine 2000 reagent according to the manufacturer’s guidelines (Thermo Fisher Scientific, Waltham, MA, USA). In brief, RAW 264.7 cells were incubated with NC, miR-21, or miR-690 inhibitors or mimics separately for 24 h in reduced serum OPTIMEM media (Invitrogen, Carlsbad, CA, USA). The expressions of NF-κB (p65) and cytokine IL-12 were analyzed (13, 32, 33).

RAW 264.7 cells (30,000 cells/100 μl in BPS medium) were plated in 96-well plates and allowed to adhere for 24 h. For transfection, 100 ng of DNA constructs [NF-κB Transient Pack (BPS Biosciences, San Diego, CA, USA) consisting of NF-κB reporter vector + constitutively expressing Renilla luciferase vector or NC reporter (non-inducible luciferase vector + Renilla luciferase vector)] along with 20 nM of miR-690 mimics or respective 20 nM of miRNA control were co-transfected into RAW 264.7 cells using Lipofectamine 2000 (Thermo Fisher Scientific). Luciferase activity was measured 24 h post-transfection with the Dual-Luciferase Reporter Assay System (BPS Biosciences, San Diego, CA, USA) as per the manufacturer’s protocol. Luciferase activity was measured as relative luciferase units (firefly luciferase/Renilla luciferase) (12, 23).

Spleen tissue samples were fixed in 10% buffered formalin, dehydrated, and paraffin embedded. These paraffin sections were stained with H&E (17, 30). Images were taken using an Olympus 1X71 microscope (Olympus, Hamburg, Germany).

The values are presented as means ± SEM. The data were analyzed using a one-way ANOVA followed by the Tukey–Kramer multiple comparisons test. An unpaired Student’s t-test was performed to find out whether there is a significant difference between the two groups (control vs. TCE). The p-values <0.05 were considered statistically significant.

The regulatory role of miRNAs in the development and normal function of immune cells involved in innate immunity is well established. MiRNAs are also involved in the adaptive immune system via activation and development of reactive T cells and B cells responding to specific antigens (20). In this study, we chose autoimmune-prone MRL+/+ mice to identify the differentially expressed miRNAs in the liver following TCE exposure. To explore the effect of TCE on the miRNA profile, we first screened for all the miRNAs in the livers of TCE-treated mice. In the microarray analysis, out of 293 miRNAs that were evaluated, 6 miRNAs were modulated by TCE. Out of 6 miRNAs, 3 were increased (mmu-miR-21a-5p, mmu-miR-690, and mmu-miR-7683-3p), whereas 3 miRNAs were downregulated (mmu-miR-207, mmu-miR-455-3p, and mmu-miR-568) ( Figure 1 and Table 1 ). We further evaluated 35 miRNAs relevant to inflammation and ADs ( Figure 2A ) following TCE and/or TCE+SFN treatment. Out of those 35 miRNAs, 8 miRNAs were modulated (mmu-miR-21a-5p, mmu-miR-690, mmu-miR-125a-5p, mmu-miR-139-5p, mmu-miR-455-3p, mmu-miR-568, mmu-miR-5100, and mmu-miR-145b; Figure 2A and Table 2 ). Interestingly, among these differentially expressed miRNAs, antioxidant SFN ameliorated the expression of 6 miRNAs (mmu-miR-21a-5p, mmu-miR-690, mmu-miR-139-5p, mmu-miR-455-3p, mmuwfi 2-miR-568, and mmu-miR-145b; Figure 2A and Table 2 ).

Out of 35 differentially modulated miRNAs, as evident from microarray analysis, we selected mmu-miR-21a-5p (miR-21) and mmu-miR-690 (miR-690) as the top candidates for further investigation ( Figures 2A–C ), as both these miRNAs not only showed ~2-fold induction in TCE-treated mice compared to the controls but also are due to their potential role in inflammatory responses (12, 34). Further validation of the increased miR-21 and miR-690 levels upon TCE treatment was done by RT-PCR, which also showed significant induction in the expression of miR-21 and miR-690 in the livers ( Figures 3A, B ). Our previous studies with an antioxidant SFN showed significant anti-inflammatory effects as well as amelioration of TCE-mediated autoimmune response (7). To determine if SFN can also provide protection through the modulation of miRNAs, we analyzed these miRNAs with or without SFN treatment along with TCE. As evident from Figures 2B, C , 3A, B , SFN treatment attenuated the effect of TCE by suppressing the expression of both miR-21 and miR-690 in the liver. Interestingly, these miRNAs (miR-21 and miR-690) were also significantly increased in the splenocytes of TCE-treated mice ( Figures 3C, D ).

To establish an association of the changes in miRNA levels with the inflammatory response, we determined the inflammatory markers in the splenocytes of TCE-treated mice. TCE treatment led to significantly increased mRNA expression of NF-kB and IL-12, and protein levels of IL-12 ( Figures 4A–D ).

Infiltration of immune cells in the liver, spleen, and kidney is associated with AIH or SLE phenotype (17, 30, 35). Based on H&E data, an abundant cluster of large macrophage-like cells and lymphocytic infiltration (pointed by red and black arrows, respectively) were seen in the spleens of TCE-treated mice ( Figure 5 ).

To further verify the findings with TCE exposure in vivo, we determined these miRNAs (miR-21 and miR-690) in vitro using macrophages (RAW 264.7 cells) (due to infiltration of macrophage-like cells that were found in the spleen; H&E staining, Figure 5 ) after their incubation with DCAC, a highly reactive metabolite of TCE with a potential to generate both inflammatory and autoimmune response (8, 36). DCAC treatment also significantly increased miR-21 expression in RAW 264.7 cells, which was attenuated by SFN ( Figure 6A ). To evaluate the target genes of miR-21 in TCE-mediated inflammation responses, RAW 264.7 cells were transfected with miR-21 mimics or inhibitors (20 nM) or respective NC. Transfection efficacy with miR-21 mimics showed significantly higher expression of miR-21 as compared to the NC group (data were not shown). MiR-21 inhibitors suppressed the effect of DCAC-mediated inflammation by suppressing p-NF-KB and IL-12 expression ( Figures 6B–D ). It is thus evident that miR-21 inhibitors can modulate mRNA expression for NF-kB (p65) and IL-12 in RAW 264.7 cells. Our data also suggest that miR-21 could play an important role in DCAC-mediated inflammation and could be a potential therapeutic target for TCE-mediated ADs.

In our previous study, we have shown that upregulation of p38 MAPK/NF-kB (p65) plays an important role in TCE-mediated autoimmunity (30). Cytokines play a crucial role in the development of inflammatory responses and ADs (37). To evaluate the target gene of miR-690 in TCE-mediated inflammation and autoimmunity, mouse RAW 264.7 cells were treated with miR-690 mimics or respective NC. Like miR-21, SFN also ameliorated DCAC-induced miR-690 expression ( Figure 7A ). MiRNA-690 mimics suppressed the expression of p-NF-kB and IL-12 in RAW 264.7 cells in the presence of DCAC ( Figures 7B–D ). Taken together, DCAC treatment induced miR-690 in RAW 264.7 cells, and these findings suggest that manipulation of miR-690 could be useful in modulating DCAC-induced inflammatory markers.

To obtain further evidence that NF-κB (p65) is a direct target of miR-690, a dual-luciferase reporter assay was used to determine the influence of miR-690 on signaling NF-κB transcriptional activation. NF-κB-luciferase reporter plasmid along with miR-690 mimics or control was co-transfected into RAW 264.7 cells. We observed that miR-690 mimics markedly inhibited the luciferase activity of cells ( Figures 7E, F ).