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

Abstract

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 β-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-1β), 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.

Keywords: Autoimmunity; Environmental pollutant; Inflammation; Microbiome; Oxidative stress; Permeability.

Figure 1.

Chronic TCE exposure leads to autoimmune and inflammatory responses in MRL+/+ mice. Mice were exposed to 0.5 mg/ml TCE for 52 weeks. The autoantibodies and cytokines were measured to monitor autoimmune disease activities. Serum levels of ANA levels (A), ASMA levels (B) and pro-inflammatory IL-6 and IL-12 levels (C). Results are mean ± SEM. n=5 *p<0.05.

Figure 2.

Splenic and hepatic T cell infiltration in CON and TCE-exposed MRL+/+ mice. Lymphocytes were harvested from the spleen and liver, then characterized for T cells. Quantification of flow cytometry analysis for CD4+ and CD8+ T cells (A), IFN-γ+ CD4+ T cells (B), PD1+ CD4+ T cells (C) in the spleen. Flow cytometry analysis for CD4+ and CD8+ T cells (D), IFN-γ+ CD4+ T cells (E), PD1+ CD4+ T cells (F) in intrahepatic lymphocytes. Results are mean ± SEM. n=5 *p<0.05; **p<0.01.

Figure 3.

TCE exposure altered gut microbiome composition. Fecal pellets from CON and TCE-treated MRL+/+ mice were collected; bacterial DNA was isolated and subjected to 16s rDNA sequencing to evaluate the composition of microbiome. A. α-diversity of gut microbiota in each group. B. Distinct microbiome patterns revealed as β-diversity in each group. C. Average relative abundances of taxa at the phylum level. D. Relative abundance at taxonomic level of family. E. Relative abundance of major genera in fecal samples from CON and TCE-treated mice. F. Quantitative PCR analysis of the fecal samples from CON and TCE groups for Akkermansia muciniphila and Faecalibacterium prausnitzii. Results are mean ± SEM. CON: n=4; TCE: n=5 # p<0.1; *p<0.05; **p<0.01.

Figure 4.

TCE exposure induced colonic oxidative stress, barrier dysfunction and inflammation. A. MDA-protein adducts in the colon of CON and TCE exposed mice at 52 weeks. B. Representative pictures for tight junction proteins in the colon. C. quantification of the signal intensity of tight junction proteins. D. Quantitative PCR analysis of inflammation related genes in the colon after TCE exposure. E. Serum Lipocalin-2 level. F. Fecal IgA levels. Results are mean ± SEM. n=5 # p<0.1; *p<0.05.

Figure 5.

Fecal microbiota transplantation from CON and TCE-treated mice in microbiome-depleted young MRL-lpr mice. A. Study design of fecal transplantation from CON and TCE-exposed mice to MRL-lpr mice, designated as CF and TF. B. α-diversity of gut microbiota in donor (CON; TCE) and recipient mice before (W0-CF; W0-TF) and after FMT (W3-CF; W3-TF). C. β-diversity of gut microbiome in donor and recipient mice after FMT. D. Average relative abundances of taxa at the family level in donor and recipient mice after FMT. Results are mean ± SEM. CF: n=4; TF: n=5 *p<0.05.

Figure 6.

Effect of FMT from CON and TCE-treated mice in microbiome-depleted young MRL-lpr mice. Serum levels of anti-dsDNA (A) and ANA (B), total lymphocyte counts (C) and CD4+ T cell number (D) in the liver of recipient mice (CF and TF). Results are mean ± SEM. n=7 # p<0.1; *p<0.05.