2,3,7,8-Tetrachlorodibenzo-p-Dioxin Alters Lipid Metabolism and Depletes Immune Cell Populations in the Jejunum of C57BL/6 Mice

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent aryl hydrocarbon receptor agonist that elicits dose-dependent hepatic fat accumulation and inflammation that can progress to steatohepatitis. To investigate intestine-liver interactions that contribute to TCDD-elicited steatohepatitis, we examined the dose-dependent effects of TCDD (0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 µg/kg) on jejunal epithelial gene expression in C57BL/6 mice orally gavaged every 4 days for 28 days. Agilent 4x44K whole-genome microarray analysis of the jejunal epithelium identified 439 differentially expressed genes (|fold change| ≥ 1.5, P1(t) ≥ 0.999) across 1 or more doses, many related to lipid metabolism and immune system processes. TCDD-elicited differentially expressed genes were associated with lipolysis, fatty acid/cholesterol absorption and transport, the Kennedy pathway, and retinol metabolism, consistent with increased hepatic fat accumulation. Moreover, several major histocompatibility complex (MHC) class II genes (H2-Aa, H2-Ab1, H2-DMb1, Cd74) were repressed, coincident with decreased macrophage and dendritic cell levels in the lamina propria, suggesting migration of antigen-presenting cells out of the intestine. In contrast, hepatic RNA-Seq analysis identified increased expression of MHC class II genes, as well as chemokines and chemokine receptors involved in macrophage recruitment (Ccr1, Ccr5, Ccl5, Cx3cr1), consistent with hepatic F4/80 labeling and macrophage infiltration into the liver. Collectively, these results suggest TCDD elicits changes that support hepatic lipid accumulation, macrophage migration, and the progression of hepatic steatosis to steatohepatitis.

Keywords: MetS; NAFLD; TCDD; jejunum; macrophage; toxicogenomics.

FIG. 1.

Microarray analysis of dose-dependent jejunal epithelial differential gene expression in female C57BL/6 mice treated by oral gavage with sesame oil vehicle or 0.01–30 µg/kg TCDD every 4 days for 28 days. A, Agilent 4X44K whole-genome microarray data were filtered using |fold change| ≥ 1.5 and P1(t) ≥ 0.999 criteria, identifying 439 genes differentially expressed at 1 or more doses. B, The number of upregulated, downregulated, and total differentially expressed unique genes at each dose.

FIG. 2.

Dose-response modeling of jejunal epithelial differentially expressed genes (DEGs) in female C57BL/6 mice treated by oral gavage with sesame oil vehicle or 0.01–30 µg/kg TCDD every 4 days for 28 days. A, Summary of ToxResponse Modeler (left) and BMDExpress (right) dose-response modeling for jejunal epithelial DEGs. B, Frequency distribution of ED₅₀ values for 339 sigmoidal features (top) and bench mark dose lower confidence limit (BMDL) (bottom) values for 161 features exhibiting hill, linear, 2° polynomial, or power responses. The distributions reveal peaks at 0.4, 1.1, and 3.2 µg/kg TCDD, suggesting a dose-dependent step-wise response in differential gene expression.

FIG. 3.

Functional analysis of jejunal epithelial differentially expressed genes (DEGs) in female C57BL/6 mice treated by oral gavage with sesame oil vehicle or 0.01–30 µg/kg TCDD every 4 days for 28 days. A, Functional clusters identified using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Scores ≥ 1.3 are considered significantly enriched. B, Heat map of DEGs associated with lipid-related processes. C, Heat map of DEGs associated with immune system processes. The number of pDREs (MS ≥ 0.8473) is indicated in the last column on the right of each heat map.

FIG. 4.

Flow cytometry analysis of TCDD-mediated effects on immune cell populations within the intestinal lamina propria. Female and male mice were treated by oral gavage with either sesame oil vehicle or 30 µg/kg TCDD every 4 days for 28 days. Single cell suspensions prepared from the lamina propria were labeled with either (A) immune cell panel or (B) dendritic cell subpopulation antibodies. Immune cell panel results are expressed as the average percentage of singlets expressing various cell surface receptors ± standard error of the mean (SEM; N ≥ 3), while dendritic cell subpopulations are expressed as the average percentage of CD11c⁺ cells co-expressing CD11b and/or CD103 ± SEM (N ≥ 12). Asterisks (*) indicate a significant difference (P ≤ .05) compared with vehicle control determined by standard t-test performed using SAS 9.3.

FIG. 5.

Comparison of differential gene expression between the jejunal epithelium and liver of female C57BL/6 mice treated by oral gavage with sesame oil vehicle or 0.01–30 µg/kg TCDD every 4 days for 28 days. A, Venn diagram of jejunal and hepatic differentially expressed genes (DEGs), identifying 53 DEGs common to both tissues. B, Heat map of common DEGs associated with immune response, oxidative and reductive processes, and lipid metabolism. The number of pDREs (MS ≥ 0.8473) is indicated in the last column on the right.

FIG. 6.

Representative images of hepatic lesions in female mice treated by oral gavage with sesame oil vehicle or 3–30 µg/kg TCDD every 4 days for 28 days. Livers were stained with hematoxylin and eosin (H&E) (left), Oil Red O (center), or anti-mouse F4/80 antibody (right). Scale bar represents 100 µm.