Involvement of Intestinal Epithelium Aryl Hydrocarbon Receptor Expression and 3, 3'-Diindolylmethane in Colonic Tertiary Lymphoid Tissue Formation

Abstract

Tertiary lymphoid tissues (TLTs) are adaptive immune structures that develop during chronic inflammation and may worsen or lessen disease outcomes in a context-specific manner. Immune cell activity governing TLT formation in the intestines is dependent on immune cell aryl hydrocarbon receptor (AhR) activation. Homeostatic immune cell activity in the intestines is further dependent on ligand activation of AhR in intestinal epithelial cells (IECs), yet whether AhR activation and signaling in IECs influences the formation of TLTs in the presence of dietary AhR ligands is not known. To this end, we used IEC-specific AhR deletion coupled with a mouse model of dextran sodium sulfate (DSS)-induced colitis to understand how dietary AhR ligand 3, 3'-diindolylmethane (DIM) influenced TLT formation. DIM consumption increased the size of TLTs and decreased T-cell aggregation to TLT sites in an IEC-specific manner. In DSS-exposed female mice, DIM consumption increased the expression of genes implicated in TLT formation (Interleukin-22, Il-22; CXC motif chemokine ligand 13, CXCL13) in an IEC AhR-specific manner. Conversely, in female mice without DSS exposure, DIM significantly reduced the expression of Il-22 or CXCL13 in iAhRKO mice, but this effect was not observed in WT animals. Our findings suggest that DIM affects the immunological landscape of TLT formation during DSS-induced colitis in a manner contingent on AhR expression in IECs and biological sex. Further investigations into specific immune cell activity, IEC-specific AhR signaling pathways, and dietary AhR ligand-mediated effects on TLT formation are warranted.

Keywords: DIM; aryl hydrocarbon receptor; colon; intestinal epithelium; tertiary lymphoid tissues.

Figure 1

Effect of the loss of AhR in IECs on clinical signs of acute colitis. Disease Activity Index (DAI), a composite measure of weight loss, stool consistency, and presence of blood in stool. Data presented indicate the mean ± SEM. For each indicated day, groups without a common letter differ; p < 0.05.

Figure 2

Serum concentration of FITC-dextran at termination. Effect diet (DIM supplementation) compared to control diet. Data presented indicate the mean ± SEM.

Figure 3

Number of tertiary lymphoid tissues (TLT/measurable area (mm²). (A) Effect of diet (DIM supplementation) compared to control diet. (B) Effect of genotype, diet, and sex in mice treated with DSS. Data presented indicate the mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 3

Number of tertiary lymphoid tissues (TLT/measurable area (mm²). (A) Effect of diet (DIM supplementation) compared to control diet. (B) Effect of genotype, diet, and sex in mice treated with DSS. Data presented indicate the mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 4

Size of Tertiary Lymphoid tissues (average area of TLT (µm²). (A) Effect of diet (DIM supplementation) compared to control diet. (B) Effect of genotype and diet (DIM vs. control) in mice treated with DSS. Data presented indicate the mean ± SEM. # p < 0.075, * p < 0.05.

Figure 5

Effect of genotype, sex, and diet in immune cell density within TLT in DSS-treated animals. Effect of genotype and diet in T-cell density within TLT (Genotype*Diet, ANOVA (KW) p = 0.0112. Data presented indicate the mean density (Cell #/pixel²) ± SEM; * p < 0.05.

Figure 6

Effect of diet, genotype, DSS, and sex in the expression of IEC-derived genes from isolated colonic crypts (ICCs). (A) Relative expression of Lcn2 (Lipocalin-2), 2-way ANOVA; male (DSS effect, p = 0.0215) and female (DSS effect, p = 0.0224). (B) Relative expression of Il-7 (IL-7), 2-way ANOVA; male (Interaction between diet/genotype and DSS, p = 0.0197). (C) Relative expression of Zo-1 (ZO-1/Tjp1). (D) Relative expression of Muc2 (Muc2). (E) Relative expression of Cldn2 (Claudin-2). (F) Relative expression of Ocln (Occludin). Data are expressed as fold change relative to WT control, n = 3–4, * p < 0.05, ** p < 0.01.

Figure 6

Effect of diet, genotype, DSS, and sex in the expression of IEC-derived genes from isolated colonic crypts (ICCs). (A) Relative expression of Lcn2 (Lipocalin-2), 2-way ANOVA; male (DSS effect, p = 0.0215) and female (DSS effect, p = 0.0224). (B) Relative expression of Il-7 (IL-7), 2-way ANOVA; male (Interaction between diet/genotype and DSS, p = 0.0197). (C) Relative expression of Zo-1 (ZO-1/Tjp1). (D) Relative expression of Muc2 (Muc2). (E) Relative expression of Cldn2 (Claudin-2). (F) Relative expression of Ocln (Occludin). Data are expressed as fold change relative to WT control, n = 3–4, * p < 0.05, ** p < 0.01.

Figure 7

Effect of genotype, diet, DSS, and sex in the expression of Il-22, Il-6, and CXCL13 from MS. (A) Relative expression of Il-22 (IL-22)/No DSS. Male (N/A, 0 = undetectable) and female (p = 0.0096, one-way ANOVA). (B) Relative expression of Il-22 (IL-22)/DSS. Male (p = 0.3469, one-way ANOVA) and female (p = 0.0021, KW). (C) Relative expression of Il-6 (IL-6)/No DSS. Male (p = 0.0021, KW) and female (p = 0.0004, one-way ANOVA). (D) Relative expression of Il-6 (IL-6)/DSS. Male (p = 0.2915, one-way ANOVA) and female (p = 0.1426, KW). (E) Relative expression of Cxcl13 (Cxcl13)/No DSS. Male (p = 0.5244, one-way ANOVA) and female (p = 0.0051, one-way ANOVA). (F) Relative expression of Cxcl13 (Cxcl13)/DSS. Male (p = 0.0015, one-way ANOVA) and female (p = 0.0106, one-way ANOVA). Data are expressed as fold change relative to WT control, n = 3–4, * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 8

Mouse model used CDX2P^CreT2 x AhR^f/f.

Figure 9

Study design.