AHR Activation Is Protective against Colitis Driven by T Cells in Humanized Mice

Abstract

Existing therapies for inflammatory bowel disease that are based on broad suppression of inflammation result in variable clinical benefit and unwanted side effects. A potential therapeutic approach for promoting immune tolerance is the in vivo induction of regulatory T cells (Tregs). Here we report that activation of the aryl hydrocarbon receptor using the non-toxic agonist 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induces human Tregs in vitro that suppress effector T cells through a mechanism mediated by CD39 and Granzyme B. We then developed a humanized murine system whereby human CD4⁺ T cells drive colitis upon exposure to 2,4,6-trinitrobenzenesulfonic acid and assessed ITE as a potential therapeutic. ITE administration ameliorated colitis in humanized mice with increased CD39, Granzyme B, and IL10-secreting human Tregs. These results develop an experimental model to investigate human CD4⁺ T responses in vivo and identify the non-toxic AHR agonist ITE as a potential therapy for promoting immune tolerance in the intestine.

Keywords: AHR; IBD; ITE; humanized mice; treg.

Figure 1. ITE induces suppressive human regulatory T cells through a mechanism mediated by AHR

(A) Suppressive activity of human naïve CD4⁺ T cells activated with plate bound αCD3/αCD28 and IL2 in the presence of vehicle (CTL), ITE, or TCDD. Percent suppression is depicted as the mean ± SEM pooled from at least 3 independent experiments (n = 21). (B) Relative expression of the AHR target CYP1A1 following stimulation with αCD3/αCD28 and IL2 in the presence of ITE or TCDD and compared to PBS vehicle control. Data are depicted as the mean ± SEM (n = 16). (C) Requirement for AHR on suppressive effect of ITE. Relative suppression following siRNA-mediated knockdown of AHR compared to scrambled siRNA as the control (left). Percent T cell suppression by ITE in the presence of 10 μM CH223191, a selective antagonist of AHR, compared to DMSO as the vehicle control. Results are depicted as the mean ± SEM pooled from two independent experiments. (D, E) qPCR analysis on RNA isolated from T cells stimulated with αCD3/αCD28 and IL2 in the presence of vehicle, ITE, or TCDD. Target fold change was calculated using vehicle control with the dashed line representing the normalized control value of 1 and the mean depicted ± SEM pooled from 3 independent experiments (n ≥ 7). (F, G) Representative flow cytometric analysis of TBX21, GATA3, RORC, FOXP3, IL-10, GZMB, and CD39 protein expression following T cell activation using αCD3/αCD28 and IL2 in the presence or absence of ITE for 6 days. Each flow panel is representative of at least 2 independent experiments. (H) Requirement for GZMB and CD39 on the suppressive activity of ITE using a blocking antibody against CD39 or the GZMB inhibitor AAD-CMK (left) and showing the requirement for GZMB and CD39 for TCDD-mediated suppression as a positive control (right) (n = 21). (I) Role of IL-10 in ITE-mediated T cell suppression using 2.5 μgml⁻¹ αIL-10 blocking antibody shown as percent suppression (left) and the ability of αIL-10 antibody to inhibit STAT3 phosphorylation in human T cells following a 20 minute stimulation with 20 ngml⁻¹ IL-10 (right). * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = not significant

Figure 2. ITE induces functional human FOXP3⁺ Tregs in the presence of TGFβ1

(A) Suppressive activity of human naïve CD4⁺ T cells activated with αCD3/αCD28 and TGFβ1 in the presence of CTL, ITE, or TCDD. Percent suppression is depicted as the mean ± SEM pooled from at least 2 independent experiments with 10 unique donors. (B) qPCR analysis on RNA isolated from T cells stimulated with αCD3/αCD28 and IL2 in the presence of CTL, TGFβ1, TGFβ1 + ITE, or TGFβ1 + TCDD. Target fold change was calculated using vehicle control with the dashed line representing the normalized control value of 1 with relative expression shown as the pooled mean ± SEM (n ≥ 10). (C) Representative flow cytometric analysis of TBX21, GATA3, and RORC protein expression in T cells following activation using αCD3/αCD28 and IL2 in the presence of CTL, TGFβ1, or TGFβ1 + ITE from 3 independent experiments. (D) qPCR analysis on RNA isolated from T cells stimulated with αCD3/αCD28 in the presence of CTL, TGFβ1, TGFβ1 + ITE, or TGFβ1 + TCDD. Target fold change was calculated using vehicle control with the dashed line representing the normalized control value of 1 with relative expression shown as the pooled mean ± SEM (n ≥ 7). ‡ P < 0.05 compared to control. (E) Representative flow cytometric analysis of FOXP3, IL-10, CD39, and GZMB protein expression in T cells following activation using αCD3/αCD28 and IL2 in the presence of CTL, TGFβ1, or TGFβ1 + ITE from 2 independent experiments. (F) Effect of CD39 blockade using CD39 blocking antibodies on the suppressive activity of TGF+ITE (left panel) or TGF+TCDD (right panel) with the mean depicted ± SEM (n ≥ 12). * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 3. TNBS-induced colitis mediated by human CD4⁺ T cells

(A) Schematic of human CD4⁺ T cell reconstitution and TNBS experimental colitis in NSGAb^oDR1 mice. (B) Change in body weight in NSGAb^oDR1 mice reconstituted with human CD4⁺ T cells 3 days following rectal administration of ethanol (EtOH) as a vehicle control (n = 6), TNBS (n = 9), or NSGAb^oDR1 mice without human T cells administered TNBS (n = 7). Each dot represents an individual animal with the mean depicted ± SEM. Data shown is pooled from 3 independent experiments and is representative of more than 6 experiments with similar results. (C) Endoscopic images of representative mice 3 days following TNBS or EtOH challenge (top) with representative H&E stained colonic sections (bottom) from 3 independent experiments (10X magnification). Scale bar = 200 μm. (D) Colitis scoring of H&E stained colon sections 3 days following the TNBS or EtOH rectal challenge. Scores for each individual mouse are shown with the mean for each group depicted ± SEM. (E) Representative microscopic images of formalin-fixed paraffin-embedded colonic sections stained for human CD3 from NSGAb^oDR1 mice previously reconstituted with human CD4⁺ T cells treated with EtOH or TNBS (left) (n = 4) and quantified (right) with bars representing the mean ± SEM (20X magnification). Scale bars = 200 μm. (F) qPCR analysis on RNA isolated from colonic tissue of reconstituted NSGAb^oDR1 mice treated with TNBS or EtOH (n = 6). Human cytokines were normalized to hypoxanthine phosphoribosyltransferase (HPRT) and the fold change was compared to a pooled human RNA control sample using the formula 2^{− (Ct(target) − Ct (HPRT))}. Bars represent the mean ± SEM. (G) Representative flow cytometric analysis of human CD4⁺ T cells isolated from the colonic lamina propria of NSGAb^oDR1 mice stimulated with phorbol myristate acetate (PMA) and ionomycin for 4 hours in the presence of GolgiStop and stained for intracellular TNF and IFNγ (top) and quantified (bottom) from 3 independent experiments (n ≥ 5). * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4. ITE prevents T cell-driven experimental colitis in humanized mice

(A) Schematic of ITE administration in TNBS experimental colitis in NSGAb^oDR1 mice reconstituted with human CD4⁺ T cells. (B) Body weight change 3 days following TNBS rectal challenge in NSGAb^oDR1 mice reconstituted with HLA-DR1 matched human CD4⁺ T cells treated with PBS (n = 8) or ITE (n = 13). Each dot represents and individual mouse with the mean depicted ± SEM pooled from 3 independent experiments using 2 unique HLA-DR1 matched healthy donor sources. (C) Representative H&E stained colonic sections 3 days post-TNBS challenge of reconstituted NSGAb^oDR1 mice treated with PBS (n = 8) or ITE (n = 13) (left) and colitis score quantified with the mean for each group depicted ± SEM pooled from 3 independent experiments with 2 unique donor sources (right). Images are 20X magnification. Scale bars = 100 μm. (D) Quantified flow cytometric data of human CD4⁺ T cells isolated from the spleen and colonic lamina propria of PBS or ITE treated NSGAb^oDR1 mice stained for human CD45 (hCD45), GZMB, CD39, FOXP3 (top), with representative dot plots shown below. Intracellular IL-10 staining was performed on T cells isolated from spleen and colonic lamina propria and stimulated ex vivo with phorbol myristate acetate (PMA) and ionomycin for 4 hours in the presence of GolgiStop. Bars represent the mean ± SEM pooled from 2 independent experiments using 2 unique HLA-DR1 matched healthy donors. (E) Schematic depicting injection of in vitro generated autologous ITE-induced suppressive T cells prior to rectal challenge with TNBS in NSGAb^oDR1 mice previously reconstituted with matched donor CD4⁺ T cells. (F) Flow cytometry dot plot showing the recovery of in vitro generated autologous Tregs (labeled with CellTrace Violet prior to intraperitoneal injection of 2×10⁶ labeled cells) from spleens of humanized mice. (G) Body weight change 3 days following TNBS rectal challenge in NSGAb^oDR1 mice reconstituted with HLA-DR1 matched human CD4⁺ T cells and injected with PBS (n = 9) or autologous in vitro ITE-generated suppressive cells (n = 7) 1 day prior to TNBS rectal challenge. Each dot represents and individual mouse with the mean depicted ± SEM pooled from 2 independent experiments using 2 unique HLA-DR1 matched healthy donor sources. (H) Representative H&E stained colonic sections 3 days post-TNBS challenge of reconstituted NSGAb^oDR1 mice injected with PBS (n = 9) or CellTrace violet-labeled autologous ITE-induced Tregs (n = 7) (left) and colitis score quantified with the mean for each group depicted ± SEM pooled from 2 independent experiments (right). Images are 20X magnification. Scale bars = 100 μm. * P < 0.05.