Activation of aryl hydrocarbon receptor by TCDD prevents diabetes in NOD mice and increases Foxp3+ T cells in pancreatic lymph nodes

Abstract

The ligand-activated transcription factor, aryl hydrocarbon receptor (AHR), is a novel inducer of adaptive Tregs. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the most potent AHR ligand, induces adaptive CD4+CD25+ Tregs during an acute graft-versus-host (GvH) response and prevents the generation of allospecific cytotoxic T lymphocytes. TCDD also suppresses the induction of experimental autoimmune encephalitis in association with an expanded population of Foxp3+ Tregs. In this study, we show that chronic treatment of NOD mice with TCDD potently suppresses the development of autoimmune Type 1 diabetes in parallel with greatly reduced pancreatic islet insulitis and an expanded population of CD4+CD25+Foxp3+ cells in the pancreatic lymph nodes. When treatment with TCDD was terminated after 15 weeks (23 weeks of age), mice developed diabetes over the next 8 weeks in association with lower numbers of Tregs and decreased activation of AHR. Analysis of the expression levels of several genes associated with inflammation, T-cell activation and/or Treg function in pancreatic lymph node cells failed to reveal any differences associated with TCDD treatment. Taken together, the data suggest that AHR activation by TCDD-like ligands may represent a novel avenue for treatment of immune-mediated diseases.

Keywords: 2,3,7,8-tetrachlorodibenzo-p-dioxin; AHR; NOD mice; TCDD; Type 1 diabetes; aryl hydrocarbon receptor; regulatory T cell.

Figure 1. Sequence analysis of exon 10 of AHR gene in NOD mice shows a low-affinity AHR^d haplotype identical to that found in DBA/2 mice

AHR: Aryl hydrocarbon receptor; NOD: Nonobese diabetic. Data adapted from [13].

Figure 2. Treatment of NOD mice with TCDD results in sustained activation of AHR as evidenced by a high level of hepatic Cyp1a1 message at 31 weeks of age

Aryl hydrocarbon receptor (AHR) activation declines when TCDD treatment is terminated and is no longer apparent after 8 weeks of VEH treatment. NOD mice were dosed orally biweekly with VEH or TCDD until 31 weeks of age. At 23 weeks of age, half of the TCDD-treated mice were switched to VEH for the remaining 8 weeks. Liver samples were obtained from mice that survived to 31 weeks and processed for RNA. Cyp1a1 expression data represent fold-change relative to β-actin message from three, 12 and three mice in VEH, TCDD and TCDD/VEH groups, respectively. NOD: Nonobese diabetic; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEH: Vehicle.

Figure 3. TCDD-treated mice are protected from diabetes by continuous treatment with TCDD

Groups of mice were treated with VEH (n = 11) or TCDD (n = 12) biweekly, beginning at 8 weeks of age. With no diabetes in any of the TCDD-treated mice at 23 weeks of age, half of the mice in the TCDD-treatment group were switched to VEH for the remainder of the study (TCDD/VEH). Blood glucose levels were measured weekly. Animals were terminated by an overdose of CO₂ when nonfasting blood glucose level exceeded 250 mg/dl for 2 consecutive days. TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEH: Vehicle.

Figure 4. NOD mice treated biweekly with TCDD from 8 to 15 weeks of age show greatly reduced insulitis

Groups of 12 mice were treated with VEH or TCDD. At 15 weeks of age, pancreata were frozen in optimal cutting temperature compound. A minimum of five 5 μm pancreatic sections, each 200 μm apart, were cut for each tissue block. Sections were fixed in formalin and stained with hematoxylin and eosin for visualization and scoring of individual islets. Data reflect scores from four representative nondiabetic mice per treatment. NOD: Nonobese diabetic; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEH: Vehicle.

Figure 5. Frequency of CD4 ⁺CD25⁺Foxp3⁺ T cells in the pancreatic lymph nodes is increased in NOD mice treated biweekly with TCDD until 31 weeks of age

Mice removed from TCDD treatment 8 weeks earlier no longer show elevated frequency of CD4⁺CD25⁺ Foxp3⁺ T cells. Data represent mean ± SEM of three, 12 and three mice in VEH, TCDD and TCDD/VEH groups, respectively. Cells were stained for flow cytometric analysis as described in Materials and Methods. NOD: Nonobese diabetic; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEH: Vehicle.