Tryptophan metabolite activation of the aryl hydrocarbon receptor regulates IL-10 receptor expression on intestinal epithelia

Abstract

IL-10 is a potent anti-inflammatory cytokine that inhibits the production of proinflammatory mediators. Signaling by IL-10 occurs through the IL-10 receptor (IL-10R), which is expressed in numerous cell types, including intestinal epithelial cells (IECs), where it is associated with development and maintenance of barrier function. Guided by an unbiased metabolomics screen, we identified tryptophan (Trp) metabolism as a major modifying pathway in interferon-γ (IFNγ)-dominant murine colitis. In parallel, we demonstrated that IFNγ induction of indoleamine 2,3-dioxygenase 1, an enzyme that catalyzes the conversion of Trp to kynurenine (Kyn), induces IL-10R1 expression. Based on these findings, we hypothesized that IL-10R1 expression on IEC is regulated by Trp metabolites. Analysis of the promoter region of IL-10R1 revealed a functional aryl hydrocarbon response element, which is induced by Kyn in luciferase-based IL-10R1 promoter assays. Additionally, this analysis confirmed that IL-10R1 protein levels were increased in response to Kyn in IEC in vitro. Studies using in vitro wounding assays revealed that Kyn accelerates IL-10-dependent wound closure. Finally, reduction of murine dextran sodium sulfate colitis through Kyn administration correlates with colonic IL-10R1 expression. Taken together, these results provide evidence on the importance of IL-10 signaling in intestinal epithelia and implicate AHR in the regulation of IL-10R1 expression in the colon.

Fig. 1. Metabolomic profiling of DSS colitic mice

(A) Principal component analysis of total metabolic differences in control mice and mice receiving 3% DSS for 7 days. Metabolites were measured by Metabolon, Inc. using LC/MS and GC/MS analysis, n=5. (B) Heat map of metabolites in the Trp pathway that are altered during DSS. (C) Summary of Trp metabolism pathway including the enzymes involved in the primary metabolism of Trp to Kyn or 5-hydroxy-tryptophan.

Fig. 2. Kynurenine profiling during active inflammation

(A) EC-HPLC analysis of Kyn in the colon of DSS mice at day 0 and day 7, n=5. (B) Concentration of Kyn in the colon of mice receiving 3% DSS for up to 5 days, n=5, *p<0.05, **p<0.01, ***p<0.001. (C) EC-HPLC analysis of Kyn in the serum of DSS mice at day 0 and day 7, n=5. (D) Concentration of Kyn in the serum of mice receiving 3% DSS for up to 5 days, n=5, *p<0.05, **p<0.01, ***p<0.001. (E) Western Blot quantification of IDO1 levels in the colons of mice receiving 3% DSS for up to 5 days. n=5, *p<0.05. (F) qPCR of ido1 transcript levels in whole colon, colonic mucosal scrapings, and enriched colonic epithelial cells after 6 days of 2.5% DSS. n=5, *p<0.05, **p<0.01.

Fig. 3. IL10R1 expression in response to AHR ligands

(A) qPCR of il10r1 and cyp1a1 transcript levels in T84 IEC in response to AHR ligands. Confluent monolayers of T84 cells were treated with Kyn (100 μM), FICZ (250 nM), or TCDD (30 nM) for 6 h. Error bars represent the S.E.M. of five replicates. (B) Luciferase-based assay of IL10R1 promoter activity in response to AHR ligands FICZ and Kyn. IFN- γ served as a positive control. Caco-2 IECs were transfected with pGL3 containing the IL10R1 promoter region or empty pGL3 vector as control, treated for 12h with 100 μM Kyn, 250 μM FICZ, or 10 ng/mL IFN- γ and luciferase activity measured, n=3 (***p<0.001). (C) Cell surface ELISA of IL10R1 expression in shControl, shRNA ARNT knockdown, and AHR inhibitor treated Caco-2 IEC. Confluent monolayers of Caco-2 IEC were treated with 100 μM Kyn, 250 nM FICZ, or 30 nM TCDD for 24h. Error bars represent the S.E.M. of triplicate samples, *p<0.05, **p<0.01. (D) Western blot analysis of IL10R1 levels in shRNA AHR knockdown Caco-2 IEC. Confluent monolayers of Caco-2 cells containing a non-template control (shNTC) or shRNA specific for AHR were treated with FICZ or Kyn for 24 h. (E) Densitometry of the western blot analysis in Fig. 3D. Relative density of the IL10R1 bands is normalized to β-actin. (F) Western blot analysis of IL10R1 levels in shRNA ARNT knockdown T84 IEC. Confluent monolayers of T84 cells containing a non-template control (shNTC) or shRNA specific for ARNT were treated with Kyn, FICZ, or TCDD for 24 h. (G) Densitometry of the western blot analysis in Fig. 3F. Relative density of the IL10R1 bands is normalized to β-actin.

Fig. 4. In vitro scratch wound assay in T84 IL10R1 kd IEC

(A) Representative photos of T84 IEC 0 h and 48h after scratch. T84 shNTC (top) and IL10R1 kd (bottom) were treated with Kyn, IL10, or both, after wounding and monitored for 48 h. (B) Restitution of scratch wounds (y-axis) in T84 shNTC (left) and T84 IL10R1 kd (right) at 0, 24, 48, and 72h (x-axis) with control, Kyn, and IL10 treatment, n=5. (C) Comparison of shNTC (black) and IL10R1 kd (gray) wound healing with FICZ and IL10. Error bars represent the S.E.M. of triplicate measurements in five independent experiments, where *** indicates p<0.001 for combination of IL10 and Kyn compared to IL10 alone.

Fig. 5. In vitro scratch wound assay in T84 ARNT kd IEC

(A) Representative photos of T84 IEC 0 h and 72 h after scratch. T84 shNTC (top) and ARNT kd (bottom) were treated with FICZ (blue), IL10 (gray), or both (red), after wounding and monitored for 72 h. (B) Restitution of scratch wounds (y-axis) in T84 shNTC (left) and T84 ARNT kd (right) at 0, 24, 48, and 72h (x-axis) with control, FICZ, and IL10 treatment, n=5. (C) Comparison of shNTC (black) and ARNT kd (gray) wound healing with FICZ and IL10. Error bars represent the S.E.M. of triplicate measurements in five independent experiments, where *** indicates p<0.0001 for combination of IL10 and FICZ compared to IL10 alone.

Fig. 6. Impact of Kyn supplementation on DSS colitis outcomes

(A) Weight curves during 2.5% DSS and recovery with and without Kyn administration (10 mg/kg every 48 h) n=5. (B) Disease activity scores during 2.5% DSS and recovery with and without Kyn administration (10 mg/kg every 48 h). Disease activity was measured by a combination of weight loss, stool consistency, and bleeding, n=5. (C) Intestinal permeability after 6 d of DSS and 2 d of recovery, with and without Kyn. Mice were gavaged with FITC-dextran and permeability measured by the concentration of FITC (y-axis) in the serum, n=5 *p<0.05. (D) Colon anatomy 8 d post DSS with or without Kyn. (E) Average colon length (y-axis) in mice receiving water or DSS with or without Kyn. n=5 *p<0.05. (F) Histology score after 8 d post DSS with and without Kyn, n=5 ***p<0.001. (G) Representative photos of H&E stained colon sections from DSS and control mice with or without Kyn.