Repression of the aryl-hydrocarbon receptor prevents oxidative stress and ferroptosis of intestinal intraepithelial lymphocytes

Abstract

The aryl-hydrocarbon receptor (AHR) is a ligand-activated transcription factor that buoys intestinal immune responses. AHR induces its own negative regulator, the AHR repressor (AHRR). Here, we show that AHRR is vital to sustaining intestinal intraepithelial lymphocytes (IELs). AHRR deficiency reduced IEL representation in a cell-intrinsic fashion. Single-cell RNA sequencing revealed an oxidative stress profile in Ahrr^-/- IELs. AHRR deficiency unleashed AHR-induced expression of CYP1A1, a monooxygenase that generates reactive oxygen species, increasing redox imbalance, lipid peroxidation, and ferroptosis in Ahrr^-/- IELs. Dietary supplementation with selenium or vitamin E to restore redox homeostasis rescued Ahrr^-/- IELs. Loss of IELs in Ahrr^-/- mice caused susceptibility to Clostridium difficile infection and dextran sodium-sulfate-induced colitis. Inflamed tissue of inflammatory bowel disease patients showed reduced Ahrr expression that may contribute to disease. We conclude that AHR signaling must be tightly regulated to prevent oxidative stress and ferroptosis of IELs and to preserve intestinal immune responses.

Keywords: AHRR; Ahr; Cyp1a1; IBD; IEL; ROS; ferroptosis; mucosal immunity; oxidative stress.

Figure 1.. Ahrr^−/− mice have reduced numbers of IEL.

(A) Numbers of CD45⁺ IEL in the small intestine of WT and Ahrr^−/− mice. (B-E) Numbers of T cells in small intestinal epithelium (B), small intestinal lamina propria (C), mesenteric lymph nodes (D) and spleen (E) of WT and Ahrr^−/− mice. (F) Representative flow cytometry plots depicting frequency of TCR-β⁺ IEL in WT and Ahrr^−/− mice. (G-J) IEL populations in small intestinal epithelium of WT and Ahrr^−/− mice, including TCR-γδ⁺ CD8αα⁺ (G), TCR-β⁺ CD8αα⁺ (H), TCR-β⁺ CD8αβ⁺ (I), and TCR-β⁺ CD4⁺ (J). (K) Immunofluorescence staining of CD8α in the small intestine of WT and Ahrr^−/− mice. (l) Quantification of CD8α+ cells per villus of WT and Ahrr^−/− mice. (M) Frequency of CD160⁺ TCR-β⁺ CD8αα⁺ IELs in WT and Ahrr^−/− mice. (N, O) Representative flow cytometry plots (N) and numbers of DP IEL (O) in WT and Ahrr^−/− mice (gated on CD45⁺, CD3⁺, TCR γδ⁻, TCRαβ⁺, CD8β⁻ CD4⁺ IEL). (P and Q) Representative flow cytometry plots plots (P) and frequency of ThPOK^low CD8αα⁺ cells (Q) in WT and Ahrr^−/− mice. Each dot represents an individual mouse. Data are pool or representative of 2–3 individual experiments. Statistical significance was determined by Mann-Whitney test. *P<0.05, **P<0.01. Please also see Figures S1 and S2.

Figure 2.. Loss of IEL in Ahrr^−/− mice is cell intrinsic.

(A) Schematic of the experiment. Purified splenic T cells from WT (CD45.1) and Ahrr^−/− (CD45.2) were transferred to Rag1^−/− mice in 1:1 ratio and IEL were analyzed after 8 weeks. (B) Representative flow cytometry plots plots showing CD4⁺ T cells and DP IEL from small intestine of reconstituted mice. (C-F) Frequency of WT and Ahrr^−/− TCR-β⁺ CD8αα⁺ (C), TCR-β⁺ CD8αβ⁺ (D), TCR-β⁺ CD4⁺ (E), and DP IEL (F), in the small intestine IEL of Rag1^−/− mice after reconstitution. (G) Rorc^cre Ahrr^fl/fl mice were generated by mating Rorc^cre with Ahrr^fl/fl mice. (H-L) Cell numbers of TCR-β⁺ CD8αα⁺ (H), TCR-β⁺ CD8αβ⁺ (I), TCR-β⁺ CD4⁺ (J), DP IEL (K), and TCR-γδ⁺ CD8αα⁺ (L), in Ahrr^fl/fl and Rorc^cre Ahrr^fl/fl mice. (M) Schematic of the experiment: IEL from the small intestine of chimeric mice reconstituted with bone marrow cells from WT (CD45.1) and Ahrr^−/− (CD45.2) mice in 1:1 ratio. (N) Representative flow cytometry plots showing CD4⁺ T cells and DP IEL from small intestine of reconstituted mice. o-s, Frequencies of WT and Ahrr^−/− TCR-β⁺ CD8αα⁺ (O); TCR-β⁺ CD8 αβ⁺ (P), TCR-β⁺ CD4⁺ (Q), DP IEL (R), and γδ (vγ7⁺) IEL (S), in small intestinal epithelium of bone marrow chimeric mice. Each dot represents an individual mouse. Data are pool or representative of 2 individual experiments. Statistical significance was determined by Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001. Please also see Figure S3.

Figure 3.. scRNAseq of WT and Ahrr^−/− IEL.

IEL (CD45⁺ CD3⁺ CD19⁻) from both WT and Ahrr^−/− mice were subjected to single cell RNA sequencing analysis. (A) UMAP plot depicting various populations of IEL. (B) Feature plots depicting expression of Cd4, Cd8b, Cd8a, Tyrobp, Klra7, Klra5, Gm156 and Tcf7. (C) Heatmap displaying the top 5 characteristic genes for each cluster of the UMAP in A. Please also see Figure S5.

Figure 4.. Ahrr deficiency induces oxidative stress in IEL.

(A) UMAP plot of stress response⁺ CD8αα⁺ IEL and (B) frequency of stress response⁺ CD8αα out of CD3⁺ T cells in WT and Ahrr^−/− mice. (C) Differential expression of Hspa1a, Hspa1b, Hsph1 and Dnajb1 transcripts in stress response⁺ CD8αα⁺ from WT and Ahrr^−/− mice. (D) Q-PCR analysis of stress response genes in CD8αα⁺ IEL from WT and Ahrr^−/− mice. (E) Violin plot depicting the expression of Gpx1 and Uba52 in stress response⁺ CD8αα⁺ IEL from WT and Ahrr^−/− mice. (F) CYP1A1 expression in ileal tissues of WT and Ahrr^−/− mice analyzed by qPCR. (G) Relative expression of CYP1A1 in IEL from WT and Ahrr^−/− mice. (H) CYP1A1 enzymatic activity in IEL from WT and Ahrr^−/− mice upon stimulation with TCDD. (I, J) DCFDA staining of IEL from WT and Ahrr^−/− mice: representative flow cytometry plots histogram of TCR-β⁺ CD8αα⁺ cells (I) and GMI of DCFDA in different IEL subsets (J). (K, L) DCFDA staining of WT and Ahrr^−/− IEL upon TCDD stimulation: representative flow cytometry plots histograms TCR-β⁺ CD8αα⁺ cells (K) and quantification in different IEL subsets (L). (M, N) C-11 BODIPY staining of WT and Ahrr^−/− IEL: representative histograms of TCR-β⁺ CD8αα⁺ cells (M) and quantification in different IEL subsets (N). Liperflou staining of WT and Ahrr^−/− IEL: representative histograms of TCR-β⁺ CD8αα⁺ cells (O) and quantification in different IEL subsets (P). Data are pool or representative of 2 individual experiments. Statistical significance was determined by Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001. Please also see Figure S6.

Figure 5.. Dietary selenium supplementation rescues IEL loss in Ahrr^−/− mice.

(A) WT and Ahrr^−/− mice were treated with selenium supplemented drinking water (2mg/l) for 4 weeks and then IELs were stained for C-11 BODIPY. (B-E) GMI of C-11 BODIPY in different IEL populations: TCR-β⁺ CD8αα⁺ (B); TCR-β⁺ CD8 αβ⁺ (C); DP IEL (D); and γδ IEL (E). (F) Representative flow cytometry plots plots showing the DP IEL in WT and Ahrr^−/− mice with and without dietary selenium supplementation. (G-M) Numbers of small intestinal CD45⁺ IEL (G), T cells in small intestinal epithelium (H); TCR-β⁺ CD8αα⁺ IEL (I), TCR-β⁺ CD8αβ⁺ IEL (J); TCR-β⁺ CD4⁺ IEL (K); DP-IEL (L); and TCR-γδ⁺ CD8αα⁺ IEL (M), of WT and Ahrr^−/− mice with and without selenium supplementation. Each dot represents an individual mouse. Data are pool or representative of 2 individual experiments. Statistical significance was determined by Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001.

Figure 6.. Dietary supplementation with Vit-E restores IEL in Ahrr^−/− mice.

(A) WT and Ahrr^−/− mice were fed a Vit-E rich diet for 5 weeks; IELs were stained for C-11 BODIPY. (B-E) GMI of C-11 BODIPY in different IEL populations: TCR-β⁺ CD8αα⁺ (B); TCR-β⁺ CD8 αβ⁺ (C); DP IEL (D); and γδ IEL (E). (F) Representative flow cytometry plots plots showing DP IEL in WT and Ahrr^−/− mice with and without dietary Vit-E supplementation. (G-M) Numbers of small intestinal CD45⁺ IEL (G), T cells in the small intestinal epithelium (H); TCR-β⁺ CD8αα⁺ IEL (I); TCR-β⁺ CD8αβ⁺ IEL (J); TCR-β⁺ CD4⁺ IEL (K); DP-IEL (L); and TCR-γδ⁺ CD8αα⁺ IEL (M), in WT and Ahrr^−/− mice with and without Vit-E supplementation. Each dot represents an individual mouse. Data are pool or representative of 2 individual experiments. Statistical significance was determined by Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001.

Figure 7.. Ahrr deficiency augments susceptibility to intestinal pathology.

(A) Schematic of C. difficile infection in WT and Ahrr^−/− mice or Ahrr^fl/fl and Rorc^cre Ahrr^fl/fl mice. (B) Body weight and c, clinical score in WT and Ahrr^−/− mice. (D) Body weight and (E), clinical score in Ahrr^fl/fl and Rorc^cre Ahrr^fl/fl mice. (F-I) Expression of Ifng, Tnfa, Il6, RegIIIg and RegIIIb in colonic and ileal tissues of WT and Ahrr^−/− mice upon infection with C. difficile. (J, K) Ahrr^−/− mice were reconstituted with WT IEL and, after 3 days, were challenged with 3% DSS for 7 days. (J) % of body weight variation and (K) colon length at day 7 in WT, Ahrr^−/− and Ahrr^−/− mice reconstituted with WT IEL. Each dot represents an individual mouse. Data are pooled or representative of 2 individual experiments. Statistical significance was determined by Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001. Please also see Figure S7.