MAIT cells exacerbate colonic inflammation in a genetically diverse murine model of spontaneous colitis

Abstract

IL-17-producing lymphocytes are involved in both tissue repair and the propagation of inflammation, with their effects highly context-dependent. Mucosal-Associated-Invariant-T-cells (MAIT), a subset of innate-like T cells with features of both Th1 and Th17 lineages, are increasingly recognized for their roles in mucosal immunity. Here, we identified the Collaborative-Cross CC011/Unc strain, which spontaneously develops chronic colitis, as being enriched for MAIT cells. This expansion coincides with an age-related loss of intestinal barrier permeability and colonic inflammation. Microbiota from CC011 mice activated MAIT cells in an MR1-dependent manner and selectively promoted the accumulation of MAIT17 cells in peripheral tissues. Single-cell transcriptomic analyses revealed colon MAIT cells from colitic CC011 mice expressed a pathogenic Th17-like signature, characterized by IL-1 and IL-23 signaling, IL-17A and IFNγ co-expression, and upregulation of IL-23R, features that correlated with inflammatory Ly6C^hi monocyte abundance. Genetic deletion of Traj33, essential for MAIT development, significantly reduced colonic inflammation in this model. These findings demonstrate that MAIT cells integrate microbial and cytokine cues to adopt a pathogenic effector phenotype that exacerbates chronic intestinal inflammation.

Keywords: Colitis; Inflammation; MAIT cells; scRNAseq.

Fig. 1.. CC011 a MAIT cell-rich collaborative cross strain at steady-state.

(A) Representative FACS plots of thymic MAIT cells (% frequency of total live cells) in CC011 and founder reference strain B6 and summarized in (B) with the Mean, and SD across 50 CC and 8 founder strains. (C) Representative FACS plots of MAIT cells (% frequency of CD3⁺ T cells) and (D) summary in tissues of B6 and CC011 mice. (E) Intracellular staining for transcription factors and (F) cell surface co-receptors in CC011 mice (% frequency of MAIT cells). Data in (B) are representative of at least 15 independent experiments with 3 CC strains acquired/experiment. ***P < 0.001, One-way ANOVA, multiple comparisons to CC011, n = 2–6 mice/group. Data in (C-F) are representative of at least 3 independent experiments. *P < 0.05; **P < 0.01, Mann-Whitney Test, n = 3–8 mice/group all mice were age 6–9 weeks old.

Fig. 2.. CC011 develop spontaneous colitis after 20 weeks of age with histological features that emulate human disease.

(A-B) Images of colons from male and female B6 and CC011 mice (week 27) and (C) Colon length (cm): Body weight (grams) of CC011. (D) Images of spleens from male and female B6 and CC011 mice (week 27). (E) % Spleen weight (grams) of body weight (grams) in B6 and CC011 strains. (F-L) Representative H & E staining of paraffin embedded sections of colon from (F) aged B6 and (G-L) CC011 mice (week 27–36). Denuding (black arrows) and hypertrophy (dark green arrows) of the surface epithelium, inflammation/transmural (white arrows), lymphocytic accumulations (red arrows), herniating gland (cyan arrow), crypt abscess (green bracket) and mucosal (green arrows) neutrophils. Dysplasia (yellow oval/rectangle) in a week 35 CC011 mouse. (M) Colon tissue inflammation and (N) Colon tissue injury scoring of H&E sections from CC011 and B6 mice based on Dieleman., et al histological grading of colitis. Data in (C), (E), (M) and (N) are representative of at least 8 independent experiments. (C, M–N), *P < 0.05, Spearman rank correlation, n = 16–68 mice/group. (E) ***P < 0.001, Mann-Whitney Test, 20–56 mice/group.

Fig. 3.. MAIT cells are the unconventional T cell subset associated with colitis onset.

(A) Representative FACS plots of colon lamina propria (LP) MAIT cells. (B) Log₁₀ transformed % MAIT Frequency of total live CD45⁺ cells and (C) MAIT cell numbers. (D) Representative Colon LP FACS plots of Treg (CD4⁺Foxp3⁺ROR-γt^+/−), and TH17 (CD4⁺Foxp3⁻ROR-γt⁺) and Log₁₀ transformed (E) Treg and (F) TH17 numbers. (G) Ratio of the %Freq of Treg:Th17 cells. (H) Representative FACS plots of Colon LP Neutrophils (CD11b⁺Ly6G⁺) and Eosinophils (CD11b⁺Sig-F⁺) in 8 week-old B6 and CC011 mice and summarized adjacent in mice aged 8–17 weeks old. Log₁₀ transformed Colon LP (I) Neutrophil and (J) Eosinophil numbers in CC011 mice. (K) FITC dextran concentration (ng/ml) in serum of orally gavaged CC011 or B6 mice. (L-O) Cytokine milleu of colon explants. Colon explant tissue was cultured overnight, and culture supernatants were analyzed for IL-1α (L), IL-1β (M), IL-17A (N) and IFNγ (O) with LEGENDplex. The dotted line represents the limit of detection for each parameter. Data in (B), (C), (E- G), and (I- J) represent at least 8 independent experiments where, *P < 0.05, ***P < 0.001, Linear regression, (B, C) n = 39; (E, F, G) n = 33; (I) n = 20; (J) n = 16. The data in (H) represents at least 3 independent experiments where **P < 0.01, Mann-Whitney Test, B6, n = 4 CC011 n = 6 mice. The data in (K) represent at least 2 independent experiments, *P < 0.05, Spearman rank correlation, n = 13; CC011, B6 n = 12. Data in L-O (Mean and SD) represent two independent experiments. The dots represent the average of the technical replicates from individual mice, *P < 0.05, Mann-Whitney test.

Fig. 4.

The microbiota of CC011 supports the peripheral expansion of MAIT cells and the MAIT17 phenotype in Germ-Free B6 mice. (A) Experimental design following oral gavage of B6 or CC011 cecal contents (>27 weeks-old donor mice) into GF-B6 3 week-old mice. (B) Representative H & E staining of paraffin embedded sections of colon from 25 week-old GF-B6 mice receiving cecal contents from B6 (GF-B6^B6) or CC011 (GF-B6^CC011). (C) Representative FACS plots of MAIT cells from spleen, liver and lung tissues (% of CD3⁺) with MAIT cell numbers summarized adjacent. (D) Representative FACS plots of MAIT1 (pink box) and MAIT17 (green box) cells from spleen, liver and lung tissues (% of MAIT) with MAIT1 numbers summarized adjacent. (E) MAIT17 numbers in spleen, (F) liver and (G) lungs of CC011, B6, GF-B6, GF-B6^B6 or GF-B6^CC011. The data in (C) represents two independent experiments pooled together with mice aged 14 and 25 weeks, **P < 0.01, ***P < 0.001, Mann-Whitney T-test. The data in (D) represents two independent experiments pooled together with mice aged 14 and 25 weeks, **P < 0.01, ***P < 0.001, Welch’s T-test. The data in (E, F, G) represents at least two independent experiments, with mice age ranging from 14- 41 weeks-old, *P < 0.05, **P < 0.01, ***P < 0.001, Welch’s ANOVA Test with Dunnetts multiple T3 comparisons.

Fig. 5.. MAIT cells from diseased colon LP show an enriched type III transcriptomic signature and signs of activation.

(A) Experimental workflow for scRNAseq of MAIT cells isolated from spleen or Colon LP of week 28 or 30 CC011 mice. (B) UMAP representation of spleen and colon scRNAseq data, colored by Seurat clusters, (C) Tissue identity or (D) Original identity. (E) Representative FACS plots of CD8α and Ki67 (Mki67) staining of MAIT cells in the spleen and colon LP of CC011 mice and summarized below. (F) Metascape derived enriched terms from the colon tissue gene signature. The top 15 enriched terms where the color denotes the p-value. (G) Feature plots of indicated genes. (H) Representative FACS histograms of ICOS expression in MAIT cells, with geometric mean intensity (gMFI) plotted adjacent. (I) Representative FACS plots of Tim-3 (Havcr2) expression in MAIT cells, with %expression in summarized adjacent. (J) Representative FACS plots of intracellular in vivo IL-17A in CC011 MAIT cells from spleen and Colon LP and summarized adjacent. The data in (E) represent two independent experiments, *P < 0.05, Paired t-Test, n = 4 mice/group. The data in (H, I) represent two independent experiments, *P < 0.05, **P < 0.01, Paired t-Test, n = 6 mice/group. The data in (J) represent two independent experiments, **P < 0.01, Mann-Whitney Test, n = 6 mice/group. All mice were aged between 28 and 41 weeks.

Fig. 6.

CC011 colonic MAIT cells are enriched for a pathogenic TH17 score and express genes consistent with IL-1R and IL-23R stimulation. (A) UMAP representation of Colon LP MAIT cells derived from B6^CAST scRNAseq integrated with Colon LP MAIT cells derived from CC011 mice, colored by Seurat clusters. (B) Percentage of cells per cluster. (C) UMAP representation of Pathogenic TH17 scores compiled from ^, on B6^CAST and CC011 Co-LP cells calculated using UCell signature score R package . (D) Box and whisker plots of Pathogenic TH17 UCell Score, Wilcoxon Test. (E) Dot plot showing selected gene expression per strain from integrated single-cell datasets in (A), scaled data is represented. (F) Representative FACS plots of intracellular in vivo IL-17A expression in young and mature adult CC011 MAIT cells and (G) numbers of IL-17A producing MAIT cells. (H) Representative FACS plots of % of IFNγ and IL-17A in MAIT, TH17 and TREG cells from Co-LP of colitic CC011 mice aged 25–35 weeks-old, cultured overnight in the presence of BFA. (I) The number of IFNγ-producing cells of indicated subset. (J) The % of IL-17A⁺IFNγ⁺ cells of indicated subset. (K) Representative FACS plots of IL-23R expression in young and mature adult Co-LP MAIT cells and (L) summarized numbers of IL-23R⁺ MAIT cells. (M) Representative FACS plots of gating for Ly6C^hi monocytes in the Co-LP of CC011. The %Freq of Live CD45⁺ cells are shown. (N) Spearman correlation of Ly6C^hi monocyte and IL-23R⁺ MAIT cell numbers. Data in (G, L) represent two independent experiments, **P < 0.01, Mann-Whitney test. Data in (I-J) represent two independent experiments, **P < 0.05, ****P < 0.00001, 1-way ANOVA. The data in (N) represent two independent experiments, **P < 0.01, R² = 0.855 Spearman-rank correlation. Young adult mice (YG) were aged 6–10 weeks old, mature adult mice were aged 26–35 weeks old.

Fig. 7.. Limited colonic pathology and immune cell infiltration in the MAIT cell deficient CC011-Traj33^−/− strain.

(A) Representative FACS plots of MAIT cells (% of CD3⁺ T cells) from tissues of 25–37 weeks-old CC011 and CC011-Traj33^−/− (Traj33^−/−), and (B) summarized. (C) Images of colons. (D) Representative H & E staining of paraffin-embedded sections of proximal and mid colon from 25-37 weeks-old CC011, Traj33^−/− and B6. (E) Colon tissue inflammation and (F) injury scoring of H&E sections from CC011 and Traj33^−/− mice, Dieleman., et al histological grading of colitis . (G) Representative myeloperoxidase immunodetection from paraffin embedded colon tissue sections from CC011 and Traj33^−/− mice and (H) summarized neutrophil counts/colon. (I) Cell number of immune cell subsets derived from FACS analyses of Colon LP from CC011 and Traj33^−/− mice. The data in (B, E, F, G and I) are representative of at least 2 independent experiments, *P < 0.05, **P < 0.01, Mann-Whitney, (B) n = 3–7 mice/group, (D-G) n = 5–7 mice/group, (I) n = 3–5 mice/group. Mouse strains were obtained from colonies maintained and housed in the same facility and room.