24-Nor-ursodeoxycholic acid improves intestinal inflammation by targeting TH17 pathogenicity and transdifferentiation

Abstract

Background: 24-Nor-ursodeoxycholic acid (NorUDCA) is a novel therapeutic bile acid for treating immune-mediated cholestatic liver diseases, such as primary sclerosing cholangitis (PSC).

Objective: Since PSC strongly associates with T helper-type-like 17 (T_H17)-mediated intestinal inflammation, we explored NorUDCA's immunomodulatory potential on T_H17 cells.

Design: NorUDCA's impact on T_H17 differentiation was assessed using a CD4⁺T_Naive adoptive transfer mouse model, and on intraepithelial T_H17 pathogenicity and transdifferentiation using an αCD3 stimulation model combined with interleukin-17A-fate-mapping. Mechanistic studies used molecular and multiomics approaches, flow cytometry and metabolic assays with pathogenic (p) T_H17. Pathogenicity of pT_H17 exposed to NorUDCA in vitro was evaluated following adoptive transfer in intestinal tissues or the central nervous system (CNS). Key findings were validated in an αCD3-stimulated humanised NSG mouse model reconstituted with peripheral blood mononuclear cells from patients with PSC.

Results: NorUDCA suppressed T_H17 effector function and enriched regulatory T cell (Treg) abundance upon CD4⁺T_Naive cell transfer. NorUDCA mitigated intraepithelial T_H17 pathogenicity and decreased the generation of proinflammatory 'T_H1-like-T_H17' cells, and enhanced T_H17 transdifferentiation into Treg and Tr1 (regulatory type 1) cells in the αCD3-model. In vivo ablation revealed that Treg induction is crucial for NorUDCA's anti-inflammatory effect on T_H17 pathogenicity. Mechanistically, NorUDCA restrained pT_H17 effector function and simultaneously promoted functional Treg formation in vitro, by attenuating a glutamine-mTORC1-glycolysis signalling axis. Exposure of pT_H17 to NorUDCA dampened their pathogenicity and expansion in the intestine or CNS upon transfer. NorUDCA's impact on T_H17 inflammation was corroborated in the humanised NSG mouse model.

Conclusion: NorUDCA restricts T_H17 inflammation in multiple mouse models, potentiating future clinical applications for treating T_H17-mediated intestinal diseases and beyond.

Keywords: AUTOIMMUNE LIVER DISEASE; BILE ACID; INFLAMMATORY BOWEL DISEASE; INTESTINAL T CELLS; PRIMARY SCLEROSING CHOLANGITIS.

Figure 1. NorUDCA decreases T_H17 frequency and enriches Tregs in multiple tissues upon CD4⁺T_Naive adoptive transfer in vivo. NorUDCA decreases T_H17 frequency and enriches Tregs in multiple tissues upon CD4⁺T_Naive adoptive transfer in vivo. (A) Experimental design. (B) Photographs of mesenteric lymph nodes and spleens from indicated groups are depicted. Quantitative analysis of colon length of indicated groups. (C) Quantitative analysis of numbers of leucocytes extracted from indicated tissues. (D, E) PAS and H&E staining of colon sections (magnifications as indicated, scale bar 200 µm). (E) Histopathological scores are shown alongside. (F, G) Representative flow cytometric plots and quantitative analysis of T_H17 and Treg cells extracted from indicated tissues. (H, I) Representative histogram plots presenting RORɣt and CCR6 expression on small intestine-IEL-infiltrating T_H17 cells. Summary of the frequency of T_H17 cells expressing RORɣt (H) or CCR6 (I) within indicated tissues. Data summarise three independent experiments. At least three mice were used per group for each experiment. Each point represents one mouse. Two samples from colonic fractions (IEL or LPL) were pooled to achieve sufficient cell numbers for flow cytometric analysis. Mean and SEM are presented. P values were calculated using the unpaired Student’s t-test (two-tailed). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. CD4⁺T_N, CD4⁺T_Naive; CCR6, C-C-chemokine receptor 6; IEL, intraepithelial lymphocytes; IL, interleukin; Inflamm, inflammation; invol, involvement; LPL, lamina propria lymphocytes; mLN, mesenteric lymph nodes; NorUDCA, 24-Nor-ursodeoxycholic acid; PAS, periodic acid Schiff; Pct, percentage; RORγt, retinoic acid orphan receptor-gamma; SI, small intestinal; Sp, spleen; T_H17, T helper-type-like 17; Treg, regulatory T cell.

Figure 2. NorUDCA targets T_H17 pathogenicity and transdifferentiation during intestinal inflammation in vivo in an αCD3 model. (A) Experimental design. (B) Photographs of intestines from IL-17A-fate-mapping mice±i.p. αCD3 fed either standard chow or NorUDCA enriched diet. (C) Absolute cell counts from leucocytes extracted from small intestines. (D) Gating strategy for small intraepithelial effector(eff)T_H17, exT_H17 and CD4⁺ lymphocytes with IL-17A producing history (YFP⁺). (E) Representative and summary of frequencies of effT_H17 cells, exT_H17 cells, YFP⁺ cells and non-T_H17 cells of indicated groups. (F) Representative expression of CCR6 on YFP⁺ cells and corresponding quantitative analysis. (G) Gating strategy for T_H17, T_H1^exTH17 and T_H1 cells transdifferentiated from exT_H17 cells (T_H1-likeT_H17). Representative flow cytometric plots and analysis of indicated cell types. (H) Representative expression of phosphorylated RPS6 (serine 235/236) on YFP⁺cells and corresponding quantitative analysis. (I, J, K) Representative flow cytometric plots and analysis of Tregs derived from exT_H17 cells and non-T_H17 cells. Data presented throughout this figure were analysed from ex vivo isolated small intestinal intraepithelial lymphocytes . Data are cumulative of two independent experiments, with at least three mice per group for each experiment. Each point represents one mouse. Mean and SEM are shown. P values were calculated using the unpaired Student’s t-test (two-tailed). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. i.p., intraperitoneal; eYFP, express yellow fluorescent protein; gMFI, geometric mean-fluorescence intensity; IFN, interferon; IL, interleukin; NorUDCA, 24-Nor-ursodeoxycholic acid; RPS6, ribosomal protein S6; T_H17, T helper-type-like 17; Treg, regulatory T cell.

Figure 3. Treg induction is essential for NorUDCA’s restriction on T_H17 pathogenicity during intestinal inflammation in vivo. (A) Experimental design. (B) H&E staining of representative histological sections (scale bar 200 µm) of indicated groups. (C) Representative flow cytometric plots and frequency of T_H17 and Treg in αCD3 challenged untreated or NorUDCA-treated DEREG mice±Treg depletion by DT administration. Data are analysed from ex vivo isolated small intestinal intraepithelial lymphocytes . Data summarises two independent experiments, with at least three mice per group per experiment. Each point represents one mouse. (D) Body weight loss data is presented, derived from one experiment with five mice per group. Results (C, D) are expressed as mean and SEM are indicated. P values were calculated using the one-way ANOVA analysis (C) or two-way ANOVA with Tukey’s multiple comparisons test (D). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. ANOVA, analysis of variance; DT, diphtheria toxin; IL, interleukin; NorUDCA, 24-Nor-ursodeoxycholic acid; Treg, regulatory T cell.

Figure 4. NorUDCA restrains pathogenic T_H17 cell differentiation and promotes functional suppressive Treg development in vitro. (A) Experimental design. (B) IL-17A production and proliferation of pathogenic (p)T_H17 cells. gMFI of IL-17A are shown. (C) Expression of RORɣt and (D) phosphorylated RPS6 (serine 235/236) in IL17A⁺Foxp3^-cells from pT_H17 culture. Rapamycin (mTORC1 inhibitor) was used for comparison (C,D). (E) Foxp3 expression and proliferation of pT_H17 cells. gMFI of Foxp3 are shown. (F) CD25 expression in IL-17A^-Foxp3⁺ cells from pT_H17 culture. (B–F) are data cumulative of two independent experiments (n=4 biologically independent samples per group). (G) Experimental design of the in vitro suppression assay. IL-17A^eGFP+Foxp3^hCD2/CD52-cells and Foxp3^hCD2/CD52+IL-17A^eGFP-cells were sorted from pT_H17 cultures, IL-17A^eGFP+Foxp3^hCD2/CD52-cells (responders) labelled with proliferation dye eF450 were mixed with Foxp3^hCD2/CD52+IL-17A^eGFP-cells (testers) or in vitro differentiated induced (i)Tregs at 1:1 ratio. (H) IL-17A^eGFP expression and dilution of proliferation dye gated on IL-17A^eGFP+Foxp3^hCD2/CD52-cells (number depicts expansion index) (n=3 biologically independent samples per group). Frequency (upper) and gMFI (lower) are shown in the representative flow cytometric plots of (B–F, H). Mean and SEM are indicated. P values were calculated using a one-way analysis of variance corrected for multiple comparisons with the Dunnett’s post hoc test. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. gMFI, geometric mean-fluorescence intensity; IL, interleukin; NorUDCA, 24-Nor-ursodeoxycholic acid; pT_H17, pathogenic T_H17; Rapa, rapamycin; RORγt, retinoic acid orphan receptor-gamma; RPS6, ribosomal protein S6; T_H17, T helper-type-like 17; Treg, regulatory T cell.

Figure 5. NorUDCA restricts glutamine metabolism that licenses mTORC1 activation and glycolysis in differentiating pT_H17 cells. (A, B) Expression of Hif1α (A) and c-Myc (B) on in vitro differentiated pathogenic (p)T_H17 cells (gated on IL-17A⁺Foxp3^-fraction). Numbers depict gMFI (A,B). (C) Expression of intracellular GLUT1 expression on differentiating pT_H17 cells. (D) Glucose uptake by differentiating pT_H17 cells. (E) Seahorse ECAR analysis in real time of activated CD4⁺T cells treated±NorUDCA. (F) Expression of ASCT2 on in vitro differentiated pT_H17 cells (gated on IL-17A⁺Foxp3^-cells). (G) Real-time PCR analysis of Gls2 expression (normalised to house-keeping Hprt) in pT_H17 cells. (H) Intracellular αKG level in pT_H17 cells. (I) Flow cytometric analysis of T_H17 and Treg within pT_H17 culture under indicated conditions. (J, K) Expression of phosphorylated RPS6 (serine 235/236) and GLUT1 on pT_H17 cells (gated on IL-17A⁺Foxp3^-fraction) under indicated conditions. (L) A model showing NorUDCA’s impact on glutaminolysis-mTORC1-glycolysis signalling in differentiating pT_H17 cells. Data summaries three independent experiments. Data shown in (A, B, C, G, H, I, J, K) n=5, (D) n=3, (E) n=10 biologically independent samples per group. Mean and SEM are shown. P values in (A, B, G, H) were calculated using the unpaired Student’s t-test (two-tailed), in (E) were calculated using a two-way ANOVA followed by Bonferroni’s multiple comparison post hoc tests and in (C, D, F, I, J, K) were calculated using a one-way ANOVA corrected for multiple comparisons with Dunnett’s post hoc test. *p≤ 0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; GLUT1, glucose transporter 1; gMFI, geometric mean fluorescence intensity; IL, interleukin; mRNA, messenger RNA: NorUDCA, 24-Nor-ursodeoxycholic acid; Oligo, oligomycin; Rapa, rapamycin; Rot, rotenone; RPS6, ribosomal protein S6; T_H17, T helper-type-like 17; Treg, regulatory T cell; 2-NBDG, 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-D-glucose; αKG, alpha-ketoglutarate.

Figure 6. pT_H17 cells briefly exposed to NorUDCA ex vivo show dampened pathogenic potential and expansion upon transfer in vivo. (A) Experimental design for adoptive cell transfer (ACT)-induced intestine inflammation model. (B) Intestines from Rag2^−/− mice reconstituted with pT_H17 cells briefly exposed±NorUDCA. (C, D) Frequency of T_H17 and Treg among small intestinal IELs ex vivo isolated from ACT-induced intestine inflammation mice. (E–G) Expression of CCR6, phosphorylated RPS6 (serine 235/236) or Ki67 on small intestinal intraepithelial T_H17 cells from ACT-induced intestine inflammation mice. Data presented in (C–G) are cumulative of two independent experiments (n=8 biologically independent samples per group). (H) Experimental design for ACT-induced experimental autoimmune encephalomyelitis model (ACT-EAE). (I) Clinical scoring. (J) Distribution of disease severity. On a scale of 1–5, No EAE=score <0.5; mild EAE=score range 0.5–2.5; severe EAE=score >3. (K) Frequency of T_H17 and Treg cells found in the CNS. (L, M) Expression of Ki67, CCR6 and CXCR3 on CNS-infiltrating T_H17 cells of ACT-EAE mice. Data presented in (I, J) is representative of two independent experiments and in (K–M) are cumulative of two independent experiments (n=8 biologically independent samples per group). Numbers depict frequencies and gMFI (E–G, L). Mean and SEM are shown. P values in (C–G, K–M) were calculated using the unpaired Student’s t-test (two-tailed) and in (I) were calculated using a two-way analysis of variance followed by Bonferroni’s multiple comparison tests. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. CNS, central nervous system; gMFI, geometric mean-fluorescence intensity; IEL, intraepithelial lymphocytes; IL, interleukin; NorUDCA, 24-Nor-ursodeoxycholic acid; pT_H17, pathogenic T_H17; SI, small intestine; T_H17, T helper-type-like 17; Treg, regulatory T cell.

Figure 7. NorUDCA represses T_H17 inflammation in humanised NSG model reconstituted with PBMCs from patients with PSC. NorUDCA represses T_H17 inflammation in humanised NSG model reconstituted with PBMCs from patients with PSC. (A) Experimental design. (B) Intestines of NSG mice reconstituted with patient with PSC-derived PBMCs±i.p. hαCD3 fed either standard chow or NorUDCA-supplementary diet. (C) Absolute cell counts of ex vivo isolated leucocyte fractions. (D) H&E staining of small intestine (scale bar 200 μm). (E) Flow cytometric analysis of splenic and hepatic T_H17 and Treg cells. (F) Expression of phosphorylated RPS6 (serine 235/236) or Ki67 on splenic and hepatic T_H17 cells. (G) Flow cytometric analysis of T_H17 and Treg cells within intraepithelial or lamina propria fractions of small intestine or colon. (H) Expression of phosphorylated RPS6 (serine 235/236) or Ki67 expression on T_H17 cells. (F, H) Depict frequency (upper) and gMFI (lower) of positive cell populations. Data are cumulative of two independent experiments, n=8 biologically independent samples per group. Every two samples from colonic fractions (IEL or LPL) were pooled to achieve sufficient cell numbers for flow cytometric analysis. Mean and SEM are shown. P values were calculated using a paired Student’s t-test (two-tailed). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. gMFI, geometric mean fluorescence intensity; IEL, intraepithelial lymphocytes; IL, interleukin; i.p., intraperitoneal; LPL, lamina propria lymphocytes; NorUDCA, 24-Nor-ursodeoxycholic acid; PBMC, peripheral blood mononuclear cell; PSC, primary sclerosing cholangitis; h, human; pT_H17, pathogenic T_H17; SI, small intestine; Sp, spleen; T_H17, T helper-type-like 17; Treg, regulatory T cell.