Interleukin-23 receptor signaling impairs the stability and function of colonic regulatory T cells

Abstract

The cytokine interleukin-23 (IL-23) is involved in the pathogenesis of inflammatory and autoimmune conditions including inflammatory bowel disease (IBD). IL23R is enriched in intestinal Tregs, yet whether IL-23 modulates intestinal Tregs remains unknown. Here, investigating IL-23R signaling in Tregs specifically, we show that colonic Tregs highly express Il23r compared with Tregs from other compartments and their frequency is reduced upon IL-23 administration and impairs Treg suppressive function. Similarly, colonic Treg frequency is increased in mice lacking Il23r specifically in Tregs and exhibits a competitive advantage over IL-23R-sufficient Tregs during inflammation. Finally, IL-23 antagonizes liver X receptor pathway, cellular cholesterol transporter Abca1, and increases Treg apoptosis. Our results show that IL-23R signaling regulates intestinal Tregs by increasing cell turnover, antagonizing suppression, and decreasing cholesterol efflux. These results suggest that IL-23 negatively regulates Tregs in the intestine with potential implications for promoting chronic inflammation in patients with IBD.

Keywords: CP: Immunology; IBD; IL-23R12; Treg cell; Tregs; colitis; colon; interleukin-23; ustekinumab.

Figure 1.. IL-23 negatively regulates colonic Treg cells

(A) qRT-PCR on Treg cells sorted from different compartments based on YFP for Il23r expression normalized to Tbp. Representative data of two independent experiments with n = 4. (B) Schematic depicting Foxp3^YFP–Cre mice injected with recombinant IL-23 or PBS i.p. every other day for a total of three injections. (C) Treg cell gating and frequency quantified by flow cytometry in Foxp3^YFP–Cre mice injected with IL-23 or PBS. Data are pooled from four independent experiments. (D) Il23r^flox/floxFoxp3^YFP–Cre (Il23r^ΔTreg) mice were generated. (E) Genotyping on DNA extracted from FACS-sorted FOXP3⁺ cells and other CD4⁺ T cell fractions to confirm Il23r deletion specific for FOXP3⁺ cells of Il23r^ΔTreg mice. IC, internal control. Irrelevant lanes and white space were cropped. (F) Representative FACS analysis examining Treg cell frequencies in the spleen, mLN, and colon lamina propria (cLP) of Foxp3^YFP–Cre and Il23r^ΔTreg mice (left) and quantified (right) where each data point is an individual mouse. Data are pooled from more than three independent experiments. (G) qRT-PCR of whole colon tissue RNA for genes associated with intestinal Treg induction or sustenance, normalized to Tbp. Histogram bars represent the mean ± SEM. (A) ANOVA with post hoc Tukey. (C, F, G) Unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.. IL-23R signaling impairs colonic Treg cell function in vitro and in vivo

(A) Naive T cells (Tn) from spleen and mLN of a Il23r^flox/flox;Cd2^Cre mice were enriched by negative selection and labeled with CellTrace Violet. Tn were activated using αCD3αCD28 beads and co-cultured with sorted colonic Treg cells from Foxp3^YFP–Cre mice in a 2:1 ratio Tn:Treg cells for 3 days in the presence or absence of IL-23. (B) Representative histogram of Tn proliferation via CellTrace Violet dilution and quantification of suppression by colonic Treg cells. Data are pooled from three independent experiments. Each data point (dot) reflects cells from four or more mice and the average of two to three technical replicates. (C) The same experiment as (B) but using Treg cells sorted from the spleen of Foxp3^YFP–Cre mice. (D) Rag1^−/− mice were injected with 500,000 wild-type naive T cells with or without 25,000 Treg cells sorted from Foxp3^YFP–Cre or Il23r^ΔTreg mice and euthanized between 6 and 8 weeks. (E) Representative H&E of colon (left) and quantification of histological injury score (right). Histogram bars represent the mean ± SEM. Scale bars, 100 μm. (B and C) Paired t test. (E) Mixed-effects model with false discovery method of Benjamini, Krieger, and Yekutieli. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.. Selective deletion of Il23r in Treg cells provides a competitive advantage during Citrobacter rodentium-induced colitis

(A) Rag1^−/− mice were sublethally irradiated (450 rads) and transplanted using a 1:1 mixture of lineage-depleted bone marrow cells from Foxp3^YFP–CreCD45.1⁺ and Il23r^flox/floxFoxp3^YFP–CreCD45.2⁺ mice. (B) Recipient mice exhibiting a 1:1 ratio of CD4⁻ T cells were analyzed 6 to 8 weeks post-transplantation with the YFP⁺ fraction gated and the CD45.2⁺/CD45.1⁺ ratio quantified. Data are pooled from three independent experiments. (C) Rag1^−/− were transplanted with bone marrow as in (A), 6 weeks later mice followed by gavage with C. rodentium (3 × 10⁸ CFU). (D) Representative IVIS images with flux quantified for each mouse. (E) CFU at experimental endpoint (14 days after inoculation). (F) Flow cytometry plots of IL-23R-sufficient and IL-23R-deficient T cells (CD4⁺FOXP3⁻) and Treg cells (CD4⁺FOXP3⁺) at endpoint by flow cytometry with corresponding CD45.2/CD45.1 ratio. Histogram bars represent the mean ± SEM. (B, F) ANOVA with post hoc Tukey. ***p < 0.001.

Figure 4.. IL-23R-deficient colonic Treg cells exhibit altered cholesterol homeostasis

(A) RNA-sequencing was performed on FACS-sorted Treg cells (CD45⁺CD4⁺FOXP3⁺ cells) from the cLP of Foxp3^YFP–Cre and Il23r^ΔTreg mice with dimensionality reduction via principal-component analysis (PCA) for samples grouped based on genotype. Each dot in the PCA is representative of at least five mice. (B) Volcano plot depicting differentially expressed genes in colonic Treg cells based on genotype. (C) Histogram of filipin-III staining on colon YFP⁺ or YFP⁻ cells from Foxp3^YFP–Cre and Il23r^ΔTreg mice (left) with MFI quantification (right). (D) Secondary bile acids were quantified for stool and serum. (E) Quantification of colonic Treg cells following treatment with vehicle control (placebo) or 30 mg kg⁻¹ SR9243 for 5 days. Data are pooled from three independent experiments. (F and G) mLN/splenic Treg cells were enriched using CD25⁺ positive selection using magnetic beads and stimulated with αCD3αCD28 in presence of IL-2 and T0901317. After 3 days, YFP⁺ cells were flow sorted followed by qRT-PCR for Il23r and Abca1 and quantified (G). Histogram bars represent the mean ± SEM. (F and G) Data are pooled data from five independent experiments. (C, E) Unpaired t test. (F, G) Paired t test. *p < 0.05, **p < 0.01.

Figure 5.. IL-23 reduces colonic Treg cell frequency via apoptosis

(A) Illustration of Foxp3^{EGFP–Cre-ERT2}Rosa26^lsl-tdTomato mice depicting excision of the floxed stop sequence and recombination post tamoxifen. This irreversibly labels Foxp3 expressing cells with tdTomato whereas GFP depends on continued expression of Foxp3. (B) Pharmacokinetics of tamoxifen-induced recombination at the Rosa26 locus assessed by examining tdTomato⁺ cells among GFP⁺ cells using flow cytometry. Representative results of two independent experiments with n = 2. Vertical dotted lines indicate tamoxifen administration. (C) Mice were gavaged with tamoxifen and concomitantly injected with recombinant IL-23 (as for Figure 1B). Representative gating and quantification of “ex-Treg” cells; cells that are tdTomato⁺ but GFP negative. Data are pooled from four independent experiments. (D) Representative flow cytometry gating and quantification of Annexin-V staining. Data pooled from four independent experiments. (E) Flow cytometry of Foxp3^YFP–Cre and Il23r^ΔTreg mice at baseline for Ki67 and (F) Annexin-V. For Ki67 staining cells were fixed and permeabilized and concomitantly stained for GFP and Ki67. Histogram bars represent the mean ± SEM. (C, F) Unpaired t test. (D, E) ANOVA with post hoc Tukey. *p < 0.05, **p < 0.01.