Homeostatic PPARα Signaling Limits Inflammatory Responses to Commensal Microbiota in the Intestine

Abstract

Dietary lipids and their metabolites activate members of the peroxisome proliferative-activated receptor (PPAR) family of transcription factors and are critical for colonic health. The PPARα isoform plays a vital role in regulating inflammation in various disease settings, but its role in intestinal inflammation, commensal homeostasis, and mucosal immunity in the gut are unclear. In this study, we demonstrate that the PPARα pathway in innate immune cells orchestrates gut mucosal immunity and commensal homeostasis by regulating the expression of IL-22 and the antimicrobial peptides RegIIIβ, RegIIIγ, and calprotectin. Additionally, the PPARα pathway is critical for imparting regulatory phenotype in intestinal macrophages. PPARα deficiency in mice led to commensal dysbiosis in the gut, resulting in a microbiota-dependent increase in the expression of inflammatory cytokines and enhanced susceptibility to intestinal inflammation. Pharmacological activation of this pathway decreased the expression of inflammatory cytokines and ameliorated colonic inflammation. Taken together, these findings identify a new important innate immune function for the PPARα signaling pathway in regulating intestinal inflammation, mucosal immunity, and commensal homeostasis. Thus, the manipulation of the PPARα pathway could provide novel opportunities for enhancing mucosal immunity and treating intestinal inflammation.

Figure 1. The absence of PPARα signaling exacerbates susceptibility to T cell-mediated colitis

(A) Relative fold induction of luciferase reporter gene activity (representing PPAR activity) in the SI and colon as compared to spleen of PPRE-luciferase reporter mice treated with or without PPARα inhibitor (GW6471) (n= 3). (B) Percent weight change compared to initial weight for Rag1KO and Rag1/αKO mice at indicated time points post CD4⁺CD25⁻CD45RB^hi T cell adoptive transfer (n=8). (C) Representative colon histology of Rag1KO and Rag1/αKO mice on 6 weeks post naïve CD4⁺ T cell transfer (H&E staining with original magnification, 100X). Histology scores are shown in (D) and data are representative of two independent experiments. (E, F) Representative FACS plots or frequencies of colonic IL-17A⁺, IFN-γ⁺, IL-17A⁺IFN-γ⁺ and IL-22⁺ IL-17A⁺ CD4⁺ T cells from Rag1KO and Rag1/αKO mice on 6 weeks post-transfer of naïve CD4⁺ T cell (n=8). (G) Excised colon samples in panel D were cultured for 2 days ex vivo, and the secreted IL-17A, IFN-γ and IL-22 cytokine levels in the culture supernatants were quantified by ELISA. The error bars indicate mean ± SEM of 8 mice/group. *p<0.05; **p<0.01; ***p<0.001.

Figure 2. Increased susceptibility of Rag1/αKO mice to DSS-induced colonic inflammation

Rag1KO and Rag1/αKO mice were treated with 3% DSS in drinking water for 6 days and at day 9 colons of mice were analyzed for inflammation. (A–D) Change in body weight, diarrhea, rectal bleeding and colon length (day 9) of Rag1KO and Rag1/αKO mice (n≥6). (E) Representative images of H&E-stained colonic sections from DSS treated Rag1KO and Rag1/αKO mice (day 9, original magnification, 100X). (F) Excised colon samples in panel D were cultured for 2 days ex vivo, and the secreted IL-6, TNF-α, IL-1β, IL-12p70, IL-10 and IL-22 cytokine levels in the culture supernatants were quantified by ELISA. The error bars indicate mean ±SEM of 5–6 mice/group. *p<0.05; **p<0.01; ***p<0.001.

Figure 3. PPARα signaling limits inflammatory responses to commensal microbiota

(A, B) FACS plot representing percentages or cumulative frequencies of CD4⁺ T cells positive for IL-17A and IFN-γ cells isolated from colon of WT (littermate control) and αKO mice treated with (Abx, bottom panels) or without (None, top panels) antibiotics treatment (n=8). (C, D) Excised colon samples in panel A were cultured for 2 days ex vivo, and then the secreted IL-17A, IFN-γ, IL-1β, TNF-α, IL-6, IL-10 and IL-22 amounts in the culture supernatants were quantified by ELISA (n=5). (E–H) Rag1/αKO mice adoptively transferred with FACS sorted CD4⁺ CD25⁻CD45RB^hi T cells from WT mice and were left untreated or treated with antibiotics for 6 weeks. (E) Percent weight change as compared to initial weight for antibiotic treated and untreated Rag1/αKO mice at various weeks post naïve CD4⁺ T cell adoptive transfer (n=8). (F,G) Representative FACS plots or frequencies of colonic IL-17A⁺, IFN-γ⁺ and IL-17A⁺IFN-γ⁺ CD4⁺ T cells from Rag1/αKO mice on 6 weeks post-transfer of naïve CD4+ T cell from panel I (n=8). (H) Excised colon samples in panel F were cultured for 2 days ex vivo, and the secreted IL-17A and IFN-γ cytokine levels in the culture supernatants were quantified by ELISA. The bar indicates mean ±SEM of 8 mice/group *p<0.05; **p<0.01; ***p<0.001.

Figure 4. PPARα signaling in colonic macrophages limits the expression of inflammatory cytokines and suppresses Th1/Th17 cells differentiation

(A) FACS plot showing intracellular expression levels of PPARα protein in DCs and macrophages isolated from the colon of the WT and αKO mice. Data are from one experiment representative of three. (B) Intracellular expression of IL-17 and IFN-γ in naïve CD4⁺OT-II T cells stimulated to differentiate in vitro by colonic macrophages isolated from WT and αKO mice, in the presence of TGF-β (1 ng/ml). Numbers in FACS plots represent percentage of cells positive for the indicated protein. Data are from one experiment representative of three (C) Cumulative frequencies of CD4⁺OT-II T cells positive for IL-17, and IFN-γ as described in panel B (n=3). (D) Sorted colonic macrophages from WT and αKO mice were cultured for 2 days ex vivo, and IL-1β, IL-12p40, IL-6 and IL-10 cytokine amounts in the culture supernatants were quantified by ELISA (n=4). The bar indicates mean ±SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 5. PPARα regulates the expression of IL-22 and anti-microbial peptides in the intestine

(A) Relative quantification of different bacterial species in the fecal material from αKO mice compared with WT mice as analyzed by quantitative RT-PCR analysis (n= 5). (B, C) Quantitative real-time PCR analysis of regIIIb, regIIIg, S100A8, S100A9 and il22 mRNA expression relative to GAPDH in colon of αKO mice and WT. (D, E) FACS plot representing percentages or cumulative frequencies of IL-22 producing cells gated on CD45⁺ CD3⁻ CD19⁻ cells isolated from colons of WT (littermate control) and αKO mice. (F) Quantitative real-time PCR analysis of il22 mRNA expression relative to GAPDH in colon of Rag1 (littermate control) and Rag1/ αKO mice. (G) Representative FACS plot and (H) frequencies for IL-22 producing cells gated on CD45⁺ CD3⁻ CD19⁻ cells isolated from colons of Rag1 and Rag1/αKO mice. Error bars show mean values ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 6. PPARα regulates IL-22 produced by NKp46⁺ innate lymphoid cells

(A) Quantitative real-time PCR analysis of il22 mRNA expression relative to GAPDH in NKp46⁺ cells (CD3⁻ CD19⁻ NK1.1⁻ NKp46⁺) isolated from colon of αKO and WT mice. (B) Frequencies for IL-22 producing cells gated on CD45⁺ CD3⁻ CD19⁻ NK1.1⁻ NKp46⁺cells isolated from colons of αKO and WT mice (n=5). (C, D) Sorted colonic NKp46⁺ innate lymphoid cells and macrophages from WT and αKO mice were co-cultured for 2 days ex vivo, and IL-22 cytokine amounts in the culture supernatants were quantified by ELISA (n=4). (E) FACS plot showing intracellular expression levels of PPARα protein in NKp46⁺ innate cells isolated from the colon of WT (solid) and αKO (dashed) mice. Error bars show mean values ± SEM. *p<0.05; **p<0.01.

Figure 7. Pharmacological activation of PPARα ameliorates mucosal inflammation

(A–F) CD45RB^hiCD4⁺ T cells from WT mice were adoptively transferred into Rag1 mice. Animals were treated with PPARα agonist orally (GW7647; 10mg/kg; on Weeks 2,3,4) and monitored over a period of time for percent weight loss compared to initial weight. (A) Percent weight change for Rag1 mice treated with PPARα agonist compared with untreated mice at various weeks post naïve CD4⁺ T cell adoptive transfer (n=6). (B, C) Representative colon histology (H&E staining with original 10X magnification) and histology scores of Rag1 mice on 6 weeks post naïve CD4⁺ T cell transfer treated with or without PPARα agonist. (D, E) Representative FACS plots and frequencies of colonic IL-17A⁺, IFN-γ⁺ and IL-10⁺ cells from Rag1 treated with PPARα agonist compared with untreated mice on week 6 post naïve CD4⁺ T cell adoptive transfer (n=6). (G) Excised colon samples in panel C were cultured for 2 days ex vivo, and then the secreted IL-17A, IFN-γ, IL-10 and IL-22 cytokine amounts in the culture supernatants were quantified by ELISA. The bar indicates mean ±SEM of 6 mice/group. *p<0.05; **p<0.01; ***p<0.001.