GPR120 Inhibits Colitis Through Regulation of CD4+ T Cell Interleukin 10 Production

doi:10.1053/j.gastro.2021.09.018

. 2022 Jan;162(1):150-165.

doi: 10.1053/j.gastro.2021.09.018. Epub 2021 Sep 16.

GPR120 Inhibits Colitis Through Regulation of CD4⁺ T Cell Interleukin 10 Production

Wenjing Yang¹, Han Liu², Leiqi Xu², Tianming Yu¹, Xiaojing Zhao³, Suxia Yao¹, Qihong Zhao⁴, Sean Barnes⁵, Steven M Cohn⁵, Sara M Dann⁶, Hongjie Zhang⁷, Xiuli Zuo⁸, Yanqing Li⁸, Yingzi Cong⁹

Affiliations

¹ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas.
² Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas; Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan, China.
³ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas; Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
⁴ Bristol Myers Squibb, Princeton, New Jersey.
⁵ Department of Gastroenterology and Hepatology, University of Texas Medical Branch at Galveston, Galveston, Texas.
⁶ Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas.
⁷ Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
⁸ Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan, China.
⁹ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas; Department of Pathology, The University of Texas Medical Branch, Galveston, Texas. Electronic address: [email protected].

PMID: 34536451
PMCID: PMC8678294
DOI: 10.1053/j.gastro.2021.09.018

GPR120 Inhibits Colitis Through Regulation of CD4⁺ T Cell Interleukin 10 Production

Wenjing Yang et al. Gastroenterology. 2022 Jan.

. 2022 Jan;162(1):150-165.

doi: 10.1053/j.gastro.2021.09.018. Epub 2021 Sep 16.

Authors

Affiliations

¹ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas.
² Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas; Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan, China.
³ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas; Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
⁴ Bristol Myers Squibb, Princeton, New Jersey.
⁵ Department of Gastroenterology and Hepatology, University of Texas Medical Branch at Galveston, Galveston, Texas.
⁶ Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas.
⁷ Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
⁸ Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan, China.
⁹ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas; Department of Pathology, The University of Texas Medical Branch, Galveston, Texas. Electronic address: [email protected].

PMID: 34536451
PMCID: PMC8678294
DOI: 10.1053/j.gastro.2021.09.018

Abstract

Background & aims: G protein-coupled receptor (GPR) 120 has been implicated in regulating metabolic syndromes with anti-inflammatory function. However, the role of GPR120 in intestinal inflammation is unknown. Here, we investigated whether and how GPR120 regulates CD4⁺ T cell function to inhibit colitis development.

Methods: Dextran sodium sulfate (DSS)-induced colitis model, Citrobacter rodentium infection model, and CD4⁺ T cell adoptive transfer model were used to analyze the role of GPR120 in regulating colitis development. The effect of GPR120 on CD4⁺ T cell functions was analyzed by RNA sequencing, flow cytometry, and Seahorse metabolic assays. Mice were administered GPR120 agonist for investigating the potential of GPR120 agonist in preventing and treating colitis.

Results: Deficiency of GPR120 in CD4⁺ T cells resulted in more severe colitis in mice upon dextran sodium sulfate insult and enteric infection. Transfer of GPR120-deficient CD4⁺CD45Rb^hi T cells induced more severe colitis in Rag^-/- mice with lower intestinal interleukin (IL) 10⁺CD4⁺ T cells. Treatment with the GPR120 agonist CpdA promoted CD4⁺ T cell production of IL10 by up-regulating Blimp1 and enhancing glycolysis, which was regulated by mTOR. GPR120 agonist-treated wild-type, but not IL10-deficient and Blimp1-deficient, T helper 1 cells induced less severe colitis. Furthermore, oral administration of GPR120 agonist protected mice from intestinal inflammation in both prevention and treatment schemes. Gpr120 expression was positively correlated with Il10 expression in the human colonic mucosa, including patients with inflammatory bowel diseases.

Conclusions: Our findings show the role of GPR120 in regulating intestinal CD4⁺ T cell production of IL10 to inhibit colitis development, which identifies GPR120 as a potential therapeutic target for treating inflammatory bowel diseases.

Keywords: Blimp1; Effector CD4(+) T cells; Glycolysis; Inflammatory Bowel Diseases; Intestinal Homeostasis.

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Conflict of interest statement

Disclosures: No authors have conflicting financial, professional, or personal interests.

Figures

**Fig. 1.. CD4⁺T cell-specific GPR120 KO mice develop more severe colitis upon DSS insult and *C. rodentium* infection.**
(A–B) GPR120 expression was measured in naïve CD4⁺T cells, activated CD4⁺T cells, Th1, Th17, Treg, bone-marrow dendritic cells (BMDCs), large bowl epithelial cells (LB IECs), and small bowel epithelial cells (SB IECs) (N=4/group). Western bolts (A) and GPR120 protein relative expression (B). (C–F) *Cd4*^creGpr120^fl/+ (WT) mice and *Cd4*^creGpr120^fl/fl (*Gpr120*^ΔCD4) mice (N=14/group) were administrated with 1.65% DSS (w/v) in drinking water for 7 days followed by drinking water alone for additional 3 days. (C) Mouse weight change. (D) Representative intestinal H&E staining. (E) Pathological score. (F) Colonic secretion of cytokines. (G–M) *Cd4*^creGpr120^fl/+ (WT) mice and *Cd4*^creGpr120^fl/fl (*Gpr120*^ΔCD4) mice (N=12/group) were orally infected with *C. rodentium* on day 0, and sacrificed on day 14. (G) Weight change. (H) Fecal *C. rodentium* counts. (I) Representative gross morphology of the colon. (J) Colon length. (K) Representative intestinal H&E staining. (L) Pathological score. (M) Colonic secretion of cytokines. All data are pooled from three independent experiments. (D and K) Scale bar, 300 μm. (A) one-way ANOVA; (C, F, G, H, J, and M) unpaired Student t-test; (E and L) Mann–Whitney U test.

**Fig. 2.. GPR120-deficient CD4⁺CD45RB^hiT cells induce more severe colitis in *Rag*^−/− mice.**
WT and GPR120-deficient CD4⁺CD45Rb^hiT cells (1×10⁵ cells/mouse) were intravenously transferred to *Rag*^−/− mice (N=12/group). (A) Mouse weight changes. (B) Representative gross morphology of the colon. (C) Colon length. (D) Representative intestinal H&E staining. (E) Pathological score. (F) Colonic secretion of cytokines. (G) Representative flow cytometry profile of LP CD4⁺T cells. (H–K) Bar charts of IFNγ⁺, IL-17A⁺, IL-10⁺, and Foxp3⁺CD4⁺T cells. All data are pooled from three independent experiments. (D) Scale bar, 300 μm. (A, C, F, and H–K) unpaired Student t-test; (E) Mann–Whitney U test.

**Fig. 3.. GPR120 promotes CD4⁺T cell production of IL-10 to suppress colitis.**
(A–C) Splenic CD4⁺T cells were activated with anti-CD3/anti-CD28±CpdA for 48 hours to analyze gene expression by RNA-Seq (N=3/group). (A) Differentially expressed genes between CD4⁺T cells treated with or without CpdA in Heatmap. Arbitrary units. (B) KEGG pathway enrichment analysis. (C) Specific gene expressions in Heatmap. Arbitrary units. (D–F) CD4⁺T cells were activated with anti-CD3/anti-CD28±CpdA under Th1 conditions (N=9/group). Flow cytometry profile of IL-10⁺CD4⁺T cells after 5 days (D–E). IL-10 in culture supernatants after 2 days (F). (G–J) WT and IL-10^−/− CD4⁺T cells were activated with anti-CD3/anti-CD28±CpdA under Th1 conditions for 5 days and transferred to *Rag*^−/− mice (N=12/group). (G) Mouse weight change. (H) Representative intestinal H&E staining. (I) Pathological score. (J) Colonic secretion of cytokines. (D–J) Data were pooled from three independent experiments. (H) Scale bar, 300 μm. (E, F, G, and J) unpaired Student’s t-test; (I) Mann–Whitney U test.

**Fig. 4.. Blimp1 mediates GPR120 induction of IL-10 through the mTOR-Stat3 pathway.**
Splenic CD4⁺T cells were activated with anti-CD3/anti-CD28±CpdA and various inhibitors under Th1 conditions. (A) *Prdm1* mRNA expression on day 2 (N=13/group). (B–C) Flow cytometry profile of IL-10⁺CD4⁺T cells in WT and Blimp^−/− CD4⁺T cells on day 5. (D) IL-10 in culture supernatants on day 2 (N=10/group). (E–F) Phosphorylated and total mTOR expressions at 30 minutes (N=5/group). (G) *Prdm1* and (H) *Il10* mRNA expression in CD4⁺T cells treated with or without CpdA±rapamycin on day 2 (N=9/group). (I–J) Phosphorylated and total Stat3 expressions at 2 hours in CD4⁺T cells treated with or without CpdA±rapamycin (N=5/group). (K) *Prdm1* and (L) *Il10* mRNA expression in CD4⁺T cells treated with or without CpdA±Static (N=9/group). (M–N) Flow cytometry profile of IL-10⁺CD4⁺T cells on day 5, and (O) IL-10 in culture supernatants on day 2 (N=9/group). (P–S) WT and Blimp1^−/− CD4⁺T cells from *Cd4*^crePrdm1^fl/+ mice and *Cd4*^crePrdm1^fl/fl mice were activated with anti-CD3/anti-CD28±CpdA under Th1 conditions for 5 days and transferred to *Rag*^−/− mice (N=15/group). (P) Mouse weight change. (Q) Representative intestinal H&E staining. (R) Pathological score. (S) Colonic secretion of cytokines. All data are pooled from three independent experiments. (A, C, D, F, J, P, and S) unpaired Student’s t-test; (G, H, N, O) one-way ANOVA; (R) Mann–Whitney U test.

**Fig. 5.. GPR120 agonist promotes IL-10 production through the glycolysis-HIF1α pathway.**
(A–B) Splenic CD4⁺T cells were activated with anti-CD3/anti-CD28±CpdA under Th1 conditions for 48 hours, and the Mito stress test kit was used to measure the parameters of mitochondrial respiration (N=7/group). The OCR profile, basal respiration, ATP-related respiration, maximum respiration, and spare respiration capacity. (C–D, and H–I) Splenic CD4⁺T cells were activated with anti-CD3/anti-CD28 under Th1 conditions for 48 hours, and a Glycolytic stress test kit was used to measure the key parameters of glycolysis (N=8/group). The ECAR profile, glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic ECAR. (E–G) CD4⁺T cells were treated with or without CpdA±2-DG (N=9/group). IL-10⁺CD4⁺T cells on day 5 (E–F), and IL-10 in culture supernatants on day 2 (G). (J) *Hif1a* mRNA expression in CD4⁺T cells treated with or without CpdA±2-DG at 48 hours (N=9/group). (K–M) CD4⁺T cells were treated with or without CpdA±YC-1 (N=9/group). Flow cytometry profile of IL-10⁺CD4⁺T cells at day 5 (K–L) and IL-10 production in culture supernatants on day 2 (M). (E–G, and J-M) Data are pooled from three independent experiments. (B and D) Unpaired Student’s t-test (F, G, I, L, and M) one-way ANOVA.

**Fig. 6.. Oral feeding of GPR120 agonist prevents and treats colitis.**
(A–F) WT CD4⁺CD45Rb^hiT cells (1×10⁵ cells/mouse) were intravenously transferred to *Rag*^−/− mice and orally administered with or without CpdA (N=12/group) daily from the day of cell transfer until the mice were sacrificed 6 weeks after cell transfer. (A) Mouse weight change. (B) Gross morphology of colon. (C) Colon length. (D) Representative intestinal H&E staining. (E) Pathological score. (F) Colonic secretion of cytokines. (G–L) WT CD4⁺CD45Rb^hiT cells (1×10⁵ cells/mouse) were intravenously transferred to *Rag*^−/− mice and orally administered with or without CpdA (N=12/group) daily from 2 weeks post cell transfer for additional 4 weeks. (G) Mouse weight change. (H) Gross morphology of colon. (I) Colon length. (J) Representative intestinal H&E staining. (K) Pathological score. (L) Colonic secretion of cytokines. All data are pooled from two independent experiments. (D and J) Scale bar, 300 μm. (A, C, F, G, I, and L) unpaired Student t-test; (E and K) Mann–Whitney U test.

**Figure 7.. *Gpr120* is positively correlated with *Il10* in human colonic mucosa.**
(A–C) The data from GSE11223 in the GEO database were retrieved. (A) Gpr120 expression in the colonic mucosa of healthy controls (HC, N=27) and patients with ulcerative colitis (UC, N=31). (B–C) The correlation between colonic *Gpr120* and *Il10* in HC (B) and UC patients (C). (D–F) Intestinal biopsies were collected from HC (N=14), UC patients (N=10), and patients with Crohn’s disease (CD, N=21). The correlation between intestinal *Gpr120* and *Il10* in HC (D), UC patients (E), and CD patients (F). (A) unpaired Student t-test; (B–F) Spearman correlation.

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References

1. Globig AM, Hennecke N, Martin B, et al. Comprehensive intestinal T helper cell profiling reveals specific accumulation of IFN-gamma+IL-17+coproducing CD4+ T cells in active inflammatory bowel disease. Inflamm Bowel Dis 2014;20(12): 2321–9. - PubMed
1. Franke A, Balschun T, Karlsen TH, et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet 2008;40(11): 1319–23. - PubMed
1. Franke A, McGovern DP, Barrett JC, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci. Nat Genet 2010;42(12): 1118–25. - PMC - PubMed
1. Begue B, Verdier J, Rieux-Laucat F, et al. Defective IL10 signaling defining a subgroup of patients with inflammatory bowel disease. Am J Gastroenterol 2011;106(8): 1544–55. - PubMed
1. Glocker EO, Kotlarz D, Boztug K, et al. Inflammatory bowel disease and mutations affecting the interleukin-10 receptor. N Engl J Med 2009;361(21): 2033–45. - PMC - PubMed

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[1] Globig AM, Hennecke N, Martin B, et al. Comprehensive intestinal T helper cell profiling reveals specific accumulation of IFN-gamma+IL-17+coproducing CD4+ T cells in active inflammatory bowel disease. Inflamm Bowel Dis 2014;20(12): 2321–9. - PubMed

[2] Globig AM, Hennecke N, Martin B, et al. Comprehensive intestinal T helper cell profiling reveals specific accumulation of IFN-gamma+IL-17+coproducing CD4+ T cells in active inflammatory bowel disease. Inflamm Bowel Dis 2014;20(12): 2321–9. - PubMed

[3] Franke A, Balschun T, Karlsen TH, et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet 2008;40(11): 1319–23. - PubMed

[4] Franke A, Balschun T, Karlsen TH, et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility. Nat Genet 2008;40(11): 1319–23. - PubMed

[5] Franke A, McGovern DP, Barrett JC, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci. Nat Genet 2010;42(12): 1118–25. - PMC - PubMed

[6] Franke A, McGovern DP, Barrett JC, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci. Nat Genet 2010;42(12): 1118–25. - PMC - PubMed

[7] Begue B, Verdier J, Rieux-Laucat F, et al. Defective IL10 signaling defining a subgroup of patients with inflammatory bowel disease. Am J Gastroenterol 2011;106(8): 1544–55. - PubMed

[8] Begue B, Verdier J, Rieux-Laucat F, et al. Defective IL10 signaling defining a subgroup of patients with inflammatory bowel disease. Am J Gastroenterol 2011;106(8): 1544–55. - PubMed

[9] Glocker EO, Kotlarz D, Boztug K, et al. Inflammatory bowel disease and mutations affecting the interleukin-10 receptor. N Engl J Med 2009;361(21): 2033–45. - PMC - PubMed

[10] Glocker EO, Kotlarz D, Boztug K, et al. Inflammatory bowel disease and mutations affecting the interleukin-10 receptor. N Engl J Med 2009;361(21): 2033–45. - PMC - PubMed

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GPR120 Inhibits Colitis Through Regulation of CD4⁺ T Cell Interleukin 10 Production

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GPR120 Inhibits Colitis Through Regulation of CD4⁺ T Cell Interleukin 10 Production

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