Fig 2: GPR120 activation induced regulatory DCs by inhibiting glycolysis.a Wild-type (WT) and GPR120-/- (GPR120 KO) mice were injected with P. acnes (P.ac). Vehicle or TUG891 (10 mg/kg) was administered i.p. to WT mice on days 0, 2, 4, and 6 after P. acnes injection (n = 7 mice per group). Glucose uptake of CD11c+ DCs in mouse livers was measured by 2-NBDG assay. Mean fluorescence intensity (MFI) of 2-NBDG was shown. b–f BMCs from WT and GPR120-/- mice were isolated and cultured to induce maturation. TUG891 (20 µM) with or without AH7614 (20 µM) pretreatment was added to the medium of BMCs from WT every 2 days. LPS (0.1 µg/mL) was added on day 7. CD11c+ cells were separated using magnetic cell sorting microbeads. b 2-NBDG was added, and glucose uptake was determined by flow cytometry. MFI of 2-NBDG was shown. c Quantitative real-time PCR was used to determine the mRNA expression of Slc2a1-5 in DCs on day 8. d, f Protein expression of G6PD, HK2, LDHA, and PDH in DCs was determined by immunoblot analysis during DC maturation or with GPR120 interference on day 8. g–k The glycolysis inhibitor bromopyruvic acid (BA, 5 µM) was added to the DC induction medium. g The numbers of CD11c+ DCs among the cultured cells were analyzed by flow cytometry on day 8. h, i The expression of CD80, CD86, and MHC II of CD11c+ DCs was analyzed by flow cytometry on day 8. j Levels of TGF-ß and IL-10 were measured by enzyme-linked immunosorbent assay. k CD4+ T cells were cocultured with DCs that underwent the indicated treatments for 16 h. CD25 and CD69 expression in CD4+ T cells was analyzed by flow cytometry. The results are representative of three to six independent experiments and presented as the mean ± SEM. Significant differences were analyzed by One-way ANOVA. **p < 0.01, ***p < 0.001, ns no significance.

Fig 3: 16:4(n-3)-induced chemotherapy resistance is mediated via GPR120 in vivo. Treatment of C26 tumor-bearing BALB/c mice with cisplatin (cis) and sCM derived from splenocytes that were stimulated with GPR120-specific agonist TUG-1197 induces chemoresistance, which indicates a central role for GPR120 in 16:4(n-3)-mediated resistance. A) GPR120-specific antagonist AH-7614 was able to completely block 16:4(n-3)-mediated resistance, whereas GPR40-specific antagonist GW1100 was ineffective. Tumors of mice that were treated with cisplatin and sCM + 16:4(n-3) showed a decrease in ?H2AX levels 4 h after treatment compared with animals treated with cisplatin alone. B) Mice that were treated with cisplatin and sCM + 16:4(n-3) + AH-7614 had ?H2AX levels similar to mcie treated with cisplatin alone. Scale bars, 50 µm. All experiments in this figure were conducted in C26 tumor-bearing BALB/c mice. All graphs show grouped results of 2 independent experiments with similar outcomes (n = 8 per group). Data are presented as mean ± sem. Statistical significance was determined by 1-way ANOVA (A) or 2-tailed Student’s t test (B); all compared with cisplatin alone unless indicated otherwise. *P < 0.05; **P < 0.01.

Fig 4: Changes in tight junction-related genes expression in the colon of DSS- and DSS+FFAR4-treated mice. Figure shows data for the expression of occludin (A), claudin 1 (B), claudin 2 (C), claudin 3 (D) claudin 4 (E), claudin 7 (F), claudin 10 (G), and claudin 12 (H). Values expressed as percent of control group. Data represent mean ± SEM, n = 12. Data were analyzed with one-way ANOVA, followed by Newman–Keuls post-hoc test. * p < 0.05, ** p < 0.01 as compared to control; # p < 0.05, ## p < 0.01 as compared to DSS.

Fig 5: Representative micrographs of immunohistochemical staining of FFARs in formalin-fixed, paraffin-embedded sections of healthy and DSS-treated mouse colon. Figure shows data for FFAR1 (A,B), FFAR2 (C,D), FFAR3 (E,F), and FFAR4 (G,H). Arrowheads indicate stained cells. Scale bar = 100 µm. The amount of FFARs (I) was presented as an epithelium surface area coverage (%). The pictures were taken in bright field setup with standard color charge-coupled device (CCD) camera (Zeiss AxioCam ICc1; Carl Zeiss, Poznan, Poland). Data are presented as means (bars) with SEM. Significance estimated with the use of unpaired t-test with Welch’s correction n = 2–8; * p < 0.05, as compared to control group.