GPR120 promotes neutrophil control of intestinal bacterial infection

Figures & data

Figure 1. Gpr120^−/− mice demonstrate altered gut microbiota composition.

Notes: (a) 16S counts were measured in fecal pellets collected from WT and Gpr120^−/− mice using qRT-PCR. (b-d) Fecal pellets were collected from WT and Gpr120^−/− mice, and 16s rRNA sequencing was performed. PCA analyses were determined using Bray-Curtis comparison (b). The differences in microbiota abundance by phylum were determined between WT and Gpr120^−/− mice (c). The differences in the Bacteroidales S24-7 group and Clostridiales vadinBB60 group were determined between WT and Gpr120^−/− mice (d). Data were expressed as mean ± SEM. Statistical significance was tested by a two-tailed unpaired Student t-test (a, c, and d) or Bray-Curtis comparison (b). *p < 0.05, **p < 0.01.

Figure 2. Gpr120^−/− mice are impaired in the clearance of intestinal Citrobacter rodentium and are susceptible to intestinal inflammation.

Notes: (a-e) WT and Gpr120^−/− mice (n = 5) were infected with Citrobacter rodentium(5 × 10⁸ colony‐forming units (CFU)/mouse) by oral gavage. Mice were sacrificed on day 10 post-infection. (a) Representative histopathology images of the colons were shown. (b) Histological scores were assessed. (c) TNF-α secretion in colons was determined. (d-e) Fecal pallets and spleens were collected on day 10, and CFU in feces (d) and spleens (e) were quantified. (f-h) WT and Gpr120^−/− mice (n = 5) were treated with 2% DSS in drinking water for 7 days and normal drinking water for another 3 days. Mice were sacrificed on day 10. (f) Representative histopathology images of the colons were shown. (g) Histological scores were assessed. (h) TNF-α secretion in colons was determined. One representative of three independent experiments was shown. Data were expressed as mean ± SEM. Statistical significance was tested by the non-parametric two-tailed Mann-Whitney U test (b and g) or the two-tailed unpaired Student's t-test (c-e, and h). *p < 0.05, **p < 0.01.

Figure 3. Depletion of neutrophils decreases the intestinal clearance of Citrobacter rodentium.

Notes: WT mice (n = 5/group) were infected with Citrobacter rodentium(5 × 10⁸ colony‐forming units (CFU)/mouse) by oral gavage. One group of mice was administered with anti-Ly6G antibody, and another was given anti-IgG antibody as a control daily. Mice were sacrificed on day 10 post-infection. (a) Representative histopathology images of the colons were shown. (b) Histological scores were assessed. (c) TNF-α secretion in colons was determined. (d-e) Fecal pallets and spleens were collected on day 10, and CFU in feces (d) and spleens (e) were quantified. One representative of three independent experiments was shown. Data were expressed as mean ± SEM. Statistical significance was tested by the non-parametric two-tailed Mann-Whitney U test (b) or the two-tailed unpaired Student t-test (c-e). *p < 0.05, **p < 0.01.

Figure 4. GPR120 agonist promotes neutrophil inhibition of enteric bacterial growth through the upregulation of ROS and NETs.

Notes: (a-b) WT or GPR120-deficient neutrophils (n = 4/group) were treated with or without CpdA (3 µM) for 1 h. ROS production was measured using the Amplex Red Hydrogen Peroxide Assay Kit (a). WT or GPR120-deficient neutrophils were then stained with Hoechst 33342 (blue), and representative NETs were shown (b). (c) WT or GPR120-deficient neutrophils (n = 5/group) were pre-treated with or without CpdA (3 µM) for 1 h, and then co-cultured with Citrobacter rodentium in the plates for 12 h. The bacterial suspensions were then transferred to solid MacConkey’s agar culture plates overnight, and CFU was counted. (d) WT neutrophils (n = 6/group) were pre-treated with or without CpdA (3 µM) for 1 h and then co-cultured with Citrobacter rodentium (or Citrobacter rodentium were cultured alone) in the presence of DPI or/and GSK484 in the plates for 12 h. The bacterial suspensions were then transferred to solid MacConkey’s agar culture plates overnight, and CFU was counted in the plates for 12 h. The bacterial suspensions were then transferred to solid MacConkey’s agar culture plates overnight, and CFU was counted. (e-f) Neutrophils were pre-treated with or without CpdA (3 µM) for 1 h, and then co-cultured with Escherichia coli O9:H4 (e) and Escherichia coli O83:H1 (f) for 12 h. The bacterial suspensions were then transferred to Luria Broth’s agar culture plates overnight, and CFU was counted. One representative of three independent experiments was shown. Data were expressed as mean ± SEM. Statistical significance was tested by the two-tailed unpaired Student’s t-test (a, c, and e-f) or one-way ANOVA (d). **p < 0.01, ***p < 0.001, ***p < 0.0001.

Figure 5. mTOR mediates GPR120 induction of neutrophil production of ROS and formation of NETs.

Notes: (a) Neutrophils were treated with or without CpdA (3 µM) for 5 min. The phosphorylated mTOR levels were determined by FACS. (b-c) Neutrophils were treated with or without CpdA (3 µM) in the presence or absence of rapamycin (2 μM) for 1 h. ROS production (b) and NETs formation (c) were assessed. (d) Neutrophils were pre-treated with or without CpdA (3 µM) in the presence or absence of rapamycin (2 μM) for 1 h, and then co-cultured with Citrobacter rodentium in the plates for 12 h. The bacterial suspensions were then transferred to solid MacConkey’s agar culture plates overnight, and CFU was counted. One representative of three independent experiments was shown. Data were expressed as mean ± SEM. Statistical significance was tested using the two-tailed one-way ANOVA (b and d). ***p < 0.001, ***p < 0.0001.

Figure 6. GPR120 regulates NETs formation in neutrophils through the upregulation of glycolysis.

Notes: (a-c) Neutrophils (n = 4/group) were treated with or without CpdA (3 µM) for 1 h, and then OCR (a and b) and ECAR (a and c) levels were detected by Seahorse XF Cell Energy Phenotype Test using a Seahorse XF96 Analyzer. (d-e) Neutrophils were pre-treated with or without CpdA (3 µM) in the presence or absence of 2DG (250 µM) for 1 h. ROS production (d) and NETs formation (e) was determined. (f) Neutrophils were pre-treated with or without CpdA (3 µM) in the presence or absence of 2DG (250 µM) for 1 h, and then co-cultured with Citrobacter rodentium in the plates for 12 h. The bacterial suspensions were then transferred to solid MacConkey’s agar culture plates overnight, and CFU was counted. One representative of two independent experiments was shown. Data were expressed as mean ± SEM. Statistical significance was tested by the two-tailed unpaired Student's t-test (b and c) or the two-tailed one-way ANOVA (d and f). *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001.

Figure 7. GPR120 regulates IL-17A and IL-22 production and IEC barrier function in neutrophils.

Notes: (a-b) WT neutrophils (n = 4/group) were treated with or without CpdA (3 µM) in the presence or absence of IL-23 (20 ng/mL) for 24 h, and IL-17A (a) and IL-22 (b) production in culture supernatants were measured by ELISA. (c) WT and GPR120-deficient neutrophils were treated with IL-23 (20 ng/mL) for 24 h, and IL-17A and IL-22 levels were determined. (d-g) Neutrophils (n = 4/group) were treated with IL-23 in the presence or absence of CpdA (3 µM)/rapamycin (2 μM, d-e) or 2DG (250 µM, f-g) for 24 h, and IL-17 (d and f) and IL-22 (e and g) production in culture supernatants were measured by ELISA. One representative of three independent experiments was shown. (d-i) Mode-K cells (n = 3/group) were cultured in the medium of control or CpdA-treated neutrophils, and the genes of Reg3g and Tjp1 were determined after 24 h. (J) Mode-K cells (n = 3/group) in the upper inserts were cultured in the medium of control or CpdA-treated neutrophils and treated with proinflammatory cytokines (10 ng/mL of LPS, 40 ng/mL of TNF-α, and 20 ng/mL of IL-1β). TEER levels were determined by Epithelial Volt-Ohm at different time points, and the percentage of original TEER was calculated. One representative of three independent experiments was shown. Data were expressed as mean ± SEM. Statistical significance was tested by the two-tailed unpaired Student's t-test (a-B) or the two-tailed one-way ANOVA (c-F). **p < 0.01, ***p < 0.001, ***p < 0.0001.

Figure 8. The schematic diagram of GPR120 regulation of neutrophil anti-bacterial function.

Notes: GPR120 activation in neutrophils induces NETs formation and ROS production, which directly enhance anti-bacterial activity. In addition, GPR120 promotes IL-17A and IL-22 production in neutrophils, which contributes to intestinal epithelial barrier integrity. These processes were differentially regulated by enhanced glycolysis and mTOR activation.

Data availability statement

16S rRNA sequencing data have been deposited in SRA under the BioProject number PRJNA716350 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA716350).