Fig 1.
Baseline parameters are comparable between wildtype and Gpr43−/−mice.
The number of circulating leukocytes (A) and neutrophils (B) in the peripheral blood of wildtype and Gpr43−/−mice were quantitated. Bone marrow leukocytes (C) and neutrophils (D) isolated from wildtype and Gpr43−/−mice were quantitated. N ≥ 8 per group. Baseline cell surface expression of L-selectin (E), and PMA-induced L-selectin shedding (F) and respiratory burst (G) was measured and compared between wildtype and Gpr43−/−bone marrow neutrophils. N ≥ 4 mice per group or independent in vitro experiments performed in duplicates, N.S. denote not statistical significant, t test.
Fig 2.
GPR43 deficiency induces migration, accelerated neutrophil rolling and adhesion following 1 h of LPS challenge.
Neutrophils were isolated from the bone marrow of wildtype and Gpr43−/−mice, and analysed for chemotaxis towards increasing concentration of CXCL1 (A) and LPS (B). N ≥ 4 independent in vitro experiments performed in duplicates, ***p < 0.001, *p < 0.05 vs Wildtype, 2-way ANOVA. Intravital microscopy was utilised to visualise and quantitate the number of intravascular neutrophils that were rolling (C) or adherent (D) following 1 h of saline or LPS challenge in wildtype and Gpr43−/−mice. N ≥ 5 mice per group, ***p < 0.001, **p < 0.01 vs corresponding Saline group, t test. #p < 0.05 vs Wildtype, t test.
Fig 3.
GPR43-deficient neutrophils display slow rolling following 4 h of LPS challenge.
Intravital microscopy was utilised to visualise and quantitate the number of intravascular neutrophils that were rolling (A), and their rolling velocity (B) or adherent (C) following 4 h of saline or LPS challenge in wildtype and Gpr43−/−mice. N ≥ 5 mice per group, ***p < 0.001 vs corresponding Saline group, t test. # p < 0.05 vs Wildtype, t test.
Fig 4.
Exacerbated intestinal inflammation in Gpr43−/−mice in response to LPS.
(A) Representative histological images of jejunum of wildtype and Gpr43−/−mice following 4 h of saline or LPS challenge. Neutrophils are denoted by the black arrowheads. Scale bar = 100 μm. (B) Histological quantification of neutrophils in the (i) duodenum, (ii) jejunum and (iii) ileum of wildtype and Gpr43−/−mice following 4 h of saline or LPS challenge. N ≥ 5 mice per group, **p < 0.01, *p < 0.05 vs corresponding Saline group, t test. #p < 0.05 vs Wildtype, t test.
Fig 5.
Acetate reduces neutrophil migration via a mechanism mediated by GPR43.
The total number of leukocytes in the peritoneal cavity was quantified in wildtype and Gpr43−/−mice at 24 h following sham or cecal ligation and puncture (CLP; (A)), or at 2 h after saline, fMLP or fMLP + acetate (B). The peritoneal neutrophil numbers of acetate-treated wildtype and Gpr43−/−mice at 2 h after saline or fMLP treatment were also measured (C). N ≥ 3 mice per group, **p < 0.01, *p < 0.05 vs corresponding Sham or Saline group, t test. ##p < 0.01, #p < 0.05 vs treated Wildtype, t test. &p < 0.05 vs fMLP-treated Wildtype, t test. N.S denotes not statistically significant vs fMLP-treated Gpr43−/−.
Fig 6.
No fibre diet prolongs intravascular rolling of neutrophils post-LPS.
Intravital microscopy was utilised to visualise and quantitate the number of intravascular neutrophils that were rolling (A) or adherent (B) following 4 h of saline or LPS challenge. (C) Neutrophil rolling velocity was calculated in Ctrl-fed and NF-fed mice at 4 h after LPS. N ≥ 5 per group, ***p < 0.001, **p < 0.01 vs corresponding Saline group, t test. #p < 0.05 vs Ctrl post-LPS, t test. Neutrophils were isolated from the bone marrow of mice fed on Ctrl or NF diet for 2 weeks, and analysed for chemotaxis towards increasing concentration of CXCL1 (D). N ≥ 4 independent in vitro experiments performed in duplicates, ***p < 0.001 vs Ctrl diet, 2-way ANOVA.
Fig 7.
Enhanced migratory behaviour of neutrophils is maintained after microbiota transfer.
Neutrophils of GF mice reconstituted with microbiota from SPF mice fed on Ctrl or NF diet were analyzed for chemotaxis towards increasing concentration of CXCL1 (A) and fMLP (B). N ≥ 4 independent in vitro experiments performed in duplicates, **p < 0.01, *p < 0.05 vs Ctrl diet, 2-way ANOVA.