Fig. 4.

Butyrate and secondary bile acids induce P-gp to limit neutrophil transmigration across the epithelium. A–C T84 cells incubated with 5 mM butyrate, 50 μM LCA, 50 μM DCA, and/or 50 μM UDCA for 24 h. A Representative western blot showing P-gp expression in lysates. B Densitometry data describe samples pooled from at least two independent experiments; *p = 0.0199, ***p = 0.0002, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. C T84 cells were treated as in A, B, prior to measurement of Rho123 retention. Data reflect inverse of geometric mean of Rho123 fluorescence intensity. Data describe samples pooled from at least two independent experiments; *p < 0.05, ****p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test of each sample compared to DMSO control. D WT germ-free mice were delivered butyrate orally for 14 days. P-gp protein expression was measured in colonic tissue by western blot. Representative western blot is shown, each lane representing a replicate mouse within each group. Densitometry data describe samples pooled from two independent experiments; N = 6–7 mice per group; ***p = 0.0004, unpaired t test. E, F WT SPF mice were delivered cholestyramine (CME) in mouse chow for 14 days. P-gp protein expression was measured by western blot in colon tissue (E) or cecum tissue (F). E, F Representative western blot is shown, each lane representing a replicate mouse within each group. Densitometry shown relative to control group without cholestyramine. Data describe samples pooled from two independent experiments. N = 8 mice per group. E *p = 0.0237, unpaired t test. F ***p = 0.0009, unpaired t test. G Schematic of neutrophil migration experiment (BioRender). T84 cells seeded on the bottom-facing surface of Transwell® plates were preincubated with metabolites for 24 h. Primary neutrophils were added to the top (basolateral) compartment, purified HXA₃ was added to the bottom (apical) compartment, and the number of primary neutrophils that migrated across the T84 cell monolayer were quantitated. H T84 cells seeded as in G were incubated with butyrate, LCA, DCA, and/or UDCA as in A–C. Migration of primary neutrophils across this cell monolayer to HXA₃ in the apical compartment was quantified. Data shown indicate individual replicate wells and are representative of at least three independent experiments; ****p < 0.0001, ns p > 0.05, one-way ANOVA with Tukey’s multiple comparisons test