Mechanical Stimuli Affect Escherichia coli Heat-Stable Enterotoxin-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Model

Mechanical stimuli increased ST-induced AP and BL cGMP secretion levels but did not alter the ST increase in intracellular cGMP content. (a) ST increased the intracellular cGMP content under all conditions although the increase was not significant under static conditions. (b) ST significantly increased AP cGMP secretion with flow and flow plus stretch but not under static conditions. There was no difference in the AP cGMP secretion levels between the flow and flow-plus-stretch conditions. (c) ST significantly increased BL cGMP secretion only under the flow-plus-stretch condition. However, there was no difference in the BL cGMP secretion levels between the flow and flow-plus-stretch conditions. (d) The ST effect on AP cGMP secretion (difference of cGMP secretion in the presence of ST minus that under basal conditions performed on paired samples) under flow and flow plus stretch was significantly greater than the ST effect under static conditions. The ST effect on BL cGMP secretion was significantly higher under flow-plus-stretch conditions than under static conditions and was increased, although not statistically significantly, under flow alone. The ST-induced increases of AP and BL cGMP secretion levels under both flow and flow plus stretch were not different from each other. Results are means ± SEM (static basal, n = 7, and ST, n = 8; flow basal, n = 6, and ST, n = 6; flow-plus-stretch basal, n = 7, and ST, n = 7). P values were determined by one-way ANOVA with Tukey’s multiple-comparison tests. F+S, flow plus stretch.

Flow significantly increased MRP4 but not MRP5 mRNA. MRP4 and MRP5 mRNA levels were determined by qRT-PCR under basal conditions and after 6 h of ST exposure. (a) Message for MRP4 was increased by flow and flow plus stretch under baseline conditions (statistically not significant), while ST significantly increased MRP4 mRNA under conditions of flow plus stretch. (b) In contrast, MRP5 mRNA levels were similar under basal conditions and did not change significantly with ST treatment. The level of MPR5 mRNA was lower than that of MRP4. Results are means ± SEM (n = 4). P values were determined by one-way ANOVA with Tukey’s multiple-comparison tests.

GCC mRNA was not altered by mechanical stimuli. GCC mRNA levels of enteroids under static, flow, and flow-plus-stretch conditions did not show any change either among these three conditions in the basal state or with the addition of ST. Results are means ± SEM (n = 30). P values were determined by one-way ANOVA with Tukey’s multiple-comparison tests.