Fig. 5

TRPV4 modulates the inflammatory response of C. difficile toxins in enteric glial cells (EGCs). A) Inhibition of TRPV4 by the antagonist RN-1734 decreases IL-6 gene expression in EGCs exposed to TcdA, but not TcdB, over an 18-hour incubation period. IL-6 gene expression in ECGs was assessed by qPCR. Data are presented as mean ± SEM (n = 6) of relative IL-6 gene expression. Statistical significance was determined using the one-way ANOVA and Tukey tests. **p < 0.001 and ***p < 0.0001. B) Exposure to TcdA and TcdB increased the IL-1β gene expression in EGCs compared to controls. TRPV4 inhibition by RN-1734 does not significantly mitigate the upregulation of IL-1β gene expression by TcdA and TcdB. IL-1β gene expression in EGCs was assessed by qPCR. Data are presented as mean ± SEM (n = 6) of relative IL-1β gene expression. Statistical significance was determined using the one-way ANOVA and Tukey tests. **p < 0.001 and ***p < 0.0001. C) This graph illustrates a quantitative assessment of TNF-α fluorescence in EGCs following 18 h of incubation with either TcdA (50ng/mL), TcdB (1ng/mL), only EGCs with only DMEM (control group), RN -1734 alone (100µM), RN-1734 plus TcdA or RN-1734 plus TcdB. Zeiss software determined the fluorescence intensity in 5 hot areas per slide (n = 6) (areas showing intense staining). Data are presented as mean ± SEM of TNF-α fluorescence intensity. Statistical significance was determined using the one-way ANOVA and Tukey tests. ***p < 0.0001.D) Representative photomicrographs illustrate TNF-α immunofluorescence (green), the nuclear staining with DAPI (blue), and the merged images (MERGED). Scale bars = 50 μm