Figure 4: TRPV4 function is critical to MSU crystal-induced NLRP3 inflammasome activation and IL-1β production.

(a) GSK101 promotes IL-1β production in LPS-primed but not in LPS-unprimed BMDMs in a concentration-dependent manner. (b) MSU crystal-induced IL-1β production from LPS-primed BMDMs was severely suppressed by TRPV4 antagonism and genetic ablation of TRPV4 function in both global Trpv4 KO mice and MΦ-specific Trpv4 cKOs compared with their respective controls. (c) MSU crystal-induced of IL-1β production from LPS-primed BMDMs was inhibited by GSK219 in a concentration-dependent manner. (d) MSU crystal-induced IL-1β production was markedly reduced in LPS-primed BMDMs isolated from the Trpv4^−/− mice when compared with the Trpv4^+/+ mice. (e) MSU crystal-induced IL-1β production was markedly reduced in LPS-primed BMDMs isolated from the Cre⁺ Cx3cr1^CreERT; Trpv4^f/f mice when compared with their Cre^- littermates. Note PBS, LPS, and MSU crystals alone did not increase IL-1β production in (d) and (e). (f) MSU crystal-induced IL-1β production in LPS-primed BMDMs from NLRP3-deficient mice and Casp-1-deficient mice and their respective control mice. (g) GSK101-induced IL-1β production in LPS-primed BMDMs from NLRP3-deficient mice and Casp-1-deficient mice and their respective control mice. (h) ELISA analysis of IL-1β in supernatants from PMA-differentiated THP-1 cells treated with various concentrations of GSK219 and then stimulated with MSU crystals (200 μg/mL). (i) ELISA analysis of IL-1β in supernatants from human PBMCs pretreated with various concentrations of GSK219 and then stimulated with MSU crystals (200 μg/mL). Statistical significance was determined using two-way ANOVA followed by Bonferroni’s post-hoc test (a), Tukey post hoc tests (multiple comparisons, c, h and i), and Student’s t test (b, d-g). ***P<.001. n=6 per group.