Fig. 4.

HDM activates the NLRP3 inflammasome and induces IL-1β production by murine macrophages. A BMDMs isolated from WT mice were treated for 24 h with LPS (100 ng/mL) or with the indicated concentrations of HDM. ATP (5 mM) was added to each well during the last hour of culture as shown in Supplemental Fig. 4A. The supernatants were collected and IL-1β production was analyzed by ELISA. Results are representative of n = 2 independent experiments performed in triplicate. B RAW 264.7 macrophages were stimulated for 6 h with indicated concentrations of HDM and the relative mRNA level of Il1b or C) different NLRs was measured by qPCR and normalized to Gapdh gene expression. The relative mRNA level in the HDM’s 0 μg/mL condition was used as a reference and assigned to 1. Results are representative of n = 2 independent experiments performed in simplicate. D BMDMs isolated from WT (n = 6), Nlrp3 (n = 2), Casp1 (n = 3), or Il1b (n = 3) KO mice were stimulated with HDM (200 μg/mL) and ATP (5 mM) as shown in Supplemental Fig. 4A, and the levels of IL-1β or E) TNF in the supernatants were analyzed by ELISA. The cytokine level in the WT control group was used as a reference and assigned to 100%. Results are the pooled data from n = 3 independent experiments performed in triplicate. F WT BMDMs were stimulated with VEH (PBS), HDM, or HI-HDM (both at 200 μg/mL) and ATP (5 mM) as described in Supplemental Fig. 4A, and the levels of IL-1β in the supernatants were analyzed by ELISA. Results are representative of n = 2 independent experiments performed in triplicate. G The cells stimulated in F were recovered and total cell lysates were prepared for western blot analysis of NLRP3 and pro-IL-1β expression. Densitometric measurements of NLRP3 and pro-IL-1β relative to β-actin are indicated below each blot. Results are representative of n = 2 independent experiments performed in simplicate. Data are presented as mean ± SEM. Statistical significance was assessed by one-way ANOVA with post hoc Bonferroni’s test. n.s: non-significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001