Figure 2.

Enhanced ORMDL3 expression level leads to more IL-1β production.A, human MDMs were treated with LPS (200 ng/ml) for indicated times and assessed for mRNA expression of ORMDL3 at different time points (n = 3 donors, similar result obtained for an additional n = 3 donor). B, human MDM cells were transfected with scrambled or siORMDL3 for 24 h, then stimulated with LPS, ATP, MSU, and nigericin alone and LPS along with MSU (150 μg/ml) and nigericin (10 μM) for 6 h and ATP (5 mM) for 30 min after 5 h of LPS treatment and analyzed for IL-1β secretion in the cell supernatants (n = 5 donor). C, human MDM cells were transfected with ORMDL3 expressing full-length plasmid and empty vector for 24 h, then stimulated with LPS(200 ng/ml) and nigericin for 6 h and analyzed for IL-1β secretion in cell supernatant (n = 6, similar result obtained for additional n = 6 donor). Human MDM were transfected with scrambled or siORMDL3 for 24 h, treated with LPS (200 ng/ml) for another 24 h and analyzed for (D) IL-10 secretion in cell supernatant (n = 8 donor, similar result was obtained for additional n = 8 donor) and (E) TNF-α secretion in the cell supernatant (n = 10 donor, similar result was obtained for additional n = 10 donor). Human MDM cells were transfected with scrambled and siORMDL3 for 24 h, treated with LPS (200 ng/ml) and nigericin for 6 h and analyzed for (F) relative IL-1β mRNA fold by qRT-PCR (n = 4 donor, similar result obtained for additional n = 4 donor) and (G) Caspase-1 activation by flow cytometry (n = 3 donor, similar result for additional n = 3 donor). Mean ± SEM; ∗p < 0.05, ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 as determined by one-way ANOVA. EV, empty vector, ns denotes non-significant. IL, interleukin; LPS, lipopolysaccharide; MDM, monocyte-derived macrophage; MSU, monosodium urate; ORMDL3, orosomucoid-like protein 3; qRT-PCR, quantitative reverse transcription PCR; TNF, tumor necrosis factor.