Fig. 4: IL-37d inhibited pro-inflammatory cytokines expression in an IL-1R8-independent manner.

a–c A549 cells were transfected with 100 nM of human siIL-1R8 or scrambled control for 24 h, followed by transfection of the mock or IL-37d plasmid, respectively, for additional 24 h and then stimulated with IL-1β (10 ng/ml) for 6 h; IL-1R8 knockdown efficiency was analyzed by RT-PCR (a) and western blot (b). The level of IL-6 in the cell culture supernatants was determined by ELISA and inhibition rate of IL-37d on IL-6 was calculated (c). d–f Peritoneal macrophages from IL-37dtg and wild-type mouse were transfected with murine siIL-1R8 or scrambled control siRNA for 48 h and followed by stimulation with LPS (100 ng/ml) for 6 h. IL-1R8 knockdown efficiency was analyzed by qRT-PCR (d) and western blot (e). Inhibition rate of IL-37d on IL-6 in the cell culture supernatants was determined by ELISA (f). g BMDMs from wild-type mouse were treated with increasing doses of recombinant IL-37b or IL-37d protein for 2 h and stimulated with LPS (100 ng/ml) for 6 h; The level of IL-6 in culture supernatants was determined by ELISA. h, i Monoclonal Ab against IL-37 (100 μg per mouse) or equal IgG2B as a control were injected intraperitoneally to IL-37dtg mice (n = 4 per group) for 3 h followed by LPS (5 mg/kg) intraperitoneal injection for additional 4 h. The levels of IL-6 in serum (h) and spleen (i) were measured by ELISA. *P < 0.05; ***P < 0.001, ns, no significant difference. Data are shown as the mean ± SEM from three independent experiments