Fig 1: miR-150 regulates the secretion of inflammatory cytokines by modulating STAT1 expression. (A-C) THP-1 cells were first transfected with either miR-NC or miR-150 mimics alone, or co-transfected with miR-NC or miR-150 mimics and pcDNA or pcDNA-STAT1 before being treated with 1 µg/ml LPS for 24 h. THP-1 cells in the con and LPS groups were not transfected whilst cells in the con group were not treated with LPS. (A) IL-1β, (B) IL-6 and (C) and TNF-α secretion into the media supernatant by THP-1 cells were measured by ELISA. (D-F) THP-1 cells were first transfected with either miR inhibitor NC or miR-150 inhibitor alone, or co-transfected with miR inhibitor NC or miR-150 inhibitor and pcDNA or pcDNA-STAT1 before being treated with 1 µg/ml LPS for 24 h. THP-1 cells in the con and LPS groups were not transfected whilst cells in the con group were not treated with LPS. (D) IL-1β, (E) IL-6 and (F) and TNF-α secretion into the media supernatant by THP-1 cells were measured by ELISA. **P<0.01 and ***P<0.001. miR, microRNA; LPS, lipopolysaccharide; miR-NC, miR mimic negative control; anti-miR-NC, miR inhibitor control; IL, interleukin; TNF-α, tumor necrosis factor-α; si, small interfering RNA; si-NC, si-negative control.
Fig 2: miR-150 regulate the secretion of inflammatory cytokines in LPS-treated THP-1 cells. THP-1 cells transfected with miR-150 mimics were treated with 1 µg/ml LPS for 24 h, following which (A) IL-1β, (B) IL-6 and (C) TNF-α levels in the cell supernatant were measured by ELISA. THP-1 cells transfected with the miR-150 inhibitor were treated with 1 µg/ml LPS for 24 h, following which (D) IL-1β (E) IL-6 and (F) TNF-α levels in the cell supernatant were measured by ELISA. ***P<0.001. miR, microRNA, LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor-α; miR-NC, negative control; anti-miR-NC, miR inhibitor negative control.
Fig 3: TIM3 modulates M2-like macrophage polarization in vitro. (A–D) THP-1 cells were incubated with complete media (THP-1 control) or treated with 8505c cell-derived CM (THP-1 (8505c CM)) in the presence of isotype control IgG or anti-TIM3 blocking antibody for 24 h. hCLEC7A (A), hCCL13 (B), or IL-6 (C) mRNA levels were measured by RT-qPCR. (D) The supernatant was collected, and IL-6 was detected by ELISA. Data are expressed as mean ± SD. *** p < 0.0005, **** p < 0.0001.
Fig 4: Lactobacillus gasseri regulates the gene expression of TNF in a cell type-specific manner. Human macrophages were infected with either Hp or in combination with L. gas or L. bre. RNA was extracted 8 h postinfection, and the expression of (A) TNF and (B) IL-6 in THP-1-derived macrophages and (C) TNF and (D) IL-6 in MDMs was analyzed by qPCR. Target mRNA levels were normalized to the reference gene rpl37A and are presented relative to the unstimulated control, which was set to 1. Data are the means and standard deviation of triplicate samples and are representative of three independent experiments. *P < 0.05 using ANOVA followed by Bonferroni posttest.
Fig 5: 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
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