Figure 1.
Effects of rbIFNT on ISG15 expression and inflammatory cytokine secretion.
(A) THP-1 macrophages were incubated for 48 h with or without rbIFNT at the indicated concentrations. Subsequently, total RNA was extracted and analyzed by real-time RT-PCR for expression of ISG15 mRNA. (B–D) THP-1 macrophages were incubated for 48 h with rbIFNT or rhIFNB at the indicated concentrations. After priming with LPS (100 ng/mL) for 3 h or Pam3CSK4 (300 ng/ml) for 10 h, cells were treated with nano-silica particles (100 µg/mL) for 6 h. IL-1β (B and D) and IL-1α (C) levels in supernatants were then determined using ELISA. (E) LDH release from THP-1 macrophages treated with rbIFNT or rhIFNB. Data are expressed as means ±SEM (n = 3); Significant differences were identified using ANOVA; *p<0.05 and ** p<0.01.
Figure 2.
Effects of rbIFNT on nano-silica uptakes and actin polymerization.
(A) THP-1 macrophages were treated with Green nano-silica for 6 h at the indicated concentrations. (B) THP-1 macrophages were incubated for 48 h with or without rbIFNT (2.7 and 27 IU/mL), and were then treated with Green nano-silica particles (30 µg/mL) for 6 h. After washing cells, uptake of Green nano-silica particles was analyzed by flow cytometry. (C) THP-1 macrophages were incubated for 1 h with or without cytochalasin D and were treated with Green nano-silica particles (30 µg/mL) for 6 h. After washing cells, uptake of Green nano-silica particles was analyzed by flow cytometry. Representative flow cytometry plots are presented. (D) THP-1 macrophages were incubated for 48 h with or without rbIFNT (27 IU/mL), and were then treated with nano-silica particles (30 µg/mL) for 6 h. Cells were stained with fluorescent-conjugated phalloidin and analyzed by flow cytometry. Nuclei were co-stained with Hoechst 33342. Representative confocal microscopic images of the F-actin assembly are presented. Quantitative analyses were performed and data are expressed as means ±SEM (n = 3); Significant differences were identified using ANOVA; *p<0.05 and ** p<0.01.
Figure 3.
Effects of rbIFNT on expression of scavenger receptors and the functional analysis of MARCO by siRNA knockdown.
(A) THP-1 macrophages were incubated for 48 h with or without rbIFNT (27 IU/mL) and total RNA was then extracted and analyzed by real-time RT-PCR for MARCO, CD36, SR-BI, MSR1 and OLR1 mRNA. (B) THP-1 macrophages were transfected with MARCO siRNA (siMARCO) or control siRNA (siN.C.) and analyzed MARCO mRNA expression by real-time RT-PCR (C). THP-1 macrophages transfected with MARCO siRNA or control siRNA were incubated with green nano-silica particles (30 µg/mL) for 6 h. After washing cells, uptake of green nano-silica particles was analyzed the amount of nano-silica (mean fluorescence intensity; MFI) by flow cytometry. After nano-silica treatment, IL-1β levels in supernatants were determined using ELISA. Data are expressed as means ±SEM (n = 3–4); Significant differences were identified using t-test; ** p<0.01 vs. control.
Figure 4.
Effects of rbIFNT on ROS generation.
(A) THP-1 macrophages were treated with nano-silica (100 µg/mL) for the indicated periods. ROS generation was assessed using DCFDA and flow cytometry. (B) THP-1 macrophages were incubated for 48 h with or without rbIFNT (27 U/mL) and were treated with nano-silica particles (100 µg/mL) for 6 h. ROS generation was assessed using DCFDA by flow cytometry. Quantitative analyses were performed and data are expressed as means ±SEM (n = 3); Significant differences were identified using ANOVA; *p<0.05 and ** p<0.01. Representative confocal microscopic images of ROS production in THP-1 macrophages; Nuclei were co-stained with Hoechst 33342.
Figure 5.
Effects of rbIFNT on IL-1β production and the role of IL-10.
(A) THP-1 macrophages were incubated for 48 h with or without rbIFNT. After priming with LPS (100 ng/mL) for 3 h, cells were treated with nano-silica particles (100 µg/mL) for 6 h. Protein levels of pro-IL-1β and mature IL-1β in supernatants were detected by Western blot analyses. Representative photographs are shown. (B) Subsequently, total RNA was extracted and analyzed by real-time RT-PCR for expression of IL-1β mRNA. (C) IL-10 levels in supernatants were then determined using ELISA. (D) THP-1 macrophages were incubated with or without rbIFNT (27 IU/mL) in the presence of antibody against human IL-10 or control IgG, and then treated with LPS and nano-silica. IL-1β levels in supernatants were then determined using ELISA. Data are expressed as means ±SEM (n = 3); Significant differences were identified using ANOVA; ** p<0.01.