CD200Fc reduces LPS-induced IL-1β activation in human cervical cancer cells by modulating TLR4-NF-κB and NLRP3 inflammasome pathway

Abstract

Chronic inflammation plays an important role in tumorigenesis of cervical cancer. CD200Fc, a CD200R1 agonist, has been found to have anti-inflammatory effects in autoimmune diseases and neuro-degeneration. However, the anti-inflammatory effect of CD200Fc on cervical cancer has not yet to be completely understood. This study investigated the anti-inflammatory effects and mechanisms of CD200Fc in LPS-induced human SiHa cells and Caski cells. SiHa cells and Caski cells were stimulated with 40 μg/ml LPS under different concentrations of CD200Fc for 90 min or 12 hours. The mRNA and protein levels of pro-IL-1β, cleaved-IL-1β and NLRP3, as well as the protein level of cleaved caspase-1, were significantly increased in LPS-induced SiHa cells and Caski cells. LPS stimulation did not change ASC and pro-caspase-1 expression. CD200Fc down-regulated protein expression of cleaved caspase-1 and mRNA and protein expression of pro-IL-1β, cleaved-IL-1β and NLRP3. In addition, the protein levels of TLR4, p-P65 and p-IκB, as well as the translocation of P65 to nucleus, were significantly increased in LPS-induced SiHa cells and Caski cells. LPS stimulation did not change t-P65 and t-IκB on protein levels, which were components of TLR-NF-κB pathway. CD200Fc down-regulated protein expression of TLR4, p-P65 and p-IκB and inhibited the translocation of P65 to nucleus in LPS-induced SiHa cells and Caski cells. These results indicated that CD200Fc appeared to suppress the inflammatory activity of TLR4-NF-κB and NLRP3 inflammasome pathway in LPS-induced SiHa cells and Caski cells. It provided novel mechanistic insights into the potential therapeutic uses of CD200Fc for cervical cancer.

Keywords: CD200Fc; IL-1β; NLRP3 inflammasome; TLR4-NF-κB; cervical cancer.

Figure 1. Effects of CD200Fc on production and activation of IL-1β in LPS-stimulated SiHa cells and Caski cells

SiHa cells and Caski cells were stimulated with 40 μg/ml LPS under different concentrations of CD200Fc for 12 hours. The protein levels of pro-IL-1β and cleaved-IL-1β were measured by western blot analysis in SiHa cells (A) and Caski cells (D). The bar chart showed the ratio of pro-IL-1β and cleaved-IL-1β to β-actin at each groups in SiHa cells (B) and Caski cells (E). The mRNA level of pro-IL-1β was measured by qRT-PCR analysis. The bar chart showed the ratio of pro-IL-1β to β-actin at each groups in SiHa cells (C) and Caski cells (F). The extracellular levels of I IL-1β in culture media were measured using commercial ELISA kits in SiHa cells (G) and Caski cells (H). Data are the mean ± S.E.M. of three independent experiments. ^#P < 0.001 vs. control group (cultured in medium alone); **p < 0.001 vs. LPS-induced group.

Figure 2. Effects of CD200Fc on the expression of NLRP3 inflammasome components in LPS-stimulated SiHa cells and Caski cells

SiHa cells and Caski cells were stimulated with 40 μg/ml LPS under different concentrations of CD200Fc for 90 min. The protein levels of NLRP3 and ASC were measured by western blot analysis in SiHa cells (A) and Caski cells (B). The bar chart showed the ratio of NLRP3 and ASC to β-actin at each groups in SiHa cells (C) and Caski cells (D). The mRNA levels of NLRP3 and ASC were measured by qRT-PCR analysis. The bar chart showed the ratio of NLRP3 and ASC to β-actin at each groups in SiHa cells (E) and Caski cells (F). Data are the mean ± S.E.M. of three independent experiments. ^#P < 0.001 vs. control group (cultured in medium alone); *p < 0.01, **p < 0.001 vs. LPS-induced group.

Figure 3. Effects of CD200Fc on cleaved caspase-1 production in LPS-stimulated SiHa cells and Caski cells

SiHa cells and Caski cells were stimulated with 40 μg/ml LPS under different concentrations of CD200Fc for 12 hours. The protein levels of cleaved-caspase-1 and pro-caspase-1 were measured by western blot analysis in SiHa cells (A) and Caski cells (C). The bar chart showed the ratio of cleaved-caspase-1 and pro-caspase-1 to β-actin at each groups in SiHa cells (B) and Caski cells (D). The expression level of cleaved-caspase-1 in SiHa cells and Caski cells was measured by immunofluorescent staining (E). Meanwhile, the phenotype of nuclei was also investigated via DAPI staining. Scale Bar = 50 μm. Data are the mean ± S.E.M. of three independent experiments. ^#P < 0.001 vs. control group (cultured in medium alone); *p < 0.01, **p < 0.001 vs. LPS-induced group.

Figure 4. Effects of CD200Fc on the protein levels of TLR4, p-P65, t-P65, p-I-κB and t-I-κB in LPS-induced SiHa cells and Caski cells

SiHa cells and Caski cells were stimulated with 40 μg/ml LPS under different concentrations of CD200Fc for 90 min. The protein levels of TLR4, p-P65, t-P65, p-I-κB and t-I-κB were measured by western blot analysis in SiHa cells (A) and Caski cells (E). The bar chart showed the ratio of TLR4 to β-actin at each groups in SiHa cells (B) and Caski cells (F). The bar chart showed the ratio of p-P65 and t-P65 to β-actin at each groups in SiHa cells (C) and Caski cells (G). The bar chart showed the ratio of p-I-κB and t-I-κB to β-actin at each groups in SiHa cells (D) and Caski cells (H). Data are the mean ± S.E.M. of three independent experiments. ^#P < 0.001 vs. control group (cultured in medium alone); *p < 0.01, **p < 0.001 vs. LPS-induced group.

Figure 5. Effects of CD200Fc on the translocation of P65 from cytoplasm to nucleus in LPS-induced SiHa cells and Caski cells

SiHa cells and Caski cells were stimulated with 40 μg/ml LPS under different concentrations of CD200Fc for 90 min. Nuclear and cytosolic extracts were isolated and the levels of P65 in each fraction were determined by western blot analysis in SiHa cells (A) and Caski cells (B). HDAC1 and β-actin were used as internal controls. The bar chart showed the ratio of P65 to HDAC1 in nuclear at each groups in SiHa cells (C) and Caski cells (D). The bar chart showed the ratio of P65 to β-actin in cytosolic at each groups in SiHa cells (E) and Caski cells (F). Data are the mean ± S.E.M. of three independent experiments. ^#P < 0.001 vs. control group (cultured in medium alone); *p < 0.01, **p < 0.001 vs. LPS-induced group.

Figure 6. Possible mechanisms by which CD200Fc inhibited production and activation of IL-1β in LPS-stimulated SiHa cells and Caski cells

LPS-stimulated activation of TLR4-NF-κB and NLRP3 inflammasome pathways were inhibited by down-regulation of TLR4, p-P65, and p-I-κB, and NLRP3, cleaved caspase-1 after CD200Fc treatment, and subsequent inhibited production and activation of IL-1β.