Figure 5
Figure 5. DV-induced IL-1β/IL-18 secretion is dependent on NLRP3 inflammasome activation in GM-Mφ. (A) Determination of cytokine mRNA levels by real-time PCR. GM-Mφ or M-Mφ were either incubated with DV directly (top panels) or primed with LPS (5 ng/mL) for 4 hours before incubation with DV (MOI = 5) for 2 hours at 37°C. Total RNAs from mock or DV-infected GM-Mφ and M-Mφ were isolated for reverse-transcription into cDNA at the indicated time point after infection. The expression levels was determined by real-time PCR and normalized with GAPDH. Fold change of gene expression in the y-axis was calculated based on the same gene expression in mock group in DV-infected macrophages (top panels), or based on the same gene expression in nontreated group (bottom panels). Data are the mean ± SEM from 3 independent experiments. *P < .05; **P < .01 (Student t tests). h.p.i indicates hours after infection. (B) Determination of NLR mRNAs by real-time PCR. The expression levels of NLRs after DV infection in both macrophages were determined by real-time PCR and normalized with GAPDH. Fold change of gene expression in the y-axis was calculated based on the same gene expression in mock group. Data are the mean ± SEM from 3 independent experiments. *P < .05; **P < .01 (Student t tests). (C) Knockdown NLRP3 by siRNA. LPS (5 ng/mL)–primed GM-Mφ were transfected with the nontargeting siRNA control (NT-i) or the NLRP3 siRNAs (NLRP3-i) as described in “RNA interference (siRNA) assay,” and then incubated with DV (MOI = 5) as described in “DV infection to macrophages.” At 24 hours after infection, cytokine levels were determined by ELISA. Data are the mean ± SEM from 3 independent experiments. **P < .01; ***P < .001 (Student t tests). (D) Potassium efflux, cathepsin B activity, and syk signaling were essential for DV-triggered IL-1β/IL-18 production. LPS (5 ng/mL)–primed GM-Mφ were preincubated with DMSO, chemical inhibitors (APDC: 10, 30, 100μM; glibenclamide: 10, 30, 100μM, CA-074 Me: 1, 3, 10μM; Syk inhibitor: 1, 3, 10μM), followed by incubation with DV (MOI = 5) as described in “Blocking assay.” The levels of IL-1 β (left panel) and IL-18 (right panel) in the supernatants were detected by ELISA. Data are the mean ± SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001. (Student t tests). Gliben. indicates glibenclamide.

DV-induced IL-1β/IL-18 secretion is dependent on NLRP3 inflammasome activation in GM-Mφ. (A) Determination of cytokine mRNA levels by real-time PCR. GM-Mφ or M-Mφ were either incubated with DV directly (top panels) or primed with LPS (5 ng/mL) for 4 hours before incubation with DV (MOI = 5) for 2 hours at 37°C. Total RNAs from mock or DV-infected GM-Mφ and M-Mφ were isolated for reverse-transcription into cDNA at the indicated time point after infection. The expression levels was determined by real-time PCR and normalized with GAPDH. Fold change of gene expression in the y-axis was calculated based on the same gene expression in mock group in DV-infected macrophages (top panels), or based on the same gene expression in nontreated group (bottom panels). Data are the mean ± SEM from 3 independent experiments. *P < .05; **P < .01 (Student t tests). h.p.i indicates hours after infection. (B) Determination of NLR mRNAs by real-time PCR. The expression levels of NLRs after DV infection in both macrophages were determined by real-time PCR and normalized with GAPDH. Fold change of gene expression in the y-axis was calculated based on the same gene expression in mock group. Data are the mean ± SEM from 3 independent experiments. *P < .05; **P < .01 (Student t tests). (C) Knockdown NLRP3 by siRNA. LPS (5 ng/mL)–primed GM-Mφ were transfected with the nontargeting siRNA control (NT-i) or the NLRP3 siRNAs (NLRP3-i) as described in “RNA interference (siRNA) assay,” and then incubated with DV (MOI = 5) as described in “DV infection to macrophages.” At 24 hours after infection, cytokine levels were determined by ELISA. Data are the mean ± SEM from 3 independent experiments. **P < .01; ***P < .001 (Student t tests). (D) Potassium efflux, cathepsin B activity, and syk signaling were essential for DV-triggered IL-1β/IL-18 production. LPS (5 ng/mL)–primed GM-Mφ were preincubated with DMSO, chemical inhibitors (APDC: 10, 30, 100μM; glibenclamide: 10, 30, 100μM, CA-074 Me: 1, 3, 10μM; Syk inhibitor: 1, 3, 10μM), followed by incubation with DV (MOI = 5) as described in “Blocking assay.” The levels of IL-1 β (left panel) and IL-18 (right panel) in the supernatants were detected by ELISA. Data are the mean ± SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001. (Student t tests). Gliben. indicates glibenclamide.

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