Inhibition of DV-activated NLRP3 inflammasomes by anti-CLEC5A mAb. (A) Blockade of CLEC5A inhibited the production of DV-induced IL-18. GM-Mφ were preincubated with anti-CLEC5A, anti-DC-SIGN, or anti-MR blocking antibodies for 1 hour, followed by incubation with DV (MOI = 5) for 2 hours at 37°C. At 48 hours after infection, culture supernatants were harvested to determine the levels of IL-18 by ELISA. Black, grey, and white bars indicated 0.1, 1, and 10 μg/well, respectively, as described in “Blocking assay.” *P < .05 (Student t test). (B) Anti-CLEC5A mAb inhibited the transcription of IL-1β and NLRP3 in DV-infected GM-Mφ. GM-Mφ were pre-incubated with CLEC5A mAb or isotype control for 1 hour, followed by incubation with DV (MOI = 5) as described in “Blocking assay.” Total RNAs from each group were isolated for reverse-transcription into cDNA at 24 hours after infection, and expression of cytokine and NLRP3 in each group was determined by real-time PCR. Gene expression was normalized by GAPDH. Fold change of gene expression in the y-axis was calculated based on the same gene expression in the mock group. ***P < .001 (Student t test). (C) Blockade of CLEC5A did not inhibit LPS-induced IL-1β mRNA transcription. Expression of cytokines, NLRP3, and Asc after priming for 4 hours in GM-Mφ was detected by real-time PCR and normalized by GAPDH (left panel). GM-Mφ were primed with LPS (5 ng/mL), followed by incubation with anti-CLEC5A mAb or isotype control before DV infection (MOI = 30) as described in “Blocking assay” (right panel). Total RNAs were harvested at 6 hours after infection for reverse-transcription into cDNA, and gene expression level was determined by real-time PCR and normalized by GAPDH. Fold change of gene expression in the y-axis was calculated based on the same gene expression in the nonpriming group. *P < .05; ***P < .001 (Student t tests). (D-E) Blockade of CLEC5A suppressed the secretion of IL-1β and IL-18 (D) and caspase-1 p20 (E) from LPS-primed GM-Mφ. LPS (5 ng/mL)–primed or nonprimed GM-Mφ were preincubated with CLEC5A mAb or isotype control, then incubated with DV (MOI = 30) as described in “Blocking assay.” At 24 hours after infection, supernatants were subjected to ELISA assay to determine cytokine levels and caspase-1 p20 amounts. Data in each figure are expressed as the mean ± SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001 (Student t tests). (F) Blockade of CLEC5A suppressed the expression and processing of pro-IL-1β and pro-IL-18. LPS (5 ng/mL)–primed GM-Mφ were preincubated with CLEC5A mAb or isotype control, then infected with DV (MOI = 30) as described in “Blocking assay.” At 24 hours after infection, cell lysates were harvested to determine the pro-form and mature form of IL-1β and IL-18. IL-1β was detected by Western blot. Actin was the internal control. IL-18 was pulled down from cell lysates by anti–IL-18 mAb before Western blot analysis. (G) CLEC5A controlled DV-induced IL-1β production. Clec5a^+/+Stat1^−/− and Clec5a^−/−Stat1^−/− mice (8 weeks) were inoculated intraperitoneally with 2 × 10⁵ PFU/mice of DV2 (New Guinea C-N) as described previously.²⁰  IL-1β (left panel) and IL-18 (right panel) in mice sera were measured by ELISA at day 7 after DV infection. Each dot represents an individual mouse; horizontal bars represent mean values for each group. ***P < .001 (Student t test).