Figure 3.

NLRC4 and NLRP3 are differentially involved in flagellin activation in HD-DC and HIV-DC. 2 × 10⁵ MDDC from healthy donors (HD-DC; n = 5) or HIV-infected patients (HIV-DC; n = 5) were stimulated with 5 μg/ml flagellin (FLG) for 3, 8, 18, and 24 hours. Cells were harvested for total RNA isolation and evaluation of relative gene expression by qPCR (A–E). (A) Basal gene expression of NLRP3, NAIP, NLRC4, CASP1, IL18, and IL1B was expressed as 2^−ΔCt. (B) FLG-induced expression of NLRP1, NLRP3, NLRC4, NAIP, CASP1, IL1B, and IL18 genes was calculated as 2^−ΔΔCt (fold-change, FC) and reported as logFC in a heat-map graph for HD-DC and HIV-DC. (C) FC values for NLRC4 are compared between HD-DC and HIV-DC at all the time-points. (D) FC values for NLRP3 are compared between HD-DC and HIV-DC at all the time-points. (E) Basal gene expression of miR223, BRCC3, NEDD8, and CARD8 was expressed as 2^−ΔCt. (F) 2 × 10⁵ MDDC from healthy donors (HD-DC; n = 3) were pre-treated with 1,000 UI/mL of IFN-α-2b (Schering-Plough) for 18 h and then with 5 μg/ml flagellin (FLG) or LPS 1 μg/mL LPS for 24 hours, or 1 μg/mL LPS for 24 hours plus 1 mM ATP for more 15 minutes. IL-1ß concentration was measured in culture supernatants of HD-DC with and without IFN-α-2b pre-treatment and compared with HIV-DC (n = 3) stimulated with FLG. Data are represented as mean ± standard error. Multiple t-test was applied to compare HD- and HIV-DC in (A,C–E). Kruskall-Wallis test was applied to compare HD-DC, HD-DC + IFN-α-2b, and HIV-DC sets in (F) (^*p < 0.05).