Figure 5. Gene silencing of NOD2 by siRNAs decreased cell secretion of IL-8 and IFN-α. THP-1 macrophage-like cells were gene-silenced with a mix of specific human NOD2-siRNAs or left intact with an irrelevant-siRNA. Subsequently, they were infected with DENV2 or transfected with L or PIC. Cell supernatants were then collected, and the cytokine levels were measured by ELISA. (A-B) The level of IL-8 was determined at 12 h and IFN-α at 24 h. Data in the bar graphs are expressed as the mean ± SD from three independent assays. Significance was examined with the unpaired t -test and Mann-Whitney U distribution: *** p<0.001, ** p<0.05, * p<0.05, and ns: not significant. (C ) Western blot on the silencing assay and levels of downregulation using NOD 2 specific siRNAs are shown.
NOD2 inhibition led to greater viral loads
An over 4-fold increase in viral load was produced in cells with NOD2 knocked down by treatment with specific siRNAs compared to cells with NOD2 unaffected by an irrelevant-siRNA (1.8x104 FFU/mL versus 4.4x103 FFU/mL, respectively). The same effect was observed in curcumin pretreated versus normal cells, in both cases later infected with DENV2. The level of viral titers rose more than 2.5-fold in the chemically treated versus normal cells (1.1x104 FFU/mL vs. 4.3x103 FFU/mL, respectively) (Fig. 6). The effect of the reduced expression or function of NOD2 on viral kinetics in infected cells (at 12, 24 and 48 hpi) was time-dependent beginning at 12 hpi, with the viral load increasing at 24 and again at 48 hpi. The level of titers at 24 hpi was ~2.8-3.3x103FFU/mL in untreated cells and those transfected with an irrelevant-siRNA, compared to ~2.0x104FFU/mL in the group transfected with NOD2-siRNAs. Similar data were found at 48 hpi, where titers were ~1.1x104 FFU/mL in untreated cells and those transfected with an irrelevant-siRNA, compared to ~2.6x104 FFU/mL in the group transfected with NOD2-siRNAs (Suppl. Fig. 5A).