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).