Figure 4. DENV2 induced NOD2-RIP2 and NOD2-MAVS protein interactions in THP-1 macrophage-like cells. The interaction of NOD2 with RIP2 and MAVS was measured by co-immunoprecipitation assays in whole protein cell lysates of the cells, which were mock treated, DENV2-infected, or given the corresponding treatment for the positive, immunoprecipitating in each case with anti-NOD2 antibody and immunoblotting with the indicated antibody. (A) Transfection with L18-MDP in positive control and the NOD2-RIP2 interaction evaluated 6 h later; and (B) transfected with PIC for positive control and the NOD2-MAVS interaction at 12 h. Subsequently, cells were harvested and lysed, and the total protein was quantified, also Western blot analysis of NOD2 in whole cell lysates alone was carried out in mock treated THP-1 cells and LPS (100 ng/mL) treated cells after 6 h(C) . Each interaction was analyzed by the co-immunoprecipitation of each molecule (by adding an anti-NOD2 antibody) followed by Western blotting. The immunoblots were performed by using specific antibodies for each molecule evaluated (RIP2 and MAVS), with each primary antibody being coupled to an anti-rabbit IgG-HRP secondary antibody. (D) Time-dependent interactions of NOD2 with RIP2 and MAVS found in the cells infected with DENV2 and interpreted in a colored map. Interaction rates are coded in shades of blue: dark blue indicates a higher interaction rate and light blue a lower interaction rate. Interaction rates were determined by co-immunoprecipitation assays and Western blot (as described in the text) at 6, 12, 18 and 24 h post-treatment (the measurement time post-infection is shown for each interaction). Each assay was repeated three times.
DENV2 infection induced the interaction of NOD2 with the effector proteins RIP2 and MAVS
The protein interactions of RIP2 and MAVS proteins with NOD2 were revealed by co-immunoprecipitation assays in the three experimental groups of THP-1 macrophage-like cells (Figs. 4A-B). The interaction of NOD2 with RIP2 was assessed in lysates of the cells. The interaction was manifested in DENV2-infected and L18-MDP-transfected cells, but not in untreated cells (Fig. 4A). Likewise, the MAVS protein was recovered by co-immunoprecipitation in lysates of DENV2-infected and PIC-transfected cells at 12 hpi, but not in untreated cells (Fig. 4B). A general overview is portrayed of the NOD2-RIP2 and NOD2-MAVS interactions over time (Fig. 4E). A time-dependent interaction pattern emerged from the analysis, with the NOD2-RIP2 interaction appearing first at 6 hpi and the NOD2-MAVS interaction later at 12 hpi.
Downregulation of NOD2 in DENV2-infected cells increased viral replication and reduced production of IL-8 and IFN-α
The effects of NOD2 downregulation were examined with two approaches: gene silencing with siRNAs and chemical inhibition using curcumin. To assure the viability of cells subjected to a double round of transfections with siRNAs and agonists, proper assays were carried out. The corresponding assays demonstrated a high percentage of viability (80-88%) in cells treated with curcumin as well as the ones exposed to double transfections (Suppl. Fig. 3). There was a decline in NOD2 expression in about 67% of the cells transfected with NOD2-siRNAs compared with those transfected with an irrelevant-siRNA or the mock control. The second approach involved a polyphenol found in the plantCurcuma longa , commonly called curcumin, which is reported to inhibit NOD2 oligomerization and consequently NOD2 downstream signaling [28]. DENV2-infected and L18-MDP-transfected cells pre-treated with curcumin secreted significantly less IL-8 than the same groups without the curcumin pretreatment. Accordingly, IL-8 was quantified at ~756 pg/mL and 1390 pg/mL, respectively, in cells pretreated or not with curcumin and then stimulated with an L18-MDP transfection. Likewise, the level of IL-8 was measured at ~697 pg/mL and 1073 pg/mL, respectively, in cells pretreated or not with curcumin and then infected with DENV2 (Suppl. Fig. 4).
Gene silencing had a very similar effect. Secretion of IL-8 was determined to be at ~845 pg/mL and 1151 pg/mL, respectively, in cells transfected with NOD2-siRNAs or the irrelevant-siRNA, in both cases later stimulated with the transfection of L18-MDP. IL-8 production was at ~688 pg/mL and 983 pg/mL, respectively, in cells transfected with NOD2-siRNAs or the irrelevant-siRNA, in both cases later infected with DENV2. The basal level of IL-8 (in untreated cells) was ~480 pg/mL (Fig. 5A). NOD2 silencing also had a negative impact on IFN-α secretion. Cells transfected with NOD2-siRNAs or an irrelevant-siRNA and then treated with PIC showed levels at ~73 pg/mL and 133 pg/mL, respectively. The same two pretreatments followed by DENV2 infection resulted in levels of IFN-α at ~53 pg/mL and 110 pg/mL, respectively. IFN-α was not detected in untreated cells (Fig. 5B).