Figure 6. THP-1 macrophage-like cells with a reduced expression of NOD2 produced a higher level of viral titers. Cells were knocked down in NOD2 expression by using a mix of NOD2-siRNAs or inhibited in NOD2 signaling with the curcumin pretreatment. After NOD2 downregulation, the cells were infected with DENV2. At 24 hpi, supernatants were collected, and viral progeny from supernatants were quantified by a focus forming assay in C6/36 cells. (A) Viral titers are represented as focus forming units per milliliter (FFU/mL). Data in the bar graph are expressed as the mean ± SD from three independent assays. Significance was determined with the unpaired t -test: *p < 0.05 and ***p < 0.001.
Discussion
PRRs are the first line of recognition for the defense against pathogens. During viral infections, two kinds of PRRs (TLRs and RLRs) are activated and potentiated. Additionally, members of the NLR family are vital for inflammatory functions during a viral infection. For example, NLRP3 [15-16] and NLRX1 regulate functions of the RIG-I/MAVS pathway [30] and NOD2 [19-22], a less studied effector, is also important in viral infections.
In monocytes, macrophages and dendritic cells, the immediate response at early stages of a DENV2 infection is rapid and coordinated, mediated by TLR3, TLR7, TLR8, RIG-I and NLRP3 [10,13-15]. Additionally, the activation of other intracellular sensors located in the cytosol contributes to the establishment of this response. One very dynamic molecule is NOD2, which interacts with several effectors that participate in either proinflammatory or antiviral activity.
According to the current experimental results, NOD2 mRNA and protein expression was enhanced in THP-1 macrophage-like cells in response to anin vitro DENV-2 infection. Singular patterns of subcellular distribution of NOD2 expression was observed in the cells following treatment with a NOD2 agonist (L18-MDP) or infection with DENV2, suggesting that NOD2 may be present in defined vesicles (e.g., endosomes), as previously reported [31]. However, further research is necessary to identify the nature of the vesicles. The expression of NOD2 in the cells was also induced by a PIC, measured at 12. Therefore, a positive regulation of NOD2 appears to be mediated by these molecules, as has been documented for PIC [20].
Interestingly, the expression of elevated levels of NOD2 was exhibited in DENV-2-infected cells but not the untreated cells, which was confirmed by the detection of viral NS3. Thus, active DENV2 replication seems to be necessary to stimulate the up-regulation of NOD2. Although the latter molecule has been significantly associated only with homeostatic regulation in intestinal epithelial cells [32], its involvement in key processes of the proinflammatory and antiviral immune response is clear [33]. Consequently, the current study aimed to investigate the participation of NOD2 in the immune response to a DENV2 infection in an experimental model of THP-1 macrophage-like cells. Macrophages represent one of the main targets of the dengue virus and have a pivotal role in the immune response to a dengue infection. During infection with various RNA viruses, according to previous reports, NOD2 is activated and triggers a protective innate immune response against pathogens, mediated by interactions with adaptors such as RIP2, MAVS, OAS2 and CARD9 [34].
The protein interactions between NOD2 and numerous effectors might be significant for the course and effect of the immune response [35]. Of all NOD2-related effectors described in the literature, only those with a substantial antiviral activity were herein evaluated, being RIP2, and MAVS. Co-localization sites were determined at several post-treatment time points for NOD2-RIP2, and NOD2-MAVS in DENV2-infected and agonist-stimulated cells. Co-location was more evident for NOD2-RIP2 and NOD2-MAVS.
To provide greater insights into these discoveries, the aforementioned interactions were analyzed by co-immunoprecipitation assays. NOD2 was found to interact with RIP2, and MAVS in infected cells as well as in agonist-stimulated cells, and at distinct times post-stimuli. Hence, NOD2 is a dynamic molecule with different functions in the current infection model, mainly during the early response against DENV2. Considering the previous information, we propose that the NOD2-RIP2 interaction led to the generation of inflammatory cytokines and the NOD2-MAVS interaction to a type I IFN response, which has been described for a variety of RNA viruses. Further research is needed on the overall mechanisms involved.
The present findings after knocking down NOD2 expression or signaling contribute to a more in-depth understanding of the overall effect of this molecule on cell function during a dengue infection. NOD2 downregulation by specific siRNAs (versus cells with normal NOD2) correlated with a reduced secretion of IL-8 and IFN-α in DENV2-infected cells. Inhibition of NOD2 signaling by pretreating THP-1 cells with curcumin (versus cells with normal NOD2) afforded a similar decline in the expression of these cytokines in DENV2-infected cells. However, only early times points were examined the possibility that DENV counteracts some antiviral molecules later in the infection must be further examined.
Subsequently, an evaluation was made as to whether NOD2 misfunction in the cells infected with DENV2 had an impact on viral loads. A higher level of viral titers was detected in cells with a decreased expression or signalization of NOD2, and the load increased with the passage of post-infection time. Overall, the results demonstrate a formerly unknown role for NOD2 during DENV2 infection of THP-1 macrophage-like cells: limiting the production of new viral progeny. This is a very significant finding since it indicates that NOD2 either potentiates or initiates the antiviral response to combat viral replication, a trait found for this protein in other viral infections. For instance, the foot-and-mouth disease virus counteracts NOD2 to antagonize antiviral activity [36].
NOD-2 activation has been a striking finding arose during SARS-CoV2 pandemic research, documenting a Nodosome activation (NOD2-RIP2) during Zika virus, DENV and SARS-CoV2 infection in cell lines [37].
In conclusion, the NOD2 receptor was herein upregulated and activated at the early stage of a DENV2 infection, concomitantly with active viral replication in macrophages derived from the THP-1 cell line. The activation of NOD2 during the infection led to its interaction with RIP2 and MAVS (measured at several post-infection times), and elicited the secretion of IL-8 and IFN-α. Through these mechanisms, NOD2 was involved in limiting the replication of new DENV2 viral particles in THP-1 macrophage-like cells.
Acknowledgments: The authors are very grateful to our sponsors: Jorge Ortega-Reyes for providing the anti-rabbit-IgG-APC, Antonina Oltra-Ramírez for the anti-human NOD2, and Alejandro Ayala-Castro for the IL-8 and IFN-α ELISA kits. We are deeply appreciative of Dr. Eneida Campos-Guzmán for all her assistance in the confocal microscopy studies, and of Inci Enid Ramírez-Bello (a student at the Posgrado en Inmunologia ) for all her help. L.-A.G., G.-P.B.E. and S.M.I. are COFAA and EDI fellows. This investigation was supported by the Secretaría de Investigación y Posgrado (SIP-IPN). Alan Larsen for critically reading and editing our manuscript.
Conflicts of Interest: The authors have no conflicts of interest to declare.
Funding:This research project was supported by grants to S.M.I. (SIP20151797 and SIP20161585) from the SIP-IPN, and the fellowship to D.-M.D.A. (356583) from the Consejo Nacional de Ciencia y Tecnología.
Author Contributions: Conceptualization, D.-M.D.A. and S.M.I; methodology, D.-M.D.A. and N.-A.D.; validation, N.-A.D., G.-P.B.E., L.-A.G. and S.M.I; formal analysis, D.-M.D.A., G.-P.B.E. and S.M.I.; investigation, D.-M.D.A and S.M.I.; resources, G.-P.B.E., L.-A.G. and S.M.I.; data curation, M.I.S.; writing of the original draft, D.-M.D.A. and S.M.I.; writing, review & editing, D.-M.D.A., N.-A.D., G.-P.B.E., C.-C.J. and S.M.I.; funding acquisition, S.M.I. and D.-M.D.A. All authors read and approved the final version of the manuscript.
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