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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