Abstract
The nucleotide-binding domain (NBD) and leucine-rich repeat receptors,
such as NOD-like receptors (NLRs), have pivotal functions in the innate
immune response to various viral infections participating during the
recognition of pathogens and activation of signaling pathways. One NLR,
NOD2, is a dynamic protein that is activated in the presence of viral
genomes and metabolites. However, its participation in combating a
dengue virus (DENV) infection remains unclear. The aim of this study was
to determine the role of NOD2 in macrophage-like THP-1 cells during anin vitro infection with DENV type 2 (DENV2). The interactions of
NOD2 with RIP2 and MAVS was examined in DENV2-infected and
agonist-stimulated cells. The effects of downregulating NOD2 expression
or signaling on virus loads was also evaluated. The cellular mRNA
expression and protein levels of NOD2 on cells under the stimuli were
quantified with RT-PCR, Western blot and indirect immunofluorescence.
Both the mRNA and protein expression of NOD2 was enhanced in response to
DENV-2 infection. Interactions of NOD2 with RIP2 and MAVS, analyzed with
confocal microscopy and co-immunoprecipitation assays, were
time-dependent and increased in the post-infection period, between 6 and
24 h. After silencing NOD2 expression, DENV2-infected cells displayed
greater viral loads and decreased expression of IL-8 and IFN-α (measured
in supernatants obtained from the cells), compared to the uninfected
(mock control) cells or those transfected with irrelevant-siRNA. Thus,
in response to a DENV2 infection, NOD2 was activated in THP-1 human
macrophage-like cells, the production of IL-8 and IFN-α was enhanced,
and viral replication was limited.
Keywords: DENV, NOD2, RIP2, MAVS, IFN, macrophages
Introduction
Dengue is one of the most challenging arthropod-borne viral diseases in
humans, causing around 390 million annual cases worldwide [1].
According to the WHO, dengue is a leading cause of serious disease and
mortality, mostly in Latin America and Asia. It has four antigenically
related serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) and various
genotypes within each one [2]. DENV belongs to theFlaviviridae family, which has viral particles approximately 50
nm in diameter with icosahedral capsids wrapped in a lipid bilayer
envelope. This family has single-stranded RNA (ssRNA) genomes of
positive polarity (~10.8 Kb long), with a single open
reading frame encoding a single polyprotein. The latter is
proteolytically processed by both viral and cellular proteases to
produce three structural proteins (C, prM and E) and seven
non-structural proteins (NS1, NS2A, NSB, NS3, NS4A, NS4B and NS5)
[3].
Although the human cells targeted by DENV are predominantly monocytes,
they also include macrophages, immature dendritic cells, mature
dendritic cells, and reactive splenic lymphoid cells [4-7]. In such
cells, DENV triggers the innate immune response (the first line of
defense against infection), activating several pattern recognition
receptors (PRRs) that mediate the rapid production of type I interferon
(IFNα/β) and cytokines. Hundreds of interferon-stimulated genes (ISGs,
induced by interferon IFN), modulate other branches of the immune
response [8]. The orchestration of these effectors is capable of
creating an antiviral state in both uninfected and infected cells
through a variety of mechanisms [9].
PRRs are receptors of innate immune cells responsible for recognizing
specific components (patterns) in pathogens. For viral components, ssRNA
is recognized by TLR7 [10], double-stranded RNA (dsRNA, formed
during viral replication) by RIG-I and MDA-5 molecules [11,12], and
the glycoprotein of the viral envelope by TLR3 [13,14]. During the
infection of Huh-7 cells, DENV is recognized by two types of PRRs that
act synergistically to restrict viral replication by triggering type I
IFNs [12,14]. These two types of PRRs are membrane-bound TLR3 and
cytoplasmic RLRs, the latter being RIG-I-like receptors (e.g., RIG-I and
MDA-5).
In DENV2-infected M1 macrophages (known to exhibit an inflammatory
phenotype), the NLRP3 inflammasome (a member of the NOD-like receptor
(NLR) family) processes caspase-1, leading to the production of the
active forms of IL-1β and IL-18 and the generation of pyroptosis
[15]. In platelets exposed to DENV, moreover, the NLRP3 inflammasome
is activated to modulate the release of IL-1β in micro-particles through
mechanisms possibly dependent on mitochondrial activity [16].
However, the role of other intracellular PRRs (e.g., some NLRs) in DENV
infections has not been clearly defined.
NOD2, a member of the NLR family, was initially described as a cytosolic
sensor for muramyl dipeptide (MDP) [17]. The activation of NOD2 has
been established in the presence of genomes and metabolites from
viruses, triggering and modulating diverse immune signaling pathways
related to nuclear factor kappa B (NF-κB), IFN regulatory factor (IRF)
3/7, mitogen-activated protein kinase (MAPK) and others [18]. NOD2
is activated when interacting with proteins involved in enhancing
antiviral signaling pathways, such as mitochondrial antiviral signaling
protein (MAVS) [19], receptor-interacting serine/threonine-protein
kinase 2 (RIP2) [20,21] and 2’-5’-oligoadenylate synthetase 2 (OAS2)
[22]. Even though NOD2 has been implicated in a response to
compounds that decrease cytokine production and viral replication in
cultured human monocytes infected by dengue, its specific role during
DENV infection has not yet been determined [23].
RIP2 has been reported to modulate the apoptotic pathway, mainly at 48 h
post-infection (hpi), in HepG2 cells infected with DENV2 at a
multiplicity of infection (MOI) of 1 [24]. Nevertheless, the
specific involvement of RIP2 during a DENV2 infection in monocytes,
macrophages and other types of cells is still unclear. In contrast, the
participation of MAVS in activating the antiviral responses of type I
IFNs is well known. Its activation occurs when RIG-1 (a cytosolic
protein) detects DENV [25]. There are descriptions in the literature
of anti-DENV activities mediated by members of the OAS family, including
OAS1 p42, OAS1 p46 and OAS3 p100. Various human OAS proteins appear to
be differentially induced in distinct types of cells and are
characterized by different subcellular locations and enzymatic
parameters [26]. A recent study revealed increased levels of mRNA of
OAS1, OAS2 and OAS3 in U-937 macrophage-like cells infected with DENV2
[27].
The aim of the current study was to determine the role of NOD2 in
macrophage-like THP-1 cells during an in vitro DENV2 infection.
The interactions of NOD2 with effector molecules (RIP2 and MAVS) was
examined in DENV2-infected and agonist-stimulated cells. The effects of
downregulating NOD2 expression or signaling was also evaluated. The
overall effect on viral replication was assessed.
Materials and Methods
DENV2 strains, culture and titration
The dengue virus employed presently is the Yuc17438 strain of serotype
2, which was isolated from a patient in Yucatán, Mexico. DENV2 was
cultivated on C6/36 confluent monolayers obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA), and cultured in
Leibovitz’s L-15 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented
with L-glutamine, MEM non-essential amino acids solution, MEM vitamin
solution, penicillin-streptomycin, 10 % of fetal bovine serum (FBS),
and sodium pyruvate solution (all from Gibco, Waltham, MA, USA).
Infections were carried out in Leibovitz’s L-15 medium with 2% FBS. A
mock uninfected control was treated under the same conditions. After 7
days, the supernatant of both virus-infected and uninfected (mock
control) C6/36 cells was harvested by centrifugation at 2,000 g for 10
min. Subsequently, both supernatants were diluted to a final
concentration of 8% in polyethylene glycol 8000 (Sigma-Aldrich)
solution with PBS 1x (Gibco) and incubated overnight at 4 °C. The next
day each solution was centrifuged at 6,000 g for 1 h. The pellets were
re-suspended at 1/10 of the total solution volume in L-15 medium
supplemented with 10% FBS, then aliquoted and frozen at −70°C until
needed.
Viral stocks and supernatant from the mock group were titrated in focus
forming assays on C6/36 cells. Briefly, 10-fold serial dilutions of
viral stock (200 µL) were used to infect cells on monolayers in 24-well
plates. After 1 h of gentle agitation, each well (containing C6/36 cells
supplemented with 2% of FBS) was covered with 1 mL of overlay medium
consisting of 1% carboxymethyl cellulose (Sigma-Aldrich) in L-15
medium. The overlay medium was gently removed 5 days later, and
monolayers were fixed with 30% acetone in cold PBS 1x for 20 min at 4
°C. The foci in C6/36 cells were then stained with a primary
anti-DENV-envelope antibody (clone 4G2) (Merck-Millipore, Burlington,
MA, USA) coupled to a secondary antibody and anti-mouse IgG-HRP
(Invitrogen, Carlsbad, CA, USA). Finally, the foci were revealed with
the substrate 3,3′-diaminobenzidine (DAB) (Diagnostic Biosystems Inc.,
Pleasanton, CA, USA). Viral titers were expressed as focus forming units
(FFU)/mL or plaque forming units (PFU)/mL. The experiments were
performed with an MOI of 10.
THP-1 cell culture and macrophage differentiation
The THP-1 (human monocytic leukemia) cell line was acquired from ATCC
and cultured in RPMI-1640 medium (Gibco) supplemented with sodium
pyruvate solution, L-glutamine, penicillin-streptomycin, MEM
non-essential amino acids solution, and 10% FBS (all from Gibco). The
macrophage-like state was optimally obtained by treating THP-1 cells for
3 days with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA,
Sigma-Aldrich) under several experimental conditions. Subsequently,
adherent cells (THP-1 macrophage-like cells) were gently washed three
times with PBS 1x (Gibco) and left to stand with fresh RPMI-1640 medium
for 5 days before further use.
L18-MDP or PIC transfections
THP-1 macrophage-like cells were transfected with a mixture of
LipofectamineTM 3000 reagent (LP3000) (Invitrogen,
Carlsbad, CA, USA) and L18-MDP (2 μg/mL) (Invivogen, San Diego, CA, USA)
or PIC (2 μg/mL) (Sigma-Aldrich), prepared according to the
manufacturer´s instructions. The cells were then incubated at room
temperature for 5 min. Afterwards, each mixture was placed on the
monolayer to reach a final volume of 1 mL of Opti-MEM medium (Gibco) and
were incubated at 37 °C in 5% CO2 for the indicated
periods of time.
Confocal microscopy
THP-1 cells (3x105 cells/mL) were seeded on glass
cover slips in a 24-well tissue culture plate (Corning, NY, USA). Upon
completion of the differentiation protocol, cells were either infected
with DENV2, transfected with L18-MDP or PIC (described above), or left
untreated (mock control). Briefly, the viral stocks were adjusted to an
MOI of 10 in a supplemented RPMI medium and incubated at 37 °C for 1 h
on monolayers, after which time the cells were washed three times with
PBS 1x (Gibco) and fixed with 2% of paraformaldehyde (Sigma) for 15 min
at rt. Subsequently, the cells were permeabilized with 0.1% Triton
X-100 (Sigma-Aldrich) in 1 x PBS for 15 min and blocked with 5% bovine
serum albumin (BSA, Sigma-Aldrich) at room temperature for 1 h. Upon
completion of this time, addition was made of the primary antibodies:
anti-NS3, anti-NOD2, anti-RIP2, anti-MAVS (Genetex Inc., Irvine, CA,
USA) and in each case incubation was carried out at room temperature for
2 h. The cells were then washed three times with PBS 1x before the
secondary antibodies were added. An anti-mouse IgG coupled to the
Dylight-488 antibody (Pierce Biotechnology Inc., Waltham, MA, USA) was
utilized for NOD2, and an anti-rabbit IgG coupled to the APC antibody
(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for NS3, RIP2 and
MAVS. All were incubated at room temperature for 30 min in the dark.
Finally, the nuclei
were labeled with DAPI (Sigma-Aldrich) for 15 min and the slides were
mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). The
images of 100 cells per condition were captured on a confocal laser
scanning system (LSM5 Pascal) attached to a confocal microscope
(Axiovert 200 M, Zeiss, Jena, Germany). For colocalization analysis in
confocal microscopy, JACoP program of Image J software was used for
different captured fields accounting in total for N=100 cells for each
different experimental condition, then an overlap coefficient was
obtained. Cell culture supernatants from those cells were collected and
frozen at -70°C to await cytokine measurement or viral titration.
mRNA analysis
Total cellular RNA was isolated with TRIzol (Invitrogen) as specified in
the manufacturer’s instructions. For reverse transcription (RT), the RNA
was quantified in a NanodropTM from Thermo Sc.
(Rockford, IL, USA) and then 1 μg of total RNA were added with 0.5 μg of
oligo-dT (Thermo Sc.) and incubated at 70 °C for 10 min. The RT reaction
included 1× single strand buffer, 500 mM of each deoxynucleotide
triphosphate (Roche, Mannheim, Germany), and RevertAid reverse
transcriptase (200 U) (Thermo Sc). Each RT reaction was incubated at 42
°C for 1 h to furnish cDNA. On each cDNA strand obtained, PCR was
performed in a commercial master mix with 2.0 mM MgCl2(Amplicon III, Odense M, Denmark). The human GAPDH gene served as the
endogenous control. The following primers sequences (Invitrogen) were
used for RT-PCR: GAPDH, forward 5´-GGTCATCCATGACAACTTTGG-3´ and reverse
5´-GTCATACCAGGAAATGAGCTTGAC-3´; NOD2, forward
5´-CAGCTGGACTACAACTCTGTGG-3´ and reverse 5´- GCAGAGTTCTTCTAGCATGACG-3´.
All readings were normalized to the mRNA level of GAPDH.
Western blot
For Western blot analysis, THP-1 macrophage-like cells differentiated in
6-well plates (2x106 cells/well) were separated into
groups: infected with DENV2, transfected with L18-MDP or PIC, and
untreated (mock control). Upon completion of the time of each treatment,
cells were lysed with RIPA buffer (50 mm Tris HCl, pH 8, 150 mm NaCl,
1% NP-40, 0.5% sodium deoxycholic acid and 0.1% SDS), which contains
a protease inhibitor cocktail (COmpleteTM, Roche
Diagnostic GmbH, Mannheim, Germany). The total protein concentration was
determined by employing a PierceTM BCA protein assay
kit (Thermo Fisher Scientific, Rockford, IL, USA) according to the
manufacturer’s instructions. Fourty µg of total protein were separated
by 10% SDS–PAGE and transferred to a nitrocellulose membrane (Bio-Rad
Laboratories, Berkeley, CA, USA). Membranes were blocked with 5%
non-fat milk in PBS 1x for 1 h and then membranes were immunostained as
described elsewhere. Proteins were visualized with Luminata Forte
substrate (Merck-Millipore) and the immunoreactive proteins were
detected by exposure to Kodak Carestream film (Sigma-Aldrich).
Co-immunoprecipitation assays
For these assays, THP-1 macrophage-like cells were differentiated in a
T-25 cell culture flask (5x106 cells/flask).
Subsequently, they were infected with DENV2, transfected with L18-MDP or
PIC, and untreated (mock control). When the incubation period of each
treatment was completed, cells were lysed with RIPA buffer containing a
cocktail protease inhibitor (previously mentioned). Total protein
lysates were quantified as previous detailed, and 150 µg of total
protein were incubated with an anti-NOD2 antibody (2 µg/reaction)
overnight at 4°C with constant rocking. Subsequently, 15 μL of protein A
agarose beads (Thermo Sc.) were added to each mixture and incubated
while gently mixing by constantly inverting the tube at 4 °C for 3 h.
The precipitates were then washed eight times with PBS 1x-protease
inhibitors, the pellets were re-suspended, and the NOD2-RIP2 and
NOD2-MAVS interactions were evaluated with an immunoblot assay.
Immunoblots were probed with the following antibodies: anti-RIP2 and
anti-MAVS (previously explained) coupled to an anti-rabbit IgG-HRP
conjugated antibody. They were visualized by using enhanced
chemiluminescence with Luminata Forte substrate (Merck-Millipore).
Small interfering RNA (siRNA) assays
A total of 2.5x106 THP-1 macrophage-like cells in
6-well plates were transfected with a mixture of two siRNAs Card4 (25
pmol) and Card6 (25 pmol) against human NOD2 (NOD2-siRNAs, catalog
number 1027415, Qiagen, Hilden, Germany). As a negative control,
AllStars negative control siRNA (irrelevant-siRNA 50 pmol, catalog
number 1027281, Qiagen) were transfected with LP3000 (Invitrogen), as
specified in the manufacturer’s protocol, for 12 h. Cell viability was
determined with the MTT assay (Sigma-Aldrich). Briefly, 15 µg of MTT
reagent (500 µg/mL) were added to each well and incubated at 37 °C for 3
h. Cells were then lysed with 100 µL of DMSO (Sigma-Aldrich) and rocked
until the purple formazan crystal was completely solubilized. Absorbance
was measured at 600 nm in a microplate reader (Thermo Sc) and the
percentage of cell viability was calculated with the resulting values,
as follows: (Atreatment −
Ablank)/(Acontrol −
Ablank) × 100%. Additionally, the efficiency of cells
with NOD2 knocked down was assessed by Western blot and indirect
immunofluorescence, as described elsewhere.
Curcumin treatments
THP-1 macrophage-like cells (3x105 cells/mL) were
seeded on 24-well tissue culture plates. After the differentiation
protocol, cells were washed twice with PBS 1x. Curcumin (Sigma Aldrich)
was diluted in ethanol according to manufacturer´s instructions, adding
30 µM/mL [28] to each well for the treated group. Ethanol was added
to control wells. Finally, the plates were incubated at 37 °C for 1 h,
and each stimulus was co-incubated as indicated elsewhere.
Determination of cytokine levels
The cytokine concentration was quantified in THP-1 cell culture
supernatants. Distinct ELISA kits were employed for human IL-8
(Peprotech, Cranbury, NJ, USA) and human IFN-α (eBioscience, San Diego,
CA, USA). Both measurements were performed as specified in the
manufacturer´s instructions.
Statistical Analysis
Differences between
groups were analyzed on GraphPrism® version 8.0 (GraphPad Software Inc.,
San Diego, CA, USA). For comparison between three groups, significance
was examined with one-way ANOVA. Comparisons between two groups of
continuous variables were made with the Student’s t -test in the
case of parametric distributions and the Mann–Whitney U test for
nonparametric distributions.
Results
DENV2 infection upregulates NOD2 expression
NOD2 expression was evaluated by confocal microscopy at distinct time
points in THP-1 macrophage-like cells infected by DENV, transfected with
L18-MDP, or untreated (mock control). There was a low expression of this
molecule in the uninfected cells (Fig. 1A). A singular pattern of
distribution was observed in the DENV2-infected cells from 6-24 hpi,
with NOD2 localized in the cytosol in a characteristic pattern in
approximately 15-20% of the cells (Fig. 1B). In contrast,
~40-50% of the cells transfected with synthetic L18-MDP
(the positive control) exhibited a more vesicle-like pattern of
distribution for NOD2 (Fig. 1C).
Macrophages derived from the THP-1 cell line are reported to be
permissive for DENV2 infection in vitro [29]. As a control
for DENV infection, the expression of viral NS3 protein (red) was
examined, being detected at 12 hpi. The NS3 and NOD2 sites were found
co-localized in 18% of double positive cells (Suppl. Fig. 1). On the
other hand, the quantification of NOD2 mRNA in untreated (control) and
DENV2-infected THP-1 macrophage-like cells showed the following
increases in expression for the latter: 1.5-fold at 3 hpi, 2.6-fold at 6
hpi and 3.5-fold at 12 hpi. Treatment with L18-MDP resulted in the
greatest increase in the expression of NOD2 of the three groups:
1.6-fold at 3 h, 4.3-fold at 6 h and 5.5-fold at 12 h (Fig. 1D). Western
blot analysis confirmed the higher level of NOD2 in DENV2-infected and
L18-MDP-transfected cells (compared to the control): 1.46-fold at 12 hpi
and 3.1-fold at 24 hpi in the infected cells; 1.4-fold at 3 h, 2.45-fold
at 6 h and 5-fold at 12 h in the transfected cells (Fig. 1E).