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).