Ethics statement

The study was approved by the Ethics Committee of the “Sede de Investigación Universitaria-Universidad de Antioquia.” Written informed consent was obtained from all individuals who voluntarily participated in this study according to the principles of the Declaration of Helsinki. Four healthy donors were included in this study.

DENV-2 stock and viral titration

The highly pathogenic DENV-2 New Guinea C (NGC) strain, characterized by its ability to induce high viremia and severe infection in non-human primates [40], was propagated in Ae. Albopictus-derived C6/36-HT cells (ATCC) using a multiplicity of infection (MOI) of 0.01. The C6/36 HT were grown in Leibovitz’s L-15 medium (L-15; Sigma-Aldrich) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Massachusetts, USA) and 1% antibiotic-antimycotic solution (Corning, New York, USA), and incubated at 34°C. DENV-2 culture supernatants were stored at −80°C and titrated by plaque assay on BHK-21 cells (clone 15, ATCC), as previously reported [41].

Cultures of primary human monocytes and differentiation into monocyte-derived macrophages

Human peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte-enriched blood units from healthy donors (n = 2–4) through a density gradient with Lymphoprep (STEMCELL Technologies Inc., Vancouver, Canada) by centrifugation at 850 x g for 21 min. Platelet depletion was performed by washing with Phosphate-buffered saline (PBS) (Sigma-Aldrich) three times at 250 x g for 10 min, and the percentage of CD14 + positive cells was determined using flow cytometry. To obtain monocyte-derived macrophage (MDM), 5x10⁵ CD14-positive cells were seeded into each well of 24-well plastic plates as described in [42,43]. Briefly, the monocytes were allowed to adhere for 2 h in RPMI-1640 medium (Sigma-Aldrich) supplemented with 0.5% autologous serum, 4 mM L-glutamine, and 0.3% NaCO₃ and cultured at 37°C and 5% CO₂. Non-adherent cells were removed by washing twice with PBS 1X, and monocytes were cultured in RPMI-1640 medium supplemented with 10% FBS, 0.3% NaCO₃, 1% antibiotic-antimycotic solution 100X, and were incubated at 37°C and 5% CO2 for 6 days to obtain MDMs, fresh media was added to the cultures every 2 days.

In vitro DENV-2 infection of monocyte-derived macrophages

MDMs were infected with DENV-2 at MOI 5 in serum-free RPMI-1640 and incubated at 37 C for 1.5 hours, as previously reported [41]. Then, the cells were washed with PBS to remove the unbound virus, and a fresh, complete medium was added. MDMs were incubated at 37 C and 5% CO₂. Culture supernatants and cell lysates were collected at 1.5-, 3-, 5.5-, and 24-hour post-infection (h.p.i) and stored at −80 C.

Flow cytometry analysis

A BD LSRFortessa flow cytometer (BD Biosciences, New Jersey, USA) was used to assess cell size (FSC-A) and cytoplasmic complexity/granularity (SSC-A) of MDMs. To quantify the expression of cell surface macrophage markers, uninfected MDMs were stained with following mouse anti-human antibodies: CD68 (PE, clone: eBioY1/82A), TLR2 (PE, clone: TL2.1), TLR4 (PE, clone: HTA125), CD14 (FITC, clone: 61D3), and MMR/CD206 (PE, clone: 19.2) (eBioscience, Massachusetts, USA, and BD Bioscience), or their respective isotypes controls. Staining was performed in PBS for 30 minutes at 4°C. The cells were then washed by centrifugation and resuspended in PBS.

DENV-2 infection in MDMs was evaluated by intracellular staining of the viral envelope (E) protein. Briefly, MDMs were harvested from culture and fixed using a fixation/permeabilization buffer (eBioscience, USA). After washing, MDMs were stained with a mouse anti-flavivirus E antibody (inhouse, clone: 4G2) for 30 minutes, followed by a goat anti-mouse IgG-PE secondary antibody (Thermo Scientific, USA) for 30 min. Positively labelled cells were defined using isotype controls and unstained cells. A compensation matrix was applied to compensate for spectral overlap. At least 20,000 events were acquired for each sample, and data were analysed using FlowJo software (version 10). Debris and dead cells were gated out using forward and side scatter gating.

RNA extraction and Bulk RNA-seq of DENV-2-infected MDMs

Total RNA was extracted using the Direct-zol RNA Miniprep Plus (Zymo Research, California, USA) according to the manufacturer’s instructions. RNA samples were treated with DNase I (Zymo Research) to remove genomic DNA. RNA concentration was determined by spectrophotometry using a Nanodrop (Thermo Scientific, Massachusetts, USA), and 300 ng of RNA was used for bulk RNA-Seq, which was performed on an Illumina HiSeq 4000 platform. After sequencing, the image data were converted into raw reads and stored in FASTQ format for each sample, with quality assessment using FastQC (google/vFqiZ), as previously described [44]. Then, clean reads were obtained by removing low-quality adapter, poly N-containing, and shorter-than-70 bp reads. The location of the reads on the reference genome was determined quickly and precisely by comparing reads with the reference genome (GRCh38 or DENV-2 [NC_001474]) using HISAT2 software [45]. The new transcripts were then assembled using String Tie software [46], and using the feature counts tool in Subread software [47], the raw count number in each sample was obtained. Next, raw counts were subject to the following workflow executed on R software (version 4.2.0). Initially, we annotated gene rows with their respective genotype (non-coding gene, pseudogene, and protein-coding gene), symbols, and EntrezID. Based on the genotype, we selected only those protein-coding genes and then generated a list with their lengths using the Homo.sapiens library. Batch effect correction on raw count data for each transcriptome was performed using ComBat seq [48]. Then, to determine the top DEG, we selected genes with a false discovery rate (FDR) < 0.05 and |Log₂ Fold Change (FC) (DENV-2-infected MDMs/control MDMs) > 0.6, using DESeq2 library [49]. Then, the raw counts were normalized to transcripts per million (TPM) using R statistical software (version 4.2.0). As previously reported, we selected the genes linked to JAK-STAT signaling, pattern recognition receptors (PRRs) signaling, and antiviral and inflammatory responses [42,50]. We plotted the data using bar plots and heatmaps with GraphPad Prism for Windows (GraphPad Software version 8.0.1) and R statistical software. Gene set enrichment analyses (GSEA) for overall DEG regulated in DENV-2-infected MDMs were performed using the ClusterProfiler package (Version 4.0) [51]. A gene co-expression network was constructed using Pearson correlation coefficients. The data were filtered to retain only significant correlations (p < 0.05), and only pairs involving DENV-RNA were selected. The network was visualized using ggraph with the “graphopt” layout algorithm. Edges were coloured according to the sign of the correlation (positive or negative) and weighted by the absolute value of the correlation. The Raw RNA-seq data have been deposited in GEO under accession number GSE297386.

RNA extraction, cDNA synthesis, and real-time qPCR

Total RNA was extracted from MDMs infected or not with DENV-2 at an MOI of 5, at 1.5, 10, and 24 h.p.i. Then, 300 ng of RNA was used to construct cDNA libraries for each experimental group using the commercial RevertAid Minus First Strand cDNA Synthesis Kit (Thermo Scientific), following the manufacturer’s protocol. RT-qPCR was used to validate the expression of 19 key DEGs identified through bulk RNA-seq, using a set of specific primers (S1 Table). RT-qPCR amplifications were performed using the Maxima SyBR-Green system (Thermo Fisher Scientific). The Bio-Rad CFX manager obtained the cycle thresholds (Ct) determined for each sample using a regression fit in the linear phase of the RT-qPCR amplification curve. The relative expression of each target gene was normalized to that of the unstimulated control and housekeeping gene GAPDH.

Cytokine quantification

Levels of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), CXCL8/IL-8, and CCL-2 were quantified in MDM culture supernatants using commercially available ELISA kits (BioLegend) following the manufacturer’s instructions. The detection limit was 2–10 pg/mL.

Transcriptomic analysis of DENV-2-infected MDDCs and monocyte subsets from acute dengue patients

To extrapolate the transcriptomic results obtained in DENV-2-infected MDMs to other in vitro and in vivo models of DENV infection, and confirm the biological relevance of biomarkers identified in DENV-2-infected MDMs, we reanalysed the publicly available Microarray dataset (GSE58278) [52], performed in human Monocyte-derived dendritic cells (MDDCs) infected with DENV-2 NGC, at MOI 20, for 24 hours. Also, we reanalysed the publicly available bulk RNA-seq dataset (GSE176079) [53], generated from sorted classical (CM, CD14⁺⁺CD16^-), intermediate (IM, CD14⁺⁺CD16⁺), and non-classical (NM, CD14^DimCD16⁺) Mon subsets isolated from six acute dengue patients (ADP) and three healthy donors. To determine the top DEG, we selected genes with a false discovery rate (FDR) < 0.05 and |Log₂ Fold Change (FC) (DENV-infected cells/Uninfected control) > 0.6, using GEO2R.

Statistical analysis

Statistical analysis was performed using GraphPad Prism for Windows (GraphPad Software version 8.0.1, San Diego, CA, USA; www.graphpad.com). Shapiro-Wilks test was performed to determine the normality of the data. Data are represented as mean ± SEM or as median with interquartile range. The statistical tests are indicated in the fig legends. Significant results were defined for multiple t-tests in the multitranscript analysis p < 0.05 (*) and in the validation experiments for Kruskal Wallis or repeated measures Anova p < 0.05 (*).

Human monocyte-derived macrophages are susceptible and permissive to DENV-2 infection and exhibit dynamic transcriptional response in vitro

Cytokine storm is a key factor in endothelial-integrity disruption, plasma leakage, and haemorrhage in severe dengue patients [8,10,18,29,54]. Mononuclear phagocytes, as macrophages, are essential components of the innate immune response that play a critical role in the control and immunopathogenesis of dengue (Reviewed in [55]). Human macrophages are target cells for DENV infection, contributing to viral replication and promoting robust inflammatory responses [41,50]. However, the role of macrophages in inducing cytokine storms during the early stages of DENV infection is still poorly understood. To investigate this, human monocytes from healthy donors were isolated and differentiated into MDMs over six days in RPMI-1640 medium supplemented with 10% FBS, as previously reported [41,50]. MDMs were then infected at MOI 5 of the highly pathogenic DENV-2 NGC strain for 0 (control), 1.5, 3, 5.5, 10, or 24 hours, after which various immunological and molecular assays were performed, as outlined in Fig 1A. First, we confirmed the macrophage immunophenotype of our in vitro MDM model by flow cytometry. MDMs expressed high levels of classical macrophage markers, including CD68 (Macrosialin) and TLR4 (Fig 1B). In addition, they expressed high levels of DENV-entry receptors, including TLR2, CD14, and macrophage mannose receptor CD206 (Fig 1B), supporting their susceptibility to DENV infection.

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Fig 1. DENV-2 infection induces a dynamic transcriptional profile in human macrophages.

(A) Schematic overview of the experimental design. The flowchart was created using BioRender. (B) Immunophenotype of human MDMs. (C) Frequency of DENV-infected MDMs at 24 h.p.i. (D) Production of infectious DENV-2 viral particles at 24 h.p.i. (E) Quantification of DENV-2 RNA in infected MDMs. (F) PCA analysis of transcriptomic data. (G) DEG, both upregulated and downregulated, in DENV-2-infected MDMs. DEGs were identified using DESeq2 with a p-value < 0.05 and |Log2 Fold Change (DENV-2-infected MDMs vs Control MDMs) | > 0.6. (H) Gene ontology analysis of DEGs upregulated by DENV-2 infection in human MDMs.

https://doi.org/10.1371/journal.pntd.0013366.g001

Next, we evaluated the permissiveness of the MDMs model to DENV-2 replication by quantifying the frequency of infected cells by flow cytometry, and the production of infectious viral particles in culture supernatants at 24 h.p.i by plaque assay using BHK-21 cells. We found that approximately 21.95 ± 1.48% of cultured MDMs were infected and expressed DENV-E antigen (Fig 1C). Furthermore, high levels of infectious viral particles were detected at 24 h.p.i (mean 4.6x10⁵ PFU/mL; Fig 1D), confirming both the susceptibility and permissiveness of our in vitro MDM model to DENV-2 infection.

To examine the kinetics of DENV-2-RNA synthesis and the associated transcriptomic changes in infected MDMs, we performed bulk RNA-seq. The results show an early peak in DENV-2-RNA accumulation at 1.5 h.p.i. However, a reduction in DENV-2-RNA copy number was observed at 3 and 5.5 h.p.i, (Fig 1E), suggesting partial degradation of internalized viral particles or RNAs by MDMs. This period was defined as the eclipse period of infection, representing the interval between viral entry and the initiation of productive RNA synthesis and virion assembly. Subsequently, viral RNA synthesis increased markedly from 10 h.p.i and reached its maximum level at 24 h.p.i (Fig 1E), consistent with the timing of viral release (Fig 1D). These results are in line with our previous observations on DENV replication in human MDMs [41,50].

In line with the kinetics of viral replication, PCA analysis of host gene expression (Fig 1F) revealed dynamic changes in the MDM transcriptome, with the most pronounced alterations occurring at 24 h.p.i. Notably, PCA also indicated low inter-individual variability between MDMs derived from two independent donors, supporting that the observed transcriptional changes were driven by DENV-2 infection rather than donor-specific genetic background. Additionally, RNA-seq analysis revealed transcriptional changes during the viral growth curve of DENV-2 infection (Fig 1G). Differentially expressed genes showed two waves: the first during the viral entry-eclipse periods (1.5 to 5.5 h.p.i), with the lowest number of transcribed genes at 10 h.p.i, and a maximum peak at 24 h.p.i. A total of 92, 378, 366, 210, and 1571 DEG were upregulated by DENV-2 infection at 1.5, 3, 5.5, 10, and 24 h.p.i, respectively, while DENV-2-infected MDMs downregulated 11, 88, 189, 149, and 955 DEG at 1.5, 3, 5.5, 10, and 24 h.p.i, respectively. DEGs induced during early phase of infection (eclipse period) were associated with robust inflammatory responses (Fig 1H), showing significant enrichment of biological processes related to granulocyte recruitment and activation, as well as the induction of pro-inflammatory pathways. In contrast, at 24 h.p.i, we observed significant enrichment of pathways linked to the activation of IFN-dependent JAK-STAT signaling and establishing an antiviral state. Together, these findings suggest that human MDMs mount a dynamic and complex immune response to DENV-2 infection, characterized by a temporal shift from early inflammation to a later antiviral response.

Active DENV-2 replication induces a differential gene expression profile of pattern-recognition receptors in human macrophages

Macrophages play a key role in the induction of innate immune antiviral response by activating different PRRs to promote the activation of signaling pathways that lead to the induction of inflammatory and antiviral factors [41,50,56,57]. As shown in Fig 2A, the expression pattern of TLRs is very dynamic and varies depending on the time of infection. While DENV-2 infection-induced up-regulation of TLR2 and TLR7 mRNA during the early stages of infection (1.5 and 3 h.p.i), the mRNA expression of TLR2, TLR3, TLR4, TLR7, and TLR8 reaches its maximum peak of expression at 24 h.p.i (Figs 2A, S1A). Interestingly, TLR5 mRNA expression was significantly downregulated starting at 5.5 h.p.i, with a peak of transcriptional repression observed at 24 h.p.i. In contrast, TLR9 mRNA expression was not induced (Figs 2A, S1A). TLRs induce inflammation through MyD88 or TRIF adaptor molecules [42,58]. Next, we determine if DENV-2 infection promotes the expression of these components in MDMs. While MYD88 showed a maximum expression peak at 24 h.p.i, TRIF exhibited two peaks: a minor peak at 1.5 h.p.i, and a maximum peak at 24 h.p.i (Figs 2A, S1A). Altogether, these results suggest that TLRs could play a crucial role in the recognition of DENV-PAMPs from the early hours of infection, with a peak response at 24 h.p.i.

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Fig 2. Active DENV-2 replication induces differential expression of pattern-recognition receptors and inflammatory mediators in human macrophages.

Human primary MDM cultures were either infected or not with DENV-2, as outlined in Fig 1A. Gene expression (mRNA) is presented as Log2FC heatmaps. Shown are the mRNA expression profiles of: (A) Toll-like receptor signalling components. (B) cytoplasmic viral RNA sensors. (C) NOD-like receptors and inflammasome components. (D) NF-kB complex and IRFs. (E) cytokines and growth factors. (F) chemokines. DEGs were identified using DESeq2, with a p-value < 0.05, and |Log2 Fold Change (DENV-2-infected MDMs vs Control MDMs) | > 0.6.

https://doi.org/10.1371/journal.pntd.0013366.g002

Next, we evaluated the expression of various PRRs involved in recognizing nucleic acids, including viral RNA sensors in DENV-2-infected MDMs (Figs 2B, S1B). mRNA levels of viral RNA sensors, including RIG-I, Melanoma differentiation-associated protein 5 (MDA5), IFIT1, PKR, and other RNA sensors increased following DENV-2-RNAs replication in MDMs, with their expression peaking at 24 h.p.i (Figs 2B, S1B). The expression pattern of viral RNA sensors was consistent with DENV-2-RNA replication observed in infected MDMs (Fig 1E). These findings suggest that the induction of RNA sensors, like to TLRs or RIG-I, may play a key role in detecting DENV-RNAs and promoting innate immune antiviral responses in infected MDMs.

DENV-2 infection promotes a dynamic transcriptional induction of NOD-like receptors and inflammasomes signaling in human macrophages

Previous reports showed that DENV infection promotes NLRP3-inflammasome complex activation in different cell populations to induce the proteolytic maturation of pro-IL-1β and pro-IL-18 into IL-1β and IL-18, respectively [59,60]. Both interleukins play a key role in dengue immunopathogenesis, and high IL-1β serum levels correlate with the severity of dengue in patients [10,18,33]. However, the dynamics of inflammasome transcription at early and late stages in DENV-infected macrophages are poorly understood. DENV-2 infection upregulated the expression of NLRP3-inflammasome components, including NLRP3, as well as pro-IL1β and pro-IL18 with an initial peak of mRNA expression at 1.5 and 3 h.p.i, followed by a second peak at peak at 24 h.p.i (Figs 2C, S1C). Additionally, DENV-2 infection upregulated the mRNA expression of other NLRs, including NOD1, and NLRC5, all of which had mRNA expression peak at 24 h.p.i (Figs 2C, S1C). Next, we determined the mRNA expression of essential components of NLRP3 signaling pathway, Caspase 1 (CASP1) and Apoptosis-associated speck-like protein (ASC). In both infected and uninfected MDMs, high mRNA levels of ASC mRNA were observed, which decreased at 5.5 and 10 h.p.i, then increased again at 24 h.p.i. In contrast, CASP1 and Gasdermin D (GSDMD) expression remains unchanged during the eclipse period of infection but increased significantly at 24 h.p.i (Figs 2C, S1C). Together, these results suggest that priming signals for induction of NLRP3, pro-IL1β and pro-IL18 mRNA expression occur at early stages during the eclipse period of DENV-2 replication in MDMs. However, the induction of effector molecules involved in inflammasome activation, including CASP1 and GSDMD, occurs later in the infection, coinciding with high levels of DENV-2 replication.

DENV-2 infection promotes a dynamic transcriptional modulation of NF-kB complex and interferon-regulatory factors in human macrophages

Activation of PRRs triggers signaling pathways that lead to the activation of transcription factors, including the Nuclear factor kB (NF-kB) complex and Interferon-regulatory factors (IRFs) [42,57]. Those, in turn, promote the production of cytokines, chemokines, and/or interferons, all of which contribute to effective inflammatory and antiviral responses [42,57]. Our transcriptomic analysis showed that DENV-2 infection promotes a dynamic modulation of NF-kB-complex components, including NF-kB1, NF-kB2, RELA, and IκBα (Figs 2D, S1D). NF-kB1 and NF-kB2 showed a maximum expression peak during the early stage of infection (3 h.p.i), followed by a decrease to a minimum at 10 h.p.i, before increasing again at 24 h.p.i. RELA significantly increase its expression at 24 h.p.i (Figs 2D, S1D). In contrast, IkBα, a negative regulator of NF-kB, was significantly induced early in the infection (1.5 h.p.i) and again at a later stage (24 h.p.i; (Figs 2D, S1D).

Like the NF-kB complex, DENV-2 infection promotes dynamic modulation of interferon-regulated factors (IRFs). Although IRF1, IRF7, and IRF8 exhibited a peak of maximum expression at 24 h.p.i, IRF1 and IRF8 mRNA levels also increased during the first 3 h.p.i before decreasing to their lowest levels between 5.5 and 10 h.p.i (Figs 2D, S1D). Together, these results suggest that DENV-2 infection induces two expression peaks for the NF-kB-complex and IRFs in infected MDMs. This modulation may be dependent on the recognition of structural PAMPs present in DENV-2 particles during the early hours of infection or, later, the recognition of DENV-2 replication intermediates (dsRNA), viral genome (ssRNA), and/or viral proteins during the peak of active viral replication and viral release.

DENV-2 infection of human macrophages promotes dynamic and robust NF-kB- and STAT1-dependent pro-inflammatory responses

In line with results presented in Fig 2D, DENV-2 infection promotes a dynamic transcription of NF-kB-target genes in MDMs, including Tumoral necrosis factor-α (TNFα), Interleukin 6 (IL6), and IL10 (Figs 2E, S1E). TNFα and IL6 exhibited two peaks of transcription, one at 1.5 h.p.i, and another at 24 h.p.i, whilst IL10 increased slightly at 1.5 h.p.i, and then drastically decreased from 3 to 24 h.p.i, suggesting that active DENV-2 replication negatively regulated IL10 gene transcription in infected MDMs. Furthermore, we found that DENV-2 infection induced the expression of different members of the IL-12 family of cytokines in MDMs (Figs 2E, S1E). While the IL-12p70 subunit, IL12p35, was not expressed during the first 10 h.p.i, its transcription reached a maximum peak at 24 h.p.i. IL12p40, however, showed an initial expression peak at 3 h.p.i, with a maximum expression peak at 24 h.p.i (Figs 2E, S1E). Similarly, IL23p19, one of the subunits of IL-23 (IL23p19/IL12p40), reached its maximum expression peak at 3 h.p.i, then decreased drastically with a lower peak at 10 h.p.i, and increased slightly at 24 h.p.i (Figs 2E, S1E). These results suggest a dynamic regulation in the induction of IL-12 family subunits during DENV-2 infection in MDMs.

Interestingly, DENV-2 infection induced mRNA expression of different growth factors in MDMs, including Macrophage colony-stimulating factor (M-CSF) and Vascular endothelial growth factor-α (VEGFα), which reaching their expression peak at 3 h p.i, with lower levels observed at 24 h.p.i (Figs 2E, S1E). Moreover, we observed that DENV-2 infection in MDMs induced a significant increase in the mRNA expression of STAT1-dependent pro-inflammatory factors, including IL7, IL15, TNF-related apoptosis-inducing ligand (TRAIL), and B cell activating factor (BAFF) (Figs 2E, S1E). The mRNA expression of these genes reached their expression peak in the later stages of infection (24 h.p.i), with low levels observed during the eclipse period of infection, suggesting that induction of those inflammatory factors is dependent on active viral replication in MDMs.

Regarding CC/CXC-chemokines, DENV-infected MDMs exhibited distinct patterns. CCL2 mRNA expression decreased at 5 h.p.i, followed by upregulation and reaching a peak at 24 h.p.i (Figs 2F, S1F). CCL4 mRNA was highly induced during the first hours of infection (1.5 to 3 h.p.i), and peaked again at 24 h.p.i (Figs 2F, S1F). CXCL1 and CXCL8/IL8 mRNA levels reached their highest peak at 3 h.p.i, with the lowest expression levels observed at 10 h.p.i, remained low until 24 h.p.i (Figs 2F, S1F). Moreover, STAT1-dependent chemokines including CCL5, CCL8, CXCL10, and CXCL11 showed a slight increase during the early stage of infection, the most significant increase occurred after 10 h.p.i, peaking at 24 h.p.i (Figs 2F, S1F). CXCL9 was not expressed during the first 10 h.p.i, reaching its maximum expression peak at 24 h.p.i. Together, these results suggest that recognizing structural DENV-2-PAMPs during the eclipse period of DENV-2 infection promotes robust NF-kB-dependent pro-inflammatory factors expression. However, later in infection (24 h.p.i), the recognition of viral replication intermediates (ss/dsRNA) and/or structural DENV PAMPs may activate both NF-kB- and STAT1-dependent pathways, potentially playing a role in the induction of cytokine storm in DENV-2-infected MDMs.

Sensing of DENV-2 RNAs promotes a dynamic, multi-type IFN-dependent antiviral response in human macrophages

As shown in Fig 1, the most prominent transcriptomic changes in DENV-2-infected MDMs coincided with the peak of viral RNA replication and induction of cytoplasmic viral RNA sensors at 24 h.p.i (Fig 2B), suggesting that the sensing of DENV-RNAs by PRRs may be a key driver of macrophage hyperactivation during infection. Supporting this hypothesis, we observed a strong and significant correlation between kinetics of DENV-2 RNA replication and induction of cellular mRNAs associated with the establishment of inflammatory and antiviral responses, including IFNs, antiviral proteins, costimulatory molecules, and other inflammatory mediators (Fig 3A). These findings indicated that PRR-mediated sensing of DENV-2 RNAs is critical for late-phase hyperactivation of innate inflammatory and antiviral pathways in infected MDMs.

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Fig 3. Active DENV-2 replication in human macrophages promotes a dynamic, multi-type IFN-dependent antiviral response.

Human primary MDM cultures were either infected or not with DENV-2, as shown in Fig 1A. Gene expression (mRNA) is presented as Log2FC heatmaps. (A) correlation analysis between DENV RNA levels and transcriptomic changes in MDMs. (B) Interferons. (C) JAK-STAT signalling components. (D) ISGs encoding antiviral proteins. (E) phagocytic receptors and macrophage effector enzymes. (F) macrophage activation markers and co-stimulatory molecules. (G) MHC class I and II molecules. DEGs were identified using DESeq2 with a p-value < 0.05, and |Log2 Fold Change (DENV-2-infected MDMs/Control MDMs) | > 0.6.

https://doi.org/10.1371/journal.pntd.0013366.g003

Interferons are key inducers of the antiviral state in the cells, promoting the expression of STAT1-dependent pro-inflammatory factors that amplify innate and adaptive immune responses [50]. In line with the significant modulation of IRFs (Fig 2D), we found that DENV-2 infection induces high and significant mRNA expression of various type I IFNs, including IFNα1, IFNα2, IFNα8, IFNα13, IFNα14, and IFNβ1, as well as type III IFNs, including IFNλ1, IFNλ2, and IFNλ3 in MDMs, but only at 24 h.p.i (Figs 3B, S2A). In contrast, both subunits of IL-27 (recently proposed by us as IFN-π, a type V IFN [50]), IFNπp28/IL27p28 and EBI3, were induced increasingly from the first h.p.i, reaching their maximum expression peak at 24 h.p.i (Figs 3B, S2A). Type II IFN was not induced at any time evaluated. These results suggest active DENV-2 replication promotes a robust multi-type IFN-dependent antiviral response in human MDMs in late stages of infection.

In line with IFN expression, DENV-2 infection does not induce significant changes in the expression profile of JAK-STAT signaling components during the early stages of infection. However, at the later stages, there is an increased mRNA expression of JAK2, JAK3, STAT1, STAT2, and STAT3, with a maximum peak observed at 24 h.p.i (Figs 3C, S2B). In the same way, mRNA expression of JAK-STAT negative regulators, including SOCS1, SOCS3, and USP18, peaked at 24 h.p.i (Figs 3C, S2B). These results suggest active DENV-2 replication promotes robust IFN-dependent JAK-STAT signaling activation in MDMs. In agreement with those results, DENV-2 infection also significantly upregulated mRNA expression of genes encoding antiviral proteins (AVPs)/ISGs, including members of APOBEC3-, GBP-, IFI-, IFITM-, MX-, OAS-, and TRIM-family, as well as Indoleamine 2,3-dioxygenase 1 (IDO1), ISG15, ISG20, Tetherin, and Viperin, all of which peak at 24 h.p.i (Figs 3D, S2C). Altogether, results confirm that active DENV-2 replication promotes a robust multi-type IFN-dependent antiviral activity, and robust STAT1-dependent pro-inflammatory response in human MDMs (Fig 2).

DENV-2 infection increases the phagocytic capacity of macrophages and promotes an adaptive immune response

The DENV-dependent cytokine storm is a complex immunopathological response in which different immune cells, including mononuclear phagocytes and T cells, play independent and complementary roles in promoting and amplifying inflammatory response in dengue patients [10,18,61]. Our results showed that DENV-2 infection induces a robust pro-inflammatory and antiviral response in human MDMs (Figs 2 and 3), promoting high expression of chemotactic factors and cytokines involved in T cell activation and differentiation (Fig 2E and 2F). These results suggest that DENV-2 infection induces macrophage activation to promote inflammatory response and antigen presentation to T cells. To explore this hypothesis, we evaluated the expression of macrophage markers associated with their activation profile and capability to promote antigen presentation to T cells.

Fig 3E notably revealed that DENV-2 infection significantly upregulated mRNA expression of genes encoding phagocytic receptors, including c-type lectin receptors such as Macrophage inducible Ca²⁺-dependent lectin receptor (MINCLE), Sialic acid binding Ig-like lectin 1 (SIGLEC1); FCγ receptors including CD16A/FCγR3A, CD32A/FCγR2A, CD64A/FCγR1A. Furthermore, scavenger receptors such as CD163 and Macrophage scavenger receptor 1 (MSR1), all of which peaked at 24 h.p.i (Figs 3E, S2D). Important, however, C-type lectin domain containing 5A (CLEC5A) mRNA expression peaks at 5.5 h.p.i, whereas CD206 was significantly induced only at 1.5 h.p.i (Figs 3E, S2D). DC-SIGN was low expressed by MDMs, and its mRNA levels were not affected by DENV infection. Additionally, DENV-2 infection also upregulated mRNA expression of macrophage enzymes involved in the induction of inflammatory response, including Cyclooxygenase 2 (COX2), which was induced during the early hours of infection (1.5 to 3 h.p.i), followed by a decrease in expression levels between 5 and 10 h.p.i, and a subsequent peak at 24 h.p.i (Figs 3E, S2D). Together, results suggest that active DENV-2 replication induces an inflammatory activation profile in MDMs, increasing their phagocytic capability.

Furthermore, DENV-2 significantly upregulated mRNA expression of genes encoding cell-surface macrophage activation markers, including AXL receptor tyrosine kinase (AXL), CD38, SLAM family member 7 (SLAMF7), all of which peaked at 24 h.p.i (Figs 3F, S2E). Moreover, DENV-2-infected MDMs significantly upregulated the expression of costimulatory molecules such as CD40, CD80, CD86, and programmed death-ligand 1 (PD-L1) with a mRNA expression peaking at 24 h.p.i (Figs 3F, S2E). In contrast, ICAM1 peaked at 3 h.p.i reaching its maximum expression at 24 h.p.i (Figs 3F, S2E). Furthermore, DENV-2 infection significantly increased the mRNA levels of 20S immunoproteasome (20S-IP) components, including Low molecular mass peptide 2 (LMP2), as well as Antigen peptide transporter (TAP) components including TAP1. These genes were exclusively transcribed at 24 h.p.i (Figs 3G, S2F). In contrast, non-significant changes were observed in the mRNA expression of HLA-A or Beta-2-microglobulin (B2M) in DENV-2-infected MDMs as compared to the control (Figs 3G, S2F). Notable, DENV-2 infection up-regulated mRNA expression of genes encoding MHC-II components, including class II major histocompatibility complex trans-activator (CIITA), CD74, HLA-DPA1, and HLA-DRA molecules, all of which peak at 24 h.p.i (Figs 3G, S2F). Together, results suggest that DENV-2-infected MDMs may contribute to antigens presentation to T cells, promoting an inflammatory macrophage activation profile and supporting the development of anti-DENV adaptive immune responses.

Validation of RNA-seq data by RT-qPCT and ELISA

To validate the RNA-Seq results, MDM cultures (n = 3) were infected with DENV-2 (MOI 5) for 1.5, 10, and 24 h.p.i, and uninfected MDMs (0) as control. The mRNA expression and protein production of selected DEG associated with inflammatory response identified by bulk RNA-Seq were measured using RT-qPCR or ELISA, respectively. The mRNA levels of TLR2 and TLR3 exhibited significantly increased expression at 24 h.p.i (Fig 4A), consistent with RNA-seq results (Fig 2). Similarly, mRNA expression of TLR7, NF-kB1 and IkBα displayed dynamic modulation, with initial peaks at 1.5 h.p.i, a decrease at 10 h.p.i, and subsequent peaks at 24 h.p.i (Fig 4A), aligning with RNA-seq results (Fig 2). However, protein levels of NF-kB-dependent pro-inflammatory factors, including TNF-α, IL-6, and CXCL-8/IL-8, were detected time-dependent, with all peaking at 24 h.p.i (Fig 4A). These findings diverged from RNA-Seq data, where mRNA expression of these factors was also evident at earlier time points (Fig 2). Together, these results suggest that the early recognition of DENV-PAMPs during the eclipse period of DENV-2 infection/replication promotes NF-kB-complex activation, driving the transcription of NF-kB-dependent pro-inflammatory factors (Fig 2E). However, based on our findings, we hypothesize that the translation and/or release of these inflammatory factors by infected cells predominantly occur later in the DENV-2 replicative cycle (Fig 4A).

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Fig 4. Experimental validation of RNA-seq results using RT-qPCR and ELISA.

Human primary MDM cultures (n = 3) were either infected or left uninfected with DENV-2 (MOI = 5). Total RNA and culture supernatants were collected at 0 (control), 1.5, 10, and 24 h.p.i., and RT-qPCR and ELISA were performed. (A) mRNA levels of NF-κB-dependent inflammatory modulators and protein production. (B) IFN-STAT1-dependent antiviral and inflammatory factors. Data are presented as mean ± standard deviation (SD). Data normality was assessed using the Shapiro-Wilk test. Statistical analysis was performed using One-way ANOVA followed by Fisher’s least significant difference post-hoc test. Significant differences between control MDMs and DENV-2-infected MDMs were defined as p-value <0.05 (*).

https://doi.org/10.1371/journal.pntd.0013366.g004

Concerning the DEG involved in the induction of antiviral response, RT-qPCR analysis confirmed significant upregulation of IFNα1, IFNβ1, IFNλ1, IFNπp28/IL27p28, and EBI3 mRNA levels, all peaking at 24 h.p.i in DENV-2-infected MDMs (Fig 4B), in agreement with RNA-seq results (Fig 3). Additionally, DENV-2 infection significantly increased transcriptional expression of genes encoding JAK-STAT signaling components, including STAT1, STAT3, SOCS1, and SOCS3, which also peaked at 24 h.p.i (Fig 4B). AVPs/ISGs such as APOBEC3A, MX2, and Viperin, along with STAT1-dependent pro-inflammatory factors like BAFF and CXCL10, reached maximum expression levels at 24 h.p.i (Fig 4B), corroborating bulk RNA-seq findings (Fig 3). Furthermore, the production of CCL-2 protein was validated in DENV-2-infected MDMs, with levels peaking at 24 h.p.i (Fig 4B). Altogether, these findings confirm the reliability of transcriptome data and demonstrate that DENV-2 infection promotes a dynamic NF-kB-dependent pro-inflammatory response, as well as a multi-type IFN-STAT1-dependent pro-inflammatory and antiviral states in human MDMs.

DENV-2-infected Dendritic cells, and monocyte subsets from acute dengue patients promote innate antiviral response and cytokine storm

Monocytes (Mon) are the primary precursors of macrophages and dendritic cells in tissues [62]. Although Mon, macrophages, and dendritic cells exhibit distinct phenotypic and functional characteristics, those populations of mononuclear phagocytes are key targets for DENV infection and play a critical role in dengue immunopathogenesis and control [14,18,27,52,53]. These cells express a broad range of PRRs essential for sensing viral infections and initiating innate pro-inflammatory and antiviral responses (57)(62) [57,62]. To better characterize transcriptomic dysregulation and indirectly confirm the biological relevance of biomarkers identified in DENV-2-infected MDMs, we reanalysed the publicly available Microarray dataset (GSE58278) [52], performed in human Monocyte-derived dendritic cells (MDDCs) infected with DENV-2 NGC, at MOI 20 for 24 h.p.i. Also, we reanalysed the publicly available bulk RNA-seq dataset (GSE176079) [53], generated from sorted classical (CM, CD14⁺⁺CD16^-), intermediate (IM, CD14⁺⁺CD16⁺), and non-classical (NM, CD14^DimCD16⁺) Mon subsets isolated from six acute dengue patients (ADP) and three healthy donors. This reanalysis aims to enhance the reliability of immunological activation state prediction in both dengue patients and in vitro models of DENV-infected macrophages and dendritic cells.

First, we evaluated the mRNA expression of activation markers in DENV-2-infected MDDCs and Mon subsets derived from ADP. As shown in Fig 5A, DENV-2-infected MDDCs only down-regulated CCR1 mRNA expression. However, IM and NM subsets from ADP significantly up-regulated CD14 mRNA expression, whereas CM exhibited significant upregulation of CD16A/FCγR3A mRNA expression compared to healthy donors. Further, IM and NM, but not CM subsets, significantly up-regulated mRNA expression of adhesion molecules, including L-selectin and ICAM1, as well as chemokine receptors involved in Mon migration from the blood into the tissues [63], including CCR1, CCR2, CXCR1, and CXCR2 (Fig 5A). Additionally, all Mon subsets from ADP showed significant upregulation of mRNA levels for phagocytic receptors, also highly expressed by macrophages (Fig 5A), including SIGLEC1, CD64A and MSR1 (Fig 5B). In contrast, MINCLE and CD163 mRNA expression was selectively upregulated in IM and NM subsets, while CD32A expression remained unchanged (Fig 5B). However, Mon subsets from ADP did not modulate CD206 and DCs activation marker DC-SIGN mRNA (Fig 5B). These results suggest that DENV infection may drive the migration of IM and NM subsets from the bloodstream into tissues, and their differentiation to macrophages during the acute phase of DENV infection in humans. This implies that those subsets of Mon could play pivotal roles in systemic DENV dissemination. Interestingly, DENV-2-infected MDDCs did not modulate the expression of c-type lectin receptors and scavenger receptors as Mon (Fig 5B) and MDMs (Fig 3A) but significantly upregulated FcγR, including CD32A and CD64A, suggesting that Mon subsets and MDMs induced a higher phagocytic activity during DENV infection as compared with MDDCs.

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Fig 5. DENV-2-infected dendritic cells and monocyte subsets from acute dengue patients modulated PRR expression and promoted a robust NF-kB-dependent inflammatory response.

We reanalysed publicly available Microarray data from human Monocyte-derived dendritic cells (MDDCs) infected with DENV-2 (MOI 20) for 24 hours. n = 3. In addition, we reanalysed a publicly available bulk RNA-seq dataset generated from sorted classical, intermediate, and non-classical Mon subsets isolated from six acute dengue patients (ADP) and three healthy donors. Gene expression (mRNA) is presented as Log2FC. The mRNA expression profiles included the following categories: (A) Monocyte activation markers. (B) Phagocytic receptors. (C) TLR signalling components. (D) viral RNA sensors. (E) Inflammasome components. (F) NF-kB complex and IRFs. (G) NF-kB-dependent inflammatory factors. (H) Interferons. (I) JAK-STAT signalling components. (J) ISGs encoding antiviral proteins. (K) STAT1-dependent inflammatory factors. DEGs were identified using GEO2R, with a p-value < 0.05 and |Log2 Fold Change (DENV-infected cells vs unstimulated cells) | > 0.6 (dotted line).

https://doi.org/10.1371/journal.pntd.0013366.g005

Consistent with RNA-seq results from DENV-2-infected MDMs, both IM and NM subsets from ADP exhibited significant upregulation of TLR2 and TLR4 mRNA levels (Fig 5C). However, non-significant changes were observed in TLR7 and TLR8 mRNA expression, whilst TLR3 mRNA expression displayed non-significant yet considerable variability among patients. Notably, both CM and IM subsets exhibited upregulation of MyD88 mRNA, whereas TRIF mRNA levels were significantly increased across all Mon subsets (Fig 5C). It is worth noting that DENV-2-infected MDDCs upregulated only TLR8 mRNA. Moreover, in line with DENV-2-infected MDMs results (Fig 2B), the induction of viral RNA sensors involved in sensing DENV-RNAs, including RIG-I, MDA5, LGP2, DDX60, IFIT1, and PKR mRNA levels were upregulated by DENV-2-infected MDDCs and across all Mon subsets derived from ADP (Fig 5D). These results suggest that MDDCs and various Mon subsets established a robust antimicrobial state to counter DENV replication through upregulation of PRRs involved in sensing DENV-PAMPs, consistent with findings in DENV-2-infected MDMs (Fig 2).

Furthermore, transcriptomic analysis of DENV-2-infected MDDCs and Mon subsets from ADP revealed differential modulation of NLRP3-inflammasome components. While DENV-2-infected MDDCs significantly upregulated NOD2 and CASP1 mRNAs, IM and NM subsets significantly upregulated NLRP3 mRNA levels. Still, CM and NM subsets exhibited significant increases in ASC mRNA expression (Fig 5E). Notably, however, MDDCs and all Mon subsets displayed significant increases in IL1β and IL18 mRNAs levels in response to DENV infection (Fig 5E). Only CM showed a slight increase in GSDMD mRNA expression (Fig 5E). These findings suggest a differential regulation of inflammasome components during DENV infection in MDDCs and Mon subsets. In addition, consistent with RNA-seq data from DENV-2-infected MDMs, DENV-2-infected MDDCs and IM and NM subsets from ADP showed significant increases in mRNAs expression of NF-kB-complex and IRFs components, including NF-kB2 and RELB, whilst IkBα and IRF7 mRNA expression was significantly upregulated across all Mon subsets and MDDCs (Fig 5F). While MDDCs, CM and IM subsets demonstrated significant increases in IRF1 mRNA levels in response to DENV infection, NF-kB1, RELA, and IRF3 mRNA levels were significantly induced in DENV-2-infected MDDCs but remained unaltered across Mon subsets (Fig 5F). Moreover, DENV-2-infected MDDCs and all Mon subsets from ADP exhibited significant upregulation of NF-kB-dependent inflammatory factors, including cytokines such as TNFα, IL6, IL10, and M-CSF, as well as chemokines including CCL4, CXCL1, and CXCL8/IL8 (Fig 5G), as was observed in DENV-2-infected MDMs (Fig 2). These results suggest that all population of mononuclear phagocytes, including Mon subsets, dendritic cells, and macrophages play a key role in sensing DENV infection to promote robust and systemic NF-kB-dependent inflammatory responses.

Notably, DENV-2-infected MDDCs significantly upregulated the expression of different types of IFNs, including IFN-I (IFNα2, IFNβ1, and IFNε), IFN-III (IFNλ1), and both subunits of IFN-V (IFNπp28/IL27p28 and EBI3) (Fig 5H), suggesting that like DENV-2-infected MDMs (Fig 4), MDDCs induce a multi-type IFN-dependent antiviral response to DENV infection. However, Mon subsets from ADP exhibited considerable variability and non-significant changes in IFNα2 and IFNβ1 mRNA levels, without induction of IFN-III (Fig 5H). Important however, all Mon subsets from ADP showed significant expression of IFN-π/IL-27 subunits (IFNπp28/IL27p28 and EBI3) (Fig 5H). In line with those results, DENV-2-infected MDDCs upregulated the expression of JAK-STAT signaling components involved in IFN-I/IFN-III/IFN-V signaling transduction, including JAK2, STAT1, STAT2, STAT3, SOCS3 and USP18 (Fig 5I). However, all Mon subsets from ADP only upregulated mRNA expression of STAT1, SOCS1, and USP18 (Fig 5I). In contrast, STAT3 and SOCS3 mRNA expression was induced only in IM and NM subsets (Fig 5I). In agreement with IFNs expression, DENV-2-infected MDDCs and all Mon subsets from ADP upregulated mRNA levels of antiviral ISGs/AVPs, including GBP1, IFITM1, IDO1, ISG15, MX1, OAS1, TRIM22, and Viperin (Fig 5J), indicating the induction of a robust antiviral state. Additionally, DENV-2-infected MDDCs and all Mon subsets significantly upregulated STAT1-dependent inflammatory factors, including cytokines such as IL15, TRAIL, and BAFF, along with chemokines CCL8, CXCL10, and CXCL11 (Fig 5K). However, CCL2 mRNA was upregulated only by Mon subset, whereas CCL5 mRNA was induced by DENV-2-infected MDDCs and NM subset from ADP (Fig 5K). These results confirm that DENV infection in both MDDCs and Mon subsets activate IFN-dependent JAK-STAT signaling and induce robust STAT1-dependent pro-inflammatory and antiviral responses, as was observed in DENV-2-infected MDMs (Fig 3)

Together, these results showed that in vitro DENV-2-infected MDDCs and all Mon subsets from ADP play a pivotal role in promoting both NF-kB- and IFN-STAT1-dependent inflammatory responses. This aligns with findings in DENV-2-infected MDMs (Figs 3 and 4), confirming the key role of all mononuclear phagocytes in promoting cytokine storm and immunopathogenesis during DENV infection in humans, both in vitro and in vivo. The results are summarized in Fig 6.

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Fig 6. DENV-2 infection triggers a robust innate immune response in human MDMs via multiple PRRs, NF-κB, and IFN-STAT1 signaling pathways.

(A) Human MDMs express receptors that facilitate DENV entry. (B) Early during infection, TLR2 and TLR7 recognized DENV-RNA and other PAMPs, initiating NF-κB-dependent inflammatory responses. (C) Following the eclipse phase, active viral replication leads to immune hyperactivation, characterized by upregulation of multiple PRRs, including TLRs, NLRs, and viral RNA sensors, further amplifying inflammation. (D) DENV-2-infected MDMs activate multiple IFN pathways, producing type I/III and V IFNs, which trigger STAT1-mediated activating response, including ISGs and STAT1-dependent inflammatory factors.

https://doi.org/10.1371/journal.pntd.0013366.g006

Limitations of the study

This study has several limitations:

• Although we used the highly pathogenic DENV-2 NGC strain as a model of severe DENV infection in MDMs, it remains to be determined whether the immune response elicited by MDMs in response to this strain is comparable to that induced by other DENV serotype or by clinical isolates from dengue patients, which may better reflect the viral fitness and immune evasion strategies of currently circulating strains.
• Most severe DENV-2 infections in humans occur during secondary infections, in which antibody-dependent enhancement (ADE) mediated by non-neutralizing heterotypic anti-DENV antibodies plays a central role in pathogenesis by promoting hyper-infection of mononuclear phagocytes through FCγ receptors (CD32, CD16). However, this study did not assess the impact of ADE on cytokine storm induction in DENV-2-infected MDMs. Although mononuclear phagocytes are key contributors to dengue immunopathogenesis, other immune cells, such as B cells, T cells, and NK cells, may also actively participate in the development of the cytokine storm. Further investigation is needed to evaluate the role of these immune subsets in the broader inflammatory response to DENV infection.