Vitamin D enhances antiviral responses in dengue virus-infected macrophages by modulating early-response gene expression

Yordi Sebastián Tamayo-Molina; Juan Felipe Valdés-López; Geysson J Fernandez; Silvio Urcuqui-Inchima

doi:10.1371/journal.pone.0330751

. 2025 Aug 21;20(8):e0330751. doi: 10.1371/journal.pone.0330751

Vitamin D enhances antiviral responses in dengue virus-infected macrophages by modulating early-response gene expression

Yordi Sebastián Tamayo-Molina ^1,^#, Juan Felipe Valdés-López ^1,^#, Geysson J Fernandez ², Silvio Urcuqui-Inchima ^1,^*

Editor: Helene Minyi Liu³

PMCID: PMC12370076 PMID: 40839599

Abstract

Dengue virus (DENV), the etiological agent of dengue fever, remains a global health concern, leading to severe illness and death in the absence of any definitive cure. Research has shown that vitamin D may reduce DENV replication in vitro and that dengue patients with low or deficient vitamin D levels are at higher risk of severe dengue. Studies indicate that viral replication is inhibited in human monocyte-derived macrophages (MDM) differentiated in the presence of vitamin D (D3MDM), suggesting that vitamin D may prevent DENV entry into host cells. However, despite these findings, the role of vitamin D in regulating the temporal expression patterns of genes as early, mid, and late transcriptional profile of DENV-infected macrophages remains unclear. Therefore, utilizing a kinetic transcriptomic profile is crucial. This approach provides detailed insights into the dynamic changes in gene expression over time, helping to clarify how vitamin D can modulate the immune response at critical stages of DENV infection. To address the transcriptional dynamics, we conducted a comprehensive analysis of gene expression patterns in MDM and D3MDM infected with Dengue virus serotype 2 (DENV-2). Utilizing bulk RNA sequencing alongside a standard viral growth curve, we systematically analyzed transcriptional kinetics by selecting key time points: 1.5, 3, 5.5, and 10 hours post-infection (h.p.i.) to monitor early viral entry and replication events and 24 h.p.i. to assess gene expression during peak viral particle production. Our temporal analysis revealed a progressive increase in cellular transcripts within the first hour of infection, with a more pronounced gene expression pattern in DENV-2-infected MDM compared to DENV-2-infected D3MDM at this early stage. Enrichment analysis indicated a reduced inflammatory response in DENV-2-infected D3MDM. Additionally, transcription factor analysis suggested diminished NF-κB signaling, but enhanced IRF5 activity was elevated in the DENV-2-infected D3MDM. High-dimensional clustering analysis identified nine unique gene clusters across both macrophage types, with notable upregulation of genes associated with antiviral activity, including IDO1, ISG20, OASL, IFI44L, RSAD2, IFIT1, MX1, EPSTI1, CXCL10, and CXCL11 in DENV-2-infected D3MDM at 1.5 h.p.i., suggesting an enhanced early antiviral response. These findings indicate that vitamin D modulates the magnitude and diversity of the early transcriptional responses, highlighting its potential as a therapeutic option to mitigate DENV severity.

Introduction

Dengue virus (DENV) is a vector-borne flavivirus transmitted by the Aedes mosquito. According to the World Health Organization (WHO), DENV has been a global public health concern since the 1960s. It remains prevalent in the Caribbean, Latin America, Southeast Asia, and the Pacific Islands [1]. DENV is the etiological agent of dengue fever (DF), or severe forms of the disease known as severe dengue (SD). The physiopathology of SD is multifactorial, but some individuals have a higher risk of developing severe symptoms, such as in cases of heterotypic infection and individuals with co-morbidities, including obesity and diabetes [2–4]. Despite the socioeconomic and public health impact of dengue outbreaks, no antiviral therapy for dengue is currently available. Therefore, further research is urgently needed to develop drugs or treatments to manage DENV infection and prevent the progression of dengue with clinical warning signs.

In the immunopathogenesis of dengue, tissue-resident macrophages are susceptible and permissive to dengue viral replication. Macrophages can detect DENV via pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs). Specifically, surface TLR2, along with its co-receptors CD14 and TLR6, has been documented as an innate sensor of DENV [5,6]. In addition, endosomal TLRs such as TLR3, TLR7, TLR8, and cytosolic RLRs, including RIG-I and melanoma differentiation-associated gene 5 (MDA5), recognize DENV viral mRNA [7]. Stimulation of nuclear factor kappa B (NF-κB)- and interferon regulatory factors (IRFs)-dependent gene transcription is a central aspect of inflammatory and antiviral activation by TLRs and RLRs [8]. The NF-κB pathway is essential for generating an inflammatory response in macrophages, including the production and secretion of cytokines, chemokines, and the release of antimicrobial peptides [9]. The IRFs, including IRF1, IRF3, IRF5, and IRF7, have been shown to coordinately regulate type I/II IFNs responses, as well as expression of IFNs stimulated genes (ISG), thereby establishing an antiviral state during DENV infection [10,11]. The establishment of antiviral state, along with the recruitment and activation of inflammatory cells are essential components of the host defense against viral infections. However, DENV can subvert these antiviral pathways, delaying the host’s defense mechanism and amplifying viral replication, which may lead to an excessively vigorous response that can be detrimental, potentially causing plasma leakage, organ dysfunction and increasing the risk of mortality [12]. Clinical data have shown an association between levels of CC chemokines in plasma and the severity of dengue [13,14]. Further, inflammatory chemokines have indeed been implicated as a contributor to the pathogenesis and severity of DENV infection [15–17]. Therefore, modulating DENV-induced chemokines may potentially prevent excessive inflammation and the induction of the so-called cytokine storm [18–20].

In this context, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), hereafter referred to as Vitamin D, has emerged as a prophylactic and a good treatment against viral infections, including DENV. Recent studies have identified a link between low vitamin D levels and heightened infection susceptibility [21]. Furthermore, it has been reported that daily supplementation with 4000 international units of Vitamin D decreased the susceptibility of monocyte-derived dendritic cells (MDDC) and monocyte-derived macrophages (MDM) to DENV-2 infection [22,23]. In addition, MDM differentiated in the presence of vitamin D (D3MDM) exhibit reduced surface expression of macrophage mannose receptor (MMR/CD206), thereby restricting DENV entry [24]. Moreover, D3MDM show higher expression of antiviral proteins and antimicrobial peptides, which interfere with DENV-2 [25,26] and Zika virus replication [27]. RNA-Seq and miRNA-Seq analysis of D3MDM revealed that Vitamin D enhances the immune response of macrophages by modulating a regulatory network of microRNAs and mRNAs, which plays a critical role in orchestrating the transcriptional reprogramming necessary to combat viral infections [28]. These antiviral effects fine-tune the immune response, ensuring that macrophages can efficiently control viral replication while avoiding excessive inflammation that could lead to severe outcomes.

The experiments using transcriptional kinetics of macrophages in response to lipopolysaccharide exposure or coronavirus infection have revealed significant early, mid, and late gene responses, underscoring the dynamic regulation of transcriptional profiles [29,30]. On the other hand, vitamin D target genes, in a time course analysis in mononuclear cells, are classified into primary target genes (4–8 h) and secondary targets (24–48 h) [31]. Accordingly, studying the transcriptional kinetics provides a framework to investigate how the temporal transcriptional response of vitamin D target genes, segregated into early, mid, and late targets, may not only mitigate inflammation but also enhance the dynamics of the macrophage responses against dengue virus.

Given the predominant role of Vitamin D in limiting virus replication, this study aims to utilize bulk RNA-Seq to analyze MDM and D3MDM infected with DENV-2. To achieve this, we used a standard viral growth curve to examine the early, mid, and late stages of infection in MDM and D3MDM. Our results show that in D3MDM, predicted early IRF5 transcription activity leads to the expression of a subset of ISGs that can restrict viral replication, which results in reduced early expression of genes involved in the pro-inflammatory response. We propose that, in D3MDM, vitamin D restricts early viral replication, thereby dampening the initial inflammatory response.

Materials and methods

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

DENV-2 stock was obtained by growing the virus in Aedes albopictus-derived C6/36-HT cells (ATCC) at a multiplicity of infection (MOI) of 0.01. C6/36 HT cells 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 harvested, stored at −80 °C, and titrated by plaque assay on BHK-21 cells (clone 15, ATCC).

Culture of primary human monocytes and differentiation into monocyte-derived macrophages in the presence (D3MDM) or absence (MDM) of vitamin D

Human peripheral blood mononuclear cells (PBMCs) from leukocyte-enriched blood units of healthy donors were isolated 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 PBS 1X (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 human monocytes, 24-well plastic plates were scratched with a 1000 μL pipette tip and seeded with 5x10⁵ CD14 positive cells per well to allow its adherence during 2 h in RPMI-1640 medium (Sigma-Aldrich) supplemented with 0.5% autologous serum, 4 mM L-glutamine, and 0.3% NaCO3 and incubated at 37°C and 5% CO₂. Non-adherent cells were removed by washing twice with 1X PBS. Monocytes were cultured in RPMI-1640 medium supplemented with 10% FBS, 4 mM L-glutamine, 0.3% NaCO3, and 1% antibiotic-antimycotic solution (100X), either with (D3MDM) or without (MDM) 0.1 nM or 1 nM of vitamin D. The cells were incubated at 37°C and 5% CO₂ for 6 days to differentiate into MDMs, with fresh media added to the cultures every 2 days (S1 Fig).

In vitro DENV-2 infection of monocyte-derived macrophages

MDM and D3MDM were infected with DENV-2 strain from New Guinea at an MOI of 5 in serum-free RPMI-1640. The samples were incubated at 37 °C for 1.5 hours, after which the cells were washed with 1X PBS to remove unbound virus, and a fresh complete medium was added. The cells were then incubated at 37 °C and 5% CO₂. Culture supernatants and cell lysates were collected at 1.5-, 3-, 5.5-, 10-, and 24-hour post-infection (h.p.i.) and stored at −80 °C (see S1 Fig).

RNA extraction and cDNA synthesis

Total RNA was extracted using the Direct-zol™ RNA Miniprep Plus kit (Zymo Research, California, USA), following the manufacturer’s protocol. RNA samples were treated with a DNase I column (Zymo Research) to remove contaminating genomic DNA. RNA concentration was measured using Nanodrop spectrophotometry (Thermo Scientific, Massachusetts, USA). For bulk RNA-Seq, 1 μg of RNA was used per sample, and sequencing was performed on an Illumina HiSeq 2000 platform (Macrogen, Seoul, South Korea). After sequencing, the image data were converted into raw reads and stored in FASTQ format for each sample, with quality assessment using FastQC [6].

Bulk RNA-Seq of DENV-infected macrophages

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 dengue virus type 2 [NC_001474]) using HISAT2 software [7]. The new transcripts were then assembled using StringTie software [8], and using the feature Counts tool in Subread software [9], the raw count number in each sample was obtained. Data was stored in the Gene Expression Omnibus (GEO) repositories under accession number: GSE297386.

Data annotation and batch effect correction

The raw counts were processed using the following workflow in R software (version 4.2.0) [10]. First, gene rows were annotated with their respective genotype (non-coding gene, pseudogene, and protein-coding gene), symbols, and Entrez IDs. We then filtered the dataset to include only protein-coding genes and generated a list of their lengths using the Homo.sapiens library [11]. Batch effect correction of the raw count data for each transcriptome was performed using ComBat-Seq [12].

Analysis of differentially expressed genes

To identify the top differentially expressed genes (DEGs), we selected genes with a false discovery rate (FDR) < 0.05 and |Log₂ Fold Change (FC) (treated/untreated) > 1. Multidimensional scaling (MDS) analysis plot was performed using Glimma [14], and Pearson correlation between samples was calculated (see S2A Fig). Gene set enrichment analysis (GSEA) for overall genes regulated in MDM and D3MDM were performed using the ClusterProfiler package (Version 4.14) [15], while transcription factor analysis was performed with the TFactS database (Version 0.99) [16] (see S2B Fig). Unique DEGs between the two transcriptomes were identified, followed by GSEA using ClusterProfiler (Version 4.14) [15] and transcription factor analysis with iRegulon (Version 1.3) [17] (see S2C Fig).

Statistical analysis

Statistical analyses and figure generation were performed using R statistical software. Anova was used to compare treatments between the groups, and for multiple comparisons, Student’s T-test with Bonferroni correction was applied. For RNA-seq statistics, we use DESeq2 library [13], and to enhance data visualization, we utilized ggplot2 (Version 3.5) [32], ggprism (Version 1.0) [33], and ComplexHeatmap (Version 2.22) [34].

Results

The expression of genes in the VDR signaling pathway and their target genes depend on vitamin D concentration

Macrophages are key target cells for DENV infection and are crucial in orchestrating the innate immune response to control viral replication. In this study, we performed a comparative analysis of macrophages differentiated in the presence of physiological doses of vitamin D (D3MDM) or not (MDM) and infected with DENV-2 serotype 2 New Guinea strain (MOI: 5). The concentration of vitamin D used in this study (0.1 nM) was selected to mimic physiological levels of circulating 1,25(OH)₂D₃, which typically range between 0.04 and 0.150 nM in serum. This concentration also corresponds to the high-affinity dissociation constant (Kd = ~0.1 nM) of 1,25(OH)₂D₃ for the VDR [35]. Furthermore, we previously reported that macrophages differentiated in the presence of 0.1 nM vitamin D (D3MDM) exhibited reduced susceptibility to DENV infection [24,25]. As shown in Fig 1A, D3MDM were consistently less susceptible to DENV-2 infection than MDM. The percentage of inhibition reached approximately 35% and 45% in D3MDM differentiated with 0.1 nM (D3[0.1 nM]MDM) and 1 nM (D3[1 nM]MDM) of vitamin D, respectively, compared to MDM at 24 h.p.i. (Fig 1B). It is worth noting that the antiviral effect of vitamin D appears to be dose dependent.

Fig 1 — **(A)** Plaque assay of MDMs differentiated in the presence of 0.1 nM (D3[0.1 nM]MDM) or 1 nM (D3[1 nM]MDM) of vitamin D and infected with DENV-2 at 24 h.p.i. **(B)** Percentage of viral inhibition in D3[0.1 nM]MDM and D3[1 nM]MDM compared to MDMs. **(C)** Number of differentially expressed genes in uninfected D3[0.1 nM]MDM and D3[1 nM]MDM. (D) Overrepresentation analysis of enriched signaling pathways. **(E)** Common and unique VDR-regulated genes in D3[0.1 nM]MDM and D3[1 nM]MDM. Data are presented as mean ± standard deviation.

To further gain a better understanding of the effect on the vitamin D signaling pathway, we analyzed the VDR signaling pathway using our bulk RNA-Seq datasets of MDM differentiated with a physiological dose of vitamin D (D3[0.1 nM]MDM), and compared them to publicly available transcriptomic data from MDM differentiated with 1 nM vitamin D ((D3[1 nM]MDM; GSE209698). The DEGs analysis revealed 16 upregulated and 43 downregulated genes in D3[0.1 nM]MDM, while 100 genes were upregulated and 56 downregulated in D3[1 nM]MDM (Fig 1C). Gene overrepresentation analysis using WikiPathways showed that both concentrations induced the expression of genes associated with the vitamin D receptor pathway (WP2877), including CLMN, G0S2, CD14, TREM1, and CAMP, which were regulated in both concentrations (Fig 1D). In contrast, the higher vitamin D concentration also induced the expression of additional genes, such as CYP24A1, MYC, ALOX5, HILPDA, and S100A8 (Fig 1E). These findings indicate that 0.1 nM of vitamin D is insufficient to drive notable transcriptional changes, whereas 1 nM exerts a more pronounced effect on VDR-regulated genes.

Analysis of differentially expressed genes during early and late stages of DENV-2 infection in macrophages differentiated in the presence of vitamin D

To obtain a broad picture of the MDM and D3MDM response to DENV-2 infection, bulk RNA-Seq was used to analyze differential gene expression at the mRNA level. The RNA was isolated at 1.5-, 3-, 5.5-, 10- and 24- h.p.i. and converted into cDNA libraries for sequencing. Two biological replicates were processed for each condition. The early time points (1.5-, 3-, and 5.5- h.p.i), and the mid time point (10 h.p.i). were selected to capture kinetic changes in the transcriptional profile associated with early viral entry and replication, independent of the effects of infectious virus release. The 24 h.p.i. time point was chosen as the late time of infection, corresponding to the assembly and release of new viral progeny. Gene counts of viral mRNA indicated a delay in viral replication at the mid time point (10 h.p.i.) in D3MDM compared to MDM (Fig 2A). However, a slightly higher count of viral mRNA was observed at earlier time points (1.5-, 3-, and 5.5- h.p.i) in D3MDM compared to MDM. Interestingly, there were no differences in the count of viral mRNA between the two macrophage types at 24 h.p.i. (Fig 2A). This suggests that vitamin D3 may have a differential effect on viral replication kinetics during the early- and mid-stages of DENV-2 infection.

Fig 2 — **(A)** Kinetics of DENV-2 genome copies over time. **(B)** Principal component analysis (PCA) showing the transcriptional trajectories of MDM (blue) and D3MDM (green) at 0-, 1.5-, 3-, 5.5-, and 10-hpi. **(C)** PCA for the early (0 h) and late (24 h) time points. **(D)** Number of upregulated and downregulated genes over time in MDMs and D3MDMs. **(E-I)** Shared and unique differentially expressed genes at 1.5- **(E)**, 3- **(F)**, 5.5- **(G)**, 10- **(H),** and 24-hpi **(I)**. Data are presented as mean ± standard deviation.

Next, we investigated the time course of macrophage transcriptomic responses to DENV-2 infection. To analyze the transcriptome data, the transcripts corresponding to protein-coding genes were subjected to principal component analysis (PCA) to identify changes in gene signatures (see S3A Fig). A PCA plot generated from differentially expressed genes (DEGs) revealed that at early- and mid-stages of infection (1.5-, 3-, 5.5-, and 10- h.p.i.), the principal components (PC) 1 and 2 accounted for 53% and 25% of the total variance, respectively (Fig 2B). However, a major transcriptional shift occurred at 24 h.p.i, reflected by a dramatic change in the variance; PC1 explained 94% of the variance, while PC2 explained only 6% (Fig 2C).

To define the top DEGs, we selected genes with a p-adj < 0.05 and |Log₂ FC (Infected MDM or D3MDM/Uninfected MDM or D3MDM) | > 1. Results show that MDM exhibited more DEGs than D3MDM at all times of infection evaluated, except at 24 h.p.i. A total of 2.941 genes exhibiting differential expression were identified following DENV-2 infection of MDM, with 1.953 upregulated and 988 downregulated genes. Specifically, at 1.5-, 3-, 5.5-, 10-, and 24- h.p.i, there were 82, 255, 215, 149, and 1.252 upregulated genes, while 3, 45, 122, 108, and 710 were downregulated genes, respectively (Fig 2D). In the same way, a total of 2.140 DEGs were identified following DENV-2 infection of D3MDM, with 1.553 upregulated and 584 downregulated. At 1.5-, 3-, 5.5-, 10-, and 24- h.p.i, 63, 132, 89, 29, and 1.240 genes were upregulated, while 5, 13, 6, 0, and 560 genes were downregulated, respectively (Fig 2D). Moreover, based on the distinct transcriptional trajectories observed in the PCA, correlation analysis between MDM and D3MDM suggests that early infection DEGs are highly correlated and time-dependent (see S3B Fig).

We compared the number of DEGs between macrophages to screen for a common or distinct transcriptional signature. Both macrophage types (MDM and D3MDM) exhibit a shared core transcriptional response following DENV-2 infection, regulating 48 (Fig 2E), 130 (Fig 2F), 84 (Fig 2G), 28 (Fig 2H), and 1.474 (Fig 2I) common genes at 1.5-, 3-, 5.5-, 10-, and 24- h.p.i, respectively. Furthermore, DENV-2-infected MDMs exhibited a more diverse response, with 39, 166, 252, 228, and 487 uniquely expressed genes compared to 22, 15, 11, 1, and 327 genes in D3MDM at the times of infection evaluated (Fig 2E-I). Overall, these results showed a higher gene expression pattern in DENV-2-infected MDM than in DENV-2-infected D3MDM at the early- and mid-stages of infection, with a delayed DENV-2 viral mRNA replication.

Vitamin D shapes the gene expression in DENV-2-infected macrophages

Gene set enrichment analysis (GSEA) using clusterProfiler revealed distinct transcriptional signatures in DENV-2-infected MDM and D3MDM (Fig 3A). GSEA of DEGs found in DENV-2-infected macrophages identified 32 enriched active GO functions. However, the set of genes associated with these GO terms showed significant differences between the two groups. In both macrophage types, across different post-infection time points, the main activated pathways involved signal transduction, inflammatory response, response to cytokines, and cytokine-mediated signaling pathway. Additional enriched pathways in DENV-2-infected MDM included response to chemokine, chemokine-mediated signaling pathway, leukocyte chemotaxis, granulocyte chemotaxis, and neutrophil chemotaxis. In contrast, these pathways were less enriched in DENV-2-infected D3MDM, particularly during the mid-stage of infection (10 h.p.i.).

Interestingly, specific pathways related to toll-like receptors signaling, interleukin (IL) 1 beta production, macrophages chemotaxis, and natural killer cell chemotaxis were enriched at various post-infection time points in DENV-2-infected MDM but not in DENV-2-infected D3MDM (Fig 3A). Conversely, the regulation of cytoskeletal organization was uniquely enriched in DENV-2-infected D3MDM. Pathways such as IL-10 production, IL-12 production, and T-cell chemotaxis were enriched in MDMs and D3MDM, but only at 24 h.p.i (Fig 3A). A notable distinction was observed in the viral response pathway. In DENV-2-infected MDMs, this pathway was enriched at multiple time points, with the most substantial enrichment at late stage (24 h.p.i.), but no enrichment at mid stage (10 h.p.i.). In contrast, DENV-2-infected D3MDM displayed enrichment of the viral response pathway only at early (1.5- and 5.5- h.p.i.) and late (24- h.p.i.) time points. Similarly, the stress response pathway was consistently enriched from the earliest time points in DENV-2-infected MDM, while in D3MDM, this pathway was enriched at three distinct peaks: at 1.5-, 5.5- and 24- h.p.i., (Fig 3A). In general, the pattern of GO functions enriched at early- and mid-time post-infection were less similar between MDM and D3MDM than the pattern observed at late stage.

In summary, while the enriched GO functions at 24 h.p.i. were broadly similar between MDM and D3MDM, significant differences were evident at earlier and mid time points, particularly at the mid stage (10 h.p.i.). This suggests that D3MDM may exhibit a unique early transcriptional response to DENV infection, which might influence the subsequent inflammatory and antiviral responses.

Global changes in gene expression may be linked to the activity of NF-κB

The distinct transcriptional patterns observed in DENV-2-infected MDM and D3MDM emphasized the dynamic and complex nature of the immune response. Global changes in gene expression, such as those linked with the delayed inflammatory response of DENV-2-infected D3MDM, may be associated with the differential accumulation of transcription factors (TFs). To address this, we used TFactS in R software to identify key TFs-driven gene expression differences between MDM and D3MDM infected with DENV-2.

The top 14 most influential TFs common to both infected macrophage types and that was part of the core enrichment group included AR, ATF3, BACH1, CEBPB, EGR1, JUN, NFKB1, RELA, SMAD3, SMAD4, TP63, and VDR (Fig 3B). Although these TFs were regulated from the early hours of infection, their activity varied significantly over time, particularly during the early- and mid-stages of infection. The most pronounced differences occurred at the mid stage (10 h.p.i.). However, at 24 h.p.i., both macrophage types exhibited a similar activity of TFs, suggesting a convergent response during the late stage of infection. Notably, the number of genes regulated by TFs associated with inflammatory responses (RELA, NFKB1, and JUN) and with vitamin D signaling pathway (VDR), peaked at 3-, 5.5- and 24- h.p.i. in DENV-2-MDM, whereas in DENV-2-infected D3MDM, peaks were observed only at 3- and 24-h.p.i. (Fig 3C). In addition, we observed temporal changes in the expression of these transcription factors during infection. In both macrophage types, VDR mRNA levels declined from 0 to 3 h.p.i., peaked at 5.5 h.p.i., and then decreased again from 10 to 24 h.p.i. (Fig 3D) Notably, VDR expression was consistently higher in D3MDMs than in MDMs between 0 and 3 h.p.i. We also found that JUN, RELA, and NFKB1 mRNA expression peaked at 3 h.p.i., declined at 10 h.p.i., and increased again at 24 h.p.i. Also, at their peak expression, the levels of these TFs were lower in D3MDMs than in MDMs, with the most pronounced difference observed for NFKB1 (Fig 3D). This cyclical pattern of NF-κB-dependent inflammatory gene expression suggests a more robust and sustained inflammatory response in MDMs during the early- and mid-stages of infection compared to D3MDM.

Unique genes are dependent on the transcription factors NF-κB and IRF5

The results presented so far show that differentiated macrophages’ gene expression levels and patterns differ in the presence or absence of vitamin D at early- and mid-stages post-infection. To investigate the mutually exclusive effect of vitamin D on gene expression, we focused on unique genes expressed in DENV-2-infected MDM and DENV-2-infected D3MDM (Fig 2E-I). We generated a network of TFs and their target genes using iRegulon to determine the relationship between unique genes highly enriched in the two groups of macrophages. We found that NFKB1 is predicted to be a TF-regulating gene expression in DENV-2-infected MDM, while both IRF5 and NFKB1 are expected to regulate the expression pattern in DENV-2-infected D3MDM, each with a normalized enrichment score (NES) > 6 (Fig 4A). The predicted motifs are shown in the inset of Fig 4A.

Fig 4 — (A) Enrichment of TFs and associated gene motifs predicted by iRegulon for *NFKB1* and *IRF5.* **(B)** The regulatory network of TFs and their regulons, highlighting unique target genes in MDM and D3MDM. (C) VDR ChIP-seq analysis in THP-1 cells at early (2 h) and late (24 h) time points showing VDR binding near *IRF5* and *NFKB1* gene loci. **(D)** mRNA-seq analysis of VDR knockout (VDR-KO) THP-1 cells showing transcript per million (TPM) levels of *IRF5* and *NFKB1* at 4 and 24 h. Data are presented as mean ± 95% confidence interval.

As indicated in the network, the NF-κB complex was enriched for regulons in both DENV-2-infected MDM and D3MDM, whereas IRF5 was exclusively predicted in D3MDM (Fig 4B). To determine whether vitamin D directly regulates IRF5 and NFKB1 gene expression, we reanalyzed VDR ChIP-Seq data (GSE89431) and transcriptomic data from wild-type (TW) and VDR-knockout (KO) THP-1 cells (GSE157514), at both early (2 and 4 h) and late (24 h) time points. VDR binding was consistently observed at the proximal promoter of IRF5 from early to late time points, whereas no VDR occupancy was detected at the NFKB1 locus (Fig 4C). Accordingly, IRF5 transcript levels (TPM) increased significantly in vitamin D-treated WT THP-1 cell (D3-VDR-THP1), but not in vitamin D-treated VDR-KO THP-1 cells (D3-KO-THP1), where IRF5 expression remained comparable to that of untreated controls (Fig 4D). This demonstrates that the gene expression differences at early stages between both macrophage types, whether differentiated in the presence or absence of vitamin D, are IRF5 dependent.

The time course of NF-κB and IRF5 target gene expression in MDM and D3MDM

We performed hierarchical clustering analysis to identify unique gene expression patterns in DENV-2-infected MDM or D3MDM, revealing nine distinct clusters across the viral growth curve (Fig 5).

Cluster 1 (n = 21 genes) displayed higher gene expression levels in DENV-2-infected MDM than in D3MDM (Fig 5A). These genes peaked in expression at 3 h.p.i. in both macrophage types, followed by a decline at 10 h.p.i. and then reached a second peak at 24 h.p.i. Notably, at 10 h.p.i, gene expression was almost completely abolished in DENV-2-infected D3MDM but recovered to levels comparable to MDM by 24 h.p.i. Genes in this cluster were mainly associated with cytokine-mediated signaling pathways, response to tumor necrosis factor, and leukocyte chemotaxis.

Cluster 2 (n = 20 genes) comprised genes exclusively transcribed in DENV-2-infected MDM at early time points, with a peak expression at 3 h.p.i. followed by near-complete suppression at 10 h.p.i. (Fig 5B). These results suggest that vitamin D treatment may downregulate the expression of these genes. Cluster 2 genes were mainly involved in cytokine-mediated signaling pathways and leukocyte chemotaxis.

Cluster 3 (n = 11 genes) comprised genes whose maximum expression peak was observed in DENV-2-infected MDM at 3 h.p.i. whereas these genes were not expressed in D3MDM at this time point (Fig 5C). Notably, the expression of genes of this cluster was absent at 5.5- and 10- h.p.i. in both macrophage types. However, by 24 h.p.i. the expression became similar in both MDM and D3MDM. This cluster includes genes associated with cytokine-mediated signaling pathways, IL-8 production, and positive regulation of programmed cell death.

Cluster 4 (n = 24 genes) included genes with low expression levels at the early infection stages, specifically in DENV-2-infected MDM (Fig 5D). However, both macrophage types displayed a maximum expression peak of these genes at 24 h.p.i. The genes of this cluster included genes involved in cytokine-mediated signaling pathways and positive regulation of programmed cell death.

Cluster 5 (n = 27 genes) and cluster 6 (n = 26 genes) included genes induced during the late stage of infection but only in DENV-2-infected MDM. In cluster 5, genes peaked in expression at 24 h.p.i. (Fig 5E), while in cluster 6, gene expression decreased at 24 h.p.i. (Fig 5F). Upregulated genes in these clusters were primarily involved in toll-like receptor signaling pathways and canonical NF-κB signal transduction. In contrast, downregulated genes were mainly associated with cytoskeleton organization and regulation of secretion.

Cluster 7 (n = 13 genes) consists of genes initially induced in DENV-2-infected D3MDM at 3 h.p.i. but not in MDM. These genes were not expressed at 5.5 h.p.i. and reached their maximum expression peak at 24 h.p.i. in both macrophage types (Fig 5G). Genes in this cluster were involved in cytokine-mediated signaling pathway and virus response, including IDO1, ISG20, OASL, IFI44L, RSAD2, IFIT1, MX1, EPSTI1, CXCL10, and CXCL11 targets of IRF5 (Fig 4C). These findings suggest that D3MDM upregulates antiviral genes early in the infection, which may influence viral entry, reducing the number of infected cells, as indicated in our previous studies [24,25].

Interestingly, in contrast to the patterns observed for clusters 5 and 6, cluster 8 (n = 27 genes) and cluster 9 (n = 26 genes) involved genes that were either upregulated or downregulated, respectively, in DENV-2-infected D3MDM starting at 10 h.p.i. and reaching their maximum expression peak at 24 h.p.i. (Fig 5H and I). The upregulated genes in cluster 8 were primarily involved in second messenger-mediated signaling, response to tumor necrosis factor, cytokine-mediated signaling pathway, and regulation of secretion. Conversely, the downregulated genes in cluster 9 were mainly associated with second messenger-mediated signaling and regulation of secretion.

Discussion

Immunomodulatory compounds have gained attention in recent years and are considered promising therapeutic options for preventing severe dengue fever. In particular, vitamin D has gained interest in this field due to its ability to modulate inflammatory responses [28,36–38]. In the present study, to further explore the mechanism of action of vitamin D in human macrophages, we differentiated primary human macrophages in the presence of physiological doses of Vitamin D (0.1 nM). Using mRNA-Seq analysis, we identified the time-course effect of vitamin D on DENV-2 replication and macrophage transcriptome. As was observed in previous studies conducted in our laboratory [24,25], we found that DENV-2-infected D3MDM exhibited a lower percentage of infected cells (~25%) compared to conventional MDMs (~15%) at MOI of 5. This reduction infection was associated with decreased mRNA expression of TLR3 and TLR7, along with increased expression of antiviral genes, including PKR and OAS1 [22–25]. Here we found in DENV-2-infected D3MDM that kinetics of mRNA levels of both chemokines and cytokines decreased at early post-infection. However, viral mRNA was higher in the presence of vitamin D at 1.5-, 3- and 5.5- h.p.i. and significantly decreased at 10 h.p.i. In addition, at later stages of infection, DENV-2 viral load decreased in D3MDM, whereas the transcriptional program and viral genome copies remain relatively similar between the two macrophage types. This may indicate that vitamin D dampens the early- and mid-inflammatory response to DENV-2 and delays viral genome replication, promoting a less inflammatory phenotype in macrophages and significantly reducing the expression of genes associated with the recruitment of inflammatory cells. Further, the shared late transcriptional response can be explained by the presence of a bystander-like phenotype [39,40].

Vitamin D is well-documented to exert genomic effects via the transcription factor activity of the VDR on target gene promoters [41]. We observed that macrophages differentiated in the presence of physiological doses of vitamin D have a minor influence on DEGs and VDR target genes. In contrast, at higher concentrations (1 nM), vitamin D upregulates more DEGs and VDR target genes, suggesting that the effects of physiological doses of vitamin D were predominantly genomic. Moreover, according to the literature, previous studies have reported non-genomic effects of vitamin D that alter the expression of genes involved in various signaling cascades, including those related to innate and adaptive immunity [25,42]. However, we could not assess these effects in the present study. On the other hand, we have reported that genomic effects of vitamin D upregulates antimicrobial peptides such LL-37 (encoded by CAMP) that restrict Zika virus (ZIKV) and DENV-2 infection in human macrophages [26,27]. However, ZIKV infection has been shown to inhibit the expression of VDR target genes in human monocytes, thereby preventing Vitamin D antiviral effects [43].

The altered transcriptional program observed in D3MDM was driven by the lower activity and expression of TFs-encoding genes such as NFKB1 and RELA, which is in accordance with the downregulation of genes involved in cytokine- and chemokine-mediated signaling pathways. In line with these results, several studies have found that vitamin D decreases DNA binding of NF-κB by increasing the expression of IκBα in keratinocytes [44], fibroblasts [45,46], dendritic cells [47], and kidney cells [48]. However, it has been shown that vitamin D inhibits p65 nuclear translocation in a VDR-dependent manner, leading to the downregulation of NF-κB target genes [49]. Furthermore, vitamin D limits chemokine expression in adipocytes and macrophages in mouse models [50]. Interestingly, clinical studies using transcriptome data from blood patients support the immune system’s involvement in the response to vitamin D3 supplementation, specifically with the downregulation of the NF-kB signaling pathway [51,52]. Therefore, further studies are necessary to show for the crosstalk between vitamin D and NF-kB activation during DENV-2 infection in human primary macrophages.

The IRF transcription factor family plays a central role in regulating immune cell activity and has consequently received growing attention. In this study, the transcription motif enrichment analysis predicted IRF5 as the TF involved in the upregulation of antiviral response genes, including IDO1, ISG20, OASL, IFI44L, RSAD2, IFIT1, MX1, EPSTI1, CXCL10, and CXCL11, in D3MDM but not in MDM. In line with this, a previous in vitro study demonstrated that VDR binds to the IRF5 gene loci in vitamin D-treated monocytes [53]. Similarly, our analysis of publicly available ChIP-Seq and RNA-Seq datasets from vitamin D-treated macrophages, revealed that vitamin D promotes VDR binding at the proximal promoter region of IRF5, while VDR-KO macrophages exhibit reduced IRF5 transcript levels. Moreover, an epigenomic study in monocytes and a transcriptomic analysis of macrophages differentiated in the presence of vitamin D showed high levels of NOD2 [53,54]. IRF5 is a downstream factor of the NOD2 and TLR3 signaling pathways and is essential for type I IFN signaling, thereby enhancing the antiviral state [55]. Although it has been reported that IRF5 cooperates with RelA to regulate the expression of inflammatory genes involved in macrophage polarization [56], and that vitamin D reduces lipopolysaccharide (LPS)-induced IRF5 phosphorylation, thereby attenuating the inflammatory activity of macrophages [57]. In addition, IRF5 has been shown to drive the expression of genes characteristic of M1 macrophage polarization, including TNFα, IL12, and IL-23, and play a critical role in regulating macrophage functional phenotypes [57]. Interestingly, M1 polarized MDM significantly restricts ZIKV replication, an effect associated with the upregulation of ISGs [58]. These findings suggest that IRF5 functions as a key mediator of the vitamin D-induced antiviral state in human macrophages. By promoting IRF5 expression and its transcriptional activity, vitamin D may prime macrophages to mount a robust early induction of ISGs upon viral infection. This early ISG activation is dependent on IFN-I production and is critical for establishing an antiviral intracellular environment, thereby delaying viral replication and reducing viral antigen levels. Collectively, our findings support the notion that vitamin D orchestrates an antiviral state via the VDR-IRF5-ISG axis, contributing to the observed restriction of DENV replication.

Clinical studies performed in patients with dengue infection [59], patients with dengue and coinfected with H. pylori [60], and hospitalized patients with dengue fever in Pakistan [61] have shown an association between vitamin D deficiency and dengue disease progression. Furthermore, vitamin D supplementation has decreased the risk of severe dengue in patients with dengue fever [62]. Here, we show that vitamin D limits DENV-2 early replication that leads to lower induction of NF-κB and, subsequently, lower gene expression of cytokines and chemokines in primary human macrophages (Fig 6). Controlling the inflammatory response to DENV infection while maintaining antiviral activity may reduce disease severity and, consequently, decrease morbidity and mortality in DENV-infected patients. Vitamin D might be a prognostic factor for developing severe dengue disease, and its supplementation would reduce the stronger inflammatory response presented in patients infected with dengue. Vitamin D is safe, inexpensive, and widely available, and could be an effective therapeutic strategy. However, despite the findings of this study and the previously discussed literature, further research is still necessary before considering vitamin D as a therapeutic strategy for controlling dengue progression.

Fig 6 — Vitamin D signaling through the VDR pathway activates the transcription factor IRF5, promoting the expression of a subset of ISGs. DENV is recognized by surface or endosomal TLRs and cytoplasmic RLRs, triggering NF-κB activation and induction of the inflammatory response. Vitamin D restricts viral entry by downregulating the mannose receptor (*CD206*) and upregulating of the antimicrobial peptide LL-37 (encoded by *CAMP*). Viral particles that enter the cell release their genome, which is further restricted at the translation level (e.g., *IFIT1*, *IDO1*) and replication (e.g., *OASL*, *MX1*, *ISG20*, *RSAD2*). The reduction in viral entry and replication results in lower availability of viral antigens and replication intermediates for TLR and RLR sensing, ultimately attenuating NF-κB signaling and decreasing the expression of pro-inflammatory cytokines and chemokines. Created with BioRender.com Tamayo Molina, Y. (2025) https://BioRender.com/740eopk.

In summary, our previous studies demonstrated that vitamin D restricts DENV-2 replication through multiple mechanisms, including the downregulation of CD206, a receptor involved in virus attachment, and the upregulation of the antimicrobial peptide LL-37, both of which contribute to reduced viral entry [24]. In line with these findings, we further show that vitamin D also regulates the expression of IRF5, which in turn induces the expression of antiviral genes, including IDO1, ISG20, OASL, IFI44L, RSAD2, IFIT1, and MX1, all of which contribute to limiting viral replication. Therefore, by restricting viral entry, limiting replication, and reducing viral particle production, vitamin D indirectly attenuates activation of the NF-κB signaling pathway, resulting in decreased expression of proinflammatory cytokines and chemokines (Fig 6).

Although our kinetic gene expression analysis identifies multiple regulatory factors, this study has several limitations. First, while our data provides temporal resolution, bulk RNA sequencing limits our ability to distinguish gene expression dynamics between infected and uninfected cells. Second, the potential non-genomic effects of VDR remain to be experimentally validated. Future studies should quantify VDR localization in nuclear and cytoplasmic compartments and clarify how vitamin D modulates gene expression through NF-κB-dependent mechanisms. Another limitation is the lack of protein-level data in the present study. However, our previous work has examined protein expression during vitamin D-induced macrophage differentiation and subsequent DENV-2 infection, yielding results consistent with our transcriptomic analysis [24,25].

Conclusion

Our findings reveal that DENV-2-infected MDM and DENV-2-infected D3MDM share a common transcriptional program following DENV-2 infection. However, vitamin D exposure modulates both the magnitude and diversity of this response, attenuating early- and mid-phase inflammatory signaling while enhancing the early antiviral activity. These results highlight a previously unrecognized role of calcitriol as a key regulator of macrophage transcriptional profile in response to DENV-2 infection.

Supporting information

S1 Fig. Experimental design.

Macrophages were differentiated or not in the presence of calcitriol 0.1 nM. Then, macrophages were infected with DENV-2 at an MOI of 5. Subsequently, cells were collected at 1.5-, 3-, 5.5-, 10-, and 24-h.p.i. Cells were used for bulk RNA sequencing, and supernatants were collected at 24 h.p.i. to quantify viral infectious particles.

(TIF)

pone.0330751.s001.tif^{(1.1MB, tif)}

S2 Fig. Piline for analysis of transcriptome data.

Transcriptome processing and analysis. Raw counts were pre-processed using batch effect correction and low-count filtering. Then, principal component analysis, differential expression analysis, and correlation analysis were performed (A). Gene set enrichment using clusterProfiler and transcription factor enrichment using TFactSR were performed for differentially expressed genes in both macrophage types (B). Unique genes in macrophages were used to search for biological processes using clusterProfiler and transcription factor enrichment by iRegulon.

(TIF)

pone.0330751.s002.tif^{(1.7MB, tif)}

S3 Fig. Analysis of all time points. Principal component analysis of all samples together (A).

Correlation analysis between samples in DENV-2 infected MDMs (B).

(TIF)

pone.0330751.s003.tif^{(1.3MB, tif)}

Acknowledgments

The authors thank the blood bank of the “Escuela de Microbiología, UdeA, Medellín, Colombia” for providing us with leukocyte-enriched blood units from healthy individuals and the personnel at the institutions where the study was performed.

Data Availability

Data was stored in the Gene Expression Omnibus (GEO) repositories under accession number: GSE297386.

Funding Statement

SUI; Minciencias, grant grant No. 111584467188, Contract No. 428-2020. https://minciencias.gov.co/sala_de_prensa/se-extiende-la-fecha-para-aplicar-la-convocatoria-icgeb-research-grants-2020-y-se. Universidad de Antioquia-CODI Acta No. 2020-34065. https://www.udea.edu.co/wps/portal/udea/web/inicio/!ut/p/z1/04_Sj9CPykssy0xPLMnMz0vMAfIjo8zi_QJNXQ2NnA18_D2NXQ0CLf1MA4zdPY1MzI31wwkpiAJKG-AAjgZA_VFgJRaWzkaGjiYGPgbehqYGjoGuAX4h_s4BLl5GUAV4zCjIjTDIdFRUBAC3KPbO/dz/d5/L2dBISEvZ0FBIS9nQSEh/. The funders played no role in the study design, data collection and analysis, publication decision, or manuscript preparation.

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PLoS One. doi: 10.1371/journal.pone.0330751.r001

Decision Letter 0

Helene Minyi Liu

5 Jun 2025

PONE-D-25-07206Vitamin D Enhances Antiviral Responses in Dengue Virus-Infected Macrophages by Modulating Early-Response Gene ExpressionPLOS ONE

Dear Dr. Urcuqui-Inchima,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers raised the questions of the role of IRF5 and the Vitamin D doses used in your experiments. Additional experiments to show the role of IRF5 and/or modification of the current statement are highly recommended before resubmission. Please carefully address all the concerns the reviewers addressed.

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Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: In this manuscript, Tamayo-Molina et. al. explore the impact of vitamin D on antiviral responses in dengue virus (DENV)-infected human monocyte-derived macrophages (MDMs). The authors apply a kinetic transcriptomic approach to characterize early, mid, and late gene expression patterns during infection, highlighting key transcriptional differences modulated by vitamin D treatment. This work addresses a timely and clinically relevant question, considering the lack of specific antiviral therapies for dengue fever. I have several concerns as follows:

1. Non-genomic vs Genomic Effects: While the authors suggest non-genomic mechanisms of vitamin D at physiological concentrations, further evidence is needed to support this claim. As acknowledged in the limitations, additional validation such as VDR localization studies or functional assays distinguishing genomic from non-genomic actions would greatly strengthen the manuscript.

2. Infection Heterogeneity: Given that bulk RNA-seq was performed, the heterogeneity of infected versus bystander cells could confound interpretations. A brief discussion or data (e.g., infection rates, percentage of infected cells) could help contextualize transcriptomic changes.

3. Vitamin D Dose Justification: The distinction between the effects of 0.1 nM and 1 nM vitamin D is intriguing. However, it would benefit the reader if the authors could justify the clinical relevance of the chosen concentration (0.1 nM) and its pharmacological or physiological basis.

Minor comments:

• In the Introduction, the background on IRF5 and its known antiviral roles could be expanded.

• A more explicit statement on how this work advances previous studies by the same group would clarify the novelty.

• Please ensure consistent gene naming (e.g., VDR, NF-κB subunits) throughout the text.

• Consider editing for minor grammatical improvements and flow in the Discussion section.

Reviewer #2: The legend of Figure 1A indicates that the concentration of VitD used was 0.1 nM. However, in Figures 1C and 1D, it is suggested that 0.1 nM is insufficient to exert an effect. This inconsistency should be clarified, and the rationale for the concentration used in each panel needs to be justified.

In Figure 2, there is a spelling error that should be corrected. Regarding Figure 2A, the data showed that the concentration of VitD used may have been insufficient to suppress viral replication. It is recommended that the authors test a broader or higher range of concentrations to fully evaluate the antiviral potential of vitamin D. Additionally, in Figure 2D, the numerical data shown in the figure do not align with the values described in the main text.

In Figure 5, several issues affect the clarity and interpretation of the data. First, there are some instances of mislabeling or mismatched labels between the figure panels and the corresponding text. Second, in Figure 5H, the data do not clearly support the conclusion stated in the text. The term “early stage of infection” is mentioned in the text but is not clearly defined in terms of specific time points, making it difficult to assess the relevance of the findings. The authors should specify the exact time frame referred to as the early stage and show corresponding data that support their claims.

Although the manuscript proposed that vitamin D modulates viral infection through the VDR signaling pathway, the current data do not provide strong support for this mechanism. The changes observed in the VDR pathway after vitamin D treatment are minimal, and it remains unclear which downstream target proteins are significantly affected. Without clear identification of these targets, it is difficult to draw conclusions about how vitamin D might influence viral replication. Furthermore, the manuscript suggested potential involvement of NF-κB and IRF5, but no direct evidence is presented to establish a mechanistic link between vitamin D signaling and these immune regulators. Additional experiments are needed to substantiate these claims and clarify the molecular pathways involved.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2025 Aug 21;20(8):e0330751. doi: 10.1371/journal.pone.0330751.r002

Author response to Decision Letter 1

2 Jul 2025

Thank you for your email and the positive feedback on our paper “Vitamin D Enhances Antiviral Responses in Dengue Virus-Infected Macrophages by Modulating Early-Response Gene Expression”. PONE-D-25-07206. We sincerely appreciate the very constructive comments of the reviewer.

Reviewer #1:

In this manuscript, Tamayo-Molina et. al. explore the impact of vitamin D on antiviral responses in dengue virus (DENV)-infected human monocyte-derived macrophages (MDMs). The authors apply a kinetic transcriptomic approach to characterize early, mid, and late gene expression patterns during infection, highlighting key transcriptional differences modulated by vitamin D treatment. This work addresses a timely and clinically relevant question, considering the lack of specific antiviral therapies for dengue fever. I have several concerns as follows:

Majos comments:

We thank the referee for the observation. Indeed, we agree with your point of view. However, we cannot access such validations involving VDR localization or functional assays. Because of that, we focus the manuscript on the genomic effects of vitamin D, supporting the role of IRF5-mediated antiviral responses. We added in the limitations of the study this concern.

In the discussion, in line 463, we added the following:

“However, we could not assess these effects in the present study.”

In the limitations, we added in lines 532 to 535 the following:

“Second, the potential non-genomic effects of VDR remain to be experimentally validated. Future studies should quantify VDR localization in nuclear and cytoplasmic compartments and clarify how vitamin D modulates gene expression through NF-κB-dependent mechanisms.”

We added the following lines to attend to the referee’s observation in the discussion section. Lines 400 to 444:

“As was observed in previous studies conducted in our laboratory [24, 25], we found that DENV-2-infected D3MDM exhibited a lower percentage of infected cells (~25%) compared to conventional MDMs (~15%) at MOI of 5. This reduction infection was associated with decreased mRNA expression of TLR3 and TLR7, along with increased expression of antiviral genes, including PKR and OAS1 [22–25].”

We thank the referee for this suggestion. We added in lines 217 to 222 the following:

The concentration of vitamin D used in this study (0.1 nM) was selected to mimic physiological levels of circulating 1,25(OH)₂D₃, which typically range between 0.04 and 0.150 nM in serum. This concentration also corresponds to the high-affinity dissociation constant (Kd = ~0.1 nM) of 1,25(OH)₂D₃ for the VDR [35]. Furthermore, we previously reported that macrophages differentiated in the presence of 0.1 nM vitamin D (D3MDM) exhibited reduced susceptibility to DENV infection [24, 25].

Minor comments:

• In the Introduction, the background on IRF5 and its known antiviral roles could be expanded.

It was done

• A more explicit statement on how this work advances previous studies by the same group would clarify the novelty.

It was done

• Please ensure consistent gene naming (e.g., VDR, NF-κB subunits) throughout the text.

It was done

• Consider editing for minor grammatical improvements and flow in the Discussion section.

It was done

Reviewer #2:

1. The legend of Figure 1A indicates that the concentration of VitD used was 0.1 nM. However, in Figures 1C and 1D, it is suggested that 0.1 nM is insufficient to exert an effect. This inconsistency should be clarified, and the rationale for the concentration used in each panel needs to be justified.

We agree with the reviewer comments and have modified Figures 1C and 1D accordingly. The updated figures provide a more precise representation of the transcriptional changes induced by Vitamin D. These modifications led to slight but noteworthy changes, which have been incorporated into the main text in lines 232 to 239, as follows:

“The DEGs analysis revealed 16 upregulated and 43 downregulated genes in D3[0.1 nM]MDM, while 100 genes were upregulated and 56 downregulated in D3[1 nM]MDM (Fig 1C). Gene overrepresentation analysis using WikiPathways showed that both concentrations induced the expression of genes associated with the vitamin D receptor pathway (WP2877), including CLMN, G0S2, CD14, TREM1, and CAMP, which were regulated in both concentrations (Fig 1D). In contrast, the higher vitamin D concentration also induced the expression of additional genes, such as CYP24A1, MYC, ALOX5, HILPDA, and S100A8 (Fig 1E). .”

2. In Figure 2, there is a spelling error that should be corrected. Regarding Figure 2A, the data showed that the concentration of VitD used may have been insufficient to suppress viral replication. It is recommended that the authors test a broader or higher range of concentrations to fully evaluate the antiviral potential of vitamin D. Additionally, in Figure 2D, the numerical data shown in the figure do not align with the values described in the main text.

We thank the reviewer for this suggestion. We have added the antiviral activity of macrophages differentiated with 1 nM of Vitamin D (Figures 1A and 1B). With these changes, we explore a broader dose of vitamin D. However, we cannot access genome replication as in Figure 2A for Vitamin D 1 nM. In this context, we added in lines 222 to 227 the following phases:

“As shown in Figure 1A, D3MDM were consistently less susceptible to DENV-2 infection than MDM. The percentage of inhibition reached approximately 35% and 45% in D3MDM differentiated with 0.1 nM (D3[0.1 nM]MDM) and 1 nM (D3[1 nM]MDM) of vitamin D, respectively, compared to MDM at 24 h.p.i. (Fig 1B). It is worth noting that the antiviral effect of vitamin D appears to be dose dependent.”

Regarding Figure 2D, we apologize for this mistake; we have corrected this inconsistency between the number of upregulated genes in the text and figure.

3. In Figure 5, several issues affect the clarity and interpretation of the data:

a. First, there are some instances of mislabeling or mismatched labels between the figure panels and the corresponding text.

b. Second, in Figure 5H, the data do not clearly support the conclusion stated in the text. The term “early stage of infection” is mentioned in the text but is not clearly defined in terms of specific time points, making it difficult to assess the relevance of the findings. The authors should specify the exact time frame referred to as the early stage and show corresponding data that support their claims.

We apologize for this mistake. We have corrected the mislabeling in Figure 5. In addition, we defined the specific time points regarding early, mid, and late effects. Therefore, in lines 250 to 254, we added which stage corresponds to these time points:

“The early time points (1.5-, 3-, and 5.5- h.p.i), and the mid time point (10 h.p.i). were selected to capture kinetic changes in the transcriptional profile associated with early viral entry and replication, independent of the effects of infectious virus release. The 24 h.p.i. time point was chosen as the late time of infection, corresponding to the assembly and release of new viral progeny.”

Importantly, we introduced through the text the analysis of the results in line with these three time points: early, mid and late.

4. Although the manuscript proposed that vitamin D modulates viral infection through the VDR signaling pathway, the current data do not provide strong support for this mechanism. The changes observed in the VDR pathway after vitamin D treatment are minimal, and it remains unclear which downstream target proteins are significantly affected. Without clear identification of these targets, it is difficult to draw conclusions about how vitamin D might influence viral replication. Furthermore, the manuscript suggested potential involvement of NF-κB and IRF5, but no direct evidence is presented to establish a mechanistic link between vitamin D signaling and these immune regulators. Additional experiments are needed to substantiate these claims and clarify the molecular pathways involved.

We thank the referee for this important suggestion. We have modified Figure 4B and added available data in GEO regarding the mechanistic link between vitamin D signaling and these immune regulators in human macrophages. Therefore, we added two new figures: Figure 4C and 4D. We hope this helps us understand the biological significance of Vitamin D IRF5-mediated antiviral response. In the text, in lines 370 to 378, we added the following:

“To determine whether vitamin D directly regulates IRF5 and NFKB1 gene expression, we reanalyzed VDR ChIP-Seq data (GSE89431) and transcriptomic data from wild-type (TW) and VDR-knockout (KO) THP-1 cells (GSE157514), at both early (2 and 4 h) and late (24 h) time points. VDR binding was consistently observed at the proximal promoter of IRF5 from early to late time points, whereas no VDR occupancy was detected at the NFKB1 locus (Fig 4C). Accordingly, IRF5 transcript levels (TPM) increased significantly in vitamin D-treated WT THP-1 cell (D3-VDR-THP1), but not in vitamin D-treated VDR-KO THP-1 cells (D3-KO-THP1), where IRF5 expression remained comparable to that of untreated controls (Fig 4D). .”

In addition, we cannot provide further evidence of the mechanistic link between NF-κB and the vitamin D pathway. However, to further expand the effect of macrophages differentiated with vitamin D on gene expression, we have conceived a new subfigure in Figure 3. We added the following in lines 345 to 352:

“In addition, we observed temporal changes in the expression of these transcription factors during infection. In both macrophage types, VDR mRNA levels declined from 0 to 3 h.p.i., peaked at 5.5 h.p.i., and then decreased again from 10 to 24 h.p.i. (Fig 3D) Notably, VDR expression was consistently higher in D3MDMs than in MDMs between 0 and 3 h.p.i. We also found that JUN, RELA, and NFKB1 mRNA expression peaked at 3 h.p.i., declined at 10 h.p.i., and increased again at 24 h.p.i. Notably, at their peak expression, the levels of these TFs were lower in D3MDMs than in MDMs, with the most pronounced difference observed for NFKB1 (Fig 3D).”

Attachment

Submitted filename: Point-by-point response to the reviewers.docx

pone.0330751.s005.docx^{(30.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0330751.r003

Decision Letter 1

Helene Minyi Liu

29 Jul 2025

PONE-D-25-07206R1Vitamin D Enhances Antiviral Responses in Dengue Virus-Infected Macrophages by Modulating Early-Response Gene ExpressionPLOS ONE

Dear Dr. Urcuqui-Inchima,

No further experiments are required. However, statistic analysis of Figure 3 and Discussion of the functional link between IRF5 and vitamin D signaling is required.

Please submit your revised manuscript by Sep 12 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Helene Minyi Liu, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

No further experiments are required. However, statistic analysis of Figure 3 and Discussion of the functional link between IRF5 and vitamin D signaling is required.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: The authors have made meaningful revisions in response to previous comments, and the manuscript has improved. However, several concerns remain that need to be addressed to strengthen the conclusions:

1. In Figures 3C and 3D, statistical analysis is not indicated. Without clear markers of significance, it is difficult to evaluate the reliability and relevance of the observed differences.

2. The study relies primarily on RNA-seq data and lacks molecular validation. Additional experiments, such as western blotting to assess IRF5-related proteins and viral proteins levels, are needed to support the claim that vitamin D has antiviral activity and could reduce inflammation.

3. The manuscript reports an increase in IRF5 expression following vitamin D treatment combined with viral infection. However, the functional link between IRF5 and vitamin D signaling remains unclear. The authors should further explain the relevance of this finding and how it contributes to the proposed antiviral mechanism.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2025 Aug 21;20(8):e0330751. doi: 10.1371/journal.pone.0330751.r004

Author response to Decision Letter 2

4 Aug 2025

Point-by-point response to reviewers

1. In Figures 3C and 3D, statistical analysis is not indicated. Without clear markers of significance, it is difficult to evaluate the reliability and relevance of the observed differences.

Answer: We thank the reviewer for this observation. We have now included statistical analysis for Figure 3D using a two-way ANOVA model (Expression ~ Treatment * Time), followed by post hoc unpaired t-tests at each time point. However, statistical analysis could not be performed for Figure 3C, as there is only a single value of regulated genes (K) per condition at each time point. Thus, no biological replicates are available for comparison.

Answer: We thank the reviewer for their insightful comment and suggestions. However, we would like to clarify that the primary objective of this study was to perform a transcriptomic analysis of MDMs differentiated in the presence or absence of vitamin D and subsequently infected with DENV. We aimed to gain a deeper molecular understanding of the functional and antiviral responses modulated by vitamin D in this context.

We have added the following lines to address the reviewer’s comment in the Discussion section (lines 499–513):

“These findings suggest that IRF5 functions as a key mediator of the vitamin D-induced antiviral state in human macrophages. By promoting IRF5 expression and its transcriptional activity, vitamin D may prime macrophages to mount a robust early induction of ISGs upon viral infection. This early ISG activation is dependent on IFN-I production and is critical for establishing an antiviral intracellular environment, thereby delaying viral replication and reducing viral antigen levels. Collectively, our findings support the notion that vitamin D orchestrates an antiviral state via the VDR-IRF5-ISG axis, contributing to the observed restriction of DENV replication. Notably, IRF5 has been shown to drive the expression of genes characteristic of M1 macrophage polarization, including TNFα, IL12, and IL-23, and play a critical role in regulating macrophage functional phenotypes [57]. Interestingly, M1 polarized MDM significantly restricts ZIKV replication, an effect associated with the upregulation of ISGs [58].

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PLoS One. doi: 10.1371/journal.pone.0330751.r005

Decision Letter 2

Helene Minyi Liu

6 Aug 2025

Vitamin D Enhances Antiviral Responses in Dengue Virus-Infected Macrophages by Modulating Early-Response Gene Expression

PONE-D-25-07206R2

Dear Dr. Urcuqui-Inchima,

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PLoS One. doi: 10.1371/journal.pone.0330751.r006

Acceptance letter

Helene Minyi Liu

PONE-D-25-07206R2

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Experimental design.

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S2 Fig. Piline for analysis of transcriptome data.

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S3 Fig. Analysis of all time points. Principal component analysis of all samples together (A).

Correlation analysis between samples in DENV-2 infected MDMs (B).

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Data Availability Statement

Data was stored in the Gene Expression Omnibus (GEO) repositories under accession number: GSE297386.

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PERMALINK

Vitamin D enhances antiviral responses in dengue virus-infected macrophages by modulating early-response gene expression

Yordi Sebastián Tamayo-Molina

Juan Felipe Valdés-López

Geysson J Fernandez

Silvio Urcuqui-Inchima

Roles

Abstract

Introduction

Materials and methods

Ethics statement

DENV-2 stock and viral titration

Culture of primary human monocytes and differentiation into monocyte-derived macrophages in the presence (D3MDM) or absence (MDM) of vitamin D

In vitro DENV-2 infection of monocyte-derived macrophages

RNA extraction and cDNA synthesis

Bulk RNA-Seq of DENV-infected macrophages

Data annotation and batch effect correction

Analysis of differentially expressed genes

Statistical analysis

Results

The expression of genes in the VDR signaling pathway and their target genes depend on vitamin D concentration

Fig 1. Virus quantification and VDR pathway analysis.

Analysis of differentially expressed genes during early and late stages of DENV-2 infection in macrophages differentiated in the presence of vitamin D

Fig 2. Early and late response of MDM and D3MDM to DENV-2 infection.

Vitamin D shapes the gene expression in DENV-2-infected macrophages

Fig 3. Vitamin D modulates gene expression in DENV-2-infected MDM and D3MDM.

Global changes in gene expression may be linked to the activity of NF-κB

Unique genes are dependent on the transcription factors NF-κB and IRF5

Fig 4. Analysis of TF target genes unique to MDM and D3MDM during DENV-2 infection.

The time course of NF-κB and IRF5 target gene expression in MDM and D3MDM

Fig 5. Hierarchical clustering analysis of unique genes in DENV-2-infected MDM and D3MDM.

Discussion

Fig 6. Effects of vitamin D on the time course of DENV-2 infection in human primary macrophages.

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Helene Minyi Liu

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Helene Minyi Liu

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