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

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.