Acta Tropica 232 (2022) 106497 Available online 1 May 2022 0001-706X/© 2022 Published by Elsevier B.V. Vitamin D modulates the expression of Toll-like receptors and pro-inflammatory cytokines without affecting Chikungunya virus replication, in monocytes and macrophages Juan Felipe Valdés-López , Paula Velilla , Silvio Urcuqui-Inchima * Grupo Inmunovirología, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia A R T I C L E I N F O Keywords: Chikungunya virus Inflammation Vitamin D3 Innate immunity Toll-like receptors Vitamin D receptor Antiviral activity A B S T R A C T Chikungunya virus (CHIKV) is a zoonotic arthropod-borne virus that causes Chikungunya fever (CHIKF), a self- limiting disease characterized by myalgia and acute or chronic arthralgia. CHIKF pathogenesis has an important immunological component since higher levels of pro-inflammatory factors, including cytokines and chemokines, are detected in CHIKV-infected patients. In vitro studies, using monocytes and macrophages have shown that CHIKV infection promotes elevated production of pro-inflammatory cytokines and antiviral response factors. Vitamin D3 (VD3) has been described as an important modulator of immune response and as an antiviral factor for several viruses. Here, we aimed to study the effects of VD3 treatment on viral replication and pro- inflammatory response in CHIKV-infected human monocytes (VD3-Mon) and monocyte-derived macrophages differentiated in the absence (MDMs) or the presence of VD3 (VD3-MDMs). We found that VD3 treatment did not suppress CHIKV replication in either VD3-Mon or VD3-MDMs. However, the expression of VDR, CAMP and CYP24A1 mRNAs was altered by CHIKV infection. Furthermore, VD3 treatment alters TLRs mRNA expression and production of pro-inflammatory cytokines, including TNFα and CXCL8/IL8, but not IL1β and IL6, in response to CHIKV infection in both VD3-Mon and VD3-MDMs. While a significant decrease in CXCL8/IL8 production was observed in CHIKV-infected VD3-Mon, significantly higher production of CXCL8/IL8 was observed in CHIKV- infected VD3-MDM at 24 hpi. Altogether, our results suggest that vitamin D3 may play an important role in ameliorating pro-inflammatory response during CHIKV infection in human Mon, but not in MDMs. Although further studies are needed to evaluate the efficacy of VD3; nevertheless, this study provides novel insights into its benefits in modulating the inflammatory response elicited by CHIKV infection in humans. 1. Introduction Chikungunya virus (CHIKV) is an arthritogenic member of the Alphavirus genus (family Togaviridae) transmitted by Aedes mosquitoes (Strauss and Strauss, 1994) and has reemerged as a global public health threat. CHIKV is responsible for a febrile illness called Chikungunya fever (CHIKF), a self-limiting disease characterized by myalgia and acute or chronic arthralgia [Reviewed in: (Valdés López et al., 2019)]. Although the first cases of patients infected by CHIKV were reported in 1952 in Tanzania, East Africa (MC, 1955), over the last decade, CHIKV infection has affected millions of people in 45 countries or territories in the Americas [Reviewed in: (Valdés López et al., 2019)]. CHIKV targets human non-hematopoietic cells including dermal and muscle fibro blasts, neuronal cells, and to less extent hematopoietic cells, such as monocytes and macrophages (Young et al., 2019; Abraham et al., 2020; Felipe et al., 2020). As innate sentinels of their host, monocytes and macrophages sense invading pathogens and trigger innate immune responses, as the first line of defense, by receptors known as pattern recognition receptors (PRRs) (Mytar et al., 2004; Orozco et al., 2021). PRRs are expressed on the cell surface, in phagocytic vesicles, and in the cytosol of various innate immune cells. The PRRs recognize diverse virus components known as Pathogen-associated molecular patterns (PAMPs), which lead to intracellular signaling and subsequently the synthesis of pro-inflammatory cytokines. In turn, these cytokines recruit other im mune cells, activate adaptive immune responses, and inhibit viral spreading [Reviewed in (Carty et al., 2021)]. Among the PPRs, Toll-like receptors (TLRs) are the most well-characterized in humans. Among * Corresponding author. E-mail addresses: felipe.valdes@udea.edu.co (J.F. Valdés-López), paula.velilla@udea.edu.co (P. Velilla), silvio.urcuqui@udea.edu.co (S. Urcuqui-Inchima). Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropica https://doi.org/10.1016/j.actatropica.2022.106497 Received 11 March 2022; Received in revised form 12 April 2022; Accepted 30 April 2022 mailto:felipe.valdes@udea.edu.co mailto:paula.velilla@udea.edu.co mailto:silvio.urcuqui@udea.edu.co www.sciencedirect.com/science/journal/0001706X https://www.elsevier.com/locate/actatropica https://doi.org/10.1016/j.actatropica.2022.106497 https://doi.org/10.1016/j.actatropica.2022.106497 https://doi.org/10.1016/j.actatropica.2022.106497 http://crossmark.crossref.org/dialog/?doi=10.1016/j.actatropica.2022.106497&domain=pdf Acta Tropica 232 (2022) 106497 2 TLRs, TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are cell surface re ceptors, while TLR3, TLR7, TLR8, and TLR9 are expressed on the surface of endosomal compartments and are involved in viral nucleic acid detection (Arababadi et al., 2018). CHIKF is an inflammatory disease where TLR-induced cytokines and chemokines have been detected in the serum of CHIKF patients, including interleukin (IL)1β, IL6, IL12p70, IL17A, IL27, CCL2, CCL5, CCL17, CXCL8/IL8, and CXCL10 (Kashyap et al., 2014). Extensive studies on host immune response during CHIKV infection suggest an association between pro-inflammatory cytokines, IL1β and IL6, during the acute phase of the disease, while high levels of IL6, IL27, CCL2, and CXCL8/IL8 are linked to the development of chronic phase or prolonged arthralgia (Ng et al., 2009; Chow et al., 2011; Dupuis-Maguiraga et al., 2012; Lohachanakul et al., 2012). Recently, we provided evidence that CHIKV infection activates the TLR signaling pathway and induces dif ferential inflammatory and antiviral response in human monocytes (Mon) and monocyte-derived macrophages (MDMs) (Felipe et al., 2020). As reported by Dutta and Tripathi (Dutta and Tripathi, 2017), polymorphisms of TLR7 and TLR8 are associated with susceptibility to CHIKV infection. Further, pretreatment of mice with Poly (I:C), a TLR3 agonist, decreased CHIKV titers in the brain and increased the produc tion of TLR3, IFNβ and antiviral genes (Priya et al., 2014). Furthermore, infiltrating monocytes and tissue-resident macrophages have been postulated to act as a vehicle and a reservoir for chronic CHIKV (Niki tina et al., 2018). Additionally, functional studies in mice and immune profiling of patients infected with CHIKV highlight phenotypic and transcriptional response which can control or contribute to pathogenesis depending on the stage of infection (J Gardner et al., 2010; Michlmayr et al., 2018; Morrison et al., 2011). Despite the potential risk of large-scale CHIKV that could severely impact public health globally, currently, there are no vaccines or anti virals available for the treatment of CHIKV infection. The limited re sources to fight against CHIKV infection and its rapid re-emergence has led to the search for new compounds that could prevent CHIKV infection or control the progression of CHIKF. Several epidemiologic studies indicate that poor vitamin D3 (VD3) status can have an impact on a range of diseases. VD3 modulates both innate and adaptive immunity (Adams and Hewison, 2008), besides its key role in calcium and phos phorus metabolism (Al-Ghamdi et al., 2012). Conversion of cholecal ciferol, the biologically inactive precursor of VD3, to its biologically active metabolite 1α,25(OH)2VD3 (calcitriol) requires two hydroxyl ation steps that are accomplished by 25-hydroxylases (CYP27A1 and CYP2R1) and by 1α-hydroxylase (CYP27B1) [reviewed in (Bikle, 2014)]. For tight control of VD3 levels, calcitriol induces a negative feedback loop to its rapid degradation, by upregulation of 24-hydroxylase (CYP24A1) and suppression of CYP27B1 expression (Pike and Meyer, 2010). Active vitamin D3 or calcitriol modulated innate immune response, by regulating gene expression, through the transcription factor vitamin D receptor (VDR) (Haussler and McCain, 1997). Thus, calcitriol-activated VDR binds to accessible genomic sites in the pro moter of its target genes and modulates their transcription. Inadequate levels of VD3 have been linked to an increased risk of malfunctions of immune response reported in several immune-related diseases, including viral infections [Reviewed in (Teymoori-Rad et al., 2019)]. Based on the pleiotropic role of VD3, several studies have evaluated the link between VD3 and TLRs in vitro and in vivo conditions. Sadeghi et al. (Sadeghi et al., 2006) reported that VD3-treatment decreased levels of TLRs in monocytes by blocking NF-kB1/RELA translocation to the nucleus. VD3 treatment has been shown to decrease pro-inflammatory cytokines, chemokine and monocyte traf ficking, as well as down-regulation of TLR2 and TLR4 expression in monocytes (Sadeghi et al., 2006; Pedersen et al., 2007; Zhang et al., 2012). Although it remains to be fully defined, the mechanisms by which VD3 modulates inflammation in viral infections may relate to the modulation of TLR signaling. Furthermore, VD3 inhibits replication of Human immunodeficiency virus type 1 (HIV-1), Dengue virus (DENV), Hepatitis C virus (HCV), and Respiratory syncytial virus (RSV), in respective host cells (Alvarez et al., 2019; Giraldo et al., 2018; Hurwitz et al., 2017). However, our understanding of the impact of VD3 on im mune regulation, specifically TLR expression and cytokine production and antiviral activity against CHIKV is unknown. In the current study, we aimed to assess the possible antiviral and immunomodulatory effects of VD3 treatment in the control of CHIKV infection and the regulation of TLRs expression and pro-inflammatory response in both VD3 treated and untreated Mon as well as in MDMs differentiated in the presence (VD3-MDMs) or the absence (MDMs) of VD3. 2. Materials and methods 2.1. Ethics statement The protocols for individual enrollment and sample collection were approved by the Committee of Bioethics Research of Sede de Inves tigación Universitaria, Universidad de Antioquia (Medellín, Colombia), and inclusion was preceded by a signed informed consent form, ac cording to the principles expressed in the Declaration of Helsinki. 2.2. RNA-seq data and bioinformatics analysis Our RNA sequencing data set performed on MDMs and VD3-MDMs (using 1 nM VD3) was reanalyzed. Counts matrix was normalized to transcripts per million (TPM). Then, gene expression (mRNA) was normalized by calculating Reads per kilobase per million mapped reads (RPKM) [TPM/Gene size (Kbs)]. Differential expression of mRNAs in each experimental group was identified using the DEseq package (version 1.8.261) implemented in R software (version 3.6.3). To deter mine the differentially expressed genes (DEG) focusing on TLRs, NF-κB and VD3/VDR signaling pathway, we used the edgeR package of R software where the false discovery rate (FDR)< 0.05 and the |Log2 Fold Change (FC) (VD3-MDMs / MDMs) |> 0.6 (|log2FC|> 0.6), were used as the threshold to determine the statistically significant difference in gene expression. n = 4. 2.3. Differential expression analysis of previously published data To evaluate the effect of VD3 on Mon and PBMCs, specifically on VD3/VDR signaling pathway, we reanalyzed the publicly available RNA-Seq GSE157514 (GEO) (Warwick et al., 2021) and the GSE179621 (GEO) (Hanel et al., 2020). The RNA-Seq dataset GSE157514 (Warwick et al., 2021) was performed from VDR knockout or control THP-1 cells treated with 100 nM of VD3 or with vehicle (EtOH), for 24 h. n = 4. The RNA-Seq dataset GSE179621 (Hanel et al., 2020) was performed in human PBMCs treated with 10 nM of VD3 or with vehicle (EtOH), for 24 h. RNA-Seqs were analyzed as described in the 2.5 section “RNA-seq data and bioinformatics analysis”. 2.4. CHIKV stocks and viral titration A clinical isolate of CHIKV obtained following the protocol described in (Pastorino et al., 2005), from CHIKF patient (kindly gifted by Pro fessor Francisco Javier Díaz, University of Antioquia), was amplified from a Colombian patient’s serum and propagated in Vero cells (ATTC CCL-81), as we previously reported (Felipe et al., 2020). Briefly, cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Massachu setts, USA), 4 mM L-glutamine (Sigma-Aldrich), 0.3% (v/v) sodium carbonate (NaCO3; Sigma-Aldrich) and 1% (v/v) antibiotic–antimycotic solution (Corning-Cellgro, New York, USA), and incubated at 37 ◦C and 5% CO2 in cell culture flasks, to a cell density of 1 × 105 -1 × 106 cells/mL. Vero cells were inoculated with CHIKV at 0.1 multiplicity of infection (MOI), incubated at 37 ◦C and 5% CO2 for 2 days, or until an J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 3 advanced cytopathic effect was observed. Next, supernatants were collected, precleared by centrifugation (1650 × g for 10 min), and stored at − 80 ◦C. CHIKV stocks were titrated by plaque assay on Vero cells, as previously described (Felipe et al., 2020). The virus titer was determined to be 2.1 × 108 PFU/mL. 2.5. Blood samples from healthy donors Venous peripheral blood samples were obtained from healthy in dividuals, aged 20–40 years, who had not been previously vaccinated against the yellow fever virus. All our experiments were performed with cells from at least six healthy donors. 2.6. Culture of human monocytes (Mon) and differentiation into monocyte-derived macrophages (MDMs) in the absence or presence of 1α,25-dihydroxyvitamin D3 (VD3) Human peripheral blood mononuclear cells (PBMCs) were isolated through a density gradient with Lymphoprep (STEMCELL Technologies Inc, Vancouver, Canada) by centrifugation at 850 x g for 21 min, from whole blood mixed with 2% EDTA, donated by healthy volunteers or leukocyte-enriched blood units from the healthy individual from the blood bank of the “Escuela de Microbiologia, UdeA, Medellín, Colombia”, as described previously (Arboleda Alzate et al., 2017). PBMCs from each healthy volunteer were worked independently. Platelet depletion was performed by washing with PBS-1X (Sigma-Al drich) three times at 250 x g for 10 min and the percentage of CD14 positive cells was determined by flow cytometry as we previously re ported (Arboleda Alzate et al., 2017). To obtain monocytes, 24-well plastic plates were scratched with a 1000 μL pipette tip and then, 5 × 105 CD14 positive cells/well were plated and allowed to adhere for 2 h in RPMI-1640 medium supplemented with 0.5 (v/v) autologous serum or plasma, 4 mM L-glutamine and 0.3% (v/v) NaCO3 and cultured at 37 ◦C and 5% CO2. Nonadherent-cells were removed by washing twice with PBS-1X and cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS, 4 mM L-glutamine, 0.3% (v/v) NaCO3 and 1% (v/v) antibiotic-antimycotic solution 100X (complete medium), and were cultured in the absence (Mon) or in the presence (VD3-Mon) of 0.1 nM of Fig. 1. Transcriptomic analysis of VD3/VDR signaling pathway. (A and B) Wild THP-1 cells and THP KO for VDR, treated or not with Vit D. (C and D) Peripheral Blood Mononuclear Cells (PBMC) treated or not with Vit D. (E and F) Differentiated MDM in the absence (MDM) or presence of Vit D (VD3-MDM). J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 4 1α,25-dihydroxyvitamin D3 (VD3; Sigma Aldrich, USA) and incubated at 37 ◦C and 5% CO2 overnight. To obtain MDMs, monocytes were cultured for 6 days without (MDMs) or with 0.1 nM of VD3 (VD3-MDMs), as we previously described (Arboleda Alzate et al., 2017; Valdés López and Urcuqui-Inchima, 2018). VD3 was replenished with fresh medium every 48 h. 2.7. Mon, VD3-Mon, MDMs and VD3-MDMs infection with CHIKV CHIKV infection of primary human monocytes (Mon and VD3-Mon) and macrophages (MDMs and VD3-MDMs) were performed at MOI of 10 or 5, respectively, in serum-free RPMI-1640 medium. Samples were incubated at 37 ◦C for 1.5 h. An hour and a half after infection the cells were washed with PBS-1X to remove unbound virus and fresh complete medium (with or without VD3) was added and left at 37 ◦C with 5% CO2. Cell culture supernatants and cell lysates were obtained at 6 and 24 h post-infection (hpi) and stored at − 80 ◦C. CHIKV replication was per formed using the supernatants of Mon, VD3-Mon, MDMs and VD3- MDMs collected at 6 and 24 hpi and evaluated by plaque assay on Vero cells, as previously described (Felipe et al., 2020). 2.8. Quantitative real-time PCR for TLRs, Cathelycidin, VDR and CYP24A1 Total RNA was extracted with TRIzol reagent (Thermo Fisher Sci entific) following the manufacturer’s instructions. For cDNA synthesis, the RevertAid Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. The mRNA quantification of TLR2, TLR3, TLR4, TLR7, TLR8, VDR, CAMP, CYP24A1, and phosphoglycerate kinase (PGK; Housekeeping gene), from uninfected and CHIKV-infected Mon, VD3-Mon, MDMs and VD3- MDMs was performed by RT-qPCR using primers previously reported (Giraldo et al., 2016; Marín-Palma et al., 2021). The BioRad CFX man ager was used to obtain the cycle thresholds (Ct) that were determined for each sample using a regression fit in the linear phase of the PCR amplification curve. The RT-qPCR was carried out using SYBR system (Invitrogen, Oregon, USA). The relative quantification of each mRNA was normalized to the housekeeping gene PGK, and uninfected control, using the ΔΔCt method. |Log2 FC|> 0.6 was used as the threshold to determine the significant difference in gene expression. n = 3. 2.9. Cytokine and chemokines quantification The ELISA test MAX™ Deluxe Set for Human was used for the Fig. 2. CHIKV replication in VD3-Mon and VD3-MDMs and expression of VDR and VDR-target genes. (A) Mon and Mon treated with Vit D (VD3-Mon) infected with CHIKV. (B) differentiated MDM in the absence (MDM) or the presence of Vit D (VD3-MDM) and infected with CHIKV. (C) Components expression of Vit D signaling pathway in Mon and VD3-MDM infected with CHIKV. (D) Components expression of Vit D signaling pathway in MDM and VD3-MDM infected with CHIKV. J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 5 detection of TNFα, IL1β, IL6, IL10 and CXCL8/IL8 (BioLegend, San Diego, CA, USA) in culture supernatants of Mon, VD3-Mon, MDMs and VD3-MDMs infected with CHIKV, following manufactureŕs instructions. The detection limit was 4–10 pg/mL. 2.10. Statistical analysis Statistical analysis was performed using GraphPad Prism 8 (Graph Pad Software Inc. San Diego, CA). Statistical tests are indicated in the figure legends. Data are represented as mean ± SEM, Log2 Fold Change (Log2 FC), or RPKM. Significant results were defined as p<0.05 (*), p<0.01 (**) and p<0.002 (***). 3. Results 3.1. Transcriptomic analysis of VD3/VDR signaling Since VDR is critical for most of VD3 actions, and to understand the significance of VDR signaling in CHIKV-infected cells, we first rean alyzed three independent RNA-Seq datasets. RNA-Seq dataset GSE157514 (Warwick et al., 2021) was performed in VDR knockout (THP-1-VDR-KO) or control THP-1 cells treated with 100 nM VD3 or with vehicle (EtOH) for 24 h; RNA-Seq GSE179621 (Hanel et al., 2020) was performed in human PBMCs treated with 10 nM of VD3 or with vehicle (EtOH) for 24 h, and our RNA-Seq dataset performed on MDMs and VD3-MDMs. RNA-Seqs were analyzed to examine the expression levels of core components of the VD3/VDR signaling and VDR-target genes. As shown in Fig. 1, we observed both down- and up-regulation of VDR signaling components and VDR-target genes. As expected, decreased expression of VDR mRNA was observed in THP-1-VDR-KO cells with or without VD3 treatment (Fig. 1A). Additionally, THP-1 + VD3, but not THP-1-VDR-KO + VD3, induced high expression of CYP24A1 mRNA in response to VD3. Further, both human PBMCs stimulated with VD3 and VD3-MDMs, showed a significant decrease in VDR mRNA, and an increase in CYP24A1 mRNA expression (Fig. 1C and D, respectively). While the transcriptomic analysis of VDR target genes showed a significant increase in mRNAs of CAMP, CD14, CD11b, FBP1, and PD-L1 in TPH-1 + VD3 (Fig. 1B), expression of these genes was not observed in TPH-1-VDR-KO + VD3, indicating that its expression is VDR-dependent. It was reported earlier that Lysozyme (Lyz) expression is enhanced in vitro by VD3 (Redecker et al., 1989). Here, we report that Lyz mRNA expression was not changed either in THP-1 or in THP-1-VDR-KO treated with VD3 (Fig. 1B). However, in both human PBMCs stimu lated with VD3 and VD3-MDMs, a significant increase of Lyz was observed (Fig. 1D and F). Furthermore, significantly higher mRNA expression of CAMP, CD14, CD11b, and FBP1 was observed in THP-1 + VD3, PBMCs + VD3 and VD3-MDMs, except CD11b for the latter, as compared with the control (Fig 1B, D and F, respectively). Even though the expression of PD-L1 mRNA remained unchanged in PBMCs stimu lated with VD3 (Fig. 1D), a significant increase was observed in THP-1 + VD3 and VD3-MDMs (Fig. 1B and F). 3.2. CHIKV replication in VD3-Mon and VD3-MDMs A comparable CHIKV titer was observed in VD3-Mon as compared with Mon, suggesting that VD3 treatment does not influence infectious virus particle production (PFU/mL), either at 6 or 24 hpi (Fig. 2A), although at 24 hpi a slight decrease in the production of CHIKV infec tious particles was observed in the VD3-MDMs. The release of CHIKV particles in the culture supernate of Mon and VD3-Mon was observed from 6 hpi and gradually increased at 24 hpi. Similarly, we found no significant difference in CHIKV replication, as determined by the release of virus particles, in VD3-MDMs as compared with MDMs (Fig. 2B). As in Mon, CHIKV particles production in both MDMs and VD3-MDMs was detected from 6 hpi and gradually increased at 24 hpi. 3.3. CHIKV infection alters the expression of VDR and VDR-target genes in Mon and VD3-Mon Based on the transcriptomic analysis, we determined that VD3 either down or upregulated expression of VD3/VDR signaling components and VDR-target genes in different immune cell populations (Fig. 1). Since our results suggest that VD3 treatment does not affect CHIKV replication in VD3-Mon and VD3-MDMs, we proceeded to determine the influence of CHIKV infection on the expression of VDR and VDR-target genes by RT-qPCR. As shown in Fig. 2C, while CHIKV infection exerts significant down-regulation of VDR mRNA expression in both Mon and VD3-Mon within 6 h of infection, the level of VDR mRNA was not altered in these cells at 24 hpi, as compared with uninfected VD3-Mon. The results suggest that CHIKV infection exerts significant down-regulation of VDR gene expression in both Mon and VD3-Mon at 6 hpi. CYP24A1 is known to regulate Vitamin D activity, and its expression is induced by VD3 through the activation of VDR (Fig. 1). We note that while CYP24A1 mRNA expression was significantly down-regulated in CHIKV-infected Mon as compared to uninfected VD3-Mon, a significant up-regulation of CYP24A1 mRNA was found in CHIKV-infected VD3- Mon at 6 hpi, compared with CHIKV-infected Mon (Fig. 2C). However, a significant down-regulation of CYP24A1 mRNA was observed in CHIKV- infected VD3-Mon at 24 hpi, as compared to CHIKV-infected Mon or uninfected VD3-Mon (Fig. 2C). Results suggest that CHIKV infection induces a significant increase in CYP24A1 mRNA expression in VD3- Mon at 6 hpi, but is downregulated at 24 hpi, although not in CHIKV- infected Mon at 24 hpi. Furthermore, CHIKV infection leads to signifi cant down-regulation of CAMP mRNA in both Mon and VD3-Mon at 24 hpi, without a noticeable effect at 6 hpi (Fig. 2C). 3.4. CHIKV infection alters the expression of VDR and VDR-target genes in MDMs and VD3-MDMs Since our results indicated that VD3 does not affect the production of CHIKV infectious particles in VD3-MDMs, we proceeded to investigate the possible influence of CHIKV infection on VDR and VDR target genes expression. We compared VDR mRNA expression in MDMs and VD3- MDM, both infected with CHIKV vs VD3-MDMs uninfected (control). VDR mRNA level was increased slightly in MDMs and VD3-MDMs at 6 hpi as compared to the control (Fig. 2D). However, the expression of VDR mRNA was down-regulated in MDMs and VD3-MDMs infected with CHIKV at 24 hpi (Fig. 2D). In addition, while CYP24A1 mRNA expres sion was significantly down-regulated in CHIKV-infected MDMs, its expression was significantly increased in CHIKV-infected VD3-MDMs, at 6 and 24 hpi, as compared with CHIKV-infected MDMs (Fig. 2D). In addition, a significant down-regulation of CAMP mRNA expression was observed in CHIKV-infected MDMs at 6 and 24 hpi, but not in CHIKV- infected VD3-MDMs, (Fig. 2D). Taken together, these results suggest that CHIKV infection dysregulated VD3/VDR signaling pathway. 3.5. Transcriptional regulation of TLRs, NF-κB-complex and NF-κB target genes in THP-1 cells, THP-1-VDR-KO cells and PBMCs treated with VD3, and VD3-MDMs Vitamin D deficiency and abnormalities of VDR expression confer susceptibility to infectious agents. Previously, we reported that monocyte-derived dendritic cells (MDDCs) from healthy donors who received 4000 IU/day of Vitamin D, showed a decreased expression of TLR3, TLR7, and TLR9 mRNAs (Martínez-Moreno et al., 2020), sug gesting a possible role of VD3 in regulating TLRs signaling in immune cells. We reanalyzed three independent RNA-Seq datasets from THP-1 cells and THP-1-VDR-KO cells treated with VD3, PBMCs treated with VD3, and VD3-MDMs, to determine whether the expression of TLRs, the NF-κB signal components and NF-κB target genes are altered in response to VD3 treatment. This is very interesting since activation of transcrip tion factor NF-kB has involved in the regulation of immune response, J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 6 including inflammation as well as cell survival, and it is considered as prototypical representative of a pro-inflammatory signaling pathway. Differentially expressed genes examined by RNA-Seq analysis showed that neither TLRs nor NF-κB-complex or NF-κB-target genes were altered in THP-1 + VD3 or THP-1-VDR-KO + VD3 (Fig. 3A, B and C). However, IκBα, CXCL1, and CXCL8/IL8, were induced in response to VD3 treatment in those cells (Fig. 3A, B and C). These results confirm that those genes are VDR-target genes. Further, basal level expression of TNFα was significantly down-regulated in THP-1 + VD3 cells as compared with THP-1 cells (Fig. 3C). The results suggest that VD3 treatment does not alter the expression of TLRs in monocytic cell lines, but significantly increased the expression of chemokines, including CXCL1 and CXCL8/IL8, and down-regulated the expression of TNFα, in a VDR-dependent manner. Although the level of TLR4 mRNA was increased, TLR1, TLR6, TLR7, and TLR8 transcripts decreased in PBMCs + VD3, compared with un treated PBMCs (Fig. 3D). Thus, VD3 treatment both down- and up- regulated the expression of TLRs in human primary cells. Similarly, VD3 treatment significantly decreased RELB mRNA level and strongly increased IκBα mRNA expression in PBMCs (Fig. 3E). Further, while the TNFα mRNA was significantly decreased, significantly higher levels of IL1β, CXCL1, and CXCL8/IL8 expression were observed in PBMCs + VD3 as compared with unstimulated PBMCs (Fig. 3F). As was observed in PBMCs + VD3, differentiation of MDMs in the presence or absence of VD3 leads to a change in the expression pattern of TLRs. Thus, while the TLR2 mRNA expression was significantly increased, the mRNA of TLR3, TLR4, TLR5, TLR6 and TLR7 was significantly decreased in VD3-MDMs as compared with MDMs (Fig. 3G). Additionally, NF-κB2 and IκBα mRNA levels were significantly higher in VD3-MDMs as compared to MDMs (Fig. 3H). Furthermore, while mRNA levels of TNFα and IL10 were significantly down-regulated, the levels of IL1β, CXCL1, CXCL8/ IL8 and COX2 mRNA were significantly up-regulated in VD3-MDMs as compared with MDMs (Fig. 3I). Altogether, the results suggest that VD3 treatment has an immunomodulatory effect not only on TLRs expression but also on NF-κB-complex and NF-κB-target genes, including both pro- and anti-inflammatory factors in human primary immune cells (PBMCs and MDMs). 3.6. TLRs and cytokines expression is altered in CHIKV-infected Mon and VD3-Mon As described in Fig. 3, VD3 can up or down-regulate the expression of several genes that play a role in the recognition of viruses, such as TLRs. Herein, we quantified using qRT-PCR mRNA expression of TLRs in un infected and CHIKV-infected Mon and VD3-Mon following 6 and 24 h of treatment with VD3. While levels of both TLR2, TLR7 and TLR8 mRNA were significantly higher in Mon and VD3-Mon at 6 hpi, when compared to uninfected VD3-Mon, the expression level was not changed at 24 hpi, compared to uninfected VD3-Mon (Fig. 4A). In contrast, the TLR4 mRNA expression was significantly lower in Mon and VD3-Mon at 6 hpi compared to uninfected VD3-Mon (Fig. 4A). However, TLR4 mRNA expression significantly increased in CKIKV-infected VD3-Mon vs CKIKV-infected Mon at 24 hpi (Fig. 4A). We next examined the expression of cytokines in Mon and VD3-Mon infected with CHIKV, to determine whether VD3 plays a role in the modulation of the inflammatory response, despite not affecting CHIKV replication. To this end, pro-and anti-inflammatory cytokines were quantified using the ELISA test. As described previously (Felipe et al., Fig. 3. Transcriptomic analysis of TLR expression and activation of the NF-kappaB complex in Mon, PBMC and MDM treated or not with Vit D. (A) Expression of TLRs, (B) components of the NF-Kappa B complex, and (C) NF-kappaB target genes in wild-type THP-1 cells or THP-1-VDR-KO treated or not with vitamin D. (D) Expression of TLRs, (E) components of the NF-Kappa B complex and (F) NF-target genes kappaB in PBMC, treated or not with vitamin D. (G) Expression of TLR, (H) components of the NF-Kappa B complex and (I) NF-kappaB target genes in differentiated MDM in the presence or absence of vitamin D. J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 7 2020), the peak of TNFα was observed in Mon at 6 hpi (Fig. 4B); how ever, a significantly lower level of TNFα was observed in VD3-Mon. At 24 hpi, we noted a decrease in TNFα level in Mon and VD3-Mon infected with CHIKV but continued to be significantly lower in VD3-Mon than Mon. Further, while a strong increase of CXCL8/IL8 was observed in both CHIKV-infected Mon and VD3-Mon at 6 hpi, significantly lower levels of CXCL8/IL8 were observed in VD3-Mon as compared to Mon at 24 hpi (Fig. 4E). As shown in Fig. 4D, a significantly higher level of IL6 was observed in CHIKV-infected VD3-Mon compared to Mon, at 6 hpi which was maintained up to 24 hpi, without noticeable differences be tween VD3-Mon and Mon. IL10 production was significantly lower in CHIKV-infected VD3-Mon at 6 hpi compared to Mon, an upward trend was observed in VD3-Mon at 24 hpi (Fig. 4F). Although these results showed an up- or down-regulated expression of TLRs and both pro- and anti-inflammatory cytokines in CHIKV-infected Mon and VD3-Mon, further studies are necessary to determine whether this effect is associ ated with viral infection or VD3 treatment. 3.7. TLRs and cytokines expression is altered in CHIKV-infected MDMs and VD3-MDMs As observed in RNA-Seq analysis (Fig. 3G), MDMs differentiated in presence of VD3 lead to down- or up-regulation of TLRs mRNA expres sion. We determined the expression of TLR mRNAs in CHIKV-infected MDMs and VD3-MDM, and uninfected VD3-MDMs. Significantly increased expression of TLR2 mRNA was observed in CHIKV infected MDMs and VD3-MDMs at 6 hpi as compared to uninfected VD3-MDMs, which was maintained up to 24 hpi in VD3-MDMs, but not in MDMs (Fig. 5A). The TLR3 mRNA expression was significantly higher in CHIKV-infected MDMs at 24 hpi, as compared to CHIKV-infected VD3- MDMs as well as with uninfected VD3-MDMs (Fig. 5A). The mRNA level of TLR7 was significantly higher in CHIKV-infected VD3-MDMs at 24 hpi, as compared to CHIKV-infected MDMs and uninfected VD3-MDMs (Fig. 5A). The expression of both TLR4 and TLR8 mRNA remained un changed in CHIKV-infected MDMs and VD3-MDMs, where the expres sion levels were similar to that of the control (Fig. 5A). The peak of TNFα production in CHIKV-infected MDMs and VD3- MDMs was observed at 6 hpi and was significantly reduced in VD3- Fig. 4. Expression of TLRs, proinflammatory cytokines, and chymosins in Mon treated or not with Vit D and infected with CHIKV. (A) Expression of TLRs in Mon treated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (B) Production of TNF-alpha in Mon treated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (C) IL1beta production in Mon treated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (D) IL6 production in Mon treated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (E) Production of CXCL8 / IL8 in Mon treated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (F) IL10 production in Mon treated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 8 MDMs as compared to MDMs (Fig. 5B). Further, a strong decrease in TNFα production was observed in both MDMs and VD3-MDMs infected with CHIKV at 24 hpi (Fig. 5B). In contrast, significantly higher levels of IL1β were observed in CHIKV-infected VD3-MDMs vs MDMs, at 24 hpi (Fig. 5C); though, peak expression was observed at 6 hpi in both CHIKV- infected MDMs and VD3-MDMs but without significant differences. CXCL8/IL8 production showed a continuous increase from 0 up 24 hpi; however, while at 0 and 6 hpi a significantly lower production was observed in CHIKV-infected VD3-MDMs as compared to CHIKV-infected MDMs, at 24 hpi a significantly increases was found in VD3-MDMs (Fig. 5E). Although no differences were found in IL6 level, it increased from 6 up 24 hpi in both CHIKV-infected MDMs and VD3-MDMs (Fig. 5D). The peak of IL10 production was observed at 6 hpi and strongly decreased at 24 hpi in both CHIKV-infected MDMs and VD3- MDMs, without significant differences (Fig. 5F). Taken together, the results suggest that CHIKV infection triggers an inflammatory response in MDMs, but the response in MDMs differentiated in presence of VD3 is higher or lower, dependent on cytokines and the conditions tested. 4. Discission Musculoskeletal and joint tissues of CHIKV-infected patients and mice are heavily infiltrated with Mon and macrophages and several studies suggest that these cell populations promote the control but also immunopathogenesis of acute and chronic CHIKV infection (Felipe et al., 2020; Valdés-López et al., 2021; J Gardner et al., 2010; Poo et al., 2014) . Previously, we reported that CHIKV replication in Mon and MDMs peaks at 18- and 24 hpi, and viral infection activates the TLR pathway to induces a robust pro-inflammatory response and antiviral response (Felipe et al., 2020). Further, induction of pro-inflammatory cytokines production by CHIKV infection starts early in Mo and MDMs. In the present study, we focused on immunomodulatory function of VD3, and its antiviral effects against CHIKV infection as a possible drug target to control CHIKF. Contrary to expectation, VD3 did not suppress CHIKV replication in either VD3-Mon or VD3-MDMs. Inter estingly, lower levels of VDR were observed in both VD3-Mon and Mon during the first hours of CHIKV infection as compared to uninfected VD3-Mon. However, the mRNA expression level of VDR was not significantly altered either in MDMs or VD3-MDMs in response to Fig. 5. Expression of TLRs, proinflammatory cytokines, and chymosins in MDM differentiated or not with Vit D and infected with CHIKV. (A) Expression of TLRs in MDM differentiated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (B) Production of TNF-alpha in MDM differentiated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (C) IL1beta production in MDM differentiated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (D) IL6 production in MDM differentiated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (E) Production of CXCL8/IL8 in MDM differentiated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. (F) IL10 production in MDM differentiated or not with Vit D and infected with CHIKV, at 6 and 24 hpi. J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 9 CHIKV. Since VDR participates in multiple signaling pathways, and low VDR levels can compromise VD3 signaling, we hypothesize that CHIKV infection attenuates VDR signaling in Mon and MDMs. In partial support of the hypothesis, we observed significantly lower expression of CAMP mRNAin Mon, irrespective of VD3 treatment at 24 hpi. Further, signif icantly lower expression of CAMP mRNA was observed in CHIKV- infected MDMs at the initial stages of infection, as compared to CHIKV-infected VD3-MDMs and uninfected VD3-MDMs. These results suggested that CHIKV can decrease the CAMP expression since the transcriptomic analysis performed in THP-1/THP-1-VDR-KO cells and PBMCs treated with VD3, and VD3-MDMs showed high levels of CAMP gene in response to VD3 treatment, further supporting our hypothesis. Several studies suggest that viruses inhibit VD3 signal transduction leading to down-regulation of VDR. VD3 acts through the VDR, and VD3/VDR deficiency is associated with various digestive diseases and plays important role in susceptibility to viral infections, including Influenza A virus, CMV (Mowry et al., 2011), and HCV [reviewed in (Villar et al., 2013)]. Further, VDR gene variations were suggested to correlate with chronic HBV infection (Bellamy et al., 1999). Gotlieb et al. (Gotlieb et al., 2018) found that HBV down-regulates VDR levels in hepatoma cells lines and prevents VD3-dependent inhibition of viral gene transcription and viral production. Additionally, Haug et al. (Haug et al., 1994) reported that HIV-1 impairs innate immune defenses by down-regulating the VDR pathway through the generation of reactive oxygen. Yenamandra et al. (Yenamandra et al., 2009) showed that both VDR mRNA and protein levels were lower in EBV-transformed cells as compared to primary B cells. Together, reports suggest that VD3/VDR signaling is target for inhibition by different viruses, suggesting that it plays an important role in the inductin of antiviral response. The changes of signaling pathways in response to the inhibition of VDR expression following CHIKV infection remain unknown. Further studies are needed to identify the factors that inhibit VDR expression following CHIKV infection. However, since we observed significantly higher levels of CYP24A1 mRNA and low levels of VDR mRNA, we suggest that dysregulation of expression of this enzyme may play an essential role in the lack of VD3 effect against CHIKV replication. Indeed, while CHIKV infection of VD3-Mon was associated with marked induction of CYP24A1 expression from the first hours of infection (6 hpi), upregulated expression was observed in VD3-MDMs later in infection (24 hpi). Furthermore, VD3 treatment without infection induced a strong mRNA expression of CYP24A1 in Mon and MDMs. These observations would explain the significantly lower levels of both VDR and CAMP mRNA expression observed in VD3-Mon and VD3- MDMs, suggesting also inhibition of the negative feedback loop by a relative deficiency of VDR-associated VD3, which implies less respon siveness to vitamin D treatment. It has been very well described that biologically active vitamin D induces a negative feedback loop by upregulation of the CYP24A1 and suppression of CYP27B1 expression for tight control of VD3 levels (Pike and Meyer, 2010). CYP24A1 is a VDR-target gene that controls VD3 levels by the conversion of both 25-OH-VD3 and 1α,25-(OH)2VD3 into 24-hydroxylated products tar geted for excretion along well-established pathways [reviewed in (Jones et al., 2012)]. Therefore, the observed up-regulation of CYP24A1 mRNA and down-regulation of VDR and CAMP mRNAs during CHIKV infection in Mon and MDMs may further indicate an specific viral interference with the VD3/VDR signaling pathway. However, further work is necessary to confirm our hypothesis and to determine the precise role of CHIKV in inhibiting VDR signaling. Innate immune response is the first line of defense against viral in fections and TLRs and related pathways are involved in virus sensing and induction of pro-inflammatory and antiviral responses [reviewed in (Schenten and Medzhitov, 2011)]. Vitamin D has been shown to down-regulate TLR2, TLR4, and TLR9 expressions in cultured mono cytes from healthy volunteers (Sadeghi et al., 2006). Additionally, VD3 has been shown to decrease TLR3, TLR7, and TLR9 mRNA expression in PBMCs from systemic lupus erythematosus patients (Yazdanpanah et al., 2017). In concordance with reports mentioned above, we found by RNA-Seq analysis both up- and down-regulation of TLRs in human pri mary cells (PBMCs and MDMs), but not in monocytic cell lines (THP-1), in response to VD3 treatment. We found that VD3 treatment down-regulate mRNA espression of TLR1, TLR6, TLR7 and TLR8 in PBMCs, and TLR3, TLR4, TLR5, TLR6 and TLR7 in MDMs. However, VD3 up-regulated mRNA expression of TLR4 and TLR2 in Mon and MDMs, respectively. In contrast, while TLR2 was up-regulated in MDMs and VD3-MDMs in response to CHIKV infection, TLR3 and TLR8 were downregulated at the first hours of infection. Interestingly, in bone marrow chimeric mice were showed that TLR3-expressing hematopoi etic cells is required for effective CHIKV clearance, but did not directly regulate CHIKV-induced joint inflammation (Her et al., 2015). Since in CHIKV-infected patients an up-regulation of TLR3 expression during the acute and early convalescent phase of the disease was observed (Her et al., 2015), the authors suggest that enhanced TLR3 expression is part of a general innate immunity against CHIKV. On the other hand, activation of TLRs up-regulates expression of VDR followed by induction of CAMP mRNA expression (Liu et al., 2006; Campbell and Spector, 2012; Shin et al., 2010). Stimulation of human macrophages with TLR1/2 heterodimer agonist (Pam3CSK4) was found to induce the expression of CYP27B1 and VDR which may lead to the induction of antimicrobial peptides and contribute to microbial infec tion susceptibility (Liu et al., 2006). Furthermore, TLR8 activation in human macrophages induces the expression of human CAMP, VDR and CYP27B1, and inhibits HIV-1 replication, supporting the important role of VD3 in the control of HIV-1 infection (Campbell and Spector, 2012). These findings highlight critical role of VD3 in host defense by altering the induction of TLRs and antimicrobial peptides in response to virus infection. Induction of TLR signaling activates NF-κB complex and induces a robust NF-κB-dependent pro-inflammatory responses (Valdés-López et al., 2022). Many pro-inflammatory cytokines and chemokines are targets of NF-κB regulation (Valdés-López et al., 2022; Bonizzi and Karin, 2004), and it has also been reported that VD3 directly decreases NF-κB activity by decreasing its translocation into the nucleus, and increasing IκBα expression levels in macrophages (Cohen-Lahav et al., 2006). In concordance with other reports, we showed that IκBα is a VDR-target gene, which was up-regulated in human PBMCs, MDMs, and THP-1 cells stimulated with VD3. Further, in cultured human adipocytes was described that VD3 protects against macrophage-induced activation of NF-κB signaling and subsequent release of several cytokines and chemokines, via increased expression of IκBα and reduced NF-κB p65 phosphorylation (Ding and Wilding, 2013). Thus, reports suggest that VD3 exerts an anti-inflammatory response through up-regulation of IκBα expression and the suppression of NF-κB activation, which may explain the down- or up-regulation of cytokines expression observed in the transcriptome profiling of PBMCs + VD3 and VD3-MDMs, as well as in CHIKV-infected VD3-Mon and VD3-MDMs. Together reports suggest that VD3 is an important immunomodulatory that could contribute to the control of pathologic inflammation observed in CHIKV-infected patients. Increased levels of pro-inflammatory cytokines, including TNFα, IL1β, IL6, and CXCL8/IL8 during CHIKV infection are associated with the severity of the disease and induction of arthralgias in patients (Ninla-Aesong et al., 2019; Lobaloba Ingoba et al., 2021). Results pre sented here shown that CHIKV infection induces a robust pro-inflammatory response dependent of high production of TNFα, IL1β, IL6, and CXCL8/IL8 in both Mon and MDMs, suggesting that both im mune cell populations could play an important role in induction of inflammation and immunopathogenesis in CHIKV-infected patients. Since VD3 plays important role in suppressing the inflammatory response (Pfeffer et al., 2018), in the current study we focused on cy tokines production in VD3-Mon, as well as in VD3-MDMs both infected with CHIKV. The effect of VD3 on cytokine responses was not uniform. J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 10 CHIKV infection of VD3-Mon leads to significantly lower production of TNFα and CXCL8/IL8 within 24 hpi as compared with Mon. Further, VD3 treatment of Mon increased the production of IL6 at the earliest stages of CHIKV infection, but slightly decreased its expression in the later stages of infection. In contrast, although VD3-MDMs expressed lower levels of TNFα and CXCL8/IL8 from the first hours of infection, significantly higher levels of IL1β and CXCL8/IL8 were found in the later stages of infection (24 hpi), as compared with MDMs. Further, It has been reported that exposure of influenza-infected human lung epithelial cells to VD3 significantly decreases the production of TNFα, IFNβ, IL6 and CXCL8/IL8 (Khare et al., 2013). Taken together, reports suggest that VD3 plays a key role in control of TNFα and CXCL8/IL8 production by CHIKV-infected Mon and MDMs. However, in CHIKV-infected MDMs, VD3 can also contribute to the induction of IL1β and CXCL8/IL8 in later stages of viral replication cycle. Thus, results show that VD3 could play a different role during CHIKV infection in a cell-dependent manner, sug gesting that VDR signaling could induces a dual role either in the control or in the induction of inflammatory response in CHIKV-infected patients. It has been reported that VD3 induces IL10 mRNA expression in regulatory T cells (Di Liberto et al., 2019). However, in the present study we reported that vitamin D did not induces significant IL10 expression in THP-1 cells and PBMCs. Further, we shown that VD3 treatment down-reguated IL10 mRNA expression in MDMs, and reduced IL10 production in CHIKV-infected VD3-Mon, but non in CHIKV-infected VD3-MDMs. Levels of IL10 are correlated with plasma leakage in DENV infected subjects and higher levels have been shown to be consistently associated with severe disease (Srikiatkhachorn and Green, 2010; Green et al., 1999). Further, in U937-DC-SIGN and THP-1 was reported that VD3 inhibited IL10 response at lower concentration while at higher concentration, IL10 response is nearly restored (Jadhav et al., 2018). However, given the important role of regulatory T lymphocytes in regulation of inflammatory and antiviral response (Zelinskyy et al., 2009), the production of IL10 during CHIKV infection may also diminish the protective effects of vitamin D. Conclusions In conclusion, we found that vitamin D does not inhibit CHIKV replication significantly in human monocytes and macrophages. Instead, CHIKV replication rapidly and efficiently modulates the mRNA expres sion of VDR, CAMP and CYP24A, and modified the expression of VD3 system significantly (Fig. 6). Modifications of this system may have extensive consequences beyond the cellular level. However, VD3 mod ulates TLRs expression and inflammatory response in CHIKV-infected VD3-Mon and VD3-MDMs (Fig. 6), suggesting that although VD3 did not control CHIKV replication, VD3 could play an important role in control inflammatory response in CHIKV-infected patients. Our obser vations in an in vitro model of CHIKV infection warrant further in vitro and in vivo evaluations for a comprehensive characterization of the consequences of VD3/VDR signaling on the innate and adaptive immune response to CHIKV infection. Author statement Authors’ contributions. Funding acquisition and project adminis tration: SUI; author contributions conceptualization: JFVL, PAV and SUI; conceived and designed the experiments: JFVL and SUI; method ology: JFVL and SUI; formal analysis: JFVL, PAV, and SUI; writing original draft: JFVL and SUI; writing review and editing: JFVL, PAV, and SUI; approval of article for publication: JFVL, PAV and SUI. CRediT authorship contribution statement Juan Felipe Valdés-López: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing, Formal analysis. Paula Velilla: Conceptualization, Writing – review & editing, Formal analysis. Silvio Urcuqui-Inchima: Concep tualization, Writing – original draft, Resources, Writing – review & editing, Supervision, Formal analysis, Project administration. Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors thank Ajit Kumar for reading the manuscript and for his valuable comments. This research was supported by Minciencias/Col ciencias [grant No. 111574455028 and contrato No. 455-2019], and Universidad de Antioquia-CODI, acta 2017-16389. The funders played Fig. 6. VD3 differentially modulates inflammatory response in Mon and MDMs, both infected with CHIKV: a model. Graphic summary of more representative effects of VD3 in control of viral replication, induction of vitamin D receptor signaling, modulation of TLRs expression, and induction of inflammatory response in CHIKV- infected VD3 Mon (up) and CHIKV-infected VD3 MDMs (down). J.F. Valdés-López et al. Acta Tropica 232 (2022) 106497 11 no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. The authors would like to thank the blood bank of the “Escuela de Microbiologia, 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. References Abraham, R., Mcpherson, R.L., Dasovich, M., Badiee, M., Leung, A.K.L., Griffin, E., 2020. Both ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain affect neurovirulence in mice. MBio 11 (1) e03253-19. Adams, J.S., Hewison, M., 2008. 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Ethics statement 2.2 RNA-seq data and bioinformatics analysis 2.3 Differential expression analysis of previously published data 2.4 CHIKV stocks and viral titration 2.5 Blood samples from healthy donors 2.6 Culture of human monocytes (Mon) and differentiation into monocyte-derived macrophages (MDMs) in the absence or presenc ... 2.7 Mon, VD3-Mon, MDMs and VD3-MDMs infection with CHIKV 2.8 Quantitative real-time PCR for TLRs, Cathelycidin, VDR and CYP24A1 2.9 Cytokine and chemokines quantification 2.10 Statistical analysis 3 Results 3.1 Transcriptomic analysis of VD3/VDR signaling 3.2 CHIKV replication in VD3-Mon and VD3-MDMs 3.3 CHIKV infection alters the expression of VDR and VDR-target genes in Mon and VD3-Mon 3.4 CHIKV infection alters the expression of VDR and VDR-target genes in MDMs and VD3-MDMs 3.5 Transcriptional regulation of TLRs, NF-κB-complex and NF-κB target genes in THP-1 cells, THP-1-VDR-KO cells and PBMCs t ... 3.6 TLRs and cytokines expression is altered in CHIKV-infected Mon and VD3-Mon 3.7 TLRs and cytokines expression is altered in CHIKV-infected MDMs and VD3-MDMs 4 Discission Conclusions Author statement CRediT authorship contribution statement Declaration of Competing Interests Acknowledgments References