High-dose of vitamin D supplement is associated with reduced susceptibility of monocyte-derived macrophages to dengue virus infection and pro-inflammatory cytokine production: An exploratory study
Introduction
According to the World Health Organization (WHO), the incidence of dengue has increased dramatically in the last three decades and has expanded to new geographical areas, becoming an important problem in public health worldwide [1]. In humans, infection with dengue virus (DENV) results in a wide spectrum of outcomes leading to diverse clinical manifestations ranging from asymptomatic infection or mild undifferentiated fever, to one of the following clinical manifestations, dengue with or without warning signs and severe dengue [2]. Although the pathology of DENV is not completely understood, one of the hallmark manifestations in dengue patients includes capillary leak syndrome caused by endothelial dysfunction associated with an alteration in the balance of secreted pro-inflammatory cytokines [3]. Diverse studies have reported that DENV-infected monocytes and macrophages release soluble mediators, including interleukin (IL)-1β, and IL-6 that exert prominent effects on the function and properties of endothelial cells [4], suggesting that these cells play a role in DENV pathogenesis. Toll-like receptors (TLRs), one of the most important categories of pattern recognition receptors (PRRs), recognize viral particles or viral components and subsequently initiate signaling for pro-inflammatory cytokine production [5]. Interestingly, the DENV NS1 protein induces cytokine secretion through TLR4 [6], and contributes to vascular leak in dengue patients [7]. Furthermore, DENV up-regulates the expression of TLR3 and TLR8 in HepG2 cells and increase the expression interferon-beta and pro-inflammatory cytokines [8]. Taken together, these results show that DENV infection induces TLR activation and consequently that the interaction of DENV-TLRs can result in dengue disease progression.
It is known that 1,25-dihydroxyvitamin D [1,25(OH)2D3], the active metabolite of vitamin D, acts at multiples levels, including cell proliferation and cell differentiation, and has potent immunomodulatory activities [9]. However, the inactive form of vitamin D (25-hydroxyvitamin D) is hydroxylated to its active form by 25(OH)D-1alfa-hydroxylase (CYP27B1) [10]. The active form of vitamin D then binds to the intracellular vitamin D receptor (VDR) and induces the expression of 1,25(OH)2D- 24-hydroxylase (24-OHase; CYP24A1) which initiates the degradation of the physiologically active form of vitamin D3 [11]. The human antimicrobial peptide LL-37, which is produced as part of the innate immunity, is induced by 1,25(OH)2D3 macrophages [12],and has received considerable attention due to its antimicrobial activity against diverse pathogens [13]. However, our understanding of the impact of vitamin D on the immune response, including cytokine production by regulating the innate immune receptors is more limited. To date it is well known that vitamin D metabolites down-regulate pro-inflammatory cytokine production, but their impact on virus or viral PRR-stimulated response is very limited. Some studies reported that patients with Hepatitis C virus (HCV) and with vitamin D deficiency present severe manifestations such as hepatic cirrhosis, chronic liver disease and severe hepatic fibrosis [14], [15]. Likewise, it was reported that vitamin D supplementation increases the probability of achieving a sustained viral response following antiviral treatment, suggesting a relationship between vitamin D and HCV infection [16]. Epidemiological studies have also provided evidence that vitamin D deficiency could be associated with a risk of viral infections including influenza virus (IV), respiratory tract infections and human immunodeficiency virus 1 (HIV-1) [17]. Other reports strongly point towards vitamin D as having significant beneficial effects in the prevention of infectious diseases [18]. This is highly interesting, since vitamin D is one of the most cost-effective supplements used to improve overall human health [19], [20]. Puerta-Guardo et al., (2012) demonstrated that vitamin D treatment resulted both in a significant decrease in the percentage of U937- and Huh-7-cells infected with DENV and a reduction in the production of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 [21]. Moreover, an association between the polymorphism of the vitamin D receptor (VDR) gene and the risk of symptomatic dengue requiring hospitalization was reported [22]. While these results show an association between vitamin D and clinical outcomes of DENV infection including decreased levels of pro-inflammatory cytokines, the importance of vitamin D supplementation in the context of dengue patients is unknown.
Although vitamin D has been assigned functions in monocytes and macrophages [23], the macrophage cell type resulting in response to vitamin D treatment has not been sufficiently characterized. It has been established that the differentiation state of monocytes and macrophages is crucial in determining the susceptibility and productive infection by different viruses [24], but the influence of these differentiations on DENV infection has been sparsely examined. We observed that monocyte-derived macrophages (MDMs) differentiated in the presence of vitamin D, in addition to expressing lower levels of mannose receptor, are less susceptible to DENV infection and produce low levels of pro-inflammatory cytokines compared with MDMs differentiated in the absence of vitamin D (submitted manuscript). Therefore, based on these results the aim of the present study was to explore if oral vitamin D supplementation exerts an effect on DENV susceptibility and pro-inflammatory cytokine production in MDMs. To accomplish this, the healthy individuals received 1000 or 4000 international units (IU)/day of vitamin D supplement during 10 days. Then, from each individual, before and after the vitamin D supplement, a PB sample was taken and the monocytes isolated. They were used to obtain MDMs in the presence of autologous serum, and were then challenged with DENV-2. The data indicated that macrophages differentiated from monocytes obtained from healthy donors who received higher doses of vitamin D (4000 IU/day), exhibited higher resistance to DENV-2 infection and produced a significant decrease of pro-inflammatory cytokines and high production of IL-10. Furthermore, a significant decrease of intracellular TLR mRNA was observed. Although the results of our study suggest that the status of vitamin D can influence the immune response and DENV resistance/susceptibility to infection, further studies are required to determine the feasibility of vitamin D as anti-DENV agent and immune modulator.
Section snippets
Ethics statement
The protocols for patient enrollment and sample collection were approved by the Committee of Bioethics Research of the Sede de Investigación Universitaria, Universidad de Antioquia (Medellín, Colombia) and an individual's inclusion was preceded by a signed informed consent form, according to the principles expressed in the Declaration of Helsinki.
Study subjects
Since two oral daily doses (4000 and 7000 IU) of cholecalciferol (D3) over a 12-week period was reported safe and effective [25], our design was to
High dose of vitamin D supplementation decreases the percentage of MDM-positives for DENV E antigen
The resistance/susceptibility to DENV infection was monitored in MDMs before (VD0) and after 10 days of vitamin D supplementation (VD10). To accomplish this, the expression of the DENV E antigen in MDMs and of the viral RNA in the challenge-MDM supernatants was measured. The effect was determined in macrophages differentiated from monocytes obtained from both groups 1000VitD and 4000VitD, as described in the Materials and methods section. MDMs were then infected with DENV-2 at a MOI 5 for 24 h.
Discussion
In this study, we observed that macrophages differentiated from monocytes and obtained from healthy donors who received high doses of vitamin D supplementation are more resistant to DENV-2 infection. Indeed, there resulted a significant reduction in the percentage of DENV-2-infected macrophages and a decrease in the number of viral RNA copies. Conversely, when the macrophages were differentiated from monocytes obtained from donors who received a low dose of vitamin D, a statistically
Conflict of interest
None of the authors has any potential financial conflict of interest related to this manuscript.
Acknowledgements
This study was supported by COLCIENCIAS grant number 111556933443 from Colombia and the Universidad de Antioquia UdeA. The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors wish to acknowledge the individuals who participated in this study and the personnel at the institutions where the study was performed. We acknowledge Anne-Lise Haenni for critically reviewing the manuscript.
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