Influence Of Vitamin A Nutrition On The Antiviral Immunity Of Children With Hand, Foot And Mouth Disease

Vitamin A (VA) is one of the most important microelements for childhood nutrition. Many epidemiological and clinical investigations have confirmed that VA play important role in maintenance of mucosal integrity and regulation of immune function. VA deficiency is a major public health nutrition problem in our country. It especially affects young children, and has been known to increase the morbidity and mortality of virus infected-diseases. Recent studies from our group have reported that VA is essential in regulating numerous key biologic processes of immune cells, including those involved in growth, maturation, differentiation and proliferation. However, further studies are clearly needed to determine the effect of VA in antiviral immune response. Hand, foot, and mouth disease (HFMD) is a viral illness commonly occurring in young children, particularly those less than5years of age. In mainland China, large outbreaks of HFMD have been reported since2008, and the main causative pathogen is Enterovirus71(EV71). As yet, there is no effective vaccine to prevent EV71infection. Without an effective antiviral treatment, patients must rely on their own immune systems to overcome the infection. In the present study, we first investigate the influence of VA status on antiviral immunity in patients with HFMD. Furthermore, we established an in vitro model to study the potential mechanism of the antiviral activity of VA. Such a study could provide evidence for rational use of VA to prevent HFMD in young children. In addition, these studies further confirm that VA plays an important role in immune function of children.Part1Influence of Vitamin A status on the antiviral immunity of children with hand, foot and mouth diseaseObjective Vitamin A (VA) deficiency has been shown to affect antiviral immunity and thus may be related to the progress and outcome of hand, foot and mouth disease (HFMD) in young children. Our objective was to determine whether children with HFMD associated with VA insufficiency displayed a decline in antiviral immunity.Methods450children with HFMD and113non-infected children were included in this study. Dietary investigations were performed using a24-hour dietary questionnaire. The serum concentrations of VA were measured by high-performance liquid chromatography. The serum levels of interferon-a (IFN-α) and enterovirus71 (EV71) IgM antibodies were detected using an enzyme-linked immunosorbent assay (ELISA).Results Of the450patients in the present analysis,303(67.3%) were male, and147(32.7%) were female. Almost92%of these patients failed to meet100%of the DRIs for VA and the deficient intake seems to be more serious in the1-<4-year age group. The mean patient age at disease onset was2.5years. We found226(50.2%) stool specimens that yielded positive EV71test results and170(37.8%) patients who had complications. The mean±SD serum VA concentration for all patients was0.73±0.26μmol/L. In total,237(52.7%) of all patients had values indicative of VA insufficiency (<0.7μmol/L). The mean VA concentration in EV71-positive patients (0.64±0.24μmol/L) was significantly lower than that in EV71-negative patients (0.83±0.25μmol/L), P<0.01. Both serum concentrations of VA and IFN-a in the patients with complications were significantly lower than in patients without complications, P<0.01. The decreased concentrations of IFN-a and EV71-IgM were positively related to lower VA levels (correlation coefficient=0.58and0.41, respectively, P<0.001).Conclusions VA status is associated with the antiviral immunity and pathogenetic condition of HFMD in young children. The children with HFMD mostly presented low VA concentrations and simultaneously had lower serum IFN-a levels, decreased immune antibody production and more severe illness. Part2Effect of all-trans-retinoic acid on enterovirus71infection in vitroObjective EV71is a pathogen that causes hand, foot, and mouth disease in young children. Altered or impaired immunity is thought to facilitate EV71pathogenesis. Vitamin A (VA) is an essential micronutrient with established role in modulating host immunity. In this study we established an in vitro model to investigate the effects and potential mechanism of the antiviral activity of VA.Methods The human monocytic U937cells were cultured in vitro and infected with EV71. All-trans-retinoic acid (atRA), the active metabolite of VA, and Ro41-5253, a retinoic acid receptor a antagonist, were used as the intervene agents. The EV71VP1+capsid proteins were detected with anti-VP1antibody using immunofluorescent microscopy. The percentage of EV71-infected cells and apoptosis induced by EV71were analyzed by flow cytometry. The level of interferon (IFN)-a in the supernatants of the cultures was detected using ELISA assay. The expression of retinoic acid receptor-a (RAR-a), IFNA1, and IFNAR1mRNA were examined by real-time quantitative PCR.Results atRA reduced the percentage of EV71-infected cells and protected cells against EV71-induced apoptosis. Correspondingly, atRA markedly increased the production of IFN-a, one of the most important antiviral cytokines, in both mRNA and protein levels in EV71-infected cells. Ro41-5253, a retinoic acid receptor (RAR)-a antagonist, abrogated the inhibitory effects of atRA on EV71infection.Conclusion Our findings suggest that atRA is an IFN-inducing agent with antiviral activity against EV71in vitro, and its actions are mediated at least in part by RAR-a activity. Part3The effect of all-trans-retinoic acid on the retinoid induced gene I signaling pathway in the antiviral immune response to enterovirus71Objective Activation of host cell antiviral responses is mediated by pattern recognition receptors, such as retinoid induced gene I (RIG-I)-like receptors (RLRs). In this study, we investigated the role of atRA play in modulating RIG-I signaling induced by EV71infection.Methods A human antiviral response PCR array (containing84key genes involved in the antiviral immune response) was used to detect the changes of the mRNA expression for RIG-I and related signaling pathway genes on peripheral blood monocyte macrophages (PBMC) obtained from EV71-infected patients and non-infected controls. By the PCR array, we also measured the antiviral-related gene expression in EV71-infected and atRA-treated human monocytic cells to understand the antiviral mechanism of atRA. Results A total of50differential expression genes (≥2fold differences) was found between EV71-infected patients and uninfected controls, among them27genes were upregulated and23genes were downregulated. Notably, the main genes, including RIG-I receptor, IPS-1(an important adaptor and interacting protein), TRAF3, TRAF6, TBK1, IRF3, IFNA1and IFNAR1, of the RIG-I signaling pathway were down regulated after EV71infected, whereas in atRA treated U937cells, the expression of these genes were regulated.Conclusion Our results show that the induction of the antiviral response to EV71involves RIG-I signaling, whose expression can be regulated by atRA.