Human macrophages differentiated in the presence of vitamin D3 restrict dengue virus infection and innate responses by downregulating mannose receptor expression

ABSTARCT: Severe dengue disease is associated with high viral loads and overproduction of pro-inflammatory cytokines, suggesting impairment in the control of dengue virus (DENV) and the mechanisms that regulate cytokine production. Vitamin D3 has been described as an important modulator of immune responses to several pathogens. Interestingly, increasing evidence has associated vitamin D with decreased DENV infection and early disease recovery, yet the molecular mechanisms whereby vitamin D reduces DENV infection are not well understood. METHODS AND PRINCIPAL FINDINGS: Macrophages represent important cell targets for DENV replication and consequently, they are key drivers of dengue disease. In this study we evaluated the effect of vitamin D3 on the differentiation of monocyte-derived macrophages (MDM) and their susceptibility and cytokine response to DENV. Our data demonstrate that MDM differentiated in the presence of vitamin D3 (D3-MDM) restrict DENV infection and moderate the classical inflammatory cytokine response. Mechanistically, vitamin D3-driven differentiation led to reduced surface expression of C-type lectins including the mannose receptor (MR, CD206) that is known to act as primary receptor for DENV attachment on macrophages and to trigger of immune signaling. Consequently, DENV bound less efficiently to vitamin D3-differentiated macrophages, leading to lower infection. Interestingly, IL-4 enhanced infection was reduced in D3-MDM by restriction of MR expression. Moreover, we detected moderate secretion of TNF-α, IL-1β, and IL-10 in D3-MDM, likely due to less MR engagement during DENV infection. CONCLUSIONS/SIGNIFICANCE: Our findings reveal a molecular mechanism by which vitamin D counteracts DENV infection and progression of severe disease, and indicates its potential relevance as a preventive or therapeutic candidate

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Surface MR expression and effect of its blockade on DENV replication in MDMs and D₃-MDMs.

<p>MR surface expression was measured by FACS detection of CD206+ cells after MDM and D₃-MDM differentiation. <b>A.</b> Flow cytometry-gating strategy for the measurement of CD206+ events from the parental region A from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005904#pntd.0005904.s001" target="_blank">S1B Fig</a>. Isotype controls were used for both MDMs and D₃-MDMs to set the CD206 positive events gate. Middle and lower panels show representative distribution of CD206+ cells in MDM and D₃-MDM in gate B and the comparison of CD206 Mean Fluorescence Intensity (MFI). <b>B</b>. Statistical comparison of CD206+ percentage cells and CD206 MFI. <b>C.</b> Correlation between the percentage of CD206 positive cells and infection percentage in MDMs and D₃-MDMs observed in 4 different donors. <b>D.</b> MR ligation to DENV-2 was blocked by incubating with methyl mannoside (MM) for 2 h prior to infection. The intracellular number of GEc was measured by RT-qPCR 24 hpi and was compared with that in control mock-treated cells. Bars represent mean ± SD from at least 3 different donors. Wilcoxon signed rank test; *<i>p</i><0.05; n.s denotes non-significant.</p

Susceptibility of MDMs and D₃-MDMs to DENV-2 infection.

<p>After differentiation, the cells were challenged with DENV-2 at a MOI of 10 and 24 hpi cells, cell lysates and supernatants were obtained. <b>A</b>. FACS measurement of DENV infection as number of positive cells for intracellular DENV-E protein. <b>B.</b> DENV infection measurement and comparison between MDMs and D₃-MDMs at a MOI of 10. <b>C.</b> GEc titers measured by RT-qPCR in cell lysates and supernatants. Bars represent mean ± SD. Wilcoxon signed rank test; *<i>p</i><0.05, **<i>p</i><0.01.</p

IL-4 induced MR expression and DENV infection in MDMs and D₃-MDMs.

<p>After MDM and D₃-MDM differentiation, the cells were stimulated with IL-4 and MR induction was allowed for an additional 48 h. <b>A.</b> Surface detection of CD206 was measured by flow cytometry. Isotype controls were used for both MDMs and D₃-MDMs. Dot plots show representative distribution of CD206+ cells in MDMs and D₃-MDMs with and without IL-4 treatment. Histograms show the comparison of the CD206 MFI. <b>B</b>. Statistical comparison of CD206 MFI and percentage of CD206+ cells for all 5 donors tested. <b>C.</b> IL-4 treated MDMs and D₃-MDMs were infected with DENV and 24 hpi, the numbers of virus genome particles were measured by RT-qPCR in cell lysates and compared with those in mock-treated cells. <b>D</b>. MR ligation to DENV-2 was blocked with MM 2 h prior to infection. The intracellular numbers of GEc were measured by RT-qPCR 24 hpi, and compared with those in control mock-treated cells. Bars represent mean ± SD from at least 3 different donors. Wilcoxon signed rank test and Mann-Whitney test. *<i>p</i><0.05, **<i>p</i><0.01, *** <i>p</i><0,001. n.s = not significant.</p

DENV-induced cytokine response in MDMs and D₃-MDMs.

<p><b>A</b>. Levels of cytokines released by the two macrophage preparations following DENV infection at 24 hpi. <b>B</b> Effect of 10mM MM treatment on the LPS (10 ng/mL)-induced TNF-α secretion in MDMs and D₃-MDMs. <b>C.</b> DENV-induced secretion of TNF-α and IL-1β after MM treatment in MDMs and D₃-MDMs. Cells were pre-incubated with 10 nM MM prior to infection with DENV. Bars represent mean ± SD. Wilcoxon signed rank test. *<i>p</i><0.05, **<i>p</i><0.01, ***. n.s denotes not significant.</p

Binding and/or internalization of DENV into MDMs and D₃-MDMs.

<p>The bound and/or intracellular virus particles were detected using RT-qPCR as described in the Methods section. The percentage of bound-internalized viral particles was calculated in relation to the GEc added (equivalent of a MOI of 10). Bars represent mean ± SD; n = 5 Wilcoxon signed rank test *<i>p</i><0.05.</p