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. 2017 Oct 11;11(10):e0005904.
doi: 10.1371/journal.pntd.0005904. eCollection 2017 Oct.

Human Macrophages Differentiated in the Presence of Vitamin D3 Restrict Dengue Virus Infection and Innate Responses by Downregulating Mannose Receptor Expression

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Free PMC article

Human Macrophages Differentiated in the Presence of Vitamin D3 Restrict Dengue Virus Infection and Innate Responses by Downregulating Mannose Receptor Expression

John F Arboleda Alzate et al. PLoS Negl Trop Dis. .
Free PMC article

Abstract

Background: 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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Susceptibility of MDMs and D3-MDMs to DENV-2 infection.
After differentiation, the cells were challenged with DENV-2 at a MOI of 10 and 24 hpi cells, cell lysates and supernatants were obtained. A. FACS measurement of DENV infection as number of positive cells for intracellular DENV-E protein. B. DENV infection measurement and comparison between MDMs and D3-MDMs at a MOI of 10. C. GEc titers measured by RT-qPCR in cell lysates and supernatants. Bars represent mean ± SD. Wilcoxon signed rank test; *p<0.05, **p<0.01.
Fig 2
Fig 2. Binding and/or internalization of DENV into MDMs and D3-MDMs.
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 *p<0.05.
Fig 3
Fig 3. Surface MR expression and effect of its blockade on DENV replication in MDMs and D3-MDMs.
MR surface expression was measured by FACS detection of CD206+ cells after MDM and D3-MDM differentiation. A. Flow cytometry-gating strategy for the measurement of CD206+ events from the parental region A from S1B Fig. 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. Statistical comparison of CD206+ percentage cells and CD206 MFI. C. Correlation between the percentage of CD206 positive cells and infection percentage in MDMs and D3-MDMs observed in 4 different donors. D. 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; *p<0.05; n.s denotes non-significant.
Fig 4
Fig 4. IL-4 induced MR expression and DENV infection in MDMs and D3-MDMs.
After MDM and D3-MDM differentiation, the cells were stimulated with IL-4 and MR induction was allowed for an additional 48 h. A. 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. Statistical comparison of CD206 MFI and percentage of CD206+ cells for all 5 donors tested. C. 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. D. 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. *p<0.05, **p<0.01, *** p<0,001. n.s = not significant.
Fig 5
Fig 5. DENV-induced cytokine response in MDMs and D3-MDMs.
A. Levels of cytokines released by the two macrophage preparations following DENV infection at 24 hpi. B Effect of 10mM MM treatment on the LPS (10 ng/mL)-induced TNF-α secretion in MDMs and D3-MDMs. C. 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. *p<0.05, **p<0.01, ***. n.s denotes not significant.

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References

    1. Wilson M.E., Chen L.H. , Dengue: update on epidemiology., Curr. Infect. Dis. Rep. 17 (2015) 457 doi: 10.1007/s11908-014-0457-2 - DOI - PubMed
    1. Schwartz L.M., Halloran M.E., Durbin A.P., Longini I.M., The dengue vaccine pipeline: Implications for the future of dengue control, Vaccine. 33 (2015) 3293–3298. doi: 10.1016/j.vaccine.2015.05.010 - DOI - PMC - PubMed
    1. Aguiar M., Stollenwerk N., Halstead S.B., The risks behind Dengvaxia recommendation, Lancet Infect. Dis. 16 (2016) 882–883. doi: 10.1016/S1473-3099(16)30168-2 - DOI - PubMed
    1. Bhatt S., Gething P.W., Brady O.J., Messina J.P., Farlow A.W., Moyes C.L., Drake J.M., Brownstein J.S., Hoen A.G., Sankoh O., Myers M.F., George D.B., Jaenisch T., Wint G.R.W., Simmons C.P., Scott T.W., Farrar J.J., Hay S.I., The global distribution and burden of dengue, Nature. 496 (2013) 504–507. doi: 10.1038/nature12060 - DOI - PMC - PubMed
    1. Shepard D.S., Undurraga E.A., Halasa Y.A., Stanaway J.D., Bhatt S., Gething P., Brady O., et al. , Simmons C., Farrar J., Nguyen V., Wills B., Beatty M., Beutels P., Meltzer M., et al. , Shepard D., Coudeville L., Halasa Y., Zambrano B., Dayan G., Shepard D., Undurraga E., Halasa Y., Brady O., Gething P., Bhatt S., et al. , Shepard D., Undurraga E., Betancourt-Cravioto M., et al. , W.H. Organization, W.H. Organization, W.H.O.W.P. Region, P.A.H. Organization, W.H.O.R.O. for S.-E. Asia, Constenla D., Garcia C., Lefcourt N., Selck F., Adalja A., Boddie C., Stanaway J., Shepard D., Undurraga E., et al. , Vos T., Barber R., Bell B., et al. , D. and H.S. (DHS), Shepard D., Halasa Y., Tyagi B., et al. , Undurraga E., Halasa Y., Shepard D., Undurraga E., Betancourt-Cravioto M., Ramos-Castañeda J., et al. , W.H. Organization, Pamplona L., de M. Braga D., da Silva L., et al. , Barnighausen T., Bloom D., Cafiero E., O’Brien J., W.H. Organization, Tiga D., Undurraga E., Ramos-Castañeda J., Martínez-Vega R., Tschampl C., Shepard D., U.C. for H. Security, Grimwood K., Lambert S., Milne R., Rheingans R., Antil L., Dreibelbis R., Podewils L., Bresee J., Parashar U., Lee B., Bacon K., Bottazzi M., Hotez P., Hampson K., Coudeville L., Lembo T., et al. , The global economic burden of dengue: a systematic analysis, Lancet Infect. Dis. 0 (2016) 504–507. doi: 10.1016/S1473-3099(16)00146-8 - DOI

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Grant support

SUI were supported by grant from the Colombian department of Science, Technology and innovation (CTeI), Colciencias (Grant number: 111556933443) and Universidad de Antioquia, UdeA. IARZ was funded by NWO- VENI grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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