Transcriptional profile regulated by 1,25-dihydroxyvitamin D (Calcitriol) in primary Human Monocyte-Derived Macrophages infected by zika virus

ZIKA virus (ZIKV) infection is characterized by alterations in gene expression profile on host cells that consequently lead to an immune response. Here, we used RNA sequencing to analyze the mRNA and miRNA expression profile in human monocyte-derived macrophages (MDMs) differenitated in presenece of 1,25-dihydroxyvitamin D (Calcitriol) and infected with a Zika virus strain COL345Si-Asian at MOI of 5 and analized 24 hpi. Overall design: Human peripheral blood mononuclear cells (PBMCs) from blood samples of 4 healthy donors mixed with 2% EDTA, were isolated through density gradient with Lymphoprep. Monocytes-derived Macrophages (MDM) were obtained from total PBMCs by adherence to the plastic and differentiation to macrophages was allowed to occur for 6 days 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), at 37 °C and 5% CO2, without growth factors. Additionally, MDMs were differentiated in presence of 1,25 di-hydroxyvitamin D3 to obtain D3-MDM. 1,25 di-hydroxyvitamin D3 was added to the culture media at a final concentration of 1 nM and replenished every 24 h for six days. MDM and D3-MDM monolayers were washed with warm PBS and were infected with Zika virus strain COL345Si-Asian at MOI of 5. Three hpi, the cells were washed with warm PBS to remove unbound virus. Then 24 hpi, macrophage monolayers were used for sample collection.

寨卡病毒（ZIKA virus, ZIKV）感染以宿主细胞基因表达谱改变为特征，进而引发免疫应答。本研究采用RNA测序（RNA sequencing）技术，分析了经1,25-二羟维生素D（1,25-dihydroxyvitamin D, 骨化三醇Calcitriol）诱导分化的人单核细胞源性巨噬细胞（monocyte-derived macrophages, MDMs）在感染寨卡病毒COL345Si-Asian毒株（感染复数MOI=5）后24小时的mRNA与miRNA表达谱。

实验设计概述：采集4名健康志愿者的外周血样本，加入2%乙二胺四乙酸（EDTA）抗凝，通过Lymphoprep密度梯度离心法分离外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）。从总PBMCs中通过贴壁培养获取单核细胞源性巨噬细胞（MDMs）：于37℃、5% CO₂环境下，在添加10%（v/v）胎牛血清（FBS）、4 mM L-谷氨酰胺、0.3%（v/v）碳酸钠（NaCO3）、1%（v/v）100×抗生素-抗真菌溶液的RPMI-1640完全培养基中培养6天，使其分化为巨噬细胞，期间不添加生长因子。

此外，部分单核细胞源性巨噬细胞在1,25-二羟维生素D₃（1,25 di-hydroxyvitamin D3）存在下诱导分化为D3-MDM：将1,25-二羟维生素D₃以终浓度1 nM添加至培养基中，每24小时换液一次，持续诱导6天。

将MDM与D3-MDM单层细胞用预热PBS洗涤后，以MOI=5感染寨卡病毒COL345Si-Asian毒株。感染后3小时，用预热PBS洗涤细胞以去除未结合的病毒；感染后24小时，收集巨噬细胞单层用于样本采集。