乳酸调控巨噬细胞功能促进登革病毒2型感染

Lactate promotes dengue virus type 2 infection by modulating macrophage function

目的探究外源性乳酸对Raw264.7细胞、小鼠骨髓来源巨噬细胞(mouse bone marrow-derived macrophages,BMDMs)及THP-1细胞中登革病毒2型(dengue virus type 2,DENV-2)复制的影响及乳酸与细胞活化之间的关系。方法培养和诱导BALB/c小鼠骨髓中的BMDMs,流式细胞术检测F4/80+CD11b+细胞纯度。qRT-PCR检测不同时间点DENV-2感染BMDMs中葡萄糖转运体1(glucose transporter type 1,GLUT1)、己糖激酶2(hexokinase 2,HK2)、乳酸转运体4(monocarboxylate transporters 4,MCT4)的mRNA表达水平,比色法检测细胞上清液中乳酸含量。使用CCK-8法评估不同浓度乳酸对Raw264.7细胞、BMDMs及THP-1细胞活力的影响,qRT-PCR检测不同浓度乳酸对不同MOI DENV-2感染细胞中病毒E基因、CD163、TGF-β、CD86、视黄酸诱导基因Ⅰ(retinoic acid-inducible geneⅠ,RIG-Ⅰ)、IFN-β、干扰素刺激基因15(interferon-stimulated gene 15,ISG15)及ISG56的mRNA表达水平,流式细胞术检测CD86和CD206蛋白的表达。结果BMDMs纯度为(87.53±1.66)%。DENV-2(MOI=3)感染BMDMs后,GLUT1 mRNA转录水平在12 h一过性增高,在24 h降低(P<0.05),而HK2 mRNA转录水平在12、24和36 h均高于空白对照组和灭活DENV-2组(P<0.01);DENV-2(MOI=1.5)感染后24 h,MCT4 mRNA转录水平及上清液中乳酸含量均上升(P<0.05)。CCK-8结果显示当乳酸浓度为31.25 mmol/L时,上述3种细胞的活力均大于80%。qRT-PCR结果显示,在MOI=1和2时,35 mmol/L乳酸可增加BMDMs中DENV E基因mRNA的表达水平(P<0.05);在MOI=1.5时,35 mmol/L乳酸上调Raw264.7和THP-1细胞中的DENV E基因mRNA表达水平(P<0.001),以及BMDMs中CD163、TGF-β、RIG-Ⅰ、IFN-β、ISG15和ISG56的mRNA表达水平(P<0.05),下调CD86 mRNA的表达(P<0.05),细胞中CD206蛋白的表达增加(P<0.01),而CD86蛋白的表达下降(P>0.05)。结论本研究发现外源性乳酸能促进DENV-2在人源及鼠源巨噬细胞中的感染,该现象与细胞M2型极化有关。

Objective To investigate the impact of exogenous lactate on the replication of dengue virus type 2(DENV-2)in Raw264.7 cells,mouse bone marrow-derived macrophages(BMDMs)and THP-1 cells and explore its association with cell activation.Methods BMDMs from BALB/c mouse bone marrow were prepared and evaluated by flow cytometry to detect the proportion of F4/80+CD11b+cells.Glucose transporter type 1(GLUT1),hexokinase 2(HK2),and monocarboxylate transporters 4(MCT4)expression at mRNA level in BMDMs at different time points after DENV-2 infection were measured by qRT-PCR.The content of lactate in the culture supernatants was quantified via colorimetric assay.CCK-8 assay was used to evaluate the impacts of different concentrations of lactate on the viability of Raw264.7 cells,BMDMs,and THP-1 cells.qRT-PCR was used to detect the expression of DENV-2 E gene,TGF-β,CD86,retinoic acid-inducible geneⅠ(RIG-Ⅰ),IFN-β,interferon-stimulated gene 15(ISG15),and ISG56 at mRNA level in cells infected with DENV-2 at different MOIs in the presence of different concentrations of lactate.Meanwhile,flow cytometry was used to analyze the expression of CD86 and CD206.Results The percentage of BMDMs was(87.53±1.66)%.GLUT1 expression at mRNA level exhibited a decrease in BMDMs at 24 h after DENV-2(MOI=3)infection following a transient increase at 12 h(P<0.05),while HK2 expression at mRNA level was higher that than in blank control and inactivated DENV-2 infection groups at 12,24,and 36 h(P<0.01).Besides,there was an increase in both MCT4 mRNA level and the content of lactate in culture supernatants at 24 h after DENV-2(MOI=1.5)infection(P<0.05).The viability of the three types of cells remained above 80%when the concentration of lactate was 31.25 mmol/L.Lactate at the concentration of 35 mmol/L increased the expression of the DENV E gene at mRNA level in DENV-2-infected BMDMs at MOI=1 or MOI=2(P<0.05).Besides,it promoted the expression of DENV E gene at mRNA level in Raw264.7 and THP-1 cells(P<0.001)as well as the expression of CD163,TGF-β,RIG-Ⅰ,IFN-β,ISG15 and ISG56 at mRNA level in BMDMs at MOI=1.5,but inhibited the expression of CD86 at mRNA level in BMDMs(P<0.05).It also up-regulated CD206 protein expression(P<0.01)and down-regulated CD86 protein expression(P>0.05)in BMDMs.Conclusions Exogenous lactate enhances DENV-2 replication in both human-and murine-derived macrophages and that might correlate with M2 macrophage polarization.