以前的研究表明,体内施用具有抗炎和免疫调节特性的土壤来源的细菌,例如母牛分枝杆菌NCTC11659,可以防止应激诱导的向炎性M1小胶质细胞免疫表型和中枢神经系统(CNS)中的小胶质细胞启动的转变。目前尚不清楚母牛分枝杆菌NCTC11659是否可以直接作用于小胶质细胞以介导这些作用。本研究旨在确定母牛分枝杆菌NCTC11659对幼稚BV-2细胞极化的影响,小鼠小胶质细胞系,和BV-2细胞随后用脂多糖(LPS)攻击。简而言之,鼠BV-2细胞暴露于100μg/mL全细胞,热灭活母牛分枝杆菌NCTC11659或无菌硼酸盐缓冲盐水(BBS)载体,跟着,24小时后,通过在细胞培养基载体(CMV)或CMV对照条件下暴露于0.250µg/mL的LPS(大肠杆菌0111:B4;n=3)。LPS或CMV攻击后24小时,收获细胞以分离总RNA。使用NanoString平台的分析表明,本身,母牛分枝杆菌NCTC11659具有“佐剂样”效应,而暴露于LPS会增加编码促炎细胞因子的mRNA的表达,趋化因子配体,补体的C3成分,和炎症小体信号传导的组分如Nlrp3。在LPS攻击的细胞中,母牛分枝杆菌NCTC11659使用1.5倍变化的阈值对差异基因表达的影响有限。使用实时逆转录聚合酶链反应(real-timeRT-PCR)评估了一个基因子集,包括Arg1,Ccl2,Il1b,Il6、Nlrp3和Tnf。基于实时RT-PCR分析,母牛分枝杆菌NCTC11659本身再次诱导“佐剂样”效应,增加Il1b的表达,Il6和Tnf同时降低Arg1的表达。LPS本身增加了Ccl2、Il1b、Il6、Nlrp3和Tnf同时降低Arg1的表达。在LPS攻击的细胞中,母牛分枝杆菌NCTC11659增强LPS诱导的Nlrp3和Tnf表达增加,与小胶质细胞启动一致。相比之下,在LPS攻击的细胞中,尽管相对于媒介物处理的对照条件,母牛分枝杆菌NCTC11659不能完全防止LPS的影响,它增加了Arg1mRNA的表达,提示母牛分枝杆菌NCTC11659诱导非典型小胶质细胞表型。因此,当直接应用于小鼠BV-2小胶质细胞时,母牛分枝杆菌NCTC11659急性(在48小时内)诱导免疫激活和小胶质细胞启动作用,与其在中枢神经系统中观察到的长期抗炎和免疫调节作用相反,母牛分枝杆菌NCTC11659的热灭活制剂在体内外周给药。
Previous studies have shown that the in vivo administration of soil-derived bacteria with anti-inflammatory and immunoregulatory properties, such as Mycobacterium vaccae NCTC 11659, can prevent a stress-induced shift toward an inflammatory M1 microglial immunophenotype and microglial priming in the central nervous system (CNS). It remains unclear whether M. vaccae NCTC 11659 can act directly on microglia to mediate these effects. This study was designed to determine the effects of M. vaccae NCTC 11659 on the polarization of naïve BV-2 cells, a murine microglial cell line, and BV-2 cells subsequently challenged with lipopolysaccharide (LPS). Briefly, murine BV-2 cells were exposed to 100 µg/mL whole-cell, heat-killed M. vaccae NCTC 11659 or sterile borate-buffered saline (BBS) vehicle, followed, 24 h later, by exposure to 0.250 µg/mL LPS (Escherichia coli 0111: B4; n = 3) in cell culture media vehicle (CMV) or a CMV control condition. Twenty-four hours after the LPS or CMV challenge, cells were harvested to isolate total RNA. An analysis using the NanoString platform revealed that, by itself, M. vaccae NCTC 11659 had an \"adjuvant-like\" effect, while exposure to LPS increased the expression of mRNAs encoding proinflammatory cytokines, chemokine ligands, the C3 component of complement, and components of inflammasome signaling such as Nlrp3. Among LPS-challenged cells, M. vaccae NCTC 11659 had limited effects on differential gene expression using a threshold of 1.5-fold change. A subset of genes was assessed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), including Arg1, Ccl2, Il1b, Il6, Nlrp3, and Tnf. Based on the analysis using real-time RT-PCR, M. vaccae NCTC 11659 by itself again induced \"adjuvant-like\" effects, increasing the expression of Il1b, Il6, and Tnf while decreasing the expression of Arg1. LPS by itself increased the expression of Ccl2, Il1b, Il6, Nlrp3, and Tnf while decreasing the expression of Arg1. Among LPS-challenged cells, M. vaccae NCTC 11659 enhanced LPS-induced increases in the expression of Nlrp3 and Tnf, consistent with microglial priming. In contrast, among LPS-challenged cells, although M. vaccae NCTC 11659 did not fully prevent the effects of LPS relative to vehicle-treated control conditions, it increased Arg1 mRNA expression, suggesting that M. vaccae NCTC 11659 induces an atypical microglial phenotype. Thus, M. vaccae NCTC 11659 acutely (within 48 h) induced immune-activating and microglial-priming effects when applied directly to murine BV-2 microglial cells, in contrast to its long-term anti-inflammatory and immunoregulatory effects observed on the CNS when whole-cell, heat-killed preparations of M. vaccae NCTC 11659 were given peripherally in vivo.