Department of Microbiology and Immunology, Center for AIDS Health Disparities Research, Meharry Medical College, School of Medicine, Nashville, TN 37208-3599, USA.
J Neuroinflammation. 2012 May 18;9:95. doi: 10.1186/1742-2094-9-95.
Congenital human cytomegalovirus (HCMV) infections can result in CNS abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss. Brain pericytes play an essential role in the development and function of the blood-brain barrier yet their unique role in HCMV dissemination and neuropathlogy has not been reported.
Primary human brain vascular pericytes were exposed to a primary clinical isolate of HCMV designated 'SBCMV'. Infectivity was analyzed by microscopy, immunofluorescence, Western blot, and qRT-PCR. Microarrays were performed to identify proinflammatory cytokines upregulated after SBCMV exposure, and the results validated by real-time quantitative polymerase chain reaction (qPCR) methodology. In situ cytokine expression of pericytes after exposure to HCMV was examined by ELISA and in vivo evidence of HCMV infection of brain pericytes was shown by dual-labeled immunohistochemistry.
HCMV-infected human brain vascular pericytes as evidenced by several markers. Using a clinical isolate of HCMV (SBCMV), microscopy of infected pericytes showed virion production and typical cytomegalic cytopathology. This finding was confirmed by the expression of major immediate early and late virion proteins and by the presence of HCMV mRNA. Brain pericytes were fully permissive for CMV lytic replication after 72 to 96 hours in culture compared to human astrocytes or human brain microvascular endothelial cells (BMVEC). However, temporal transcriptional expression of pp65 virion protein after SBCMV infection was lower than that seen with the HCMV Towne laboratory strain. Using RT-PCR and dual-labeled immunofluorescence, proinflammatory cytokines CXCL8/IL-8, CXCL11/ITAC, and CCL5/Rantes were upregulated in SBCMV-infected cells, as were tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6). Pericytes exposed to SBCMV elicited higher levels of IL-6 compared to both mock-infected as well as heat-killed virus controls. A 6.6-fold induction of IL-6 and no induction TNF-alpha was observed in SBCMV-infected cell supernatants at 24 hours postinfection. Using archival brain tissue from a patient coinfected with HCMV and HIV, we also found evidence of HCMV infection of pericytes using dual-label immunohistochemistry, as monitored by NG2 proteoglycan staining.
HCMV lytic infection of primary human brain pericytes suggests that pericytes contribute to both virus dissemination in the CNS as well as neuroinflammation.
先天性人类巨细胞病毒（HCMV）感染可导致新生儿中枢神经系统异常，包括视力丧失、智力迟钝、运动缺陷、癫痫发作和听力损失。脑周细胞在血脑屏障的发育和功能中起着至关重要的作用，但它们在 HCMV 传播和神经病理学中的独特作用尚未得到报道。
将原代人脑血管周细胞暴露于一种名为“SBCMV”的原发性临床分离的 HCMV 中。通过显微镜检查、免疫荧光、Western blot 和 qRT-PCR 分析感染性。进行微阵列分析以鉴定 SBCMV 暴露后上调的促炎细胞因子，并通过实时定量聚合酶链反应（qPCR）方法验证结果。通过 ELISA 检测 HCMV 暴露后周细胞的原位细胞因子表达，并通过双标记免疫组织化学显示脑周细胞中 HCMV 感染的体内证据。
使用临床分离株 HCMV（SBCMV），感染的周细胞表现出病毒产生和典型的巨细胞病毒细胞病变。这一发现通过主要早期和晚期病毒蛋白的表达以及 HCMV mRNA 的存在得到了证实。与人类星形胶质细胞或人脑血管内皮细胞（BMVEC）相比，培养 72 至 96 小时后，脑周细胞对 CMV 裂解复制完全允许。然而，SBCMV 感染后 pp65 病毒蛋白的时间转录表达低于与 HCMV Towne 实验室株的表达。使用 RT-PCR 和双标记免疫荧光，SBCMV 感染的细胞上调了促炎细胞因子 CXCL8/IL-8、CXCL11/ITAC 和 CCL5/Rantes，以及肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）。与 mock 感染和热灭活病毒对照相比，SBCMV 感染的周细胞产生的 IL-6 水平更高。在感染后 24 小时，SBCMV 感染细胞上清液中观察到 IL-6 的诱导增加了 6.6 倍，而 TNF-α 没有诱导。使用 HCMV 和 HIV 合并感染患者的存档脑组织，我们还通过 NG2 蛋白聚糖染色监测，通过双标记免疫组织化学发现 HCMV 感染周细胞的证据。
HCMV 对原代人脑血管周细胞的裂解感染表明，周细胞既有助于病毒在中枢神经系统中的传播，也有助于神经炎症。
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