Majumdar Manasi, Ratho R K, Chawla Yogesh, Singh Mini P
Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India.
Department of Hepatology, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India.
J Clin Virol. 2015 Dec;73:8-13. doi: 10.1016/j.jcv.2015.09.011. Epub 2015 Oct 9.
The clinical manifestations of Hepatitis E virus (HEV) range from self-limiting acute viral hepatitis (AVH) to acute liver failure (ALF). The varied clinical course is thought to be immune-mediated. Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral-response and downstream signaling of TLRs modulates cytokine production, thereby playing an important role in determining the disease course.
The present study was designed to elucidate the role of TLRs and cytokine production in the immunopathogenesis of HEV.
Peripheral blood mono-nuclear cells were separated from 50 AVH-HEV, 30 ALF-HEV patients and 50 healthy-controls. One-part of the PBMC was processed for RNA-extraction another pulsed with HEV-ORF2-peptide. Gene-expression levels of TLR (2-4, 7, and 8) were checked using semi-quantitative Real-time-PCR. Cytokine levels were analyzed using Cytokine-Bead-Array. TLR3-silencing experiments were performed and post-silencing cytokine levels were estimated.
TLR3 gene-expression in AVH was significantly higher than ALF (202.4±36.36 Vs 13.71±5.01; p<0.0001). Higher amount of both anti-and pro-inflammatory cytokines; IFNγ, TNF-α, IL10 and TGF-β were detected in the PBMC culture-supernatant of AVH Vs ALF (p<0.0001, p=0.0008, p=0.0002, p<0.0001 respectively). Post-silencing TLR3, significant decrease in IFNγ level was observed in the PBMC culture-supernatant (4.08±1.06 Vs 23.20±12.51; p=N0.0213).
TLR3 and IFNγ were found to play an important role in HEV disease pathogenesis. Patients capable of expressing high levels of TLR 3 and robust IFNγ response are able to limit the disease and recover uneventfully; while the patients with lower expression of TLR3 and IFNγ progress to ALF.
戊型肝炎病毒（HEV）的临床表现从自限性急性病毒性肝炎（AVH）到急性肝衰竭（ALF）不等。这种多样的临床病程被认为是免疫介导的。Toll样受体（TLR）在感知和启动先天性抗病毒反应中起核心作用，TLR的下游信号传导调节细胞因子的产生，从而在决定疾病进程中起重要作用。
本研究旨在阐明TLR和细胞因子产生在HEV免疫发病机制中的作用。
从50例AVH-HEV患者、30例ALF-HEV患者和50例健康对照者中分离外周血单个核细胞。一部分PBMC用于RNA提取，另一部分用HEV-ORF2肽脉冲处理。使用半定量实时PCR检测TLR（2-4、7和8）的基因表达水平。使用细胞因子珠阵列分析细胞因子水平。进行TLR3沉默实验，并估计沉默后细胞因子水平。
AVH中TLR3基因表达显著高于ALF（202.4±36.36对13.71±5.01；p<0.0001）。在AVH的PBMC培养上清液中检测到的抗炎和促炎细胞因子（IFNγ、TNF-α、IL10和TGF-β）的量均高于ALF（分别为p<0.0001、p=0.0008、p=0.0002、p<0.0001）。TLR3沉默后，在PBMC培养上清液中观察到IFNγ水平显著降低（4.08±1.06对23.20±12.51；p=0.0213）。
发现TLR3和IFNγ在HEV疾病发病机制中起重要作用。能够表达高水平TLR3和强烈IFNγ反应的患者能够限制疾病并顺利康复；而TLR3和IFNγ表达较低的患者则进展为ALF。
