HCV Infection and Innate Immunity

丙型肝炎病毒是一个主要的公共卫生问题，全世界有超过1.7亿人感染。大多数丙型肝炎病毒感染病例持续存在，最终可能导致慢性肝病、肝硬变和肝细胞癌。虽然肝细胞是病毒复制的主要部位，但慢性丙型肝炎病毒感染与广泛的肝外并发症和疾病有关，包括混合性冷球蛋白血症、非霍奇金淋巴瘤、皮肤血管炎、肾小球肾炎、神经病变和淋巴增生性疾病。丙型肝炎病毒复制的肝外储存库的存在，特别是在外周血单个核细胞中，仍然存在很大的争议。目前尚不清楚在慢性丙型肝炎病毒感染过程中B细胞是如何变得失调的。 1)本课题组已经证实，游离的丙型肝炎病毒被补体调理并与红细胞CR1结合(ECR1)；丙型肝炎病毒特异性抗体的存在显著增加了这种结合。游离的丙型肝炎病毒与红细胞的结合是否与丙型肝炎病毒的清除或致病机制有关尚无研究。混合性冷球蛋白血症(II型)是与慢性丙型肝炎病毒感染相关的最常见的IC疾病之一。尽管近一半的丙型肝炎患者有可检测到的冷球蛋白，但只有不到10%的患者会发展为临床明显的疾病。我们已经证明了HCVIC与红细胞结合，因此，影响HCVIC/红细胞相互作用的因素可能会影响临床上明显的ICR相关疾病的表达。(2018年《肝病》)。 2)为了进一步探讨丙型肝炎病毒相关免疫复合体疾病的发病机制，我们检测了慢性丙型肝炎患者和健康对照组血浆中补体激活免疫复合体(IC)、类风湿因子和几种趋化因子和细胞因子的水平，以阐明丙型肝炎病毒-IC/红细胞相互作用可能的生物学反应和后果。我们还研究了与红细胞CR1表达相关的补体受体1(CR1/CD35)基因的遗传多态性，以及这种CR1表达水平与IC水平之间的关系。我们发现：a)慢性丙型肝炎患者血液中几种趋化因子包括CXCL10、CXCL12和BAFF水平明显高于健康对照组；b)慢性丙型肝炎患者循环免疫复合物(CIC)和类风湿因子水平也同样升高；c)CR1基因HH基因型的慢性丙型肝炎患者的血液CIC水平显著低于HL型的慢性丙型肝炎患者。(手稿正在准备中)。 3)我们观察到丙型肝炎病毒与CD19+B细胞的结合是由补体系统介导的。此外，利用细胞表面标志物的抗体，我们发现结合复合体主要涉及CD21(补体受体2)、CD19、CD20和CD81。在人类B细胞中，CD21与CD19和CD81形成共刺激复合体。B细胞抗原受体(BCR)与这种共刺激复合体的共连接可以降低BCR介导的B细胞激活和增殖所需的阈值。流行病学研究表明，慢性丙型肝炎患者发生B细胞非霍奇金淋巴瘤的风险增加。抗病毒治疗对丙型肝炎病毒相关淋巴瘤的消退，以及抗病毒治疗对丙型肝炎相关淋巴瘤患者总存活率的有利影响，都加强了流行病学、临床和病理生理学研究推测的丙型肝炎感染和非霍奇金淋巴瘤之间的病因学联系(《肝病学》2016)。 我们的目标是进入下一步，阐明补体介导的丙型肝炎病毒附着/感染可能导致B细胞恶性转化的途径。 4)由于B细胞在抗原刺激或多克隆激活的刺激下增殖，易发生遗传变异(突变、基因易位、基因融合、染色体扩增或缺失)，我们使用RNA测序技术，获得了来自HCV+、HBV+、HDV+和B-NHL+的患者的B细胞的突变和基因表达数据，并与丙型肝炎病毒、乙肝病毒-、HDV-、NHL+、丙型肝炎病毒+、乙肝病毒+、HDV+、NHL-患者和健康对照进行了比较。然后，我们将验证/确认成对石蜡包埋淋巴组织的结果。采用RNA测序已被证明在临床肿瘤学中是有用的，因为大多数临床相关的体细胞突变也在RNA水平上表达。对基因进行测序以鉴定突变状态，同时获得结构变异和基因表达紊乱相关疾病的信息，是一项很有前途的技术。我们是NIAID-NCI赠款的获得者：“蒙古丙型肝炎病毒和乙肝病毒介导的B细胞恶性转化的调查和丙型肝炎相关非霍奇金淋巴瘤的优先治疗”。我们从蒙古收到了200多份样本(PBMC和匹配的石蜡包埋淋巴组织)。我们已经处理并完成了从PBMC中分离的100个BCells的RNA测序。RNA-seq分析发现了与恶性肿瘤相关的差异表达基因和异常信号通路，包括PAX5、AHR、MYC、TP53以及bcr、eIF2和mTOR通路。RNPC1的上调可能通过抑制p53在肿瘤发生中发挥作用。功能分析数据将与基因组数据相结合。下一步将确认在匹配的组织样本上的发现。

HCV is a major public health problem, infecting more than 170 million people worldwide. Most cases of HCV infection become persistent and may eventually lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma. Although hepatocytes are the major site of viral replication, a broad clinical spectrum of extrahepatic complications and diseases are associated with chronic HCV infection, including mixed cryoglobulinemia, non-Hodgkins lymphoma, cutaneous vasculitis, glomerulonephritis, neuropathy, and lymphoproliferative disorders. The existence of extrahepatic reservoirs of HCV replication, particularly in PBMCs, remains highly controversial. It is unclear how B cells become dysregulated during the course of chronic HCV infection. 1) Our group has demonstrated that free HCV is opsonized by complement and binds to CR1 on erythrocytes (ECR1); the presence of HCVspecific antibody significantly increased this binding. Whether binding of free HCV to erythrocytes is related to HCV clearance or pathogenesis has not been investigated. Mixed cryoglobulinemia (type II) is among the most common IC diseases associated with chronic HCV infection. Whereas nearly half of patients with HCV have detectable cryoglobulins, less than 10% develop clinically apparent disease. We have demonstrated that HCVIC binds to erythrocytes, and it is therefore plausible that factors affecting HCVIC/erythrocyte interaction could influence the expression of clinically evident ICrelated disease. (Hepatology 2018). 2)To further explore the pathogenesis of HCV-related immune complex disease, we tested the levels of complement-activated immune complexes (IC), rheumatoid factors, and several chemokines and cytokines in plasma samples from chronic HCV patients and matched healthy control in order to elucidate the possible biological responses and consequences of HCV-IC/erythrocyte interaction. We also investigate the genetic polymorphism of complement receptor 1 (CR1/CD35) gene associated with CR1 expression on erythrocytes from both groups of individuals, and how this CR1 expression level correlates with IC level. We have found that: a) several chemokine levels including CXCL10, CXCL12, and BAFF in blood from patients with chronic HCV infection are significantly elevated when compared to those from healthy controls; b) Similar elevated levels of circulating immune complexes (CIC) and rheumatoid factors are also found in patients with chronic HCV infection; c) levels of CIC in blood from chronic HCV patients with HH genotype in CR1 gene are significantly lower than those from chronic HCV patients with HL genotype. (Manuscript in preparation). 3)We observed that the association of HCV with CD19+ B cells is mediated by the complement system. In addition, using antibodies against cell surface markers, we showed that the binding complex mainly involved CD21 (complement receptor 2), CD19, CD20, and CD81. In human B cells, CD21 is known to form a costimulatory complex with CD19 and CD81. Co-ligation of the B cell antigen receptor (BCR) with this costimulatory complex can lower the threshold required for BCR-mediated B cell activation and proliferation. Epidemiological studies have demonstrated an increased risk of developing B-cell non-Hodgkin lymphoma in patients with chronic HCV infection. Both the regression of HCV-associated lymphoma with antiviral treatment and the beneficial effects of antiviral treatment on overall survival of patients with HCV-related lymphoma have strengthened the aetiological link between HCV infection and NHL presumed from epidemiological, clinical and pathophysiological studies (Hepatology 2016). Our goal is to move to the next step and elucidate the pathways through which complement mediated HCV attachment/infection might lead to malignant transformation of B-cells. 4) Since B-cells proliferation, in response to antigenic stimulation or polyclonal activation, may predispose to genetic aberrations (mutation, gene translocation, gene fusion, chromosomal amplification or deletion) we are using RNA-sequencing, to obtain both mutational and gene expression data from B-cells obtained from patients who are HCV+, HBV+, HDV+ and B-NHL+, and compare to patients who are HCV-, HBV-, HDV-, NHL+; HCV+, HBV+, HDV+, NHL-, and healthy controls. We will validate/ confirm the results on paired Paraffin Embedded lymphatic tissues then. The adoption of RNA-sequencing has been proven useful in clinical oncology as most of the clinically relevant somatic mutations are also expressed on the RNA level. It is a promising technology to sequence genes for the identification of mutational status, while simultaneously obtaining information on structural variations and gene expression perturbations-related diseases. We are recipient of an NIAID-NCI grant: "Investigation of HCV and HBV-Mediated B-Cell Malignant Transformation and Prioritization of Treatment for HCV-Related NHL in Mongolia. We have received more than 200 samples from Mongolia (PBMCs and matched Paraffin Embedded lymphatic tissues). We have processed and completed the Rna Sequencing for 100 BCells isolated from PBMCs. RNA-seq analysis identified differentially expressed genes and dysregulated pathways commonly involved in malignancy, including PAX5, AHR, MYC, TP53 and the BCR, EIF2, and mTOR pathways. Upregulation of RNPC1 could play a role in tumorigenesis by repressing p53.Funactional analysis data will combined to the genomic data. The next step will be confirm the finding on the matched tissue samples.