HCV Infection and Innate Immunity

HCV 感染和先天免疫

• 批准号: 10467900
• 负责人: Valeria de Giorgi
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467900
• 关键词: ASPM gene; Adoption; Affect; Allergic Cutaneous Vasculitis; Antibodies; Antigen-Antibody Complex; Antiviral Agents; B Cell Proliferation; B-Cell Activation; B-Cell Antigen Receptor; B-Cell NonHodgkins Lymphoma; B-Lymphocyte Subsets; B-Lymphocytes; Binding; Bioinformatics; Biological; Blood; CD19 gene; CD81 gene; CDC2 gene; CXCL10 gene; CXCL12 gene; Cell Communication; Cell surface; Cells; Cellular Structures; Chromosomal Duplication; Chromosome Deletion; Chronic Hepatitis C; Cirrhosis; Clinical; Clinical Oncology; Complement; Complement 3d Receptors; Complement Activation; Complement Receptor; Complex; Cryoglobulinemia; Cryoglobulins; Data; Diagnosis; Disease; EZH2 gene; Epidemiology; Epigenetic Process; Erythrocytes; Extrahepatic; Future; Gene Expression; Gene Fusion; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genotype; Glomerulonephritis; Grant; HMMR gene; Hepatitis B Virus; Hepatitis C; Hepatitis C Therapy; Hepatitis C virus; Hepatocyte; Hepatology; Host Defense; Human; Immune Complex Diseases; Individual; Innate Immune Response; Investigation; Lead; Ligation; Link; Lymphoid Tissue; Lymphoma; Lymphoproliferative Disorders; MS4A1 gene; Manuscripts; Mediating; Molecular; Mongolia; Mutation; National Institute of Allergy and Infectious Disease; Natural Immunity; Neuropathy; Non-Hodgkin' s Lymphoma; Output; Paraffin Embedding; Pathogenesis; Pathway interactions; Patients; Peripheral; Peripheral Blood Mononuclear Cell; Plasma; Preparation; Primary carcinoma of the liver cells; Prognosis; Proteins; Public Health; RNA; RRM2 gene; Rheumatoid Factor; Risk; Role; Sampling; Site; Somatic Mutation; Testing; Tissue Sample; Up-Regulation; Viral; Viral Proteins; Virus; Virus Replication; Virus-Related Lymphoma; Whole Blood; base; cancer cell; chemokine; chronic infection; chronic liver disease; clinically relevant; comparison group; complement system; cytokine; differential expression; epidemiology study; gene translocation; interest; large cell Diffuse non-Hodgkin' s lymphoma; monocyte; non-Hodgkin' s lymphoma patients; overexpression; programs; response; transcriptome sequencing; transcriptomics; viral RNA

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). 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. 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, including PBMC, plasma, and matched paraffin embedded lymphatic tissues, and have performed RNA-sequencing on 75 peripheral B cell samples isolated from PBMC. Based on the RNA-sequencing output, we have identified differentially expressed genes (DEGs) between all comparison groups, and bioinformatic analysis has been performed on DEGs to help identify biologically relevant molecular pathways of interest. Our data suggest that the transcriptional changes occurring in the peripheral B cells of both B-NHL+ and HCV+ patients relative to healthy controls may be mediated by overexpression of multiple epigenetic modifier proteins. Bioinformatic investigation of DEGs among diagnosis groups has revealed key transcriptional similarities and differences. While increased expression of cell structure-related genes is a feature of B-NHL+ peripheral B cells regardless of HCV status, upregulation of proliferation-associated genes appears to be specific to B-NHL+/HCV+ patients. In particular, we have observed significant upregulation of genes associated with poor prognosis or progression of diffuse large B cell lymphoma (DLBCL), including ASPM, CDC20, CDK1, EZH2, and HMMR, as well as upregulation of genes known to interact directly or indirectly with HCV viral proteins, including ASPM, RRM2, and PCLAF. Altogether, our differential expression analysis has revealed subtle but distinct transcriptional changes in peripheral B cells from B-NHL+ patients with or without chronic HCV and has identified molecular pathways by which HCV infection may be contributing to B cell malignant transformation. Future directions include following up with functional studies and investigating circulating cytokine levels in matched patient plasma samples to support and inform our gene expression data. Additionally, we plan to investigate the presence of HCV RNA in lymphoma tissue samples as well as perform transcriptomic analyses in these samples.

HCV是一个主要的公共卫生问题，在全球感染了超过1.7亿人。大多数HCV感染的病例变得持久，最终可能导致慢性肝病，肝硬化和肝细胞癌。尽管肝细胞是病毒复制的主要部位，但肝外并发症和疾病的广泛临床谱与慢性HCV感染有关，包括混合的冰冻球蛋白血症，非霍奇金斯淋巴瘤，皮肤血管，皮肤血管炎，肾小球肾上腺炎，肾小球肾炎，神经性肿瘤疗法和乳腺肿大。 HCV复制的肝外储藏的存在，尤其是在PBMC中，仍然存在很大的争议。目前尚不清楚B细胞在慢性HCV感染过程中如何失调。 1）我们的小组证明了自由HCV通过补体调整并与红细胞上的CR1结合（ECR1）； HCVSpecific抗体的存在显着增加了这种结合。游离HCV与红细胞的结合是否与HCV清除或发病机理有关。混合的冷冻球蛋白血症（II型）是与慢性HCV感染相关的最常见的IC疾病之一。近一半的HCV患者患有可检测的冷冻球蛋白，而不到10％的患者患有临床明显的疾病。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伊克斯疾病的表达。 （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.我们还研究了补体受体1（CR1/CD35）基因的遗传多态性与两组的红细胞上的CR1表达相关，以及该CR1表达水平与IC水平的相关性。我们发现：a）与健康对照组相比，慢性HCV感染患者的血液中包括CXCL10，CXCL12和BAFF在内的几种趋化因子水平显着升高； b）在慢性HCV感染的患者中，还发现了类似的循环免疫复合物（CIC）和类风湿因素的升高； c）CR1基因中HH基因型的慢性HCV患者的血液中CIC水平明显低于慢性HCV HL基因型患者的CIC水平。 （准备中的手稿）。 3）我们观察到HCV与CD19+ B细胞的关联是由补体系统介导的。另外，使用针对细胞表面标记的抗体，我们表明结合复合物主要涉及CD21（补体受体2），CD19，CD20和CD81。在人B细胞中，已知CD21与CD19和CD81形成了共刺激络合物。 B细胞抗原受体（BCR）与该共刺激复合物的共同结合可以降低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HCV感染患者患B细胞非霍奇金淋巴瘤的风险增加。 HCV相关淋巴瘤的回归与抗病毒治疗的回归以及抗病毒治疗对HCV相关淋巴瘤患者总体存活的有益作用都加强了HCV感染与NHL之间的病因联系，而NHL假定来自流行病学，临床和病理生理学研究（Hepatotogology 2016）。 4）由于B细胞增殖响应于抗原刺激或多克隆活化，因此可能会易于遗传畸变（突变，基因易位，基因融合，融合，染色体扩增或缺失），我们正在使用RNA及定位，从并与HCV-，HBV-，HDV-，NHL+的患者进行比较； HCV+，HBV+，HDV+，NHL-和健康对照。当时，我们将验证/确认成对的石蜡嵌入淋巴组织的结果。 RNA测序的采用已被证明在临床肿瘤学中有用，因为大多数临床相关的体细胞突变也在RNA水平上表达。 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, including PBMC, plasma, and matched paraffin embedded lymphatic tissues, and have performed RNA-sequencing on 75 peripheral B cell从PBMC分离的样品。 基于RNA测序输出，我们已经确定了所有比较组之间差异表达的基因（DEG），并且在DEG上进行了生物信息学分析，以帮助识别生物学上相关的分子途径。我们的数据表明，相对于健康对照组的B-NHL+和HCV+患者的外围B细胞发生的转录变化可能是通过多种表观遗传修饰蛋白的过表达来介导的。诊断组之间对DEG的生物信息学研究表明，关键的转录相似性和差异。虽然与细胞结构相关基因的表达增加是B-NHL+外周B细胞的特征，而不论HCV状态如何，但增殖相关基因的上调似乎是B-NHL+/HCV+患者的特定的。特别是，我们已经观察到与弥漫性较差或弥漫性大B细胞淋巴瘤（DLBCL）相关的基因的显着上调，包括ASPM，CDC20，CDK1，EZH2和HMMR，以及已知与HCV病毒蛋白直接相互作用的基因上调，包括ASPM2和PCCM2。总的来说，我们的差异表达分析揭示了来自或没有慢性HCV的B-NHL+患者的外周B细胞中的细微但明显的转录变化，并确定了HCV感染可能导致B细胞恶性转化的分子途径。 未来的方向包括跟踪功能研究并研究匹配的患者血浆样本中的循环细胞因子水平，以支持和告知我们的基因表达数据。此外，我们计划研究淋巴瘤组织样品中HCV RNA的存在，并在这些样品中进行转录组分析。