Role of the HTLV-1A and HTLV1-C inflammatory profile in disease

HTLV-1A 和 HTLV1-C 炎症谱在疾病中的作用

• 批准号: 10014283
• 负责人: Genoveffa Franchini
• 金额: $ 22.27万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/10014283
• 关键词: Aborigine; Affect; Alveolar; Animal Model; Animal Testing; Animals; Anti-inflammatory; Australia; Autophagocytosis; B-Lymphocytes; Biopsy; Blood; Body Weight decreased; Bronchiectasis; CD4 Positive T Lymphocytes; CD8B1 gene; Caring; Cell Line; Cell physiology; Cells; Cerebrospinal Fluid; Cessation of life; Clonality; Coculture Techniques; Collaborations; Communities; Complementary DNA; Coupled; Cytarabine; Data; Disease; Exons; Foundations; Future; Genetic Polymorphism; HTLV-1 Infection; High-Throughput Nucleotide Sequencing; Hospitals; Human; Human Genome; Human T-lymphotropic virus 1; IL2RA gene; IL8 gene; Immune; In Vitro; Indigenous; Individual; Infection; Infiltration; Inflammasome; Inflammatory; Initiator Codon; Interleukin-6; Irrigation; Knock-out; Knowledge; Link; Lung; Lung Inflammation; MHC Class I Genes; Macaca; Maps; Measures; Melanesia; Messenger RNA; Methods; Microbial Biofilms; Modeling; Molecular Cloning; Mus; Nanotubes; Natural Killer Cells; Northern Territory; Open Reading Frames; Organ; Pathway interactions; Peripheral Blood Mononuclear Cell; Pharmacotherapy; Physicians; Plasma; Population; Primary Infection; Proteins; Protocols documentation; RNA Splicing; Reagent; Research; Research Personnel; Resistance; Role; Sampling; Seroprevalences; Signal Transduction; Spleen; Subfamily lentivirinae; T-Cell Proliferation; T-Lymphocyte; Taxes; Testing; Tokyo; United States National Institutes of Health; Vertebral column; Viral; Viral Load result; Virus; Virus Integration; animal data; chemokine; chronic infection; cytokine; cytotoxic; human data; humanized mouse; in vivo; inflammatory lung disease; integration site; leukemia; lymph nodes; macrophage; monocyte; mortality; mutant; nonhuman primate; nucleoside analog; pathogen; response; viral DNA; viral transmission; virological synapse; virtual

Accomplishments 1. By performing integrated studies on orf-I sequence in 160 HTLV-1A infected individuals and animal testing on the infectivity of molecular clones carrying polymorphism that result in preferential expression of p8 or p12, we established that efficient HTLV-1A persistence and spread in vivo requires the combined functions of the orf-I proteins. We also found that p8 is essential for productive infection of monocytes and that the expression of both proteins renders infected cells completely resistant to the MHC-class I restricted cytotoxic CD8 killing. 2. Cell-associated HTLV-1 can be transmitted by at least three different mechanisms: virological synapse, cellular conduits including Tunneling Nano Tubes, and biofilm. However, the contribution of each of these mechanisms to viral transmission remains unknown. We have demonstrated that the HTLV-1 p8 increases viral transmission by increasing both cellular conduits and Tunneling Nano Tubes. More recently, we found that inhibition of p8-medaited TNT formations by the nucleoside analog cytarabine (cytosine arabinoside, AraC) decreases viral transmission by 30%, thereby providing a treatment to partly curb the spread of HTLV-1 in vivo. Current Research & Future Plans 1. Functional studies with HTLV-1A and HTLV-1A/C viruses and orf-I cDNAs. We have synthesized the cDNAs derived from the doubly spliced rex-orf-I mRNAs of HTLV-1C that juxtapose the first exon of Rex in frame with orf-I and demonstrated that it produces p16, a protein that increases autophagy. We constructed a chimeric virus by swapping the Cla-1-Sal-1 fragment, which contains both the entire orf-I and most of the orf-II of HTLV-1A, with that of HTLV-1C. The resultant molecular clone is a replicating virus designated as HTLV-1A/CDF. We plan to create a second HTLV-1A/C by substituting the entire 3' end (HTLV-1A/C) and generating mutants of both chimeric viruses to inhibit the splicing of the rex-orf-I mRNA (HTLV-1A/CDFd16, and HTLV-1A/CDFd16,) as a control. The viral DNA clones will be transduced in the 729 B cell line that supports HTLV-1 replication and will be used to infect primary CD4+ T cells. We plan to perform functional studies on monocytes with lentiviruses that express p8 or p16 on primary monocytes and are infected by HTLV-1A WT and the orf-I knockout mutant, the chimeric HTLV-1A/CDF WT, or rex-orf-I knock-out virus (HTLV-1A/CDFd16). We will assess inflammasome activation, autophagy, and the level and type of inflammatory cytokines and chemokines produced by primary monocytes and T cells in vitro. 2. HTLV-1A and HTLV-1A/C infection of humanized mice. We have preliminary data that demonstrate that our HTLV-1A molecular clone can be transmitted to humanized NOD/SCID-yc-/- mice using infected, irradiated CD4+ T cells. We observed proliferation of human CD25+ CD4+ T cells engrafted in the humanized mice that causes extensive infiltration of these CD4+ T cells in vital organs such as the spleen, high viral burden, weight loss, and death. To explore HTLV-1 clonality in hu-Mice, we applied an optimized high-throughput sequencing (HTS) method to map viral integration sites in the human genome and simultaneously measure the abundance of the corresponding clones. The CD4+ T cell proliferation is polyclonal, as expected. We do not anticipate differences in the ability of HTLV-1A and HTLV-1A/C to cause this proliferative disease in mice, since the Tax is virtually identical in the two viruses. However, these studies will be prerequisite to demonstrate viral infectivity of the chimeric HTLV-1A/C in vivo before they can be used in studies in non-human primates. In addition, this small animal model may be foundational in testing the extent of inhibition of viral transmission by cytarabine in vivo. 3. Study the inflammatory profile of HTLV-1A and HTLV-1A/C infected macaques and humans. We have demonstrated that HTLV-1A WT and the HTLV-1 orf-I knockout viruses infect monocytes in vitro and macaquesin vivo, but the HTLV-1 orf-I knockout does not appear to persist. We obtained PBMCs infected with HTLV-1A WT or the HTLV-1A orf-I knockout virus, cultured them for 3 days, and measured the ability of adherent cells (macrophages) to produce cytokines. The blood was collected at a timepoint when both animals were positive for viral DNA in PBMCs (weeks 8-10). We found higher levels of IL-1B, IL-6, and IL-8 in the animal infected with the HTLV-1A orf-I knockout than that with the HTLV-1A WT, demonstrating that the absence of orf-I results in a qualitatively different inflammatory profile in vivo, as also demonstrated in vitro. We plan to extend this study by infecting 4 macaques with HTLV-1A and 4 with HTLV-1A orf-I knockout viruses to follow the inflammatory profiles caused by the two viruses in detail. We also plan to infect additional macaques with the HTLV-1A/CCS and the HTLV-1A/CCSd16 if warranted by the data. In all the animal studies, we will collect lung biopsies, bronchial alveolar lavage, blood, lymph nodes, gut biopsies, and spinal fluid to quantitate viral burden and differences in systemic inflammatory profiles. To compare the inflammatory profiles of ex vivo monocytes from macaques and humans infected by HTLV-1A and HTLV-1C, we have established collaborative efforts with Australian physicians at the Alice Springs Hospital that care for HTLV-1C infected Aborigines and with the IMSUT Hospital in Tokyo and Steve Jacobson at the NIH, who both care for HTLV-1A infected people, to study their inflammatory profiles directly in plasma or by short term cultures of PBMCs. Our collaboration with Australian researchers is a large pan-Australian consortium to share PBMCs, culture protocol, Luminex data, and reagents for the comparison of human and animal data.

成绩1。通过对160名HTLV-1A感染者的orf-Ⅰ序列进行综合研究，并对携带导致p8或p12优先表达的多态性的分子克隆的感染性进行动物试验，我们确定了有效的HTLV-1A体内持久性和传播需要orf-Ⅰ蛋白的组合功能。我们还发现p8对于单核细胞的生产性感染是必不可少的，并且两种蛋白质的表达使得感染的细胞完全抵抗MHC-I类限制性细胞毒性CD 8杀伤。2.细胞相关的HTLV-1可以通过至少三种不同的机制传播：病毒学突触，细胞管道（包括Tunnel纳米管）和生物膜。然而，这些机制中的每一个对病毒传播的贡献仍然未知。我们已经证明，HTLV-1 p8通过增加细胞导管和Tunnel纳米管来增加病毒传播。最近，我们发现通过核苷类似物阿糖胞苷（阿糖胞苷，AraC）抑制p8介导的TNT形成使病毒传播减少30%，从而提供了部分抑制HTLV-1在体内传播的治疗。当前研究和未来计划1. HTLV-1A和HTLV-1A/C病毒和orf-I cDNA的功能研究。我们已经合成了来自HTLV-1C的双重剪接的rex-orf-I mRNA的cDNA，其将雷克斯的第一外显子与orf-I并列在框中，并证明其产生p16，一种增加自噬的蛋白质。我们通过将HTLV-1A的Cla-1-Sal-1片段与HTLV-1C的Cla-1-Sal-1片段交换构建了嵌合病毒，Cla-1-Sal-1片段包含HTLV-1A的整个orf-I和大部分orf-II。所得分子克隆是命名为HTLV-1A/CDF的复制型病毒。我们计划通过取代整个3'端（HTLV-1A/C）并产生两种嵌合病毒的突变体以抑制rex-orf-I mRNA的剪接（HTLV-1A/CDFd 16和HTLV-1A/CDFd 16）来产生第二种HTLV-1A/C作为对照。病毒DNA克隆将在支持HTLV-1复制的729 B细胞系中转导，并将用于感染原代CD 4 + T细胞。我们计划用慢病毒对单核细胞进行功能研究，这些慢病毒在原代单核细胞上表达p8或p16，并被HTLV-1A WT和orf-I敲除突变体、嵌合HTLV-1A/CDF WT或rex-orf-I敲除病毒（HTLV-1A/CDFd 16）感染。我们将评估炎性小体激活，自噬，以及体外原代单核细胞和T细胞产生的炎性细胞因子和趋化因子的水平和类型。2.人源化小鼠的HTLV-1A和HTLV-1A/C感染。我们有初步的数据表明，我们的HTLV-1A分子克隆可以使用感染的，辐射的CD 4 + T细胞传递到人源化NOD/SCID-yc-/-小鼠。我们观察到移植到人源化小鼠中的人CD 25 + CD 4 + T细胞的增殖，这导致这些CD 4 + T细胞在重要器官如脾脏中的广泛浸润、高病毒负荷、体重减轻和死亡。为了探索人类小鼠中HTLV-1的克隆性，我们应用了优化的高通量测序（HTS）方法来绘制人类基因组中的病毒整合位点，并同时测量相应克隆的丰度。如预期的，CD 4 + T细胞增殖是多克隆的。我们预计HTLV-1A和HTLV-1A/C在小鼠中引起这种增殖性疾病的能力没有差异，因为这两种病毒的Tax几乎相同。然而，这些研究将是在嵌合HTLV-1A/C可用于非人灵长类动物研究之前证明其体内病毒感染性的先决条件。此外，这种小动物模型可能是检测阿糖胞苷体内抑制病毒传播程度的基础。3.研究HTLV-1A和HTLV-1A/C感染猕猴和人类的炎症特征。我们已经证明，HTLV-1A WT和HTLV-1 orf-Ⅰ敲除病毒在体外感染单核细胞，在体内感染猕猴，但HTLV-1 orf-Ⅰ敲除似乎并不持久。我们获得了用HTLV-1A WT或HTLV-1A orf-I敲除病毒感染的PBMC，将它们培养3天，并测量粘附细胞（巨噬细胞）产生细胞因子的能力。在两只动物PBMC中病毒DNA均呈阳性的时间点（第8-10周）采集血液。我们发现，在HTLV-1A orf-1敲除感染的动物中，IL-1B、IL-6和IL-8的水平高于HTLV-1A WT感染的动物，这表明orf-1的缺失导致了体内和体外性质不同的炎症特征。我们计划通过用HTLV-1A感染4只猕猴和用HTLV-1A orf-I敲除病毒感染4只猕猴来扩展这项研究，以详细了解这两种病毒引起的炎症特征。我们还计划用HTLV-1A/CCS和HTLV-1A/CCSd 16感染其他猕猴，如果数据证明的话。在所有动物研究中，我们将收集肺活检、支气管肺泡灌洗、血液、淋巴结、肠道活检和脊髓液，以定量病毒负荷和全身炎症特征的差异。为了比较来自被HTLV-1A和HTLV-1C感染的猕猴和人类的离体单核细胞的炎症特征，我们与爱丽丝泉医院的澳大利亚医生建立了合作关系，他们照顾HTLV-1C感染的土著人，与东京的IMSUT医院和NIH的Steve Jacobson建立了合作关系，他们都照顾HTLV-1A感染的人，直接在血浆中或通过PBMC的短期培养来研究它们的炎症特征。我们与澳大利亚研究人员的合作是一个大型的泛澳大利亚联盟，分享PBMC，培养方案，Luminex数据以及用于比较人类和动物数据的试剂。