Immune Regulation In Toxoplasmosis And Other Opportunistic Infections

弓形虫病和其他机会性感染的免疫调节

• 批准号: 8555781
• 负责人: Alan Sher
• 金额: $ 55.3万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/8555781
• 关键词: Acute; Animals; Biological Assay; CD4 Positive T Lymphocytes; Cell Differentiation process; Cells; ChIP-seq; Collaborations; Defect; Dendritic Cells; Development; Disease; Dose; Elements; Epigenetic Process; Equilibrium; Evaluation; Exposure to; Genes; Genus Mycobacterium; Glucocorticoids; Growth; HIV Infections; HIV Seropositivity; Helper-Inducer T-Lymphocyte; Host resistance; Human; IL10 gene; Immune; Immune response; Immunocompetent; Immunocompromised Host; In Vitro; Individual; Infection; Infection Control; Inflammatory; Interferon Type II; Interferon-alpha; Interferons; Interleukin-10; Interleukin-12; Life; Mediating; Messenger RNA; Modeling; Modification; Molecular; Mus; Myeloid Cells; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Natural Killer Cells; Opportunistic Infections; Parasite Control; Parasite resistance; Parasites; Pathway interactions; Peripheral Blood Mononuclear Cell; Phase; Phenotype; Physiological; Play; Population; Predisposition; Process; Production; Property; Proteins; Regulation; Relative (related person); Reporter; Reporting; Role; Signal Pathway; Signal Transduction; Study models; T cell response; T-Lymphocyte; TCR Activation; TNF gene; Terminator Codon; Th1 Cells; Th2 Cells; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transgenes; Universities; Viral; Virulence; Work; chemokine; comparative; cytokine; design; histone modification; hypothalamic-pituitary-adrenal axis; immunopathology; in vivo; macrophage; monocyte; mortality; novel; overexpression; pathogen; peripheral blood; profilin; receptor; receptor expression; response; steroid hormone

Our previous studies showed that TLR11 recognizes a profilin protein from T. gondii (TgPRF) and is required for optimal production of p40 IL-12as well as optimal control of infection. We have now extended these observations and in collaboration with Dr. Sankar Gosh (Columbia University, NY) demonstrated that TLR12, a previously uncharacterized TLR12, also recognizes TgPRF. Importantly, TLR12-deficient mice display markedly increased susceptibility to T. gondii infection relative to TLR11 KO animals and this defect is associated with decreased IFN-alpha production and reduced NK cell activity. Taken together, these studies establish a dual role for TLR11/TLR12 signaling in both innate recognition of T. gondii and host resistance to the parasite The recognition of T. gondii by human cells, which do not express TLR11 and TLR12 (because of the presence of a stop codon in the genes), is still, however, poorly understood. For this reason, as proposed in our last years report, we have initiated a new project aimed at characterizing the cytokine/chemokine response elicited in human peripheral blood myeloid cells upon exposure to Toxoplasma gondii tachyzoites and identifying the signaling pathways involved. When the response of elutriated monocytes and in vitro differentiated dendritic cells or macrophages was compared, the production of proinflammatory cytokines (e.g. TNF and p40 IL-12) and chemokines was largely restricted to monocytes and, in particular, to the CD14high subset stimulated following parasite exposure. Our results also indicate that while infection with live parasites is required for the induction of cytokine/chemokine responses by monocytes, similar responses are obtained regardless of the virulence properties of the particular T.gondii strain employed in the assay. As a potent inducer of Th1 effectors, T. gondii infection provides a unique model for studying the process of Th1 differentiation. In collaboration with Dr. John OShea group (NIAMS), we showed that activated CD4+ T lymphocytes rapidly acquire and maintain the expression of the transcriptional factor T-bet (a master regulator of Th1 cells), which in turn suppresses the expression of the transcriptional factor Bcl-6 required for the development of T follicular helper cells. In a separate collaboration with Drs. William Paul and Jeff Zhou, we employed mice that while lacking expression of endogenous T-bet, express as a transgene a T-bet reporter, to demonstrate that T-bet promotes the development of Th1 cells during T. gondii infection by repressing the expression of GATA-3 (a master regulator of Th2 cell differentiation). As summarized in previous reports, we have found that Th1 cells coexpress IFN-gamma and IL-10 in toxoplasma-infected mice. Since our initial description of IL-10+Th1 cells, CD4+ T lymphocytes with this phenotype has been demonstrated in numerous other Th1 inducing parasitic, bacterial and viral animal infection models, as well as in human PBMC cultures. However, while INF-gamma is a stable property of Th1 cells, IL-10 secretion is transient. The latter finding makes it particularly difficult to study the molecular mechanism(s) that regulate IL-10 expression in Th1 cells. In order to facilitate this task, the work completed this year, has focused on subdivision of in vivo generated Th1 cells into three populations: those that express IL-10, those poised to express the cytokine and those that are IL-10 negative. This was successfully achieved by employing two different types of IL-10 reporter mice. Comparative analysis of the three Th1 subpopulations revealed that IL-10 mRNA can be only detected in cells expressing IL-10, while evaluation of histone modifications by ChIP-seq demonstrated three distinct epigenetics configurations of the IL-10 gene specific for each Th1 phenotype. Since our earlier studies indicated that Stat1, but not Stat3, is required for IL-10+ expression in Th1 cells, we are currently investigating the role Stat1 plays in epigenetic modifications (e.g. induction of H3K4me3 (permissive) and suppression H3K27me3 (inhibitory) marks) associated with the fully activate Il10 gene. Immune responses triggered by pathogens are part of the physiological response of the host as a whole and as such may be under the influence of extrinsic controlling elements. For example, glucocorticoids (GS) a class of steroid hormones regulated by hypothalamic-pituitary-adrenal (HPA) axis are known to exert pleiotropic effects on immune cells. As reported last year, we showed that GC are induced during the late acute phase of T. gondii infection and that mice lacking GC receptor expression in T lymphocytes display rapid mortality despite efficient control of parasite growth and uncompromised expression of the regulatory cytokines (e.g. IL-10 and IL-27). To understand their increased susceptibility, we have now demonstrated that in mice that lack GC receptor in T cells Th1 cells display in vivo, but not in vitro, a hyperactive phenotype associated with increased immunopathology. Interestingly, while GC have been described to suppress IFN-γ, and augment IL-10, secretion by CD4 T lymphocytes, the lack of GC signaling in T. gondii-induced Th1 cells resulted in general overexpression of IFN-γ, TNF as well as IL-10. The latter result strongly suggests that, under the highly polarized Th1 conditions of toxoplasma infection, GC exert a type of general rheostat, rather than cytokine-targeted, regulation on Th1 effectors by increasing the threshold of Ag dose required for TCR activation. Importantly, we also showed that the CD4+ T cell response to T. gondii infection is itself required for increased GC levels. Taken together, our results demonstrate a novel regulatory circuit involving cross-talk between CD4 T lymphocytes and GC production. This pathway which acts independently of innate immune cell cytokine production plays an important role in balancing Th1 mediated pathogen clearance and tissue immunopathology.

我们先前的研究表明，TLR11识别出gondii（TGPRF）的酸性蛋白蛋白，并且是最佳生产P40 IL-12AS及其最佳控制感染所必需的。现在，我们已经扩展了这些观察结果，并与Sankar Gosh（纽约州哥伦比亚大学）合作，证明TLR12先前未表征的TLR12也认可了TGPRF。重要的是，相对于TLR11 KO动物，TLR12缺陷型小鼠对T. gondii感染的敏感性显着增加，并且该缺陷与IFN-Alpha产生降低和NK细胞活性降低有关。综上所述，这些研究确定了TLR11/TLR12信号传导的双重作用，在先天的gondii识别和对寄生虫的宿主抗性中 然而，人类细胞对未表达TLR11和TLR12的人类细胞（由于基因中存在终止密码子）的识别仍然很差。因此，正如我们最近几年的报告所提出的那样，我们启动了一个新项目，旨在表征人类外周血血细胞髓样细胞在暴露于弓形虫巨型tachyzoite时引起的细胞因子/趋化因子反应，并确定涉及的信号通路。当比较洗脱的单核细胞和体外分化的树突状细胞或巨噬细胞的反应时，促炎细胞因子（例如TNF和P40 IL-12）的产生和趋化因子和趋化因子和趋化因子在很大程度上仅限于单核细胞，尤其是限于CD14HIGH的子群曝光。我们的结果还表明，虽然单核细胞诱导细胞因子/趋化因子反应所必需的感染是必需的，但无论该测定法中采用的特定T.gondii菌株的毒力特性如何，都会获得相似的反应。 作为TH1效应子的有效诱导剂，T. gondii感染提供了一个独特的模型，用于研究Th1分化过程。与John Oshea Group博士（NIAMS）合作，我们表明激活的CD4+ T淋巴细胞迅速获取并维持转录因子T-BET（TH1细胞的主要调节剂）的表达，这又抑制了T follicular helper cell所需的转录因子BCL-6所需的转录因子BCL-6。在与Drs的另一次合作中。威廉·保罗（William Paul）和杰夫周（Jeff Zhou）采用了小鼠，尽管缺乏内源性T-bet的表达，但作为Transgene t-bet报告基因表达，以证明T-BET通过抑制GATA-3的表达来促进T. gondii感染期间Th1细胞的发展（Th2细胞分化的主要调节剂）。 正如先前报道中总结的那样，我们发现Th1细胞在弓形虫感染的小鼠中共表达IFN-GAMMA和IL-10。自从我们对IL-10+ Th1细胞的最初描述以来，在许多其他TH1诱导寄生虫，细菌和病毒动物感染模型以及人类PBMC培养物中，已经证明了具有该表型的CD4+ T淋巴细胞。但是，尽管Inf-Gamma是Th1细胞的稳定特性，但IL-10分泌是短暂的。后一个发现使研究调节Th1细胞中IL-10表达的分子机制变得特别困难。为了促进这项任务，今年完成的工作集中在体内生成Th1细胞分为三个种群的细分：那些表达IL-10的人群，那些有准备表达细胞因子的人和IL-10负的人群。这是通过使用两种不同类型的IL-10记者小鼠成功实现的。对三个TH1亚群的比较分析表明，只能在表达IL-10的细胞中检测IL-10 mRNA，而通过CHIP-SEQ对组蛋白修饰的评估表明，对每种Th1表型的IL-10基因的三种不同的表观遗传学构型。由于我们较早的研究表明，TH1细胞中IL-10+表达需要STAT1而不是STAT3，因此我们目前正在研究STAT1在表观遗传修饰中的作用（例如，H3K4ME3（允许）H3K4ME3（允许）和抑制H3K27ME3（抑制）标记（抑制）与完全活化的IL10基因相关。 病原体触发的免疫反应是宿主整体生理反应的一部分，因此可能受外在控制元件的影响。例如，已知糖皮质激素（GS）由下丘脑 - 垂体 - 肾上腺（HPA）轴调节的类固醇激素对免疫细胞产生了多效应作用。如去年所报道的那样，我们表明，在弓形虫感染的急性阶段诱导了GC，尽管有效控制了寄生虫生长和调节性细胞因子的表达，但在T淋巴细胞中缺乏GC受体表达的小鼠表现出快速的死亡率（例如IL-10和IL-10和IL-27）。为了了解它们的易感性提高，我们现在已经证明，在T细胞中缺乏GC受体的小鼠中，Th1细胞在体内表现出，但在体外不显示与免疫病理增加有关的多活跃表型。有趣的是，尽管已经描述了GC抑制IFN-γ和增强IL-10，CD4 T淋巴细胞分泌，但T. gondii诱导的Th1细胞中缺乏GC信号传导，导致IFN-γ，TNF，TNF和IL-10的过度表达。后者的结果强烈表明，在高度极化的TH1条件下，GC通过增加TCR激活所需的Ag剂量阈值来调节Th1效应子的一种普通风湿病，而不是对Th1效应子进行调节。重要的是，我们还表明，CD4+ T细胞对T. gondii感染的反应本身是GC水平升高所必需的。综上所述，我们的结果表明了一个新的调节回路，涉及CD4 T淋巴细胞和GC产生之间的串扰。这种独立于先天免疫细胞因子产生的途径在平衡Th1介导的病原体清除率和组织免疫病理学方面起着重要作用。