Analysis of Host-Pathogen Interactions

宿主-病原体相互作用分析

• 批准号: 8946432
• 负责人: Ronald N Germain
• 金额: $ 23.36万
• 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/8946432
• 关键词: 3-Dimensional; Address; Animals; Antigen Presentation; Antigen Presentation Pathway; Antigens; Bacteria; Behavior; Biological; Blood; Bone Marrow; CD4 Positive T Lymphocytes; Cell Communication; Cell Fraction; Cells; Cellular biology; Complex; Data; Dendritic Cells; Development; Effector Cell; Employee Strikes; Environment; Event; Genus Mycobacterium; Goals; Granuloma; Granuloma by Site; Hepatic Granuloma; Host Defense; Image; Immune; Immune response; Immune system; In Vitro; Individual; Infection; Infectious Agent; Infective cystitis; Influenza; Injection of therapeutic agent; Interferon Type II; Invaded; Kidney; Laboratories; Leukocytes; Limb structure; Link; Liver; Location; Lung; Lymph; Lymph Node Subcapsular Sinus; Lymphocyte; Lymphoid Cell; MHC antigen; Mediating; Memory; Methods; Microbe; Modeling; Morbidity - disease rate; Morphologic artifacts; Movement; Mycobacterium tuberculosis; Myeloid Cells; Organ; Organism; Parasites; Pathogenicity; Pathologic; Peptides; Peripheral; Physiology; Play; Population; Process; Production; Pseudomonas aeruginosa; Publishing; Reporting; Research; Research Personnel; Role; Sinus; Site; Skin; Staining method; Stains; Stream; Structure; System; T-Lymphocyte; Therapeutic Uses; Time; Tissues; Uropathogenic E. coli; Vaccination; Virus; Work; cell behavior; cell motility; cellular imaging; cytokine; density; design; imaging modality; immune function; in vivo; insight; interest; lymph nodes; macrophage; microorganism; migration; mortality; neutrophil; pathogen; research study; residence; response; two-photon

Using intravital 2-photon imaging methods developed in the LBS over the past several years, we are now able to routinely image peripheral organs and tissues such as the liver, kidney, bone marrow, gut, and skin so that immune effector cell behavior and to some extent effector functions during infectious processes can be observed. Using these new methods, we previously described lymphoid and myeloid cell dynamics in BCG-induced granulomas in the liver and showed that only a small fraction of the antigen-specific T cells within a granuloma undergo migration arrest at any time. This small fraction of such arrested cells correlates quantitatively with the fraction of specific cells making the key effector cytokine IFNgamma, as assessed by isolation and intracellular staining of the cells from the infected liver. Confocal imaging data showed that these T cells polarize their secretion of the effector cytokine towards sites of antigen load (bacteria). The limited migration arrest of antigen-specific T cells and the low number of cytokine secreting cells making just detectable levels of IFNgamma is not an artifact of the analytic method, because injection of high levels of the cognate antigenic peptide into the infected animals results in both migration arrest of nearly all the T cells and production of 1-2 logs more IFNgamma per cell by 80-90% of the T cells under these conditions. These data suggested that during normal immune responses to mycobacteria in liver granulomas, there is very limited antigen presentation just sufficient at any moment to activate a small fraction of all available effector cells into a cytokine-secretory state, and to do so just at the margin of quantitative response potential. These findings provide entirely new insights into the way in which effector T cells operate in the natural in vivo setting and point to the large differences between in vitro evoked responses and the actual behavior of effector cells at sites of infection. Many of these observations have been repeated in M tuberculosis-infected animals, arguing that it is not the low pathogenicity of the BCG that leads to such limited effector responses, or the differential dynamic behavior of macrophages or T cells within granulomas. The finding that only a small fraction of the cells being imaged in a tissue are actually engaged in effector function at any time and that these have a dynamic behavior distinct from the bulk of the imaged cells raises critical questions about many existing and ongoing studies in other laboratories using intravital 2-photon imaging, in which the bulk or average behavior of clonally-related cells is taken as representative of the functional population; our data suggest this is a very dangerous way to interpret these images and that in many cases, investigators may be missing the distinct behavior of a small percent of the cells that contribute all or most of the relevant biological activity in the system under study. These findings also point to the possibility that using therapeutic vaccination in Mtb-infected individuals to generate more effector T cells may not be as effective as desired unless ways to enhance the display of antigen needed to trigger these cells in the infected tissue sites (granulomas) can be devised to accompany the vaccination response. In addition to the liver granuloma model, during the past year we have completed and published studies of T-cell motility in infected lungs. Recent progress in lung explant imaging has permitted us to compare the response of CD4+ T cells to influenza vs. BCG infection in the lungs and address the question of whether in this site, the same limited activation of effector function is seen as in the liver and whether this varies with the pathogen. We suspected that it would, as different organisms have different mechanisms for evading the immune system; mycobacteria try to diminish antigen presentation, among other immune manipulations, whereas influenza mainly seeks to circumvent the innate immune response and does not seem to target antigen presentation. These studies confirm the conclusions of our liver granuloma model, in that only a very small fraction of the mycobacteria-specific T cells arrest movement and produce cytokine (IFN-gamma) in this infected tissue setting. In striking contrast, the influenza-expressed antigen specific T cells showing much greater fractional migration arrest and a correspondingly greater fraction of cytokine producing T-cells. These latter results both confirm the relatively close relationship we previously reported between antigen-induced stopping and effector activity of T-cells in tissues, and also emphasize that the extent of the effector response varies greatly denuding on the microbe, in apparent concordance with the density of presented antigen (influenza >> mycobacteria). In other studies, we are examining how the structure of lymph nodes is organized to limit access of invading organisms to the blood stream. Because viruses and bacteria can access lymph nodes via the lymph and from there, enter the blood stream, there are cellular and structural features of lymph nodes that capture and eliminate these organisms as they enter in the afferent lymph. Our recent data have identified both myeloid cells in the subcapsular sinus that play an important filtering role, as well as innate and adaptive lymphoid cells that take up residence near the sinus to mediate effective and rapid responses to organisms reaching this site. Loss of these barriers results in substantial systemic dissemination of pathogens such as P aeruginosa. In another model, we are using uropathogenic E coli in a model of bladder infection in which we are assessing the location and function of various myeloid cell populations, including macrophages, dendritic cells, and neutrophils, in an effector to better understand how these organisms are sensed and handled by the innate immune system.

现在使用在LBS中开发的插入式2光子成像方法，我们现在能够常规地图像外周器官和组织，例如肝脏，肾脏，骨髓，肠道，肠道和皮肤，从而可以观察到免疫效应细胞行为以及在感染过程中在某种程度上效应的功能。使用这些新方法，我们先前在BCG诱导的肝脏中描述了淋巴样和髓样细胞动力学，并表明在肉芽肿室内只有一小部分抗原特异性T细胞在任何时候都会迁移迁移。这种被捕的细胞的这一小部分与特定细胞的比例进行了定量相关，如通过分离和细胞内从感染肝脏中细胞的细胞内染色所评估的，使关键效应细胞因子IFNGAMMA相关。共聚焦成像数据表明，这些T细胞对效应细胞因子的分泌极化，向抗原负荷（细菌）的部位分泌。抗原特异性T细胞的迁移停滞有限和仅可检测到的IFNGAMMA水平的细胞因子分泌细胞数量不是分析方法的伪像，因为将高水平的同源抗原肽注入高水平的感染动物中会导致几乎所有T细胞迁移的迁移导致1-2型细胞的迁移量，并在1-2型细胞中受到80-290的限制。这些数据表明，在肝肉芽肿的正常免疫反应期间，在任何时候，抗原表现都非常有限，足以将所有可用效应细胞的一小部分激活到细胞因子分泌状态，并且在定量反应潜力的缘缘就这样做。这些发现为效应T细胞在天然体内环境中运作的方式提供了全新的见解，并指出了体外诱发的反应与感染部位效应细胞的实际行为之间的巨大差异。这些观察结果中的许多观察结果已经在感染的结核病感染动物中重复出现，认为不是BCG的低致病性导致了如此有限的效应反应，或者粒状粒细胞中巨噬细胞或T细胞的差异动态行为。实际上，只有一小部分被成像在组织中成像的细胞实际上都参与了效应功能，并且它们具有与大部分成像的细胞不同的动态行为，这对使用浸润性2-PHOTON成像中的许多现有实验室进行的许多现有研究提出了关键的问题，而在这种情况下，则将其作为Clonally-Fellied细胞的构成或平均脱光的细胞行为代表性;我们的数据表明，这是一种解释这些图像的非常危险的方式，在许多情况下，研究人员可能会缺少一小部分细胞的独特行为，这些细胞在所研究系统中贡献了所有或大多数相关的生物学活动。这些发现还指出，在MTB感染的个体中使用治疗性疫苗接种产生更多的效应T细胞可能不如所需的有效性，除非可以增强在感染组织（颗粒瘤）中触发这些细胞所需的抗原表现的方法，以伴随疫苗接种响应。 除了肝肉芽肿模型外，在过去的一年中，我们已经完成并发表了有关感染肺T细胞运动性的研究。肺部外植体成像中的最新进展使我们能够比较肺中CD4+ T细胞对肺中的流感与BCG感染的反应，并解决了在该部位中是否看到肝功能的有限激活与肝脏中的相同的有限激活以及与病原体的不同。我们怀疑这将是，因为不同的生物具有逃避免疫系统的不同机制。分枝杆菌试图减少抗原表现，除其他免疫操纵外，而流感主要试图避免先天免疫反应，并且似乎并不靶向抗原呈递。这些研究证实了我们的肝肉芽肿模型的结论，因为在这种感染的组织环境中，只有一小部分分枝杆菌特异性T细胞阻止运动并产生细胞因子（IFN-GAMMA）。在引人注目的对比中，流感表达的抗原特异性T细胞显示出更大的分数迁移停滞和相应更大的细胞因子产生T细胞的比例。这些后者的结果既证实了我们先前在组织中抗原诱导的停止和T细胞效应的活性之间报道的相对紧密的关系，并且还强调，效应子响应的程度在微生物上的范围很大，显然与呈现的抗原的密度一致（actagenza >> mycobapteria）。在其他研究中，我们正在研究如何组织淋巴结结构以限制入侵生物进入血流的访问。由于病毒和细菌可以通过淋巴进入淋巴结，然后从那里进入血液，因此淋巴结的细胞和结构特征可捕获和消除这些生物在传入淋巴中。我们最近的数据已经确定了在囊下窦中起着重要过滤作用的髓样细胞，以及在鼻窦附近居住的先天和适应性淋巴样细胞，以介导对到达该部位的生物的有效和快速反应。这些障碍的丧失导致对病原体（如铜绿）的大量全身传播。 在另一个模型中，我们在膀胱感染的模型中使用肝癌E大肠杆菌，在该模型中，我们正在评估包括巨噬细胞，树突状细胞和中性粒细胞在内的各种髓样细胞群体的位置和功能，以更好地了解这些生物体如何通过先天免疫系统感知和处理这些生物体。