ANALYSIS OF T CELL RESPONSES IN HUMAN LEISHMANIASIS

人类利什曼病 T 细胞反应分析

• 批准号: 6431576
• 负责人: David Sacks
• 金额: --
• 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/6431576
• 关键词: Macaca mulatta; Psychodidae; T lymphocyte; cellular immunity; enzyme linked immunosorbent assay; flow cytometry; gastrointestinal disorder; host organism interaction; human tissue; interferon gamma; interleukin 12; laboratory mouse; leishmaniasis; macrophage; microorganism immunology; nonhuman therapy evaluation; protozoal vaccine; tissue /cell culture; tumor necrosis factor alpha; vaccine development

We previously established a model of cutaneous leishmaniasis due to L. major infection combining two main features of natural transmission; inoculation of a low number of metacyclic promastigotes into the mouse ear dermis. In this model, the evolution of small, healing dermal lesions occurs in three distinct phases that have not been appreciated before: 1) an initial ?silent? phase, lasting 4-5 wks, favoring the establishment of the peak load of parasites in the dermis in the absence of lesion formation; 2) an acute phase, lasting 5-10 wks, corresponding to the development and resolution of a lesion that is associated with an acute infiltration of neutrophils, macrophages, and eosinophils into the dermis, and is coincident with the onset of immunity and the killing of parasites in the site; and 3) a chronic phase, lasting for the life of the animal, during which a low number of parasites persist in the skin in the absence of overt pathology. Adaptive immunity in this model confirmed a role for Th1 cells, and in addition revealed a requirement for CD8+ T cells, based on the results obtained in ?2 microglobulin KO mice, CD8 KO mice, and CD8 depleted mice, which in each case failed to control infection in the skin. The conditions favoring the persistence of low numbers of parasites in the skin following healing have also been studied. The chronic site is characterized by a high number of both CD8+ and CD4+ T lymphocytes in the dermis that are able to produce IFN g in response to the antigen. In vivo treatment with either anti-IFNg, anti-CD4+, or anti-CD8+ antibodies resulted in a dramatic increase in parasite numbers and reactivation of dermal lesions. Thus IFNg pressure is necessary to maintain a low parasite burden but not sufficient to clear it. The persistence of the parasite depends upon the production of IL-10 in the site, as evidenced by a) the presence of IL-10 staining cells in the ear and draining lymph node, b) treatment with anti-IL-10R antibodies resulted in complete clearance of parasites from the skin, and c) the complete clearance of L. major from the skin in IL-10 KO and IL-4/IL-10 KO mice. The low dose, intradermal infection model has been extended to L. tropica, which is responsible for anthroponotic cutaneous leishmaniasis. While the growth of the parasite is contained, there is no dermal pathology, parasite clearance does not occur, and a transmissible level of parasitemia persists in the skin for the life of the animal. This equilibrium was again found to be established by counter-balanced levels of IFNg and IL-10. The chronic infections due to L. tropica were cleared from the skin in IL-10 deficient mice, and in mice treated with anti-IL-10R antibodies. The natural challenge model was used to compare the potency and durability of vaccination with a cocktail of plasmid DNAs encoding the antigens LACK, M15, and MAPS, with that of heat killed promastigotes plus recombinant IL-12 (rIL-12). While both vaccines conferred complete protection against dermal leishmaniasis , this protection lasted longer in the DNA vaccinated mice. Furthermore, only the DNA vaccine reduced the parasitic burden in the skin during the acute and chronic stages to the low levels achieved in healed mice, and only the DNA vaccine eliminated the capacity of challenged mice to serve as infection reservoirs for vector sand flies. The clinical forms of leishmaniasis in humans range from self-healing cutaneous lesions to often fatal visceral disease. These diverse clinical outcomes are attributed primarily to differences in the Leishmania species initiating the infections. Animal models using a species associated with self-limiting cutaneous disease, L. major, have revealed that protective immunity requires CD40/CD40L-dependent, IL-12-driven Th1 responses. We have found that in contrast to L. major, Leishmania species responsible for visceral disease (L dononvani), as well as species associated with persistent, cutaneous lesions and occasional systemic disease (L. tropica), do not prime human dendritic cells for CD40L induced IL-12p70 production, and these intrinsic differences in parasite interactions with DC may account, at least in part, for the evolution of healing and non-healing forms of leishmanial disease.

我们之前建立了由大型利什曼病感染引起的皮肤利什曼病模型，结合了自然传播的两个主要特征；将少量后循环前鞭毛体接种到小鼠耳真皮中。在这个模型中，小的、正在愈合的真皮损伤的演变发生在三个不同的阶段，这些阶段以前没有被认识到：1）最初的“沉默”阶段。阶段，持续 4-5 周，有利于在没有病变形成的情况下在真皮中建立寄生虫的峰值负载； 2）急性期，持续5-10周，对应于与中性粒细胞、巨噬细胞和嗜酸性粒细胞急性浸润真皮相关的病变的发展和消退，并且与免疫和杀伤的发生同时发生现场的寄生虫； 3) 慢性阶段，持续动物的一生，在此期间，在没有明显病理的情况下，皮肤中持续存在少量寄生虫。 该模型中的适应性免疫证实了 Th1 细胞的作用，此外根据在 ?2 微球蛋白 KO 小鼠、CD8 KO 小鼠和 CD8 耗尽小鼠中获得的结果，还揭示了对 CD8+ T 细胞的需求，这些小鼠在每种情况下均未能控制皮肤感染。 还研究了有利于愈合后皮肤中少量寄生虫持续存在的条件。慢性部位的特征是真皮中存在大量 CD8+ 和 CD4+ T 淋巴细胞，能够响应抗原产生 IFN g。 使用抗 IFNg、抗 CD4+ 或抗 CD8+ 抗体进行体内治疗会导致寄生虫数量急​​剧增加以及真皮损伤的重新激活。因此，IFNg 压力对于维持低寄生虫负荷是必要的，但不足以清除它。寄生虫的持久性取决于该部位 IL-10 的产生，a) 耳朵和引流淋巴结中存在 IL-10 染色细胞，b) 用抗 IL-10R 抗体治疗导致完全清除皮肤上的寄生虫，以及 c) 在 IL-10 KO 和 IL-4/IL-10 KO 小鼠中完全清除皮肤上的 L. Major。 低剂量皮内感染模型已扩展到热带利什曼病，它是导致人源性皮肤利什曼病的原因。 虽然寄生虫的生长受到抑制，但不存在皮肤病理学，不会发生寄生虫清除，并且在动物的一生中皮肤中持续存在可传播水平的寄生虫血症。 再次发现这种平衡是通过 IFNg 和 IL-10 的平衡水平建立的。 在 IL-10 缺陷小鼠和用抗 IL-10R 抗体治疗的小鼠中，由热带乳杆菌引起的慢性感染从皮肤上被清除。使用自然攻击模型来比较编码抗原 LACK、M15 和 MAPS 的质粒 DNA 混合物与热灭活前鞭毛体加重组 IL-12 (rIL-12) 的疫苗接种的效力和持久性。虽然这两种疫苗都能提供针对皮肤利什曼病的完全保护，但这种保护作用在接种 DNA 疫苗的小鼠中持续时间更长。 此外，只有 DNA 疫苗才能将急性和慢性阶段的皮肤寄生虫负担降低到治愈小鼠所达到的低水平，并且只有 DNA 疫苗才能消除受攻击小鼠作为媒介白蛉感染储存库的能力。人类利什曼病的临床形式包括自愈性皮肤损伤和常常致命的内脏疾病。 这些不同的临床结果主要归因于引发感染的利什曼原虫物种的差异。使用与自限性皮肤病相关的物种 L. Major 进行的动物模型表明，保护性免疫需要 CD40/CD40L 依赖性、IL-12 驱动的 Th1 反应。我们发现，与硕大利什曼原虫不同，导致内脏疾病的利什曼原虫 (L. dononvani) 以及与持续性皮肤病变和偶发全身性疾病相关的利什曼原虫 (热带利什曼原虫) 不会为人类树突细胞提供 CD40L诱导 IL-12p70 的产生，而寄生虫与 DC 相互作用的这些内在差异可能至少部分解释了利什曼病治愈和非治愈形式的演变。