IMMUNE REGULATION AND VACCINE DEVELOPMENT IN LEISHMANIASIS

内源性IL-10是免疫稳态的中心介质，对抑制局部组织中伴随着抗微生物免疫表达的强烈炎症反应是必要的。宿主免疫和病理之间达到平衡的结果可能是慢性或持续性感染，而IL-10的缺失已被证明能够更有效地清除各种病原体，包括大利什曼原虫。我们已经解决了IL-10依赖的免疫抑制的主要来源，该菌株在C57BL/6小鼠中面对强烈的Th1反应时产生不可愈合的皮肤损伤。感染可诱导天然细胞、CD4+CD25+Foxp3+天然Treg和CD4+CD25-Foxp3诱导的Treg局部募集和产生IL-10。后一类细胞似乎与Th1效应器反应密切相关，因为它们也产生大量的干扰素-γ。在RAG-/-重组感染小鼠中，产生IL-10的Th1细胞是在没有天然Treg或来自天然来源的IL-10的情况下产生的，最重要的是，发现在抑制保护性免疫中发挥了必要和充分的作用。利用IL-10报告小鼠，来自慢性感染小鼠的IFNG+IL-10-CD4+T细胞可以在体外用抗原进一步刺激产生IL-10，这表明抗原的持续刺激推动Th1细胞通过一种包括IL-10分泌作为反馈控制机制的发育程序。IL-12，尤其是IL-27可增强Th1细胞对IL-10的诱导。 内脏利什曼病(VL)是一种威胁生命的疾病，其特征是脾、肝和骨髓无法控制地寄生。在人类内脏利什曼病(VL)中，未能控制利什曼原虫生长和全身传播的机制尚不清楚。虽然VL患者PBMC中缺乏抗原特异性Th1反应被认为与疾病进展有关，但这些患者在皮损组织中也表达高水平的IFNGamma mRNA，以及血清促炎细胞因子水平，这表明他们的免疫缺陷不能简单地用免疫耐受或Th2极化来解释。作为控制持续感染引起的炎症的一种可能的动态平衡机制，调节细胞因子IL-10水平的升高在VL的临床研究中一再被报道。我们在印度的临床研究的主要目的是确定人类VL中IL-10的细胞来源，并确定CD4+CD25+(Foxp3High)调节性T细胞是否与活动性疾病有关。我们分析了印度VL患者在两性霉素B治疗前和治疗后3-4周的PBMC和脾抽吸物中的表面标志和基因表达。结果并不表明自然的CD4+CD25+(Foxp3High)Treg细胞在人类VL中起到重要作用：它们不会积聚在脾中，也不是脾中IL-10的主要来源，它们的去除也不会挽救抗原特异性的干扰素-伽马反应。相比之下，去除CD25+细胞的脾T细胞表达IL-10mRNA的水平最高，是VL脾中的主要淋巴细胞群。VL血浆中IL-10水平的升高显著促进人巨噬细胞中杜氏钩端螺旋体无鞭毛体的生长。VL患者PBMC中的CD4+T细胞可被抗原和PMA/离子霉素刺激产生IL-10和干扰素-γ。最后，对PBMC的分析表明，CD8+T细胞在活动期表达IL-10mRNA的水平最高，治疗后显著下调。我们的结果提示，利什曼病驱动的产生IL-10的Foxp3-CD4+和CD8+T细胞可能在抑制VL患者保护性免疫反应中发挥重要作用。 热带利什曼原虫是旧世界人细菌性皮肤利什曼病(ACL)的病原体，其特点是皮损需要很长时间才能痊愈，通常会导致毁容的疤痕，而且比主要利什曼原虫引起的利什曼病更难治疗。涉及热带乳杆菌感染的实验动物模型的免疫学研究几乎是不存在的。在本研究中，我们用感染期的半环热带乳杆菌建立了C57BL/6和BALB/c小鼠的皮肤感染模型。在两种菌株中，病变发展缓慢，表现出最轻微的病理变化。尽管如此，它们在一年多的时间里仍然含有稳定数量的10,000到100,000种寄生虫，这些寄生虫被一种名为Phlebotomus Sergenti的天然沙蝇媒介有效地感染了。寄生虫生长的控制依赖于Th1反应的发展，因为IL-12、干扰素-γ或诱导型一氧化氮合酶基因缺陷的C57BL/6小鼠，或接受干扰素-γ抗体治疗的BALB/c小鼠，携带的寄生虫明显更多。相比之下，IL-10缺乏的小鼠在整个感染过程中携带的寄生虫明显较少。为了进一步研究阻止寄生虫有效清除的免疫学机制，在野生型C57BL/6小鼠感染的慢性期取消了IL-10和转化生长因子-β信号转导。与慢性大型钩端螺旋体感染不同，IL-10单独阻断对热带钩端螺旋体没有影响，但需要同时使用抗转化生长因子-β抗体来促进寄生虫从感染部位有效清除。因此，热带乳杆菌似乎利用多种抑制因子来维持慢性化，并将宿主确立为媒介沙蝇的长期感染宿主。

Endogenous IL-10 is a central mediator of immune homeostasis, necessary to keep in check the strong inflammatory reactions that can accompany the expression of anti-microbial immunity in local tissues. A consequence of the balance struck between host immunity and pathology can be chronic or persistent infection, and the absence of IL-10 has been shown to result in more efficient clearance of a variety of pathogens, including Leishmania major. We have addressed the dominant source of IL-10 dependent immune suppression induced by a strain of L. major that produces non-healing dermal lesions in the face of a strong Th1 response in C57BL/6 mice. The infection induced localized recruitment and production of IL-10 from innate cells, as well as from CD4+CD25+Foxp3+ natural Treg and CD4+CD25-Foxp3- inducible Treg. The latter cells appear to be closely linked to the Th1 effector response, in that they also produced high amounts of IFN-gamma. In Rag-/- reconstituted infected mice, the IL-10-producing Th1 cells were generated in the absence of either natural Treg or IL-10 from innate sources, and most importantly, were found to play a necessary and sufficient role in the suppression of protective immunity. Using IL-10 reporter mice, IFNg+IL-10-CD4+ T cells from chronically infected mice could be further stimulated in vitro with antigen to produce IL-10, suggesting that persistant stimulation with antigen drives Th1 cells through a program of development that includes IL-10 secretion as a mechanism of feedback control. IL-12 and especially IL-27 were found to enhance IL-10 induction in committed Th1 cells. Visceral leishmaniasis (VL) is a life threatening disease characterized by uncontrolled parasitization of spleen, liver, and bone marrow. The mechanisms underlying the failure to control the growth and systemic spread of Leishmania parasites in human visceral leishmaniasis (VL) are not well understood. While the absence of antigen-specific Th1 responses in PBMC from VL patients is thought to be causally related to disease progression, the finding that these patients also express elevated IFNgamma mRNA in lesional tissue, as well as elevated serum levels of pro-inflammatory cytokines, suggests that their immunologic defect cannot be simply explained by immune tolerance or Th2 polarization. As a possible homeostatic mechanism to control persistent infection-induced inflammation, elevated levels of the regulatory cytokine IL-10 have repeatedly been reported in clinical studies of VL. The main aim of our clinical studies in India has been to identify the cellular source of IL-10 in human VL, and determine if CD4+CD25+(Foxp3high) regulatory T cells are associated with active disease. We analyzed surface marker and gene expression in PBMC and splenic aspirates from Indian VL patients pre- and 3-4 weeks post-treatment with Amphotericin B. The results did not point to an important role for natural CD4+CD25+(Foxp3high) Treg cells in human VL: they did not accumulate in and were not a major source of IL-10 in the spleen, and their removal did not rescue antigen-specific IFN-gamma responses. By contrast, splenic T cells depleted of CD25+ cells expressed the highest levels of IL-10 mRNA, and were the predominant lymphocyte population in the VL spleen. The elevated levels of IL-10 in VL plasma significantly enhanced the growth of L. donovani amastigotes in human macrophages. CD4+ T cells in PBMC from VL patients could be stimulated by antigen followed by PMA/ionomycin to produce both IL-10 and IFN-gamma. Finally, analysis of PBMC indicated that on a per cell basis CD8+ T cells expressed the highest levels of IL-10 mRNA during active disease, with significant down-regulation observed post treatment. Our results suggests that Leishmania driven, IL-10 producing Foxp3- CD4+ and CD8+ T cells may play an important role in suppression of protective immune responses in VL patients. Leishmania tropica is the causative agent of old-world anthroponotic cutaneous leishmaniasis (ACL), which is characterized by lesions that take an extended period of time to heal, often resulting in disfiguring scars, and are more refractory to treatment than leishmaniasis caused by L. major. Immunologic studies involving experimental animal models of L. tropica infection are virtually non-existent. In the current study, infectious-stage, metacyclic L. tropica were used to establish dermal infections in C57Bl/6 and BALB/c mice. In both strains the lesions were slow to develop and showed minimal pathology. They nonetheless contained a stable number of between 10,000 and 100,000 parasites for over one year, which were efficiently picked up by a natural sand fly vector, Phlebotomus sergenti. Control of parasite growth depended on the development of a Th1 response, as C57Bl/6 mice genetically deficient in IL-12, IFN-gamma, or inducible nitric oxide synthase, or BALB/c mice treated with antibodies to IFN-gamma, harbored significantly more parasites. By contrast, IL-10 deficient mice harbored significantly fewer parasites throughout the course of infection. To further study the immunologic mechanisms that may prevent efficient clearance of the parasites, IL-10 and TGF-beta signaling were abrogated during the chronic phase of infection in wild type C57Bl/6 mice. Distinct from chronic L. major infection, IL-10 blockade alone had no effect on L. tropica, but required simultaneous treatment with anti-TGF-beta antibodies to promote efficient parasite clearance from the infection site. Thus, L. tropica appears to exploit multiple suppressive factors to maintain chronicity, and to establish the host as a long-term reservoir of infection for vector sand flies.