NOS2 and arginase in visceral leishmaniasis

描述（由申请方提供）：叙利亚仓鼠再现了人类杜氏利什曼原虫感染的两种主要形式。全身感染后，这些动物发展为进行性疾病，与人类VL的临床病理特征相似。然而，皮肤感染的仓鼠控制局部感染，并获得对全身攻击的免疫力，类似于亚临床感染个体对内脏疾病产生免疫力。这两个广泛的特点促使我们开始研究，剖析分子免疫发病机制的L。杜氏感染在这个独特的动物模型。在初步研究中，我们发现在VL的仓鼠模型中进行性疾病期间，1型T细胞应答是建立的，但是无效的并且伴随着不可检测的NOS 2表达。此外，与小鼠巨噬细胞相反，IFN-γ激活的仓鼠巨噬细胞不产生可检测的NO，并且不能控制细胞内感染。仓鼠和小鼠之间的这些区别提供了直接比较和对比这些物种中巨噬细胞活化/失活机制的动力，特别关注NOS 2-脱氢酶代谢途径。我们将把体外研究结果扩展到仓鼠的体内原理验证研究，再次关注NOS 2-脱氢酶代谢途径在抵抗疾病和保护性免疫中的作用。我们将从两个相反但互补的方向探讨NOS 2和NOS酶在杜氏乳杆菌感染中的作用。在具体目标1和2中，我们将通过感染仓鼠和小鼠巨噬细胞的体外研究以及全身感染仓鼠的体内研究，确定NOS 2和NOS表达在内脏感染发病机制中的作用。具体目标1将检验仓鼠巨噬细胞控制L. Donovani感染是N 0 S2对IFN-γ介导的活化的低应答性的结果，并且这种N 0 S2低应答性是通过转录机制介导的。具体目标2将检验以下假设：受损的NOS 2表达为受感染仓鼠或人巨噬细胞中的腺苷酸酶主导的替代活化或失活途径奠定了基础，这导致寄生虫杀灭受损。在特定目标3中，我们将确定原发性皮肤感染的控制和获得免疫后对继发性内脏攻击的保护是否是通过逆转默认的低NOS 2和高NOS表达介导的。我们假设，NOS 2-独立的机制有助于耐药性，我们将在发现一个基因表达谱，其特征在于在仓鼠的原发性和继发性感染的阻力开辟新的天地。这些在仓鼠中的研究将通过定义与鼠类模型显著不同的疾病和免疫机制来填补知识空白。

DESCRIPTION (provided by the applicant): Syrian hamsters recapitulate the two major forms of infection with Leishmania donovani seen in man. After systemic infection, these animals develop a progressive disease that mimics the clinical-pathological features of human VL. However, hamsters infected in the skin control the infection locally, and acquire immunity to systemic challenge, akin to sub-clinically infected individuals who develop immunity against visceral disease. These two broad features prompted us to initiate studies to dissect the molecular immunopathogenesis of L. donovani infection in this unique animal model. In preliminary studies we found that during progressive disease in the hamster model of VL, a Type 1 T cell response is mounted, but is ineffective and accompanied by undetectable NOS2 expression. Furthermore, in contrast to mouse macrophages, IFN-gamma-activated hamster macrophages do not produce detectable NO and are unable to control intracellular infection. These distinctions between hamsters and mice provide the impetus to directly compare and contrast mechanisms of macrophage activation/deactivation in these species, with particular focus on the NOS2-arginase metabolic pathways. We will extend in vitro findings to in vivo proof-of-principle studies in the hamster, again with the focus on the role of the NOS2-arginase metabolic pathways in resistance to disease and protective immunity. We will approach the role of NOS2 and arginase in infection with L donovani from two opposite, but complementary directions. In Specific Aims 1 and 2 we will determine the role of NOS2 and arginase expression in the pathogenesis of visceral infection through in vitro studies of infected hamster and mouse macrophages, and in vivo studies of systemically infected hamsters. Specific Aim 1 will test the hypothesis that the impaired capacity of hamster macrophages to control L. donovani infection is a result of the hypo-responsiveness of NOS2 to IFN-gamma-mediated activation, and that this NOS2 hyporesponsiveness is mediated through transcriptional mechanism(s). Specific Aim 2 will test the hypothesis that the impaired NOS2 expression sets the stage for an arginase-dominated alternative activation or deactivation pathway in infected hamster or human macrophages, which leads to impaired parasite killing. In Specific Aim 3 we will determine if the control of primary cutaneous infection and protection against secondary visceral challenge following acquisition of immunity is mediated by a reversal of the default toward low NOS2 and high arginase expression. We hypothesize that NOS2-independent mechanisms contribute to resistance and we will break new ground in the discovery of a gene expression profile that characterizes resistance to primary and secondary infection in the hamster. These studies in the hamster will fill a knowledge gap by defining mechanisms of disease and immunity that are strikingly different from those of the murine model.