NOS2 and arginase in visceral leishmaniasis-FIRCA supplement

描述（由申请人提供）：美国研究者（Dr. Peter Melby， UTHSCSA, San Antonio）和外国研究者（Dr. Sara Robledo, University of Antioquia, Medellin, Colombia）之间拟议的合作是NIH拨款R01AI61624的延伸，其中Dr. Melby是PI。实验动物研究已经确定了选择性活化巨噬细胞（AAMs）在皮肤和内脏利什曼病发病机制中的主导作用，但它们在人类易感性和疾病中的作用尚未确定。拟议的FIRCA拨款将通过调查先前研究的人群，将实验动物研究扩展到人类，这些人群要么被分类为耐药（没有临床感染，但对利什曼原虫抗原有阳性的DTH反应），要么被分类为易感（慢性非愈合皮肤利什曼病）。先前的体外研究确定，来自易感个体的巨噬细胞与来自耐药个体的巨噬细胞相比，控制利什曼原虫感染的能力降低，但这种易感性的机制尚不清楚。该建议的总体假设是，不愈合或进展的利什曼病是通过替代巨噬细胞激活介导的，这削弱了寄生虫的杀伤作用。对于所有拟议的研究，将使用从耐药或易感个体分离的利什曼原虫感染单核细胞来源的巨噬细胞（MDMs）的体外模型。第一个特异性目标将测试假设，即来自易感个体的巨噬细胞通过替代激活程序直接对利什曼原虫感染作出反应。MDMs将感染L. (V.) panamensis（皮肤型菌株）或L. (L.) donovani（嗜内脏型菌株），并使用经典标记（NO产生、NOS2表达和CCL3产生）和替代巨噬细胞激活（精氨酸酶活性、IL-10、CCL17和CCL22产生以及CD23表达）来确定利什曼原虫感染是否直接诱导替代激活，以及它是否与易感性相关。第二个特异性目标将检验来自易感个体的巨噬细胞，当感染利什曼原虫时，对2型细胞因子的替代激活作用变得更加敏感的假设。将未感染和感染利什曼的MDMs暴露于IL-4、IL-10或IL-13将确定寄生虫和细胞因子在STAT6激活和AAM基因上调中是否存在相加或协同作用。第三个特异性目标将使用siRNA介导的STAT6敲低来验证巨噬细胞的选择性激活程序依赖于STAT6的假设，以及抑制易感人巨噬细胞中STAT6的激活将增强其ifn -3诱导的抗利希曼反应。公共卫生相关性：利什曼病是一种病媒传播疾病，在世界大部分地区流行，控制战略在很大程度上是不成功的或不可持续的。皮肤利什曼病在世界许多地方是一种新出现和再出现的疾病，在哥伦比亚，皮肤利什曼病的病例数在过去4年中急剧增加。最近内脏利什曼病的流行已在印度和苏丹造成数十万人死亡。对化疗的不良反应和可用药物的缺乏使得对疾病机制的调查以及确定预防或治疗干预措施至关重要。

DESCRIPTION (provided by applicant): The proposed collaboration between the U.S. investigator (Dr. Peter Melby, UTHSCSA, San Antonio) and the foreign investigator (Dr. Sara Robledo, University of Antioquia, Medellin, Colombia) is an extension of NIH grant R01AI61624, on which Dr. Melby is the PI. Experimental animal studies have identified a dominant role for alternatively activated macrophages (AAMs) in the pathogenesis of cutaneous and visceral leishmaniasis, but their role in human susceptibility and disease has not been determined. The proposed FIRCA grant will extend the experimental animal studies to humans by investigating a previously studied population of individuals who were either classified as resistant (no clinical infection but positive DTH reaction to Leishmania antigen) or susceptible (chronic non-healing cutaneous leishmaniasis). Previous in vitro studies determined that macrophages from susceptible individuals had reduced capacity to control Leishmania infection compared to macrophages from resistant individuals, but the mechanism of this susceptibility is unknown. The overall hypothesis of this proposal is that nonhealing or progressive leishmaniasis is mediated through alternative macrophage activation, which impairs parasite killing. For all of the proposed studies, an in vitro model of Leishmania- infected monocyte-derived macrophages (MDMs), isolated from resistant or susceptible individuals, will be used. The first Specific Aim will test the hypothesis that macrophages from susceptible individuals respond directly to Leishmania infection through a program of alternative activation. The MDMs will be infected with L. (V.) panamensis (dermatropic strain) or L. (L.) donovani (viscerotropic strain), and markers of classical (NO production, NOS2 expression, and CCL3 production) and alternative macrophage activation (arginase activity, IL-10, CCL17, and CCL22 production, and expression of CD23) will be used to determine if Leishmania infection directly induces alternative activation, and if it is associated with susceptibility. The second Specific Aim will test the hypothesis that macrophages from susceptible individuals, when infected with Leishmania, become more sensitive to alternative activation effect of type 2 cytokines. Exposure of uninfected and Leishmania-infected MDMs to IL-4, IL-10, or IL-13 will determine if there is an additive or synergistic effect of the parasites and cytokines in the activation of STAT6 and upregulation of AAM genes. The third Specific Aim will use siRNA- mediated knockdown of STAT6 to test the hypothesis that the program of alternative macrophage activation is STAT6-dependent, and that inhibition of STAT6 activation in susceptible human macrophages will enhance their IFN-3-induced anti-leishmanial response. PUBLIC HEALTH RELEVANCE: Leishmaniasis is a vector borne disease that is endemic throughout much of the world, for which control strategies have largely been unsuccessful or non-sustainable. Cutaneous leishmaniasis is an emerging and re-emerging disease in many parts of the world, and in Colombia the number of cases of cutaneous leishmaniasis has increased dramatically in the past 4 years. Recent epidemics of visceral leishmaniasis have resulted in several hundred thousand deaths in India and Sudan. The poor response to chemotherapy and paucity of available drugs make investigation into the mechanisms of disease, and the identification of preventive or therapeutic interventions, of paramount importance.