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，圣安东尼奥）和外国研究者（Dr. Sara Robledo，University of安蒂奥基亚，Medellin，Colombia）之间的拟定合作是NIH研究授权R01AI61624的扩展，Melby博士是主要研究者。实验动物研究已经确定了替代性激活的巨噬细胞（AAM）在皮肤和内脏利什曼病的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赠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，这些人群被归类为耐药（无临床感染但对利什曼原虫抗原有阳性DTH反应）或易感（慢性非愈合性皮肤利什曼病）。先前的体外研究确定，与来自抗性个体的巨噬细胞相比，来自易感个体的巨噬细胞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。这一建议的总体假设是，不愈合或进行性利什曼病介导的替代巨噬细胞激活，这损害了寄生虫杀死。对于所有拟定研究，将使用从耐药或易感个体中分离的利什曼原虫感染单核细胞衍生巨噬细胞（MDM）的体外模型。第一个特定目的将测试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的假设。MDM将感染L。（五）panamensis（亲皮性菌株）或L.（L.）杜氏利什曼原虫（亲内脏株），以及经典巨噬细胞活化标志物（NO产生、N0S2表达和CCL3产生）和替代巨噬细胞活化标志物（趋化酶活性、IL-10、CCL17和CCL22产生和CD23表达）将用于确定利什曼原虫感染是否直接诱导替代活化，以及是否与易感性相关。第二个特定目的将检验易感个体的巨噬细胞在感染利什曼原虫时对2型细胞因子的替代激活效应变得更敏感的假设。将未感染和利什曼原虫感染的MDM暴露于IL-4、IL-10或IL-13将确定寄生虫和细胞因子在STAT6活化和AAM基因上调中是否存在累加或协同效应。第三个具体目标将使用siRNA介导的STAT6敲低来测试以下假设：替代性巨噬细胞活化的程序是STAT6依赖性的，并且在易感的人巨噬细胞中抑制STAT6活化将增强其IFN-3诱导的抗利什曼原虫应答。公共卫生相关性：利什曼病是一种媒介传播的疾病，在世界大部分地区流行，其控制策略在很大程度上是不成功或不可持续的。皮肤利什曼病是世界许多地区新出现和重新出现的疾病，在哥伦比亚，皮肤利什曼病病例数在过去4年中急剧增加。最近内脏利什曼病的流行在印度和苏丹造成数十万人死亡。对化疗反应差和缺乏可用药物使得调查疾病机制和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.