NOS2 and arginase in visceral leishmaniasis-FIRCA supplement

描述(由申请人提供)：美国研究员(Peter Melby博士，UTHSCSA，圣安东尼奥)和外国研究员(Sara Robledo博士，哥伦比亚麦德林安蒂奥基亚大学)之间的拟议合作是NIH拨款R01AI61624的延伸，Melby博士是PI。实验动物研究已证实交替激活的巨噬细胞(AAM)在皮肤和内脏利什曼病的发病机制中起主导作用，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个体群体将实验动物研究扩展到人类，这些个体被归类为抵抗者(没有临床感染，但对利什曼原虫的DTH反应呈阳性)或易感个体(慢性不可治愈的皮肤利什曼病)。以前的体外研究表明，与抗药性个体的巨噬细胞相比，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，无法愈合或进行性的利什曼病是通过替代的巨噬细胞激活来介导的，巨噬细胞激活会损害寄生虫的杀灭。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衍生巨噬细胞(MDM)的体外模型。第一个具体目标将检验这样一个假设，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做出反应。MDM将感染L.(V.)Panamens(亲皮株)或L.(L.)Donovani(嗜内脏菌株)以及经典(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)的标志物将用于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有关。第二个特定目标将检验这样的假设，即来自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作用变得更加敏感。将未感染和感染利什曼原虫的MDM暴露于IL-4、IL-10或IL-13，将确定寄生虫和细胞因子在激活STAT6和上调AAM基因方面是否存在相加或协同作用。第三个特定目的将使用siRNA介导的STAT6基因敲除来检验这样的假设，即交替激活巨噬细胞的程序是依赖于STAT6的，并且抑制易感的人巨噬细胞中的STAT6激活将增强其干扰素-3诱导的抗利什曼反应。与公共卫生相关：利什曼病是一种媒介传播的疾病，在世界大部分地区流行，对这些疾病的控制策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区是一种新出现和再次出现的疾病，在哥伦比亚，皮肤利什曼病的病例数量在过去4年中急剧增加。最近内脏利什曼病的流行已导致印度和苏丹数十万人死亡。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.