Macrophage and T-Cell Gene Expression in a Model of Early Infection with the Protozoan Leishmania chagasi

Visceral leishmaniasis is a potentially fatal infectious disease caused by the protozoan parasite Leishmania infantum/chagasi in the New World, or by L. donovani or L. infantum/chagasi in the Old World. Infection leads to a variety of outcomes ranging from asymptomatic infection to active disease, characterized by fevers, cachexia, hepatosplenomegaly and suppressed immune responses. We reasoned that events occurring during the initial few hours when the parasite encounters cells of the innate and adaptive immune systems are likely to influence the eventual immune response that develops. Therefore, we performed gene expression analysis using Affymetrix U133Plus2 microarray chips to investigate a model of early infection with human monocyte-derived macrophages (MDMs) challenged with wild-type L. chagasi parasites, with or without subsequent co-culture with Leishmania-na?ve, autologous T-cells. Microarray data generated from total RNA were analyzed with software from the Bioconductor Project and functional clustering and pathway analysis were performed with DAVID and Gene Set Enrichment Analysis (GSEA), respectively. Many transcripts were down-regulated by infection in cultures containing macrophages alone, and the pattern indicated a lack of a classically activated phenotype. By contrast, the addition of autologous Leishmania-na?ve T cells to infected macrophages resulted in a pattern of gene expression including many markers of type 1 immune cytokine activation (IFN-��, IL-6, IL-1��, IL-1��). There was simultaneous up-regulation of a few markers of immune modulation (IL-10 cytokine accumulation; TGF-�� Signaling Pathway). We suggest that the initial encounter between L. chagasi and cells of the innate and adaptive immune system stimulates primarily type 1 immune cytokine responses, despite a lack of classical macrophage activation. This local microenvironment at the site of parasite inoculation may determine the initial course of immune T-cell development.内脏利什曼病（Visceral leishmaniasis）是一种潜在致死性传染病：在新大陆由原生动物寄生虫婴儿利什曼原虫/恰加斯利什曼原虫（Leishmania infantum/chagasi）引发，在旧大陆则由杜氏利什曼原虫（L. donovani）或婴儿利什曼原虫/恰加斯利什曼原虫导致。感染可产生多种转归，从无症状感染到活动性疾病，后者以发热、恶病质、肝脾肿大及免疫应答受抑为特征。我们推测，寄生虫初次接触固有免疫与适应性免疫细胞的最初数小时内发生的事件，极有可能影响最终形成的免疫应答。因此，本研究采用Affymetrix U133Plus2微阵列芯片（Affymetrix U133Plus2 microarray chips）开展基因表达分析，以探究野生型恰加斯利什曼原虫感染人类单核细胞衍生巨噬细胞（monocyte-derived macrophages, MDMs）的早期感染模型，并设置是否后续与利什曼原虫未致敏自体T细胞共培养的两组对照。从总RNA中获取的微阵列数据通过Bioconductor项目（Bioconductor Project）的软件进行分析，功能聚类与通路分析则分别借助DAVID工具与基因集富集分析（Gene Set Enrichment Analysis, GSEA）完成。仅含巨噬细胞的培养体系中，感染可使大量转录本表达下调，该表达模式提示巨噬细胞未呈现经典活化表型。与之相反，在感染的巨噬细胞中加入自体利什曼原虫未致敏T细胞后，基因表达模式中出现诸多1型免疫细胞因子活化标志物，包括干扰素-γ（IFN-γ）、IL-6、IL-1α、IL-1β。同时，部分免疫调节标志物的表达出现上调，例如IL-10细胞因子积累、转化生长因子-β（TGF-β）信号通路。我们认为，恰加斯利什曼原虫与固有免疫、适应性免疫细胞的初次接触主要触发1型免疫细胞因子应答，尽管巨噬细胞未出现经典活化。寄生虫接种部位的局部微环境或可决定免疫T细胞发育的初始进程。