Human DC & macrophages in dysregulation of lung innate immunity by F

土拉热弗朗西丝菌（Francisellatularensis，Ft）是一种兼性胞内致病菌，是引起兔热病的病原体。 虽然很明显，吸入少至10英尺的亚种。土拉热可能是致命的，并且巨噬细胞是这种生物体在体内的主要储存库，Ft如何逃避消除才刚刚开始被定义，并且我们对这种病原体对其他细胞类型（包括树突状细胞（DC））的影响了解得更少。 由于DC在抗原呈递中起核心作用，阐明Ft的人类致病菌株改变DC功能的程度对于新疫苗的开发具有重要意义，并且在这方面值得注意的是，许多其他病原体在巨噬细胞和DC中的命运是不同的。我们的长期目标是在分子水平上剖析Ft破坏人类吞噬细胞功能的机制。在之前的资助期间，我们鉴定了蛋白激酶Ca作为该病原体靶向的巨噬细胞因子，以在早期阶段，在胞质溶胶中的Ft排出和复制之前破坏吞噬体成熟。PKC α活性在整个感染的巨噬细胞中下降，并且因为这种激酶还控制INF γ调节过程的子集，包括MHC II类抗原呈递，我们假设Ft可能使用类似的策略来破坏人DC的功能。然而，Ft在该细胞类型中的命运尚未描述，并且先天免疫应答可被操纵以有利于Ft清除的程度尚不清楚。为了解决这一知识缺口，我们将定义Ft在人DC中的命运，包括通过与肺泡上皮细胞共培养而“调节”的DC，并量化Ft对抗原呈递的影响。在以前的工作中，我们还定义了甘露糖受体（MR）在Ft摄取中的作用。Ft的一个显著特征是富含甘露糖的表面胶囊样物质（CLM），其与LPS一起保护生物体免受补体介导的裂解。CLM是否含有或掩盖MR配体尚不清楚，CLM对Ft生命周期其他方面的影响仍然不清楚。到目前为止，在项目1中已经分离到Ft菌株SchuS 4中的9个CLM突变体，并且我们对FTT 1238 C、FTT 1447 C、FTT 1512 c、FTT 1291、FTT 0673 C-0674中的突变体的初步表征首次表明CLM结构或丰度的改变可以从根本上影响Ft摄取和细胞内生长。因此，本研究的具体目的是：1）阐明强毒Ft subsp.土拉菌SchuS 4和Ft subsp. 在人DC中的holarctica菌株1547-57; 2）评估 巨噬细胞和DC的功能，以增加细菌杀伤和有利于抗原呈递;和3）开始定义体内和体外巨噬细胞和DC中的Ft命运中的胶囊样物质的作用。

Francisella tularensis (Ft) is a facultative intracellular pathogen and the causative agent of tularemia. Although it is clear that inhalation of as few as ten Ft subsp. tularensis can be fatal, and that macrophages are a major reservoir of this organism in vivo, how Ft evades elimination is only beginning to be defined, and we know even less about the effects of this pathogen on other cell types, including dendritic cells (DC). Because DC play central role in antigen presentation, elucidating the extent to which human pathogenic strains of Ft alter DC function has important implications for the development of new vaccines, and in this regard it is noteworthy that the fate of many other pathogens in macrophage and DC are distinct. Our longterm goal is to dissect at the molecular level the mechanisms by which Ft disrupts the function of human phagocytes. During the previous funding period we identified protein kinase Ca as a macrophage factor targeted by this pathogen to disrupt phagosome maturation at an eariy stage, prior to Ft egress and replication in the cytosol. PKCa activity declines throughout infected macrophages, and because this kinase also controls a subset of INFy-regulated processes including MHC Class II antigen presentation, we hypothesize that Ft may use a similar strategy to disrupt the function of human DC. Nevertheless, the fate of Ft in this cell type has not been described, and the extent to which the innate immune response can be manipulated to favor Ft clearance is unclear. To address this knowledge gap, we will define Ft fate in human DC, including DC that were 'conditioned' by co-culture with alveolar epithelial cells, and quantify effects of Ft on antigen presentation. In previous work we also defined a role for the mannose receptor (MR) in Ft uptake. A distinguishing feature of Ft is a mannose-rich surface capsule-like material (CLM) that together with LPS protects the organism from complement-mediated lysis. Whether CLM contains or masks MR ligands is unclear, and effects of CLM on other aspects of the Ft lifecycle remain obscure. To date, 9 CLM mutants in Ft strain SchuS4 have been isolated in Project 1, and our preliminary characterization of mutants in FTT1238C, FTT1447C, FTT1512c, FTT1291, FTT0673C-0674 suggests for the first time that alterations in CLM structure or abundance can diflFerentially effect Ft uptake and intracellular growth. Thus, the Specific Aims of this study are: 1) to elucidate the fate of virulent Ft subsp. tularensis strain SchuS4 and Ft subsp. holarctica strain 1547-57 in human DC; 2) to evaluate the potential for translational modulation of macrophage and DC function to increase bacterial killing and favor antigen presentation; and 3) to begin to define the role of capsule-like material in Ft fate in macrophages and DC in vivo and in vitro.