Activation of Macrophages by Lipoteichoic Acid

由于革兰氏阳性（G+）感染很常见，可能危及生命，因此彻底了解这些致病菌及其释放的各种分子如何与宿主免疫系统相互作用至关重要。长期以来，人们一直认为影响宿主炎症反应的一种细菌成分是脂磷胆酸（LTA），这是一种几乎所有G+细菌都表达的表面成分，在细菌生长过程中以及使用某些抗生素治疗后会释放到周围环境中。LTA在刺激先天免疫系统细胞，特别是单核细胞、树突状细胞和巨噬细胞中的作用得到了大量研究的支持。此外，我们实验室和其他人的体外实验表明，LTA在刺激免疫系统细胞启动促炎反应的同时，还具有与宿主大分子协同作用的能力，大大增强了这种细胞因子反应。本提案中概述的实验将检查LTA及其协同伙伴之一血红蛋白（Hb）在体外和体内的相互作用。具体目标，根据两年的ARRA资金限额进行修订，如下：确定LTA如何与宿主蛋白Hb协同作用。这一目标将集中于了解LTA和Hb协同作用导致巨噬细胞细胞因子分泌10倍增强的机制，并将特别关注TLR4在这一过程中的作用。我们将确定LTA+Hb刺激对巨噬细胞表面各种受体募集的影响，并确定哪些TLR信号通路用于转导这些信号。通过观察巨噬细胞受LTA+Hb刺激时转录激活的tlr应答基因集，并将这些数据与WT和TLR4-/-巨噬细胞中单独由LTA激活的基因进行比较，将进一步了解这一过程。最后，我们将开始确定哪些刺激分子负责这种反应的tlr4依赖性。2. 确定LTA和Hb在体内协同作用的后果。我们最近发现Hb增强了野生型小鼠对LTA的IL-6和tnf - α反应，这表明我们在体外研究的现象在体内也是有效的。我们假设这种增加的细胞因子反应会阻碍或加剧化脓性链球菌感染。我们将研究在Hb存在和不存在的情况下，腹腔注射LTA的小鼠血液和腹膜腔中细胞因子的释放。最后，我们将确定这些预处理是否改善或加剧化脓性葡萄球菌感染。

Because Gram-positive (G+) infections are common and can be life-threatening, it is critical to have a thorough understanding of how these pathogenic bacteria and the various molecules that they release interact with the host immune system. One bacterial component long thought to affect the host inflammatory response is lipoteichoic acid (LTA), a surface component expressed by virtually all G+ bacteria and released into the surrounding environment during bacterial growth and as a result of treatment with some antibiotics. LTA’s role in stimulating cells of the innate immune system, particularly monocytes, dendritic cells and macrophages, is supported by a large body of work. In addition, in vitro experiments from our laboratory and others have shown that, while LTA can stimulate cells of the immune system to initiate a pro-inflammatory response, LTA also has the ability to synergize with host macromolecules to greatly enhance this cytokine response. The experiments outlined in this proposal will examine both the in vitro and in vivo interactions of LTA and one of its synergistic partners, hemoglobin (Hb). The specific aims, revised according to the 2-year ARRA funding limit, are to: 1. Determine how LTA synergizes with the host protein Hb. This aim will focus on understanding the mechanisms by which LTA and Hb synergize to result in a 10-fold potentiation of macrophage cytokine secretion and will specifically focus on understanding the role of TLR4 in this process. We will determine the effects of LTA+Hb stimulation on the recruitment of various receptors to the macrophage surface and define which TLR signaling pathways are utilized to transduce these signals. Further understanding of this process will be obtained by looking at the set of TLR-responsive genes that are transcriptionally activated when macrophages are stimulated by LTA+Hb and comparing these data to the genes activated by LTA alone, both in WT and TLR4-/- macrophages. Finally, we will begin to determine what stimulatory molecule is responsible for the TLR4-dependent nature of this response. 2. Determine the consequences of synergistic interactions of LTA and Hb in vivo. We have recently found that Hb potentiates the the IL-6 and TNF-alpha responses of wildtype mice to LTA, indicating that the phenomenon we have studied in vitro is also operative in vivo. We hypothesize that this increased cytokine response will either impede or exacerbate S. pyogenes infections. We will study the release of cytokines into the blood and peritoneal cavities of mice injected i.p. with LTA in the presence and absence of Hb. Lastly, we will determine whether such pretreatments ameliorate or exacerbate a S. pyogenes infection.