Targeting professional APCs using Fasciola hepatica FABP to suppresses inflammation

摘要 在过去十年中，越来越多地使用“蠕虫疗法”来预防或改善炎症性疾病， 有前途和新颖的方法。肝片形吸虫是一种全球流行的国内寄生蠕虫， 动物，特别擅长控制其宿主的免疫反应。在感染的早期阶段， 诱导与抑制Th 1应答一致的显性Th 2/T调节型免疫应答。 最近的研究表明F.肝感染减弱小鼠实验性肝癌的临床症状 自身免疫性脑脊髓炎[1]，并防止非肥胖糖尿病小鼠发生1型糖尿病 模型[2]。然而，与F.肝病缺乏特异性，导致 免疫系统不能有效地应对旁观者感染[3，4]。更好的药物替代品 发展将是鉴定具有免疫调节能力的寄生虫分子， 精确的作用机制。脂肪酸结合蛋白（FABPs）是在细胞内的代谢中起重要作用的蛋白质。 寄生虫的脂质代谢，最近被归类为抗氧化分子[5]。我们有 证明了属于脂肪酸结合蛋白（Fh 15）的重组14.5kDa蛋白能够 在暴露于致死剂量的LPS后1小时治疗性应用时显著抑制细胞因子风暴， 提示Fh 15可能具有TLR 4拮抗剂的作用。此外，我们还发现，Fh 15处理增加了 与对照组相比，大和小腹膜MΦ（LPM和SPM）在腹膜腔（PerC）中的蓄积 [6]关于老鼠。此外，我们还通过邻位连接试验（PLA）证明了天然F。肝片 FABP变体（Fh 12）与人CD 14辅助受体结合[7]，这与活化信号传导相关。 各种TLR的级联[8-11]。由于TLR 4直接参与脓毒症期间的炎症反应， 溃疡性结肠炎（UC），Fh 15的这种作用可能对人类健康产生重大影响[12，13]。我们的中央 假设是通过注射（i. p.）FH 15足以 预防暴露于致死剂量LPS的小鼠的高死亡率，并预防或改善肠道 在DSS诱导的UC小鼠模型中的炎症。这两种治疗效果将直接与 在动物的PerC中产生M2型MΦ和诱导DC的致耐受性特性，这将是 严重依赖于CD 14。这项研究在概念上是创新的，因为这是第一次， F.肝FABP家族，特别是Fh 15，被研究为抗炎分子， 目前还没有关于MΦs和DC与体内或体外Fh 15暴露相关的研究报道。了解Fh 15如何 在先天免疫的早期阶段调节MΦs和DC并与之相互作用可能有助于 抗败血症和其他炎性疾病的抗炎药。具体目标是：1）确定 Fh 15抑制小鼠感染性休克和溃疡性结肠炎（UC）病理作用的能力 模型和，2）确定Fh 15是否诱导DC的致耐受性并确定机制- Fh 15引起的潜在免疫抑制。拟议的研究将大大推进反- 通过提供明确的F.肝片 分子作为药物靶点的作用机制。

ABSTRACT The growing use of `helminth-therapy' in the last decade to prevent or ameliorate inflammatory diseases is a promising and novel approach. Fasciola hepatica, one of the most globally prevalent parasitic helminths of domestic animals, is particularly adept at controlling the immune response of its host. At early stages of infection the parasite induces a dominant Th2/T-regulatory-type immune response coincidental with suppression of Th1 responses. Recent studies have demonstrated that F. hepatica infection attenuates the clinical signs of murine experimental autoimmune encephalomyelitis [1] and prevents the development of Type-1 diabetes in a non-obese diabetic mouse model [2]. However, the immunoregulation associated to F. hepatica lacks specificity and results in a compromised immune system unable to respond effectively to bystander infections [3, 4]. A better alternative for drug development would be to identify parasite molecules with immune-modulatory capacity and to characterize their precise mechanism of action. Fatty acid binding proteins (FABPs) are proteins that play an important role in the parasite's lipid metabolism and have been recently categorized as anti-oxidant molecules [5]. We have demonstrated that a recombinant 14.5kDa protein belonging to the fatty acid binding protein (Fh15) is able to significantly suppress the cytokine storm when is applied therapeutically 1h after exposure to lethal doses of LPS, which suggest that Fh15 could act as a TLR4-antagonist. Moreover, we also found that Fh15-treatment increased accumulation of large and small peritoneal MΦs (LPMs and SPMs) into the peritoneal cavity (PerC) compared to the of septic mice [6]. Moreover, we also demonstrated by proximity ligation assay (PLA) that the native F. hepatica FABP variant (Fh12) binds to the human CD14-coreceptor [7], which has been associated to the activation-signaling cascade of various TLRs [8-11]. Because TLR4 is directly involved in the inflammatory responses during sepsis and ulcerative colitis (UC), this role for Fh15 could have a significant impact on human health [12, 13]. Our central hypothesis is that the suppression of pro-inflammatory cytokines induced by injection (i.p.) with Fh15 is enough to prevent the high mortality in mice exposed to a lethal dose of LPS and to prevent or ameliorate the intestinal inflammation in a DSS-induced mouse model of UC. Both therapeutic effects will be directly associated with the production of M2-type MΦs in the PerC of animals and the induction of tolerogenic properties of DCs, which will be critically dependent on CD14. The proposed research is conceptually innovative because it is the first time that a protein of the F. hepatica FABP family, specifically Fh15, is investigated as an anti-inflammatory molecule and there are no reported works on MΦs and DCs in relation to Fh15 exposure in vivo or in vitro. Understanding how Fh15 modulate and interact with MΦs and DCs at early stages of innate immunity could contribute to the development of anti-inflammatory drugs against sepsis and other inflammatory diseases. The specific aims are: 1) Determine the capacity of Fh15 to suppress the pathologic effects of septic shock and ulcerous colitis (UC) in the mouse model and, 2) Determine whether Fh15 induces the tolerogenic properties of DCs and identify mechanism- underlying immunosuppression caused by Fh15. The proposed studies significantly will advance anti- inflammatory drug development based on helminth-antigen derived therapy by providing a well-defined F. hepatica molecule as drug target mechanism of action.