Targeting professional APCs using Fasciola hepatica FABP to suppresses inflammation

摘要 在过去的十年里，越来越多地使用“蠕虫疗法”来预防或改善炎症性疾病，这是一种 前景看好且新颖的方法。肝片吸虫，全球最流行的寄生蠕虫之一 特别擅长控制宿主的免疫反应。在感染早期，寄生虫 诱导主要的Th2/T调节型免疫反应，同时抑制Th1反应。 最近的研究表明，肝片吸虫感染可以减轻小鼠实验性肝病的临床症状。 自身免疫性脑脊髓炎[1]和预防非肥胖糖尿病小鼠的1型糖尿病的发展 模型[2]。然而，与肝片吸虫相关的免疫调节缺乏特异性，导致一种折衷的 免疫系统无法对旁观者感染做出有效反应[3，4]。一种更好的药物替代品 发展将是识别具有免疫调节能力的寄生虫分子，并表征它们的 精确的作用机制。脂肪酸结合蛋白(FABP)是一种在体内发挥重要作用的蛋白质 寄生虫的脂肪代谢，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(Fh15)的14.5 kDa重组蛋白能够 在暴露于致命剂量的内毒素后1小时进行治疗性应用，显著抑制细胞因子风暴， 提示Fh15可能作为TLR4拮抗剂。此外，我们还发现FH15-处理增加了 大小腹膜MΦS(LPM和SPM)在腹膜腔内蓄积的比较 脓毒症小鼠[6]。此外，我们还通过邻近结扎试验(Pla)证明了本地肝片吸虫 FABP变异体(FH12)与人CD14辅受体[7]结合，该受体与激活信号有关 各种TLR的级联[8-11]。因为TLR4直接参与脓毒症时的炎症反应， 溃疡性结肠炎(UC)，Fh15的这种作用可能会对人类健康产生重大影响[12，13]。我们的中央 假说是注射诱导的促炎细胞因子的抑制(I.P.)使用FH15就足以 预防致死剂量内毒素暴露小鼠的高死亡率，并预防或改善肠道 DSS诱导的UC小鼠模型中的炎症反应。这两种治疗效果都将直接与 M2型M-ΦS在动物体内的产生及其对DC耐受性的诱导 严重依赖CD14。这项拟议的研究在概念上是创新的，因为这是第一次 肝片吸虫FABP家族的蛋白，特别是Fh15，作为一种抗炎分子被研究，在那里 关于M-Φ、S和DC与FH15在体内或体外暴露的研究尚未见报道。了解Fh15如何 在先天免疫的早期阶段调节并与M-Φ、S和DC相互作用可能有助于 抗脓毒症等炎症性疾病的抗炎药。具体目标是：1)确定 Fh15对感染性休克和溃疡性结肠炎(UC)小鼠的抑制作用 模型和，2)确定Fh15是否诱导DC的耐受特性，并确定其机制。 Fh15引起的潜在免疫抑制。拟议的研究将极大地促进反 基于蠕虫抗原衍生疗法的炎性药物开发 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.