Novel Immunomodulators from Hard Ticks Selectively Reprogramme Human Dendritic Cell Responses

Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission.硬蜱可通过破坏脊椎动物宿主的免疫应答，实现相较于其他吸血外寄生虫更长的取食时长；这或许是硬蜱能够传播节肢动物媒介中最多样化病原体的原因之一。蜱诱导的免疫调节由唾液成分介导，其中部分组分可中和固有免疫组分或抑制适应性免疫的发育。由于树突状细胞（dendritic cells，DC）能够触发并调控适应性免疫反应，因此其成为免疫调节的理想靶点。然而，此前已报道的蜱唾液免疫活性成分，要么在细胞与分子靶点上呈现高度混杂性，要么对DC的调控效果十分有限。本研究旨在解答以下科学问题：全球体量最大、临床意义最为重要的硬蜱类群——硬蜱后突类群（ixodid metastriates）是否能够分泌可特异性调控DC活性的唾液分子。我们通过色谱分离技术，从棕耳蜱（Rhipicephalus appendiculatus）体内分离得到一种唾液腺蛋白（Japanin）；随后完成了该蛋白的克隆，并利用杆状病毒表达系统制备了重组Japanin蛋白。实验结果显示，Japanin可在体外特异性重编程DC对多种刺激的应答反应，从根本上改变其共刺激与共抑制跨膜分子的表达水平（通过流式细胞术检测），以及促炎、抗炎及T细胞极化细胞因子的分泌量（通过Luminex多重检测分析）；此外，Japanin还可有效抑制单核细胞向DC的分化过程。序列比对与酶法去糖基化实验证实，Japanin是一种分子量为17.7 kDa的N-糖基化脂质运载蛋白。通过分子克隆与数据库检索技术，我们在棕耳蜱（Rhipicephalus appendiculatus）及其近缘物种中鉴定出一组同源蛋白，其中3种已完成重组表达，并被证实具备DC调控活性。本研究所有实验数据均源自至少4名健康献血者的外周血DC样本，且经过了严格的统计学分析。本研究结果揭示了一种此前未被发现的寄生虫介导的适应性免疫逃逸机制，该机制或同样可促进病原体的传播。