Identification of interferon stimulated genes that control Toxoplasma in pig macrophages

刚地弓形虫是一种重要的食源性寄生虫，可引起牲畜和人类的疾病和死亡。弓形虫是牲畜流产的主要原因，据估计，每年使英国畜牧业损失超过1500万美元。弓形虫是导致艾滋病毒/艾滋病患者昏迷和早期死亡以及儿童失明的主要原因，据估计，弓形虫每年使美国经济损失超过80亿美元。猪是最常感染的牲畜品种之一，在繁殖母猪和育肥猪中的流行率分别超过30%和60%。猪感染可引起呼吸窘迫，死亡率超过57%。在子宫内感染的仔猪可能出生时死亡或在出生后三周内死亡。慢性感染还会增加猪对其他破坏性病原体的易感性，例如猪繁殖与呼吸综合征病毒（PRRSV），该病毒可杀死80%以上的感染猪。猪的慢性弓形虫感染也对通过受污染的猪肉感染人类弓形虫造成重大风险。超过40%的人类弓形虫感染暴发通常与受感染的猪肉有关。目前还没有针对猪弓形虫的药物或已批准的疫苗，因此，为制定控制猪弓形虫的新策略提供工具和数据是生物医学研究的一个重要优先事项。控制弓形虫感染需要干扰素（IFN）细胞因子，特别是干扰素γ (IFNg)。例如，缺乏干扰素刺激基因（ISGs）如诱导型一氧化氮合酶2 （iNOS2）的小鼠或IFNg受体缺陷的人巨噬细胞对弓形虫非常敏感。然而，我们对IFNg如何控制猪细胞中的弓形虫知之甚少。由于小鼠、人类和猪的免疫系统存在显著差异，我们不能总是推断一个物种对另一个物种的抗弓形虫反应。例如，与小鼠和人类不同，猪的单核细胞不会产生刺激T细胞产生IFNg的白细胞介素12。然而，就像在人类和老鼠身上一样，有证据表明猪细胞中的isg控制着弓形虫。例如，人类细胞中许多已知抑制弓形虫的isg的表达与猪细胞中的寄生虫负荷相关。然而，目前仍缺乏系统的研究来确定控制猪弓形虫的isg。在初步研究中，我们观察到重组IFNg可以抑制弓形虫，并且弓形虫在猪巨噬细胞中诱导了超过100种ISGs的差异表达。然而，由于IFN信号通路的冗余，以及具有潜在抗弓形虫特性的isg的数量，可能无法快速测试所有差异表达的isg的抗弓形虫特性。在罗斯林研究所，我们开发了高通量基因筛选系统，使我们能够在96孔板的细胞中过度表达单个isg。我们还将开发一种高通量系统，使我们能够敲除所有已知的猪isg。我们建议使用这些高通量筛选系统来快速识别哪些以及如何在猪巨噬细胞中表达差异的isg控制弓形虫，而这些细胞恰好也是寄生虫在自然感染期间喜欢居住的细胞。这项工作的成果将提高我们对干扰素如何控制猪弓形虫的认识，并可以在长期内加速开发减轻猪弓形虫疾病负担的工具。此外，我们将在本提案中开发的ISG敲除系统将成为研究其他影响养猪业的病原体的有用工具，包括PRRSV和猪瘟病毒，它们也由ifn控制。此外，由于弓形虫在猪和人类中的发病机制被认为具有高度可比性，本研究的结果可以为以猪为动物模型的人类弓形虫感染研究开辟新的领域。

Toxoplasma gondii is an important food-borne parasite that causes illness and death in both livestock and humans. Toxoplasma is a major cause of abortions in livestock and is estimated to cost the UK livestock industry over $15 million per year. Toxoplasma is the main cause of coma and early death in HIV/AIDS patients as well as childhood blindness, and is estimated to cost the USA economy over $8 billion per year. Pigs are among the most frequently infected livestock species with prevalence rates of over 30% and 60% in breeding sows and fattening pigs, respectively. Infection in pigs can cause respiratory distress and over 57% mortality. Piglets infected in utero may be born dead or die within three weeks of birth. Chronic infection can also increase the susceptibility of pigs to other devastating pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV) that can kill over 80% of infected pigs. Chronic Toxoplasma infection in pigs also poses significant risk for human Toxoplasma infections through contaminated pork. Over 40% of Toxoplasma infection outbreaks in humans are often linked to infected pork. No drugs or approved vaccines exists for Toxoplasma in pigs, making the generation of tools and data to prime the development of new strategies to control Toxoplasma in pigs an important priority for biomedical research.Interferon (IFN) cytokines, particularly interferon gamma (IFNg), are required to control Toxoplasma infections. For example, mice that lack interferon-stimulated genes (ISGs) such as inducible nitric oxide synthase 2 (iNOS2) or human macrophages with defects in the IFNg receptor are highly susceptible to Toxoplasma. Yet, we know very little on how IFNg controls Toxoplasma in pig cells. Due to significant differences between mice, human, and pig immune systems, we cannot always extrapolate anti-Toxoplasma responses in one species to another. For example, unlike in mice and humans, pig monocytes do not produce interleukin 12 that stimulates T cells to produce IFNg. Nevertheless, like in humans and mice, there is evidence that ISGs control Toxoplasma in pig cells. For example, the expression of many ISGs known to inhibit Toxoplasma in human cells correlate with parasite burden in pig cells. However, systematic studies to identify ISGs that control Toxoplasma in pigs are still lacking.In preliminary studies, we have observed that recombinant IFNg can inhibit Toxoplasma, and that Toxoplasma induces differential expression of over 100 ISGs, in pig macrophages. However, because of the redundancies in the IFN signalling pathway, and the number of ISGs with potential anti-Toxoplasma properties, it may not be feasible to rapidly test the anti-Toxoplasma properties of all differentially expressed ISGs. At the Roslin Institute, we have developed high throughput genetic screening systems that can allow us to over-express individual ISGs in cells in 96-well plates. We will also develop a high throughput system that will allow us to knockout all known pig ISGs. We are proposing to use these high throughput screening systems to rapidly identify which and how the differentially expressed ISGs control Toxoplasma in pig macrophages, which also happen to be the cells that the parasite prefers to live in during natural infections.The outputs from this work will advance our knowledge on how IFNs control Toxoplasma in pigs and can, in the long-term, accelerate the development of tools to reduce the disease burden of Toxoplasma in pigs. In addition, the ISG knockout system that we will develop in this proposal, will be a useful tool to study other pathogens that affect the pig industry, including PRRSV and swine fever virus that are also controlled by IFNs. Moreover, because Toxoplasma pathogenesis in pigs and humans are thought to be highly comparable, the results from this study can open new areas of research in human Toxoplasma infections using pigs as animal models.