Identification of interferon stimulated genes that control Toxoplasma in pig macrophages

猪巨噬细胞中控制弓形虫的干扰素刺激基因的鉴定

• 批准号: BB/W014807/1
• 负责人: Musa Hassan
• 金额: $ 60万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW014807%2F1
• 关键词: Identification; interferon; stimulated; genes; control

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.

弓形虫是一种重要的食源性寄生虫，可引起家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，英国畜牧业每年的损失超过1500万美元。弓形虫是HIV/AIDS患者昏迷和早期死亡以及儿童失明的主要原因，据估计，每年给美国经济造成超过80亿美元的损失。猪是最常见的感染家畜之一，繁殖母猪和育肥猪的感染率分别超过30%和60%。猪的感染可导致呼吸窘迫和超过57%的死亡率。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。慢性感染还可以增加猪对其他破坏性病原体的易感性，例如猪生殖与呼吸综合征病毒（PRRSV），它可以杀死80%以上的受感染猪。猪的慢性弓形虫感染也通过受污染的猪肉对人类弓形虫感染构成重大风险。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批准的疫苗，因此，生物医学研究的一个重要优先事项是生成工具和数据，以推动新策略的发展，以控制猪弓形虫感染。干扰素（IFN）细胞因子，特别是干扰素γ（IFNg），需要控制弓形虫感染。例如，缺乏干扰素刺激基因（ISG）如诱导型一氧化氮合酶2（iNOS 2）的小鼠或IFNg受体缺陷的人巨噬细胞对弓形虫高度易感。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、人和猪的免疫系统之间存在显著差异，我们不能总是将一个物种的抗弓形虫反应外推到另一个物种。例如，与小鼠和人类不同，猪单核细胞不产生刺激T细胞产生IFNg的白细胞介素12。然而，与人类和小鼠一样，有证据表明ISG控制猪细胞中的弓形虫。例如，已知在人细胞中抑制弓形虫的许多ISG的表达与猪细胞中的寄生虫负荷相关。然而，目前尚缺乏系统的研究来鉴定ISGs对猪弓形虫的控制作用。在初步研究中，我们观察到重组IFNg可以抑制弓形虫，并且弓形虫诱导猪巨噬细胞中超过100个ISGs的差异表达。然而，由于IFN信号通路中的冗余，以及具有潜在抗弓形虫特性的ISG的数量，快速测试所有差异表达的ISG的抗弓形虫特性可能是不可行的。在罗斯林研究所，我们开发了高通量遗传筛选系统，可以让我们在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鉴定哪些差异表达的ISGs以及如何控制猪巨噬细胞中的弓形虫，这些巨噬细胞也恰好是寄生虫在自然感染期间喜欢居住的细胞。这项工作的结果将推进我们关于IFN如何控制猪弓形虫的知识，并且从长远来看，加快开发工具，以减少猪弓形虫的疾病负担。此外，我们将在本提案中开发的ISG敲除系统将成为研究影响养猪业的其他病原体的有用工具，包括也由IFN控制的PRRSV和猪瘟病毒。此外，由于猪和人的弓形虫发病机制被认为是高度可比的，这项研究的结果可以打开新的研究领域，在人类弓形虫感染使用猪作为动物模型。