Identification of interferon stimulated genes that control Toxoplasma in pig macrophages

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.

Toxoplasma gondii, the principal cause of abortion in livestock, cost the UK livestock industry about $15 million per year. Acute infection in pigs can cause fever and respiratory distress, and over 50% mortality rates in infected pigs. Piglets infected in utero may be born premature, dead, weak, or die within three weeks of birth. Toxoplasma in pigs is also important for human health, who often get infected by consuming pork from chronically infected pigs. In humans, Toxoplasma is the main cause of childhood blindness and early death in HIV/AIDS patients. No drugs or approved vaccines exists for Toxoplasma in pigs, making the development of new anti-Toxoplasma tools for pigs an import biomedical priority.

Interferon gamma (IFNg) cytokine is required to control acute and chronic infections. However, we know relatively little about how IFNg control Toxoplasma in the pig. including which interferon-stimulated gene (ISGs) directly inhibit Toxoplasma in pig cells. In preliminary experiments, we have observed that Toxoplasma induce differential expression of over 1000 ISGs, and recombinant IFNg inhibits Toxoplasma, in pig macrophages. Guided by these and other preliminary data, this project will address the gap in our current knowledge of how ISGs control Toxoplasma in pig cells by systematically identifying which and how the differentially expressed ISGs directly control Toxoplasma in pig macrophages using pig pluripotent stem cell-derived macrophages and high throughput ISG functional screening tools that we have recently developed. The fundamental insights that we will obtain will provide a deeper understanding ISG-Toxoplasma interactions in pig macrophages, which in the long-term, may prime one health strategies reduce the disease burden and zoonotic potential of Toxoplasma in pigs. The tools and data from this work will also have broad relevance to other pig pathogens controlled by IFNs such as African swine fever.