MicroRNA coupling of the sterol metabolic network to the antiviral immune response

Virus infections remain a globally important cause of disease and death. If we can understand how individuals respond to an infection within hours after exposure to a pathogen it will help in the development of vaccines and therapies. Cholesterol has been extensively studied in the context of heart disease. Importantly, however, new evidence shows a connection between immune responses to infection and the regulation of cholesterol metabolism. Specifically, cholesterol can alter how we respond to a virus and, conversely, immune responses to infection have been shown to influence cholesterol transport, storage and removal from the body.

Hypocholesterolemia, abnormally low levels of cholesterol in the blood, often accompanies infectious diseases and has been suggested as a prognostic marker in hospitalized patients. Notably, the reasons for how and why cholesterol levels change in infection are not known. We have previously shown in tissue culture and animal models that suppressing the cholesterol synthesis pathway plays a role in defence against infection and this requires an immune hormone, interferon. Here we explore the mechanism for linking lower cholesterol levels and interferon.

Preliminary data suggests that this response is at least partly due to the action of a specific microRNA. MicroRNAs are a recently discovered family of small molecules produced by cells that act to regulate a wide-range of processes such as embryonic development or the development of disease. MicroRNAs function to decrease the abundance of molecules known as messenger RNAs that are used as templates for protein production in the cell.

In tissue culture models, we have shown that interferon induces the production of a microRNA in immune cells called macrophages. Macrophages are multi-functional white blood cells critical to the development and maintenance of immunity to a wide range of infections. Our microRNA of interest acts to reduce the abundance of a protein called SREBP2 - a master regulator of cholesterol synthesis. This leads to a decrease in the amount of cholesterol in the cell and, in doing so, creates a virus resistant state.

Here, we propose an investigation focused on characterising the mechanisms underlying these observations. In brief, we will characterise how interferon induces the specific microRNA in immune cells. We will then proceed to experiments focused on elucidating: the role of this miRNA during a viral infection in vivo, effects of the microRNA on metabolic and other cellular pathways and antiviral functions of the microRNA.
This will advance our understanding of responses to infection and will aid in the design and development of new therapeutic and diagnostic approaches in the treatment of infections.

Metabolic pathways, especially those responsible for lipid biosynthesis, are fundamental to cellular survival and are often co-opted by viruses for the enhancement of replication. Recently, we have shown that the regulation of the sterol metabolic pathway is an integral component of the interferon (IFN) antiviral response and, through a systems biology approach, have identified a previously uncharacterised microRNA that potently suppresses sterol biosynthesis and markedly inhibits viral multiplication. Here, we seek to advance our understanding of the interplay between sterol metabolism and the innate immune response against virus infection. We will test the hypothesis that IFNs stimulate the production of miRNAs that directly regulate the sterol metabolic network and, as a result, contribute to host protection against infection. We propose three specific aims. Aim 1 seeks to determine the mechanisms coupling IFN to miRNA regulation and sterol metabolism through a systematic chromatin immunoprecipitation analysis of transcription factors and PCR analysis of epigenetic modifications involved in expression of the miRNA in T or B lymphocytes or macrophages. Aim 2 will explore the in vivo regulation of the miRNA and sterol metabolism in an acute infection model. We will use murine Herpesvirus infection and analyse virus replication/spread alongside a comprehensive analysis of miRNA and sterol-pathway gene/protein expression and a systematic mass-spectrometry (MS) analysis of virus or miRNA mimic/inhibitor effects on sterol metabolites in plasma/tissues. Finally, Aim 3 will use selective metabolic rescue experiments, MS analyses of miRNA effects on sterol pathway intermediates in vitro and genome-wide HITS-CLIP experiments to identify specific targets of the miRNA host and virus and determine the causal link between miRNA inhibition of viral growth and sterol metabolism.

Coupling between metabolic pathways and the immune system is increasingly recognised as fundamentally important to health. This proposal focuses on understanding the functions of a microRNA and aims to show that an alteration of metabolism by the host inflammatory response can have a beneficial effect in combating viral infection. Work to-date suggests that the miRNA detailed in this proposal holds promise as an inhibitor of virus spread - a characteristic of importance to both public health and epidemiology. Results from this work will contribute to a relatively small body of existing knowledge describing links between lipid metabolism and immunity and, in the short-term, will benefit academic, governmental and commercial scientists. (1) Health and well-being: Infectious diseases have an enormous global impact and cholesterol regulation is critically important to diseases such as atherosclerosis. Our research will serve as a basis for new insights related to cholesterol regulation and lead to innovative "metabolic modifiers" with utility in the fields of infectious disease and inflammation. The time frame for the realisation is estimated to be around 8 to 10 years. The interest generated by this work and potential for commercialisation of new targets for the treatment of infectious or sterol-related disease will contribute to the UK's scientific reputation and productivity. (2) Training: The post-doctoral scientist will enhance their experience via the analysis and integration of new data (Mass Spectrometry and sequencing) and will use this project as a platform to develop their independent research career via publications, international presentations, collaborations and networking. (3) Communication and Engagement: The PI has a long track record in publishing at a high-level and regularly delivers talks and seminars in Europe and the USA. The science described in this proposal will attract industry interest in addition to its publication in high-impact journals and dissemination at international conferences. Together, this will lead to lead to quantifiable new academic/industrial collaborations and interactions within and outwith the UK. By working with the Edinburgh University press office, our publications on immune regulation of cholesterol have already attracted coverage from high-profile media organisations such as the BBC. We are experienced, therefore, in communicating the implications of our research to a diverse non-specialist audience. Since interest in cholesterol regulation is unlikely to diminish, our miRNA-sterol related publications will be accompanied by press releases to inform the wider public about our research. Currently, a DPM administrator logs all our interactions with the press and public. Links to available content are then made available on the DPM, SynthSys or University websites. The PI has extensive experience of scientific communication to a lay audience. For example, he has recently communicated the importance of our pathway-related work to a lay audience at the Edinburgh Science Festival. Our engagement with events and opportunities such as these will be maintained throughout the course of our funding. (4) Intellectual Property and Commercialisation: The PI has extensive experience of academic-commercial interactions. At the outset of this project, in collaboration with Edinburgh Research and Innovation, we will work to ensure mechanisms are in place for the exploitation of our findings in the UK through, for example, the acquisition of patents. Significantly, we also have close links to and are able to collaborate closely with the Edinburgh BioQuarter. The Technology Transfer team at Edinburgh BioQuarter provides an outstanding team of business development personnel and will facilitate business creation, commercial partnerships and collaborations. Should the opportunity arise, therefore, we are well placed to explore and develop commercial opportunities for our findings.