Elucidating the molecular mechanism of intracellular DNA recognition by the innate immune sensor IFI16

In this project, we would like to investigate how the cells in our body can protect themselves by detecting 'stranger' and a 'danger' signals. As part of the innate immune system, most cells in our body notice when they have been infected by viruses or other 'strangers' by recognising tell-tale signs of infection, such as the presence of viral DNA. Once a virus has been detected, the cells respond immediately by releasing alarm signals, such as interferons. These then help to combat the virus locally and call specialised immune cells to the site of infection.
Over the last decade an intensive research effort has lead to the discovery of several proteins that can sense signs of infection, called pathogen recognition receptors. We have recently identified a new receptor, IFI16, that can sense viral DNA inside cells, and protects the cell from infection with herpesviruses. In this project, we would like to investigate in more detail how this DNA receptor works, and how cells recognise different kinds of DNA.

We will address three major questions:
1.) Which other proteins co-operate with IFI16 in the recognition of viral DNA?
2.) How can a cell distinguish between viral DNA and the cell's own DNA genome?
3.) How is DNA sensed as a 'danger' as well as a 'stranger' signal?

We will try and answer these questions by using isolated human cells grown in vitro. These cells can be infected with DNA viruses, and then the alarm responses can be measured. We will also use large-scale experimental approaches to find new players that help in the recognition of DNA. For instance, we are planning to use a high-throughput robotic platform to screen more than 20,000 genes of the human genome for their involvement in DNA sensing.
While this is a basic research project not aimed at immediately providing new cures, it may nevertheless provide crucial knowledge that can help in combating disease in the future. For instance, knowing how the body recognises viral DNA may be important for the development of new vaccines. Also, the recognition of DNA as a 'danger' signal has important clinical implications. The body's own DNA can activate the innate immune system under some circumstances, for instance when broken up DNA from dead cells is not cleared effectively. This can then lead to the development of autoimmune diseases, where an excessive immune response attacks the body's own tissues. Knowing which pattern recognition receptors cause this response would provide potential targets for treatment, so that this unwanted immune response can be dampened. Another instance where DNA is recognised as a 'danger' signal is when DNA has been damaged by ultraviolet light or by toxic chemicals. In this case, the innate immune system may be alerted to the danger posed by these damaged cells, and eliminate them, in order to prevent the development of cancer. Studying how DNA is sensed in these various circumstances would provide crucial insights for the development of treatments that modulate the innate immune response.

The overall aim of this project is to investigate the molecular mechanisms that govern the innate immune response to intracellular DNA. During infection by DNA viruses, exogenous DNA can be sensed as a pathogen-associated molecular pattern (PAMP) by intracellular pattern recognition receptors (PRRs), which then promote the production of interferon-beta. This in turn acts to establish an antiviral state in the infected cell and its neighbours, and to recruit immune cells to the site of infection. We recently identified a novel intracellular receptor for viral DNA, IFI16, which is involved in the sensing of DNA viruses such as Herpes Simplex Virus 1. In this project, we will examine the function of IFI16 in human cells in more detail, and address some fundamental questions regarding the molecular mechanisms of DNA recognition.

The specific objectives of this study are:

1.) Which cellular factors contribute to the sensing of DNA by IFI16?
We will employ two large-scale unbiased approaches (a genome-wide siRNA screen and a proteomics approach to find IFI16 interaction partners) in order to identify novel factors that play a role in the recognition of viral DNA.

2.) How does the cell distinguish between viral DNA and its own DNA genome?
We will test the hypotheses that exogenous DNA is either sensed by virtue of its cytosolic localisation, or by the recognition of particular DNA features as signs of viral replication.

3.) Is IFI16 invovled in the sensing of DNA as danger-associated molecular pattern (DAMP)?
The innate immune system can also be activated by the body's own DNA, e.g. during DNA damage or when DNA from apoptotic cells is insufficiently cleared. We will determine whether IFI16 can sense DNA as a DAMP, and explore the mechanistic links between DNA damage, viral infection and innate immunity.
This work will provide fundamental insights into the innate immune response to DNA, with relevance to viral infection, vaccination and autoimmunity.

The impact of this project on society at large will be two-fold: It will contribute to the training of a new generation of scientists, and will generate knowledge about the workings of the immune system, with implications for viral infection, vaccination, tumourigenesis and autoimmunity.

1.) Training and educating the next generation of scientists

This award would allow me to establish my own laboratory and take up a Principal Investigator position at the College of Life Sciences in the University of Dundee. As such, training the next generation of scientists in research skills would be a key part of my role. I will mentor a postdoctoral research assistant funded by this award, supervise undergraduate and PhD students, and participate in undergraduate lecture courses. I will also participate in outreach activities involving secondary school students, to give them an opportunity to experience life in a research laboratory first hand. An important aspect of the training and mentoring process would also be my position as a role model, as a successful scientist and mother of two young children. This award would provide key support at this crucial stage of transition to independence, and thus demonstrate to other early career scientists that it is possible to fully participate in academic research while raising a family. The retention of female scientists in leadership positions is of crucial importance, as the prevailing 'leaky pipeline' presents a major loss of talent in the fields of science, engineering and technology.

2.) Future impact of studying the molecular mechanisms of DNA sensing in innate immunity

While this is a basic research project investigating fundamental molecular mechanisms, it has important implications for a range of medical conditions, above and beyond the innate immune response to viruses. Translating the molecular findings into health benefits will require effective communication and collaboration with immunologists from other fields and with clinicians. A detailed understanding of the molecular mechanisms of DNA sensing may have an impact on the development of DNA vaccines, as this would propose which DNA ligands are optimal for stimulating the innate immune system as adjuvant. DNA sensing by the innate immune system also underpins the development of autoimmune conditions such as systemic lupus erythematosus (SLE), a disease characterised by the presence of anti-DNA antibodies and high levels of interferon. Identifying key players in the recognition uncleared DNA from apoptotic cells, which is thought to be an underlying cause of this disease, may give rise to the identification of drug targets in the future. The Drug Development Unit at the University of Dundee has outstanding facilities for compound screening in an academic setting and would be perfectly placed for the discovery of drug candidates, should some of the factors discovered in this project turn out to be of clinical significance.
DNA that has been damaged, e.g. by ultraviolet light or genotoxic agents, can also be sensed as a 'danger' signal by the innate immune system. As IFI16 has previously been implicated in the DNA damage response, we will test whether it is involved in the DNA damage-induced innate immune response. In this context, DNA sensing may be of importance for the early detection and removal of pre-cancerous cells by the immune system.
This is an important and timely project that addresses one of the MRC's major strategic aims in the research area of "Resilience, repair and replacement", as it investigates the molecular basis of our resistance to viral infection, as well as to chemical or physical damage.