The Role of SPLUNC1/BPIFA1 in the Host Response to Respiratory Virus Infection

The PLUNC/BPIF family are rapidly evolving glycoprotein molecules that are produced continuously in the respiratory tract of mammals. They show structural similarity to BPI, a protein that is known to combat bacterial infections and to LBP, a molecule critically important in transducing signals from bacterial LPS through the TLR innate defence pathway. This led to the hypothesis that PLUNC/BPIF proteins would exhibit similar antimicrobial defence functions to BPI and LBP. However, the function of BPIF proteins is unknown and as yet there is no compelling published support for a significant antimicrobial role, and our own data has failed to show a direct antimicrobial/bacteriostatic role for the proteins, both in vivo and in vitro. In this grant we are focussing on SPLUNC1 or BPIFA1. BPIFA1 is the prototypic family member that is found in all mammals. It is one of the major secretory molecules is secreted into the respiratory tract. We have shown that BPIFA1 is produced in the cells lining the respiratory tract, and that after infection with viruses, its production is increased. Using genetically modified mice that are unable to produce BPIAF1, we have shown that it is involved in the defence against influenza A virus. We therefore believe that BPIAF1 is involved in the early response to virus infection. The aims of this project are to:
1. Discover the precise way that BPIFA1 acts in the defence mechanisms to MHV-68 infection. This will involve comparing the progress of viral infection in genetically modified mice deficient in BPIFA1 with normal mice. We will also study whether BPIFA1 influences inflammatory responses, various types of immune responses (antibody and cytotoxic T cell) as well as other anti-viral responses such as interferons.
2. Use fully differentiated cells that have been cultured in the laboratory from the respiratory tract of BPIFA1-deficient mice to look at how this protein can protect the respiratory tract from virus infection.
3. Discover the mechanisms of action of BPIFA1 by comparing differences in the expression of genes in mice and cells lacking BPIFA1 before and after infection.
The results will significantly enhance our understanding of fundamental aspects of defence to virus infection as well as aspects of respiratory biology.
The research will be carried out at the Universities of Liverpool and Sheffield by a multi-disciplinary team comprising members of the Medical and Veterinary Faculties using well-equipped facilities currently situated at these sites.

The PLUNC/BPIF family of glycoproteins are constitutively expressed in the respiratory tract of mammals. They show structural similarities to BPI and LBP, proteins critical to host defence against bacterial pathogens. However, the function of BPIF proteins is unknown. In this grant we are focussing on SPLUNC1 or BPIFA1. We have shown that BPIFA1 is produced in respiratory epithelial cells, and that after infection with Murine gamma-herpesvirus 68 (MHV-68) and Influenza A virus (IAV), its production is modulated. Using bpifa1-/- mice, we have also shown that it is involved in the defence against IAV. We therefore believe that BPIAF1 is involved in the host defence to virus infection in the respiratory tract. The aims of this project are:
1). To elucidate the temporal and spatial expression of BPIFA1 and its function in vivo.
2). To determine the functions of BPIFA1 in epithelial host defence in vitro.
3). To dissect the mechanisms involved in the regulation of SPLUNC1 expression and action.
We will use bpifa1-/- mice, an in vitro air-liquid interface culture system based on these mice and transcriptional profiling to achieve these aims. We will use two complimentary experimental pathogens. IAV, an RNA virus that produces an acute, lytic infection and MHV-68, a DNA virus that is less cytopathic and forms a persistent/latent infection. Both are relevant to disease in animals and have good sets of specific reagents that will allow the study of the host response in mice. The results will significantly enhance our understanding of fundamental aspects of host responses to virus infection in the lung as well as respiratory biology. In turn this may lead to novel approaches to improving antiviral therapies or therapeutic interventions against the consequences of viral infection.

Respiratory virus infections are of global significance to both the human and animal populations. The zoonotic potential of influenza A virus and the implications of emerging new strains are well recognised by both the scientific community and the general public. Improved understanding of the pathogenesis of infection and the host response to these pathogens is critical in improving treatment and management of respiratory disease and the associated morbidity and mortality. This work aims to further this understanding by looking at the role of SPLUNC1/BPIFA1 in the innate responses of the host respiratory tract following infection with influenza virus and gamma-herpesviruses.

The academic impact of this work therefore will be to further the knowledge of the host response to viral infection, providing the foundations of academic knowledge and understanding on which future advancements in treatment and disease control can be built. The use of the multi-disciplinary approach as proposed here brings together expertise in respiratory cell and molecular biology, molecular virology and veterinary pathology, thereby maximising the potential outputs of the research. This is also advantageous to the RAs in providing the experience and training involving multiple fields fostering a multi-disciplinary approach and its advantages for enhanced, productive science as well as allowing them to develop a range of transferable skills that will enhance their career development. This is achieved by collaboration between the Universities of Sheffield and Liverpool and is furthered by collaboration and knowledge exchange with staff of Investigative and Translational Pathology at AstraZeneca. This cooperation will enable optimal use of imaging techniques and image analysis, allowing staff and students at the Universities to benefit from AstraZeneca's expertise, strengthening existing links between the University of Liverpool and AstraZeneca and establishing a mutually beneficial relationship for future collaboration.

Cooperation between academic and industrial institutions also contributes to the economic impact of research by enhancing the research capabilities, knowledge exchange and skills of workers in both sectors. Exposure to both academic and industry in the course of this work will be of benefit to postgraduate students who will gain experience of both workplaces and the opportunities of career development in both sectors.

The societal impact of furthering the understanding of the host response to infection with Influenza virus on the health of the individual and the wider population is significant. Likewise, gamma-herpesviruses cause significant morbidity and mortality in humans and animals. Contribution to the improved treatment of individual high risk patients where infection with Influenza virus has a higher morbidity and mortality than the general population, or increased understanding of the risk factors associated with epidemic strains, both are important in the future of prevention and control of Influenza infection, in both veterinary species and the human population.