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

Lead Research Organisation: University of Liverpool
Department Name: Institute of Infection and Global Health

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description 1. We have determined that SPLUNC1 is expressed and secreted by non-ciliated cells in the respiratory tract. This secretion is modulated by infection with viruses but the degree of modulation varies depending upon the severity of infection
2.We have developed transgenic mouse models that are deficient in BPIFA1 and have combined this with in vitro 3D tracheal epithelial cultures (mTEC) to investigate its antiviral role. Here, we show for the first time that BPIFA1 plays a significant part in the mucosal defence against Influenza A virus (IAV) infection. BPIFA1 secretion was highly modulated after IAV infection and mice deficient in BPIFA1 supported a higher viral load and virus spread more rapidly to the deep lung, indicative of a defect in control of infection. Analyses using mTEC showed that entry of IAV RNPs and binding of virus particles was significantly increased in the absence of BPIFA1. Our results indicate a critical role for BPIFA1 in the initial phase of infection by inhibiting the binding and entry of IAV into mucosal epithelial cells.
3. Infection of BPIFA1-deficient mice showed that as early as day 1 post infection., there was a 1 log increase in IAV titre and a more rapid spread of virus. There was greater inflammation in BPIFA11 KO mice as compared with wild-type controls that was largely due to a 3-fold increase in neutrophils in the lungs and BAL. A significant decrease of the IAV-specific antibody response was seen in KO mice as was a decrease in IAV-responding, cytokine secreting CD8 T cells. RNAseq and high resolution label-free quantitative proteomics followed by pathway analysis reflected this, revealing a modulation in cell movement and humoral immune responses in the KO mice. Gene set enrichment analysis showed a significantly reduced enrichment in the expression of B and T lymphocyte surface markers in KO compared to wild-type mice, hence identifying molecular signatures associated with BPIFA1 action. Our data indicate that BPIFA1 is a crucial component of the mucosal host defence against Influenza A virus and provide further support for its function in influencing inflammation and adaptive immunity.
Exploitation Route The variability in expression and secretion of SPLUNC1 during infection could be exploited in terms of point-of-care diagnostics

The effects of BPIFA1 on the immune response suggest that over-expression or addition of BPIFA1 might enhance vaccine responses to IAV
Sectors Pharmaceuticals and Medical Biotechnology

 
Description BPIFA1: from anti-viral peptide to immunomodulator
Amount £480,932 (GBP)
Funding ID BB/R018863/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 10/2021
 
Description Digard 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Infection model of 3D tracheal epithelial cell cultures derived from wild type and BPIFA1 KO mice
Collaborator Contribution Reagents and expertise in terms of labelled IAV and highly concentrated preps of IAV
Impact Determination that BPIFA1 is involved in binding and entry of IAV into epithelial cells
Start Year 2015
 
Description SPLUNC1 Bingle 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution The Stewart group are experts in virus infection and animal models
Collaborator Contribution The Bingle group in Sheffield are experts in pulmonary biology and making in vitro cultures
Impact 1. Leeming, G.H., Kipar, A., Hughes, D.J., Bingle, L., Bennett, E., Moyo, N., Tripp, R.A., Bigley, A., Bingle, C.D., Sample, J.T., Stewart, J.P. Gammaherpesvirus infection modulates the temporal and spatial expression of SCGB1A1 (CCSP) and BPIFA1 (SPLUNC1) in the respiratory tract. Laboratory Investigation, In Press.
Start Year 2006
 
Description SPLUNC1 Tripp 
Organisation University of Georgia
Country United States 
Sector Academic/University 
PI Contribution The Stewart group are investigating the host response to virus infection in mouse models
Collaborator Contribution The Tripp group have supplied expertise and access to reagents such as influenza strains as well a performed infections in mice with highly pathogenic influenza strains.
Impact 1. Leeming, G.H., Kipar, A., Hughes, D.J., Bingle, L., Bennett, E., Moyo, N., Tripp, R.A., Bigley, A., Bingle, C.D., Sample, J.T., Stewart, J.P. Gammaherpesvirus infection modulates the temporal and spatial expression of SCGB1A1 (CCSP) and BPIFA1 (SPLUNC1) in the respiratory tract. Laboratory Investigation, In Press.
Start Year 2011
 
Description Presentations a Big Bang North West 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact One day participation from Dr N. Moyo plus considerable preparation time

Not aware of any directly to date
Year(s) Of Engagement Activity 2014
 
Description Television Interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview on BBC Breakfast (x2) in Oct 2017 discussing the influenza vaccination and the importance of having the vaccination for the 2017/18 season. Target audience was the general public but including carers and health professional. Feedback afterwards was positive and encouraged a greater uptake of vaccine in those that fed back.
Year(s) Of Engagement Activity 2017