Oral Barrier Immuno-Surviellance; alterations across the life-course

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

As we age, the systems of our bodies become less well able to function; rendering fit and active people increasingly infirm. It has been predicted that by 2050 there will be ~19 million people in the UK over 65 years of age. With enhanced numbers of elderly people, there becomes an increasing need to develop strategies to promote "healthy ageing", limiting the negative consequences of ageing.

One important system affected by ageing, is our immune system, influencing our ability to both fight infection and maintain tolerance. This is a particular problem at barrier sites, interfaces between the internal body and the external world (such as the gut and oral cavity). The immune system at these sites has to mediate a delicate balance; it must protect against pathogen entry and also be able to distinguish these bad invaders from commensal/friendly bacteria. This is a difficult challenge and to achieve this the immune system is carefully tailored to barrier sites creating highly specialized networks of immune cells that enforce this balance and ensure the integrity of barrier sites. Maintaining this balance becomes increasingly difficult with advancing age.

Although age-driven changes in immune function have been assessed in the gut, the influence of age on the immune network present at the oral barrier remains minimally explored. This is a significant oversight given that advancing age is accompanied by loss of oral barrier integrity and an increased incidence and severity of oral inflammation. Moreover, recent data has shown that oral inflammation is a risk factor for a plethora of disorders associated with unhealthy aging, including cardiovascular disease and diabetes. As such it becomes increasingly important to understand the age-associated changes occurring at the oral barrier.

The objectives of this grant are to determine the functional changes to oral barrier immune responses with age and to define the oral-specific factors that educate the oral barrier immune network across a life-time. By employing novel techniques that I have developed alongside cutting edge-technology, this work will transform our understanding of tissue-specific control of immune homeostasis at the oral barrier throughout a healthy life-time. In particular this work will assess the factors that promote immune dys-regulation at the oral barrier and undermine barrier homeostasis with age.

This research will be undertaken at the University of Manchester (UoM), in the Faculty of Life Sciences (FLS) and the new Manchester Collaborative Centre for Inflammation Research. Employing human samples and complimentary mouse models, this research programme will interrogate the core mechanisms underlying immune tailoring at the oral barrier. This work will be supported by a number of collaborators, both within the UK and globally, whose expertise will greatly enhance the efficiency with which this work is undertaken and whose resources will increase the scope of the work undertaken. I have established a clinical collaboration with Prof. J. Yates (at the UoM), who has helped me to develop clinical cohorts allowing me to obtain tissue samples from the oral barrier of healthy people of different ages. Complimentary to this, a key international collaborator is Prof. D. Bowdish (McMaster University, Canada), an expert in immunological ageing, who will provide me with access to aged-mouse cohorts. Additional collaborators in this research programme are experts in their fields Dr. Y Belkaid (NIAID, NIH, USA) and Prof. W. Muller (UoM, UK).

These innovative studies will elevate our fundamental understanding of the immune cell network promoting oral barrier integrity. Identifying mechanisms that change during ageing and the factors that promote these changes presents a window of therapeutic opportunity to reinforce the integrity of an aged oral barrier, limit oral inflammation and therefore safeguard systemic health, throughout the life-span of an organism.

Technical Summary

Ageing affects immune function and alters immune homeostasis at barrier sites. This is particularly important at the oral barrier, where loss of barrier integrity with age is associated with an increased incidence and severity of oral inflammation. Recent data has increasingly shown that oral inflammation is a risk factor for a plethora of systemic inflammatory conditions associated with unhealthy ageing. Despite this, the immuno-surveillance network policing the oral barrier and how this network evolves with age to preserve barrier integrity remains minimally understood.

This application aims to address this gap in knowledge by functionally understanding the unique immunological network present at the oral barrier. The studies outlined will:
(1) Functionally define the changes to the oral barrier immunological network with age. By employing both human samples and mouse models, I will determine, on a systems level, the age-dependent changes to the oral barrier immuno-surveillance network.
2) Delineate the impact of commensal microflora on the development of the oral barrier immunological network, specifically assessing the role of commensal-derived signals in establishing the oral barrier T cell network.
3) Define the mechanisms promoting age-dependent changes in the oral barrier immuno-surveillance network. By employing targeted and genome-wide approaches, I will outline the factors promoting age-associated changes at the oral barrier.

This research program will transform our understanding of tissue-specific control of immune homeostasis at the oral barrier throughout a healthy life-time. Understanding the mechanisms behind immune specialization at the oral barrier will outline possible therapeutic opportunities to intervene to support oral barrier homeostasis, and therefore safeguard systemic health, throughout the life-span of an organism.

Planned Impact

Academia; These are the primary beneficiaries of my research, which will be disseminated to this group by the publication of papers and reviews, by giving talks and by presenting posters. As with all good research, the data generated in this proposal will be assimilated by scientists and will then form a foundation upon which future work can be based. This research also builds upon collaborations with clinicians and therefore promotes cross-disciplinary interactions between scientists and clinicians. This will allow both groups to acquire new skill sets and approach their research in new ways. Myself, and my staff, will development new experimental skill-sets whilst undertaking this work, broadening our technical skills and scientific knowledge supporting us as we apply for additional grant funding and new positions within academia and/or industry.
Timescale: Interactions with these beneficiaries will occur throughout the fellowship.

General Public; The UoM engages in science out-reach programmes to disseminate research findings to the general public. This public engagement will be reinforced by the use of social media platforms such as Twitter and by posting news on my webpages. Through these mechanisms, both myself and lab members, will engage the general public. By achieving this, members of the general public will be able to: (1) find out about my research; (2) learn about science careers; (3) understand the principals of scientific research; (4) learn about the immune system; (5) gain an understanding of good oral health; (6) understand what healthy ageing is, and its importance in an ageing society.
Timescale: Interactions with these beneficiaries will occur throughout the fellowship. In addition, annual participation in UoM public outreach programmes will also occur.

Biomedical Industry; My findings will be of interest to the biomedical industry by providing a deeper understanding of how the immune system operates at the oral mucosal barrier. At the UoM I am an affiliate of the Manchester Collaborative Centre for Inflammation Research (MCCIR), a joint venture between the UoM, AstraZeneca and GlaxoSmithKline. I will meet yearly with representatives from industry, and myself and lab members will present at monthly MCCIR-industry seminars.
Timescale: Interactions with these beneficiaries will occur throughout the fellowship in the form of talks, presentations and industry site visits.

Dentists, Dental Organizations and Dental Charities; Performing the research laid out in this proposal will provide insight into mechanisms of immune homeostasis at the oral barrier. Dental practitioners, organisations and charities will benefit from this research by gaining a better understanding of the complex immunological network operating at this barrier. Collectively, they will be engaged through social media platforms and through publication of primary papers, by giving talks and by the targeted use of press releases.
Timescale: Interactions with these beneficiaries will be enhanced by my interactions with Prof. Yates bringing myself and my lab, into continued contact with dental practitioners. Interactions will this group at large will also be increased around the time that I publish papers.

Organisations promoting healthy aging; Ageing promotes loss of oral barrier integrity and increases in oral barrier inflammation. As inflammation at the oral barrier is a risk factor for a plethora of diseases associated with un-healthy ageing, my work will be of interest to those promoting healthy aging. My work will provide basic mechanistic insight into oral barrier homeostasis and outline possible therapeutic opportunities to intervene to reinforce oral barrier homeostasis, and therefore safeguard systemic health, throughout the life-span of an organism.
Timescale: Interactions with these beneficiaries will occur around the time that I publish papers.

Publications

10 25 50
 
Description One important system affected by ageing, is our immune system, influencing our ability to both fight infection and maintain tolerance. At barrier sites, interfaces between the internal body and the external world such as the gut and oral cavity, this is an increasingly important problem. The immune system at these sites has to mediate a delicate balance; it must protect against pathogen entry and also be able to distinguish these bad invaders from commensal/friendly bacteria. This is a difficult challenge and to achieve this the immune system is carefully tailored to barrier sites creating highly specialized networks of immune cells that enforce this balance and ensure the integrity of barrier sites. Maintaining this balance becomes increasingly difficult with advancing age.
Although age-driven changes in immune function have been assessed in the gut, the influence of age on the immune network present at the oral barrier remains minimally explored. This is a significant oversight given that advancing age is accompanied by loss of oral barrier integrity and an increased incidence and severity of oral inflammation.
We have recently identified a key immune population at the oral barrier which changes with age; Th17 cells. These cells increase with age and have been shown to undermine oral barrier integrity. Investigating this population of cells we have identified that novel factors, acting specifically at the oral barrier, modulate their development and function. We recently published these findings in a high impact journal, Immunity, demonstrating that barrier damage is a key cue educating T cell function at the oral barrier. Upon publication this paper received much attention both within the scientific world (being covered by two news and views pieces - one in Immunity, one in Nature Immunology) and also in the general press (being covered by tabloid newspapers and health magazines). Our data raise our fundamental understanding of immune functionality at the oral barrier, highlighting that different rules govern immune responses at this barrier compared to other barrier sites, as well as presenting a therapeutic window to modulate these immune mediators during ageing.
More recently we have been exploring a role for oral gamma delta T cells in oral barrier immune homeostasis during ageing. Through use of gnotobiotic animals, RNA-sequencing technology and developmental studies, we have defined a novel network of gamma delta T cells which are vital for maintaining immune homeostasis at the oral barrier. In the absence of this network, homeostasis is rapidly lost during ageing and immune dysfunction results. We have identified that this unique population of gamma delta T cells produce the reparative cytokine amphiregulin at steady-state. We have shown that production of this cytokine is vital to maintain oral barrier health. Identificatopm this health associated factor has subsequently allowed us to demonstrate that administration of amphiregulin during inflammation was able to restore oral barrier homeostasis and limit oral inflammation. This work is therefore the first to demonstrate a key role for oral gamma delta T cells in safeguarding oral barrier health and identify a key factor they produce which limits oral inflammation.

In addition to this work on the oral barrier we have contribute to the work of others looking at the development of immune cells across the life-course at another barrier site, the gastrointestinal (GI) tract. Supporting the work of the Dr John Grainger we have also demonstrated that a novel long-lived macrophage population, is present in the GI tract.
Exploitation Route Identifying mechanisms that change during ageing and the factors that promote these changes, presents a window of therapeutic opportunity to reinforce the integrity of an aged oral barrier. This would guard against oral inflammation and also promote healthy ageing. On a basic level, our data provide fundamental insight in immune functionality at the oral barrier - a much understudied barrier site.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description 3 year project Grant: Population Health
Amount £678,200 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2018 
End 10/2021
 
Description A novel monocyte network in the gingiva
Amount £250,000 (GBP)
Organisation Lister Institute of Preventive Medicine 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2019 
End 10/2024
 
Description ARUK PhD Studentship
Amount £108,400 (GBP)
Organisation Versus Arthritis 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 10/2021
 
Description CRUK Immunology Innovation
Amount £51,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 02/2019
 
Description ISSF University of Manchester Pump-Prime Award
Amount £23,616 (GBP)
Organisation University of Manchester 
Sector Academic/University
Country United Kingdom
Start 03/2016 
End 03/2017
 
Description Industrial Funding
Amount £297,115 (GBP)
Organisation Colgate-Palmolive Company 
Sector Private
Country United States
Start 12/2016 
End 12/2018
 
Description Infrastructure Grant
Amount £436,438 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2018 
End 11/2019
 
Title Biological samples - Characterising the oral microbiome of the mouse 
Description We have previously reported on the oral microbiome of the mouse and were subsequently invited to submit a detailed protocol on how we generate such data to the journal of Bio-Protocols (Oral Microbiome Characterization in Murine Models (2018). Abusleme L, Hong BY, Hoare A, Konkel JE, Diaz PI, Moutsopoulos NM). 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact None as yet 
 
Title Isolation of Immune cells from murine gingiva 
Description I have developed a method to examine immune populations resident in the oral barrier. Subsequently to this I was invited by JOVE to submit a methods article to their journal detailing this protocol. Please see publication list for this paper. 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact 1) Paper recently published in Mucosal Immunology. (2016) 2) Paper published in JOVE. (2016) 
URL http://www.jove.com/video/53736/isolation-characterization-functional-examination-gingival-immune
 
Description Amphiregulin Control of Immune Functions at the oral Barrier 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution My group has been exploring how gamma delta T cells contribute to immune control at the oral barrier during ageing. Our RNA-Seq data highlighted a potential role for the cytokine amphiregulin. As such we have subsequently set up a collaboration with an expert on amphiregulin; Prof Dietmar Zaiss at the University of Edinburgh.
Collaborator Contribution Prof Zaiss has provided us with access to transgenic animals that will allow us to explore (a) the role of amphiregulin at the oral barrier during ageing and (b) the cellular targets of amphiregulin at this novel immune barrier.
Impact (1) Paper submitted to PNAS: Amphiregulin producing ?d T cells are vital for safeguarding oral barrier immune homeostasis Siddharth Krishnan, Ian E. Prise, Kelly Wemyss, Louis P. Schenck, Hayley Bridgeman, Tamsin Zangerle-Murray, Conor O'Boyle, Thomas Barbera, Faiza Mahmood, Dawn. M. E. Bowdish, Dietmar Zaiss, John R. Grainger, Joanne E. Konkel
Start Year 2017
 
Description Examination of Human Oral Barrier Immune cells 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution We have collaboratively developed techniques to be able to examine oral barrier immune populations, leading to publication of a paper examining this in human health.
Collaborator Contribution Through this collaboration we have access to samples from an approved NIH clinical protocol in which gingival and buccal samples are obtained from healthy human patients as well as patients with monogenic diseases. These samples supplement those already available to us in Manchester as well as allowing for some examination of gingiva from patients with specific genetic mutations.
Impact 1) Paper published in Mucosal Immunology 2016. 2) Paper published in JOVE 2016. 3) Abstract submitted to BSI, December 2014. 4) Abstract submitted to BODS, November 2014.
Start Year 2015
 
Description Immunological ageing 
Organisation McMaster University
Country Canada 
Sector Academic/University 
PI Contribution Hosted in my laboratory a student from Prof D. Bowdish's lab. Patrick Schenk was awarded a travel fellowship to visit my lab and learn immunological techniques to aid him in his assessment of oral immune parameters.
Collaborator Contribution Prof. D. Bowdish provides me with access to aged cohorts of mice, including SPF and GF mice. Now that Patrick Schenk is trained is able to undertake some experiments for me in the Bowdish lab with these aged mice.
Impact Output: On-going Mechanical Damage from Mastication Drives Homeostatic Th17 Cell Responses at the Oral Barrier. Dutzan N, Abusleme L, Bridgeman H, Greenwell-Wild T, Zangerle-Murray T, Fife ME, Bouladoux N, Linley H, Brenchley L, Wemyss K, Calderon G, Hong BY, Break TJ, Bowdish DM, Lionakis MS, Jones SA, Trinchieri G, Diaz PI, Belkaid Y, Konkel JE, Moutsopoulos NM. Immunity. 2017 Jan 17;46(1):133-147. doi: 10.1016/j.immuni.2016.12.010. Multidisaplinary: Immunology, microbiology, bioinformatics.
Start Year 2016
 
Description Oral Health Parameters in Human Cohorts 
Organisation University of Manchester
Department Faculty of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I am a Co-PI on this grant examining samples for a Human Volunteer Study to Elucidate Beneficial Effects of oral hygiene routines. 15 patients have been returned with 6 examination time-points. My group will examine oral rinses from each patient by flow cytometry, defining and characterising the cellular components.
Collaborator Contribution The grant with an industrial partner was awarded to Prof A. McBain, who pioneered and secured the grant with the industrial partner.
Impact Mulitdisaplinary; immunology, microbiology, bioinformatics.
Start Year 2016
 
Description National/International press coverage of published paper 
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 We released a press release to co-incide with the publication of our paper in Immunity (Dutzan et al). The press release was picked up by a number of news outlets that led to articles being published in: the Daily Mail, the Mirror, the Sun, Mens Health, the Daily Express. As well as a number of online news outlets. The URL given below is an example of one of the news pieces.
Year(s) Of Engagement Activity 2017
URL http://www.express.co.uk/life-style/health/756271/Chewing-food-fights-illness-boosting-mouth-immune-...
 
Description Science Spectacular Manchester Museum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The MCCIR ran a stall at the Manchester Museum Science Spectacular Day. I operated the "Good and Bad Bacteria" stand aiming to introduce bacteria to middle school aged children. This was a very interactive day with games and a "make your own microbe" station, allowing the children to ask many questions.
Year(s) Of Engagement Activity 2015
URL http://www.engagement.manchester.ac.uk/highlights/manchester_science_festival/science_spectacular/