Function of CXCR2 signalling within the articular cartilage

Lead Research Organisation: Queen Mary, University of London
Department Name: William Harvey Research Institute

Abstract

Cartilage is the tissue that covers the ends of our long bones, allowing the frictionless motion of our joints. This tissue is lost (degraded) during osteoarthritis, leading to joint pain and incapacity to walk or perform simple tasks. Osteoarthritis is the leading cause for disability allowance in the UK; it affects greater than 1 in 3 of the population over the age of 50 and therefore is a major factor affecting well-being, particularly in the elderly.

To-date, we do not have any treatment that stops progression of osteoarthritis and the only remedies are pain killers and joint replacement. Although generally effective, joint replacement does not live up to patients' expectations in ~20% of cases, and has a limited life span often requiring complex and challenging revision surgery (with a poor success rate). These serious limitations of joint replacement are becoming more prominent due to the fact that people live much longer and because more people are developing osteoarthritis at a younger age (e.g. due to obesity, or following sports injuries).

Following injury, cartilage responds to maintain its integrity, however, cartilage breakdown progresses when this response is insufficient. Excitingly, recent data have shown that supporting these intrinsic protective responses can be very successful in animal models, preventing cartilage breakdown and even regenerating damaged tissue.

We have discovered that molecules called ELR+CXC chemokines are involved in these protective responses. Previously known for attracting immune cells to sites of inflammation, we have discovered that, when present within healthy cartilage, these molecules have a totally different role supporting cartilage integrity following injury. As a consequence, mice that have been engineered to lack this protective response are more susceptible to experimental osteoarthritis. We discovered that ELR+CXC chemokines are retained in the cartilage tissue because they are bound to complex sugar molecules called HSPGs. Therefore, ELR+CXC chemokines present in cartilage cannot attract inflammatory cells and, instead, help cartilage to stay healthy and recover from minor injury. When, during osteoarthritis, cartilage becomes badly damaged, ELR+CXC chemokines are lost from the tissue so that they can no longer protect cartilage and they may also contribute directly to inflammation.

In this study we intend to answer the following questions:
1) How exactly do these chemokines protect cartilage during osteoarthritis? To do this we will make a mouse model that will enable us to separate the effects of chemokines on cartilage from those on other aspects of joint function and inflammation. Importantly, it will also provide information on which point during the development of osteoarthritis these molecules function, which is essential if we are to harness this protective activity for therapeutic applications.

2) What is the most potent ELR+CXC chemokine to support cartilage health? There are different ELR+CXC chemokines and there is data to suggest that some may be better than others at supporting cartilage integrity. Identifying the best chemokine is very important for therapeutic applications.

3) Can we modify chemokines so that they become unable of attracting inflammatory cells and yet retain their capacity to protect cartilage? We will achieve this by modifying the way they bind to HSPGs so that they cannot adhere to blood vessels where they would recruit inflammatory cells.

4) We will build a biomaterial containing and retaining ELR+CXC chemokines to test the potential of tissue engineering to repair/protect cartilage without causing inflammation.

This project develops from our discovery of this unexpected function of chemokines and identifies a way we can protect/repair cartilage in osteoarthritis: a leading cause of disability for which we still do not have a cure.

Technical Summary

Osteoarthritis (OA) is characterized by cartilage breakdown and is a leading cause of disability for which we do not have a cure. We discovered that ELR+CXC chemokines retained within cartilage through binding to heparan sulphate proteoglycans (HSPGs) have an unsuspected homeostatic function. Disruption of their receptor CXCR2 predisposed mice to experimental OA with chondrocyte dedifferentiation and apoptosis; similar data were obtained in human chondrocytes by blocking both CXCR1 and 2. CXCL6 supported human chondrocyte differentiation in vitro and, during embryogenesis, was expressed in the future permanent articular cartilage. The scope of this application is to understand the mechanism by which ELR+CXC chemokine signalling protects cartilage in osteoarthritis without causing inflammation, with the final goal of developing a therapeutic application.

Aims:

1) To isolate the function of CXCR2 within adult cartilage from developmental or inflammation phenotypes we will induce OA in a cartilage-specific, tamoxifen-inducible CXCR2 knockout mouse.

2) We will compare the cartilage homeostatic potency of different CXCR1/2 ligands.

3) We will study the biochemistry and function of ELR+CXC-HSPGs interactions in the context of cartilage homeostasis by HS digestion, mutagenesis of the HSPG binding residues, and by binding assays. We will identify the chemokine-HSPGs binding partners by co-immunoprecipitation followed by western blotting of candidate HSPG or mass spectrometry.

4) We will test if we can inhibit the capacity of ELR+CXC chemokines to cause inflammation without altering their homeostatic properties by disrupting HSPG binding (necessary for the formation of haptotactic gradients and neutrophil extravasation) and incorporating these mutant molecules in a bioscaffold. The efficacy of this strategy will be tested in an in vivo cartilage formation assay involving the adoptive transplantation of human chondrocytes and neutrophils in nude mice.

Planned Impact

Greater knowledge of the homeostatic (and intrinsic protective) mechanisms in adult cartilage will likely facilitate the development of new therapeutics for osteoarthritis with the potential for huge socio-economic impact (i.e. with benefit for the UK pharmaceutical industry, OA sufferers and their carers). One in 3 people over the age of 45 suffer from this debilitating condition with increased incidence in older individuals (e.g. 42% of men and 49% of woman age 75 and over).

The costs to society are estimated over $185 billion yearly in the USA only for medical care (Kotzlar & Rizzo, Arthritis Rheum 2007). In the UK OA caused the loss of 36 million working days in 1999-2000 costing the economy nearly 3.2 billion in lost production (NICE 2012 OA guidance draft).

Therefore, the beneficiaries will include:

1) Pharmaceutical industry active in the field of osteoarthritis and cartilage regeneration for developing on one hand selective chemokine blockade in inflammatory diseases, or, on the other hand, harnessing such homeostatic role to achieve chondroprotection in OA.

2) Biotech companies involved in musculoskeletal tissue engineering. The pathway will be similar to that in point one, but in vitro treatment of cellular products could be made readily possible and available for clinical practice and human trials.

3) The medical and orthopaedic community, and clinical investigators involved in osteoarthritis and cartilage regeneration.

4) Patients with osteoarthritis and cartilage defects.

5) Society at large for the spectacular direct and indirect costs related to osteoarthritis, in terms of medical costs, disability and loss of productivity.

6) veterinary surgeons involved in cartilage repair in race horses.

Publications

10 25 50
 
Description classification criteria for early osteoarthritis of the knee
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Title Adoptive model of cartilage formation in nude mice 
Description We previously used an ectopic cartilage formation assay in which human chondrocytes form human cartilage when implanted intramuscularly in nude mice. This model was great to test molecules for their effects on HUMAN cartilage in vivo, but had several limitations including large variability, low throughput, inability to use stored chondrocytes or mesenchymal stem cells. Sara Caxaria, employed in this grant, has optimized a new model in which cells are implanted in a collagen scaffold under the skin of nude mice. This has dramatically decreased the costs, improved the reproducibility and now enables to study the capacity of molecules to induce cartilage formation in stem cells. The sensitivity of the assay is now much improved. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact At the moment this method is being used in our laboratory in 3 different projects one of which funded by AR-UK, another by AR-UK and MRC and another one by a charity internal to Barts Hospital. It will be published, in due course, in the context of a paper focused on the role of GCP2 in cartilage biology 
 
Title Histomorphometric assessment of osteoarthritis in mice 
Description We have identified a more sensitive method to objectively quantify osteoarthritis in mice. This histomorphometrical method is described in Nalesso, G., Thomas, B. L., Sherwood, J. C., Yu, J., Addimanda, O., Eldridge, S. E., Dell'Accio, F. (2016). WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis. Annals of the Rheumatic Diseases. https://doi.org/10.1136/annrheumdis-2015-208577. The increased sensitivity and consistency significantly increases the power compared to traditional scoring system thereby reducing the use of experimental animals. The increased sensitivity and consistency significantly increases the power compared to traditional scoring system thereby reducing the use of experimental animals. In addition, compared to other methods, which are subjective in nature, this method is objective and more reliable. 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2016 
Provided To Others? Yes  
Impact The increased sensitivity and consistency significantly increases the power compared to traditional scoring system thereby reducing the use of experimental animals. In addition, compared to other methods, which are subjective in nature, this method is objective and more reliable. 
 
Title Model of osteochondral defect regeneration 
Description Several years ago we optimised a model of osteochondral healing in mice. While this model has been useful to study spontaneous healing of cartilage and bone, it was unsuitable to test how exogenous molecules alter this process because the defect was so small that nothing could be injected. We have now generated a new model where a much larger and much better controlled defect is generated in the femoral condyle of immunocompetent mice and such defects can be filled with a gel containing either cells or bioactive molecules. This new model is much more sensitive to change than the previous one and has enabled to study in vivo the function of GCP2 in cartilage. This model is also being used in our laboratory to study the function of other molecules in cartilage repair. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? Yes  
Impact This model enables investigators to study the effect of exogenous bioactive molecules on cartilage and bone repair. 
 
Description Generation of mutant GCP2 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution I wrote the project
Collaborator Contribution This partnership provided expertise to develop a mutant of GCP2 that does not bind to heparansulphate proteoglycans
Impact Manuscript in preparation
Start Year 2016
 
Description Jo Sherwood 
Organisation University of M√ľnster
Country Germany 
Sector Academic/University 
PI Contribution Transgenic mice
Collaborator Contribution To avoid rederiving transgenic mice, they are been bread there and crossed as required.
Impact The mice are now ready for experimentation. This will happen in December 2017
Start Year 2015
 
Description Katie Chambers, Frances Balkwill, Suzanne Eldridge - Centre for Public Engagement Large Award, "Muscling in! 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Suzanne Eldridge, Employed by me on the UKRMP grant, was awarded a Large Public Engagement grant award from QMUL Centre for Public Engagement (PI F. Balkwill, S. Eldridge and K. Chambers co-I) in 2015 (http://www.qmul.ac.uk/publicengagement/activities/items/164606.html and http://www.qmul.ac.uk/lifesciences/news-events/news/2015/164893.html). In partnership with the Centre of Cell Suzanne worked with approximately 250 children across four schools in an area of social deprivation (Tower Hamlets) to create and deliver a new school science show, 'Muscling in!' which communicates the physiology of the musculoskeletal system of the human body and engaged pupils with her research on osteoarthritis (key stage 2&3). The show now runs as a permanent educational show at the Centre of the Cell and receives bookings from schools nationally and internationally. Suzanne Eldridge was recently shortlisted as one of three finalists for the QMUL 'Interact Award - Engagement and Enterprise Awards 2017' (video summary can be found at 2.15min https://www.youtube.com/watch?v=lmOdYS5PV3k).
Year(s) Of Engagement Activity 2015,2016
URL http://www.qmul.ac.uk/publicengagement/activities/items/164606.html
 
Description Participation as scientist to the "Research into Arthritis Summer BBQ" 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Patients, carers and/or patient groups
Results and Impact Scientists at the William Harvey Research Institute describe their science to the general public (patients, children, general public). Subsequently the scientists took them through a tour in the labs and answered questions while having a barbecue.
Year(s) Of Engagement Activity 2017
 
Description Research showcase - Centre of Excellence in Osteoarthritis Pathogenesis Oxford 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Patients, carers and/or patient groups
Results and Impact This meeting was organized within the Versus Arthritis-funded Centre of Excellence in Osteoarthritis Pathogenesis Oxford of which we are members.
It was primarily aimed at showcasing to patient representatives new projects being designed within the Centre and obtaining feedback. Such feedback is then utilised to improve the applications. Therefore, while the primary objective was to obtain feedback on grant applications, the event also showcased new research to the public.
Year(s) Of Engagement Activity 2018