Rheumatology and Metabolic Medicine

Rheumatology and Metabolic Medicine - Research

Members of the Research Team:

Dr Oksana Kehoe, Dr Alasdair Kay, Mr Andrei Stefan and Miss Rebecca Morgan

Professor Jim Middleton (Honorary Clinical Scientist)

Clinical Support:

Dr Roshan Amarasena (Lead), Dr Mark Garton and Dr Ayman Askari

Our team has continued to be active this year carrying out basic science work into understanding mechanisms of rheumatoid arthritis (RA) progression and possible treatments.

We have been publishing our findings in Open Journal of Rheumatology and Autoimmune Disease, European Cells and Materials, Rheumatology.  We published a book chapter in Bone and Cartilage Regeneration volume of the Stem Cells in Clinical Applications series, by Springer Science. 

We have presented our work at several national and international meetings such as Tissue and Cell Engineering Society, London, July 2016; FIRM, Girona (Spain), September 2016; UK Mesenchymal Stem Cell meeting, York, December 2016;  Mercia Stem Cell Alliance, Manchester, December 2016; British Society for Rheumatology conference, Birmingham, April 2017 and 18th Annual Research day at the RJAH.

 

Members of the Research Team:

Dr Oksana Kehoe, Dr Alasdair Kay, Mr Andrei Stefan and Miss Rebecca Morgan

Professor Jim Middleton (Honorary Clinical Scientist)

Clinical Support:

Dr Roshan Amarasena (Lead), Dr Mark Garton and Dr Ayman Askari

Our team has continued to be active this year carrying out basic science work into understanding mechanisms of rheumatoid arthritis (RA) progression and possible treatments.

We have been publishing our findings in Open Journal of Rheumatology and Autoimmune Disease, European Cells and Materials, Rheumatology.  We published a book chapter in Bone and Cartilage Regeneration volume of the Stem Cells in Clinical Applications series, by Springer Science. 

We have presented our work at several national and international meetings such as Tissue and Cell Engineering Society, London, July 2016; FIRM, Girona (Spain), September 2016; UK Mesenchymal Stem Cell meeting, York, December 2016;  Mercia Stem Cell Alliance, Manchester, December 2016; British Society for Rheumatology conference, Birmingham, April 2017 and 18th Annual Research day at the RJAH.

 

Members of the Research Team:

Dr Oksana Kehoe, Dr Alasdair Kay, Mr Andrei Stefan and Miss Rebecca Morgan

Professor Jim Middleton (Honorary Clinical Scientist)

Clinical Support:

Dr Roshan Amarasena (Lead), Dr Mark Garton and Dr Ayman Askari

Our team has continued to be active this year carrying out basic science work into understanding mechanisms of rheumatoid arthritis (RA) progression and possible treatments.

We have been publishing our findings in Open Journal of Rheumatology and Autoimmune Disease, European Cells and Materials, Rheumatology.  We published a book chapter in Bone and Cartilage Regeneration volume of the Stem Cells in Clinical Applications series, by Springer Science. 

We have presented our work at several national and international meetings such as Tissue and Cell Engineering Society, London, July 2016; FIRM, Girona (Spain), September 2016; UK Mesenchymal Stem Cell meeting, York, December 2016;  Mercia Stem Cell Alliance, Manchester, December 2016; British Society for Rheumatology conference, Birmingham, April 2017 and 18th Annual Research day at the RJAH.

 

 

Pictured: Dr Alasdair Kay, Miss Rebecca Morgan, Dr Oksana Kehoe and Mr Andrei Stefan

 

The projects which we are currently working are explained below;

Stem Cell Conditioned Medium as a Therapeutic Agent in Arthritis.

Alasdair Kay, Andrei Stefan, Jim Middleton (Bristol University), Oksana Kehoe

Funded by Dowager Countess Eleanor Peel Trust, Orthopaedic Institute and Oswestry Rheumatology Association

Alasdair completed work on understanding the therapeutic potential of mesenchymal stem cell conditioned medium. RA is characterised by increased autoimmune responses that disrupt the natural balance of pro- and anti-inflammatory T cell production so that excessive pro-inflammatory T cells affect joints, with insufficient regulation by anti-inflammatory T cells. Therapeutic products generated by MSC are secreted into growth medium during cell culture and this stem cell conditioned medium (CM-MSC) can be collected and applied as a therapy. We assessed the ability of CM-MSC to moderate the immune reaction by restoring the normal balance of T cells and reducing the effects of an autoimmune response. This project demonstrated that isolated T cells isolated from spleens of mice with experimental arthritis develop more anti-inflammatory T cells when treated with either MSC or CM-MSC than without treatment. The production of anti-inflammatory signals was also increased following CM-MSC treatment. Overall the ratio of ‘good’ regulatory T cells to ‘bad’ Th17 cells was restored towards the levels expected in non-disease conditions (Figure 1).

A restoration of T cell balance and increased anti-inflammatory signals suggests that stem cells act through generation of direct signals with therapeutic effects. Further work will focus on the components produced by stem cells and present in CM-MSC that may be isolated to act as a therapeutic agent, in particular small membrane bound vesicles called ‘exosomes’.

 

Figure 1.  Moderated immune response following MSC conditioned medium treatment. (A) Increased FOXP3 expression representing increased anti-inflammatory activity of Tregs, (B) Increased anti-inflammatory cytokine IL-4 in culture medium of T cells with CM-MSC and (C) increased expression of IL4 on T cells isolated from spleens of mice with experimental arthritis following CM-MSC treatment. (D) Improved ratio of anti-inflammatory regulatory T cells (FOXP3) to pro-inflammatory Th17 cells (IL17a) following CM-MSC treatment.

 

Evaluation of Mesenchymal Stem cell-derived extracellular vesicles as a therapy for Rheumatoid Arthritis.

Alasdair Kay, Rebecca Morgan, Andrei Stefan, Oksana Kehoe

Funding was provided by the RJAH Orthopaedic Hospital Charity, Oswestry Rheumatology Association and Orthopaedic Institute

MSCs function predominantly through paracrine mechanisms, via growth factors, cytokines, chemokines and bioactive, membrane-bound extracellular vesicles (including exosomes) found in mesenchymal stem cell conditioned medium (CM-MSC). Our previous project demonstrated that CM-MSC reduces swelling and cartilage destruction through modulation of the immune response in a murine model of inflammatory arthritis. This current study is assessing the contribution of exosomes to this therapeutic outcome, and examining how the culture microenvironment of MSCs can be manipulated to enhance their therapeutic effectiveness. Exosomes are applied as a treatment in our pre-clinical model of experimental arthritis, looking at their effects on inflammation, histological outcomes of disease progression and T cell polarisation towards either pro- or anti-inflammatory cell types.

Stem cells isolated from human bone marrow aspirate were used to derive exosomes (~30-100nm diameter). Following the application of exosomes, knee joint swelling significantly reduced at both 24 and 48 hours after treatment in our pre-clinical model (Figure 2). Reductions were highly significant in comparison to control animals where knee joints continued to swell in the absence of exosomes. Histological analysis and effects on the immune system are currently being investigated.

The significant reduction in joint swelling following exosome treatment demonstrates their effectiveness as a new therapeutic intervention. Effects are similar to those seen with living stem cell therapy however it is vital to identify how exosomes are able to act in an anti-inflammatory manner. Further investigation will identify the mechanisms involved. Cell free treatments such as exosomes have advantages over living cells and conditioned medium, including having a membrane that protects constituents from degradation. The use of cell-free therapy enables standardisation of dose and biological activity in treatments to ensure every therapy is at an effective standard. Characterisation of exosomes is also less complex than for CM‑MSC. These benefits will aid more rapid translation from research in the laboratory to clinical use.

 

Figure 2. Change in knee diameter (swelling) of AIA mice following exosome treatment (or PBS injection as control) shows significantly greater alleviation of swelling at days 2 and 3 post arthritis induction (n=4 per group, **p<0.01, independent samples t-test, error bars = SEM).

 

The in vitro effect of syndecan-3 gene knockout on bone marrow derived murine mesenchymal stem cells structure and adhesion properties.

Andrei Stefan, Alasdair Kay, Nick Forsyth, Oksana Kehoe

Funded by Keele University School of Medicine, Oswestry Rheumatology Association and Orthopaedic Institute

Andrei has progressed with his PhD work and showed that the absence of syndecan-3 molecule has a significant impact on stem cell morphology (Figure 3). Further research will determine whether deletion of syndecan-3 has effect on cytoskeletal proteins for both wild type (WT) and syndecan-3 knocked out (SDC-3-/-) mMSCs.

 

 

Figure 3. Morphometric results – the spread surface area of SDC-3-/- mMSCs is significantly lower than the one of WT.

In addition, Andrei showed that when different substrates are present (collagen and fibronectin), the adhered mMSCs number is significantly higher for the knocked out group (Figure 4). This implies that syndecan-3 plays an important role in cell adhesion by keeping the adhesion process at constant levels when different substrates are present.

Figure 4. The percentage of adhered SDC-3-/- mMSCs is significantly higher when the collagen and fibronectin coating is present compared to the non-coated control. Also, in presence of collagen the SDC-3-/- mMSCs adhered in a significantly higher proportion than the WT cells.

 

Uncovering the role of proteoglycans in exosome biogenesis for improved rheumatoid arthritis therapies

Rebecca Morgan, Alasdair Kay, Cathy Merry (Nottingham University), Oksana Kehoe

Funded by EPSRC Centre for Doctoral Training in Regenerative Medicine

Rebecca’s PhD work focused on a particular component of cells conditioned medium known as exosomes. These are membrane-bound vesicles released from cells including MSCs. They contain many proteins, lipids and RNA collectively known as cargo.  It may be possible to increase exosome production and alter their cargo (which defines the activity of the exosomes), to provide more effective rheumatoid arthritis therapies. This could be done by altering a proteoglycan called syndecan which is found on the surface of MSCs and their exosomes.

Work carried out to date, involves isolating human MSCs from three bone marrow aspirates (commercial source, Lonza). Cells were characterised by the ability to produce bone, cartilage and fat (typical trait of MSCs) (Figure 5).

 

 

Figure 5. Functional assay to determine capacity of human MSCs to form fat (A), bone (B) and cartilage (C).

Work has also been carried out to isolate exosomes from hMSC conditioned media. This was done using differential centrifugation where the conditioned media was subjected to multiple centrifugation steps with increasing speeds and time. Successful isolation of exosomes was determined using flow cytometry where markers including typical exosomal surface proteins (CD9, CD61 and CD83) were assessed. Figure 6 illustrates successful isolation of exosomes due to the presence of these three markers.

  

 

Figure 6. Flow cytometry analysis to confirm presence of exosomes after differential centrifugation.

Metabolic Medicine and Bone

Bone Health Clinical Service, Charles Salt Laboratories.

The bone health service currently has eight members of staff who between them cover 6.5 whole time equivalent jobs.  Two work part time in the clinical measurement side of the service as well as in the DXA (bone densitometry) unit and six members of staff work solely in the DXA unit. The service also has 3 part time consultants and a nurse who does intravenous treatments both at the RJAH and in outreach clinics. In the last financial year we scanned over 8600 patients and 1400 patients received treatment from the nurse for their bones. 

Last year in the laboratory over 1900 patient samples were analysed for the marker uNTx, a long established marker of bone breakdown as part of the clinical service.  The marker is used to monitor treatment efficacy, monitor treatment holidays and to determine baseline bone turnover.  Our current research interest is primarily in the use of uNTx to monitor a drug holiday.  Bisphosphonates are the first line drug treatment for patients with osteoporosis but these and other osteoporosis treatments have been linked to rare but serious side-effects.   Following the recommendation of a treatment holiday in patients on long-term bisphosphonates we are continuing to study the effect of a treatment holiday on bone turnover and bone mineral density.  Preliminary analysis suggests that as expected changes in uNTx levels are seen earlier than changes in bone density and that the bone density in the spine is maintained for longer than at the hip following discontinuation.  We have a large cohort of over 300 subjects who have been on a bisphosphonate holiday and we are hoping to evaluate if we can use uNTx to predict those subjects whose bone density will fall the fastest or those at greatest risk of fracture.  We will also be presenting data at the Bone Research Society Meeting in June 2017 on a group of subjects who have been on a treatment holiday for more than 3 years without significant changes to their bone health.

Research in the Charles Salt Laboratories.

The study on bone deficiency in newly diagnosed coeliac disease in men was completed and published. The work showed that bone density was particularly low in the trabecular regions in the lower limb. More importantly the study found that bone deficiency was not related to parathyroid hormone levels, but rather correlated with body weight1. Coeliac disease is known to have an association with bone deficiency and this has been ascribed to high parathyroid hormone levels. This study suggests that other factors are important, possibly inflammation, and concur with previous work in which we showed that women were at greater risk of fracture for up to ten years before clinical diagnosis of coeliac disease2. We are currently investigating whether the same conclusions can be made in women and in particular whether there is a relationship between bone, lean body mass and simple measures of inflammation. We also hope to be able to follow up women who have been treated for coeliac disease to investigate whether the known improvement in bone density at the spine and hip is found in the more distal parts of the lower limb and whether improvement in lean body mass also occurs.

To investigate changes in bone and lean mass over time and to relate these to those in coeliac subjects, we are also analysing data from a study on otherwise healthy women who have been followed up over eight years. Data from this study are also giving an insight into differences between longitudinal changes in bone and appendicular lean and cross sectional changes with time. Additionally the data are also providing information about changes in bone density between different vertebrae in the lumbar spine in healthy women and these data will be of use in deciding whether a particular vertebra is giving misleading readings in patients.

 

  1. Davie MW, Evans SF, Sharp CA. Lower Limb Metaphyseal Bone Is Lost in Men with Coeliac Disease and Does Not Relate to Parathyroid Status. J Osteoporos. 2016;2016:4131794.
  2. Davie MW, Gaywood I, George E, Jones PW, Masud T, Price T, Summers GD.  Excess non-spine fractures in women over 50 years with celiac disease: a cross-sectional, questionnaire-based study. Osteoporos Int. 2005 16:1150-5