Oksana Kehoe

Intra-articular injection of mesenchymal stem cells leads to reduced inflammation and cartilage damage in murine antigen-induced arthritis.

BackgroundRheumatoid arthritis (RA) is a debilitating and painful disease leading to increased morbidity and mortality and novel therapeutic approaches are needed. The purpose of this study was to elucidate if mesenchymal stem cells (MSCs) injected in the joints of mice with arthritis are therapeutic, reducing joint swelling and cartilage destruction.MethodsMurine mesenchymal stem cells (mMSCs) were isolated from bone marrow of C57Bl/6 mice and expanded in culture. Cells were tested for immunophenotype and their ability to form colonies and to differentiate into chondrocytes, osteocytes and adipocytes. Antigen-induced arthritis (AIA) was induced by intra-articular injection of methylated bovine serum albumin into the knee joints of preimmunized C57Bl/6 mice. After one day, when peak swelling occurs, 500,000 mMSCs labelled with red fluorescent cell tracker CM-DiI were injected intra-articularly in the right knee joint. Left knee joints were treated as controls by receiving PBS injections. Differences between groups were calculated by Mann Whitney U test or unpaired t tests using GraphPad Prism software version 5.ResultsKnee joint diameter (swelling) was measured as a clinical indication of joint inflammation and this parameter was significantly less in MSC-treated mice compared to control-treated animals 48 hours after arthritis induction. This difference continued for ~7 days. CM-DiI-labelled MSCs were clearly visualised in the lining and sublining layers of synovium, in the region of the patella and femoral and tibial surfaces. By day 3, parameters indicative of disease severity, including cartilage depletion, inflammatory exudate and arthritic index were shown to be significantly reduced in MSC-treated animals. This difference continued for 7 days and was further confirmed by histological analysis. The serum concentration of tumour necrosis factor α was significantly decreased following MSC administration.ConclusionsOur results reveal that MSCs injected in the joints of mice with AIA are therapeutic, reducing inflammation, joint swelling and cartilage destruction. These cells also integrate into the synovium in AIA.

A Chemokine Self-Presentation Mechanism Involving Formation of Endothelial Surface Microstructures

Endothelial surface microstructures have been described previously under inflammatory conditions; however, they remain ill-characterized. In this study, CXCL8, an inflammatory chemokine, was shown to induce the formation of filopodia-like protrusions on endothelial cells; the same effects were observed with CXCL10 and CCL5. Chemokines stimulated filopodia formation by both microvascular (from bone marrow and skin) and macrovascular (from human umbilical vein) endothelial cells. Use of blocking Abs and degradative enzymes demonstrated that CXCL8-stimulated filopodia formation was mediated by CXCR1 and CXCR2, Duffy Ag/receptor for chemokines, heparan sulfate (HS), and syndecans. HS was present on filopodial protrusions appearing as a meshwork on the cell surface, which colocalized with CXCL8, and this glycosaminoglycan was 2,6-O– and 3-O–sulfated. Transmission electron microscopy revealed that CXCL8-stimulated filopodial and microvilli-like protrusions that interacted with leukocytes before transendothelial migration and removal of HS reduced this migration. iTRAQ mass spectrometry showed that changes in the levels of cytoskeletal, signaling, and extracellular matrix proteins were associated with CXCL8-stimulated filopodia/microvilli formation; these included tropomyosin, fascin, and Rab7. This study suggests that chemokines stimulate endothelial filopodia and microvilli formation, leading to their presentation to leukocytes and leukocyte transendothelial migration.

Syndecan-3 is selectively pro-inflammatory in the joint and contributes to antigen-induced arthritis in mice.

IntroductionSyndecans are heparan sulphate proteoglycans expressed by endothelial cells. Syndecan-3 is expressed by synovial endothelial cells of rheumatoid arthritis (RA) patients where it binds chemokines, suggesting a role in leukocyte trafficking. The objective of the current study was to examine the function of syndecan-3 in joint inflammation by genetic deletion in mice and compare with other tissues.MethodsChemokine C-X-C ligand 1 (CXCL1) was injected in the joints of syndecan-3−/−and wild-type mice and antigen-induced arthritis performed. For comparison chemokine was administered in the skin and cremaster muscle. Intravital microscopy was performed in the cremaster muscle.ResultsAdministration of CXCL1 in knee joints of syndecan-3−/−mice resulted in reduced neutrophil accumulation compared to wild type. This was associated with diminished presence of CXCL1 at the luminal surface of synovial endothelial cells where this chemokine clustered and bound to heparan sulphate. Furthermore, in the arthritis model syndecan-3 deletion led to reduced joint swelling, leukocyte accumulation, cartilage degradation and overall disease severity. Conversely, CXCL1 administration in the skin of syndecan-3 null mice provoked increased neutrophil recruitment and was associated with elevated luminal expression of E-selectin by dermal endothelial cells. Similarly in the cremaster, intravital microscopy showed increased numbers of leukocytes adhering and rolling in venules in syndecan-3−/−mice in response to CXCL1 or tumour necrosis factor alpha.ConclusionsThis study shows a novel role for syndecan-3 in inflammation. In the joint it is selectively pro-inflammatory, functioning in endothelial chemokine presentation and leukocyte recruitment and cartilage damage in an RA model. Conversely, in skin and cremaster it is anti-inflammatory.

Mesenchymal stem cell-conditioned medium reduces disease severity and immune responses in inflammatory arthritis

We evaluated the therapeutic potential of mesenchymal stem cell-conditioned medium (CM-MSC) as an alternative to cell therapy in an antigen-induced model of arthritis (AIA). Disease severity and cartilage loss were evaluated by histopathological analysis of arthritic knee joints and immunostaining of aggrecan neoepitopes. Cell proliferation was assessed for activated and naïve CD4+ T cells from healthy mice following culture with CM-MSC or co-culture with MSCs. T cell polarization was analysed in CD4+ T cells isolated from spleens and lymph nodes of arthritic mice treated with CM-MSC or MSCs. CM-MSC treatment significantly reduced knee-joint swelling, histopathological signs of AIA, cartilage loss and suppressed TNFα induction. Proliferation of CD4+ cells from spleens of healthy mice was not affected by CM-MSC but reduced when cells were co-cultured with MSCs. In the presence of CM-MSC or MSCs, increases in IL-10 concentration were observed in culture medium. Finally, CD4+ T cells from arthritic mice treated with CM-MSC showed increases in FOXP3 and IL-4 expression and positively affected the Treg:Th17 balance in the tissue. CM-MSC treatment reduces cartilage damage and suppresses immune responses by reducing aggrecan cleavage, enhancing Treg function and adjusting the Treg:Th17 ratio. CM-MSC may provide an effective cell-free therapy for inflammatory arthritis.

Novel Anti-Inflammatory Peptides Based on Chemokine–Glycosaminoglycan Interactions Reduce Leukocyte Migration and Disease Severity in a Model of Rheumatoid Arthritis

Inflammation is characterized by the infiltration of leukocytes from the circulation and into the inflamed area. Leukocytes are guided throughout this process by chemokines. These are basic proteins that interact with leukocytes to initiate their activation and extravasation via chemokine receptors. This is enabled through chemokine immobilization by glycosaminoglycans (GAGs) at the luminal endothelial surface of blood vessels. A specific stretch of basic amino acids on the chemokine, often at the C terminus, interacts with the negatively charged GAGs, which is considered an essential interaction for the chemokine function. Short-chain peptides based on this GAG-binding region of the chemokines CCL5, CXCL8, and CXCL12γ were synthesized using standard Fmoc chemistry. These peptides were found to bind to GAGs with high affinity, which translated into a reduction of leukocyte migration across a cultured human endothelial monolayer in response to chemokines. The leukocyte migration was inhibited upon removal of heparan sulfate from the endothelial surface and was found to reduce the ability of the chemokine and peptide to bind to endothelial cells in binding assays and to human rheumatoid arthritis tissue. The data suggest that the peptide competes with the wild-type chemokine for binding to GAGs such as HS and thereby reduces chemokine presentation and subsequent leukocyte migration. Furthermore, the lead peptide based on CXCL8 could reduce the disease severity and serum levels of the proinflammatory cytokine TNF-α in a murine Ag-induced arthritis model. Taken together, evidence is provided for interfering with the chemokine–GAG interaction as a relevant therapeutic approach.

Therapeutic Benefit for Late, but Not Early, Passage Mesenchymal Stem Cells on Pain Behaviour in an Animal Model of Osteoarthritis

Background Mesenchymal stem cells (MSCs) have a therapeutic potential for the treatment of osteoarthritic (OA) joint pathology and pain. The aims of this study were to determine the influence of a passage number on the effects of MSCs on pain behaviour and cartilage and bone features in a rodent model of OA. Methods Rats underwent either medial meniscal transection (MNX) or sham surgery under anaesthesia. Rats received intra-articular injection of either 1.5 × 106 late passage MSCs labelled with 10 μg/ml SiMAG, 1.5 × 106 late passage mesenchymal stem cells, the steroid Kenalog (200 μg/20 μL), 1.5 × 106 early passage MSCs, or serum-free media (SFM). Sham-operated rats received intra-articular injection of SFM. Pain behaviour was quantified until day 42 postmodel induction. Magnetic resonance imaging (MRI) was used to localise the labelled cells within the knee joint. Results Late passage MSCs and Kenalog attenuated established pain behaviour in MNX rats, but did not alter MNX-induced joint pathology at the end of the study period. Early passage MSCs exacerbated MNX-induced pain behaviour for up to one week postinjection and did not alter joint pathology. Conclusion Our data demonstrate for the first time the role of a passage number in influencing the therapeutic effects of MSCs in a model of OA pain.

SAT0554 Intra-Articular Injection of Mesenchymal Stem Cells Improves Pain Behaviour in A Model of OA Pain

Background Osteoarthritis (OA) is a degenerative joint disease affecting around 8 million people in the UK. Pain is a prominent and often disabling feature of OA, the relationship between pain and histopathological features of OA are poorly understood. Currently, there are no reparative or disease-modifying treatments for OA and thus many patients will undergo joint replacement surgery for pain relief. Objectives To explore the effect for intra-articular mesenchymal stem cells (MSC) treatment on pain behaviour in a rodent surgical model of OA. Methods Under isoflurane anaesthesia (1.5L/min O2, 2.5% isoflurane) joint pathology was induced in adult male Sprague Dawley Rats (160-200g) by transecting the medial collateral ligament and making a full thickness cut through the meniscus (day 0) of the left knee (Mapp et al., 2008). Baseline pain behaviour were taken immediately prior to surgery (day 0) and then from 3 until 42 days post-surgery. Pain measures were weight bearing asymmetry, assessed using an incapacitance tester, and the lowering of hindpaw mechanical withdrawal thresholds, quantified using von Frey monofilaments (1-15 g). 14 days post-surgery, rats were stratified according to behavioural pain responses and under brief isoflurane anaesthesia (3% 1L/min O2), received an intra-articular injection (through the infrapatellar ligament of the left knee) 50μl of 1.5x106 mesenchymal stem cells (n=7 rats) or serum free media (SFM; n=4 rats). Pain behaviour was quantified for a further four weeks post intra-articular injection and then the experiment was terminated. Data are expressed as mean weight bearing % (weight on ipsilateral paw – weight on contralateral paw/total weight on hindpaws) x 100% ± SEM. Statistical analysis between groups was performed using a Mann Whitney test. Results Consistent with previous studies, from 3 days following medial meniscal transection (MNX) surgery rats exhibited increased weight bearing asymmetry and decreased ipsilateral paw withdrawal thresholds, compared to pre-surgery values. MNX rats which received the control treatment (intra-articular injection of SFM) exhibited a gradual increase in pain behaviour up to day 42. Intra-articular injection of MSCs at day 14 prevented a further increase in weight bearing asymmetry, which was significant when compared to OA rats treated with SFM, up to day 42 (weight bearing % SFM =19.5±0.6, MSC =9.5±3.5; p<0.05). MSC treatment had no effect on the hindpaw mechanical withdrawal thresholds in MNX treated rats. Conclusions Intra-articular injection of MSCs attenuated the further development of weight bearing asymmetry in rats with established joint pathology in the MNX model of OA. There was no effect of this treatment on the development of hindpaw mechanical withdrawal thresholds. These data suggest that intra-articular injection of MSCs may alter peripherally-driven pain, but may not affect centrally-mediated pain responses in established OA. References Mapp PI, Avery PS, McWilliams DF, Bowyer J, Day C, Moores S, Webster R, Walsh DA (2008). Angiogenesis in two animal models of osteoarthritis. Osteoarthritis Cartilage 16(1): 61-69 Acknowledgements This work was funded by EPSRC and ARUK Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3731

An initial investigation into endothelial CC chemokine expression in the human rheumatoid synovium

&NA; Rheumatoid arthritis (RA) is a destructive and chronic autoimmune inflammatory disease. Synovial inflammation is a major feature of RA and is associated with leukocyte recruitment. Leukocytes cross the endothelial cells (ECs) into the synovial tissue and fluid and this migration is mediated via a range of chemokines and adhesion molecules on the ECs. As important mediators of leukocyte extravasation, a number of chemokines from each of the chemokine families have been established as expressed in the RA joint. However, as little information is available on which chemokines are expressed/presented by the ECs themselves, the purpose of the study was to ascertain which of the CC chemokines were localised in RA ECs. Immunofluoresence was used to assess the presence of the CC‐family chemokines in RA synovial ECs using von‐Willebrand factor (VWF) as a pan‐endothelial marker and a range of human chemokine antibodies. The percentage of VWF positive vessels which were positive for the chemokines was determined. The presence of the four most highly expressed novel chemokines were further investigated in non‐RA synovial ECs and the sera and synovial fluid (SF) from patients with RA and osteoarthritis (OA). Statistical analysis of immunofluorescence data was carried out by Students t‐test. For analysis of ELISA data, Kruskal‐Wallis ANOVA followed by Dunns multiple comparison test was utilised to analyse differences in sera and SF levels for each chemokine between RA and OA. Spearman rank correlations of sera and SF chemokine levels with a range of clinical variables were also performed. Chemokine detection varied, the least abundant being CCL27 which was present in 8.3% of RA blood vessels and the most abundant being CCL19 which was present in 80%. Of the 26 chemokines studied, 19 have not been previously observed in RA ECs. Four of these novel chemokines, namely CCL7, CCL14, CCL16 and CCL22 were present on ≥60% of vessels. CCL14 and CCL22 were shown to be increased in RA ECs compared to non‐RA ECs, p = 0.0041 and p = 0.014 respectively. EC chemokines CCL7, CCL14, CCL16 and CCL22 also occurred in RA synovial fluid and sera as established by ELISA. CCL7 was shown to be significantly increased in sera and SF from RA patients compared to that from osteoarthritis (OA) patients (p < 0.01), and to have a highly significant correlation with the level of anti‐CCP (R = 0.93, p = 0.001). Less abundant chemokines shown to be present in RA ECs were CCL1‐3, CCL5, CCL10‐13, CCL15, CCL17, CCL18, CCL20, CCL21 and CCL23‐28. In conclusion, this initial study is the first to show the presence of a number of CC chemokines in RA ECs. It provides evidence that further validation and investigation into the presence and functionality of these novel chemokines expressed at RA synovial ECs may be warranted. HighlightsComparison of the presence of 26 of the CC‐chemokines in RA synovial ECs.The chemokines CCL7, CCL14, CCL16 and CCL22 were established as being present at RA synovial ECs for the first time.CCL8, CCL14, CCL19 and CCL22 are significantly increased in RA compared to non‐RA.Synovial fluid CCL7 may be a novel RA disease marker.

Mesenchymal Stem Cell Therapy in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is the second most common form of arthritis and the most common inflammatory joint disorder in the UK, affecting more than 400,000 people with around 12,000 new cases diagnosed every year. The condition is chronic and degenerative, causing pain and swelling, stiffness and fatigue. The disorder affects females to males at a ratio of approximately 3:1 with an overall incidence of around 1 in 100. It is most commonly reported in people aged >40 years with most first diagnoses occurring at age <60 years and life expectancy may be reduced by up to 18 years. With such high prevalence, extensive work has been done to investigate causes, early diagnosis and treatments for RA, yet much about the disease remains elusive, no cure currently exists and treatment options are limited. Drug treatment and physiotherapy are first line approaches. Non-steroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) are common treatments. Recent research for novel treatments has led to the introduction of biological antibodies, such as anti-tumour necrosis factor alpha (anti-TNF-α). Whilst significant impact has been made in alleviating symptoms, 50 % of RA sufferers prescribed with biological treatments discontinue the use after 2 years due to declining efficacy or adverse side effects. In untreatable cases, the last resort is joint replacement. It is advantageous in treating multifaceted disorders to utilise a multifaceted therapy and mesenchymal stem cells (MSC) infusion offers this potential. This chapter reviews the application of mesenchymal stem cells in the treatment of RA and current research perspectives. The advantages of MSC therapies are discussed alongside evaluation of optimum models for use in in vivo testing of novel treatments. Outcomes of both preclinical and the few clinical trials conducted to date are discussed to develop both the potential and future of stem cell therapy for autoimmune conditions such as RA.