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Objectives Cardiovascular (CV) mortality in RA patients is 50% higher than in the general population. There is increasing recognition that systemic inflammation is a major driver of this. IL-6 is implicated in cardiovascular disease (CVD) in the general population but its role in CVD in RA is undefined. Of the two modes of IL-6 signalling, trans-signalling is pro-inflammatory whereas classical signalling is linked with inflammation resolution. This study examines the role of IL-6 trans-signalling in CVD in a mouse model and patients with RA. Methods Myography determined the effect of IL-6 trans-signalling blockade, using sgp130Fc, on aortic constriction in murine collagen-induced arthritis. Serum CCL2 and sVCAM-1 as soluble biomarkers of sIL-6R trans-signalling were investigated in a human cross-sectional study. An observational longitudinal study investigated the association between these biomarkers and progression of subclinical atherosclerosis in early RA by measuring carotid intima-media thickness (CIMT). Results sgp130Fc reduced arthritis severity, serum CCL2 and sVCAM-1 and restored vascular function in collagen-induced arthritis (CIA). In established RA, sVCAM-1 correlated with the 28-joint DAS (DAS28) and CV risk. In early RA, baseline DAS28 was associated with CIMT change at 6 months. CIMT ‘rapid progressors’ at 12 months had higher baseline sVCAM-1, haemoglobin A1c, cholesterol:high-density lipoprotein cholesterol ratio and LDL cholesterol. Conclusions IL-6 trans-signalling plays a pivotal role in vascular dysfunction in CIA. In early RA, sVCAM-1 was associated with progression of subclinical atherosclerosis. Inflammation from RA onset in CVD-susceptible individuals may accelerate atherosclerosis. IL-6 trans-signalling blockade may be beneficial to RA patients and perhaps for atherosclerosis in the general population.

Background\ud \ud Oestrogen-deficiency induced by menopause is associated with reduced bone density and primary osteoporosis, resulting in an increased risk of fracture. While the exact etiology of menopause-induced primary osteoporotic bone loss is not fully known, members of the tumour necrosis factor super family (TNFSF) are known to play a role. Recent studies have revealed that the TNFSF members death receptor 3 (DR3) and one of its ligands, TNF-like protein 1A (TL1A) have a key role in secondary osteoporosis; enhancing CD14+ peripheral blood mononuclear cell (PBMC) osteoclast formation and bone resorption. Whether DR3 and TL1A contribute towards bone loss in menopause-induced primary osteoporosis however, remains unknown.\ud \ud Methods\ud \ud To investigate this we performed flow cytometry analysis of DR3 expression on CD14+ PBMCs isolated from pre- and early post-menopausal females and late post-menopausal osteoporotic patients. Serum levels of TL1A, CCL3 and total MMP-9 were measured by ELISA. In vitro osteoclast differentiation assays were performed to determine CD14+ monocyte osteoclastogenic potential. In addition, splenic CD4+ T cell DR3 expression was investigated 1 week and 8 weeks post-surgery, using the murine ovariectomy model.\ud \ud Results\ud \ud In contrast to pre-menopausal females, CD14+ monocytes isolated from post-menopausal females were unable to induce DR3 expression. Serum TL1A levels were decreased approx. 2-fold in early post-menopausal females compared to pre-menopausal controls and post-menopausal osteoporotic females; no difference was observed between pre-menopausal and late post-menopausal osteoporotic females. Analysis of in vitro CD14+ monocyte osteoclastogenic potential revealed no significant difference between the post-menopausal and post-menopausal osteoporotic cohorts. Interestingly, in the murine ovariectomy model splenic CD4+ T cell DR3 expression was significantly increased at 1 week but not 8 weeks post-surgery when compared to the sham control.\ud \ud Conclusion\ud \ud Our results reveals for the first time that loss of oestrogen has a significant effect on DR3; decreasing expression on CD14+ monocytes and increasing expression on CD4+ T cells. These data suggest that while oestrogen-deficiency induced changes in DR3 expression do not affect late post-menopausal bone loss they could potentially have an indirect role in early menopausal bone loss through the modulation of T cell activity.

Background\ud \ud The influence of patient demographics and mode of admission on the ‘weekend effect’ remains unclear. This study examins the relationship between day of admission, patient demographics, mode of presentation and survival.\ud \ud \ud Methods\ud \ud Hospital admissions over a three-year period were studied. Patients with an inpatient stay less than 24 h and those who were discharged from the emergency department were excluded. In-hospital mortality was correlated with day of admission, age, gender and mode of presentation in a binary logistical regression analysis.\ud \ud \ud Results\ud \ud There were 448,827 admissions, of which 350,648 (85.7%) occurred during a weekday. 256,777 (62.7%) were emergency presentations, which was closely related to a weekend admission (92.3% vs 57.8%, p < 0.001). There were 8099 deaths of which 6336 (78.2%) related to a weekday admission and 1736 (21.4%) related a weekend admission. Mortality for elective admissions was 78 (0.05%) compared to 8021 (3.12%), p < 0.001 in emergency admissions. Univariable regression analysis revealed a weekend admission (Odds Ratio (OR) 1.68 (95% confidence interval (CI) 1.60–1.78, p < 0.001) and emergency presentation (OR 63.02 (95%CI 50.42–78.77), p < 0.011) were associated with weekend mortality. On multivariable analysis the OR for weekend admission reduced to 1.07 (95%CI 1.01–1.13), p = 0.013 and the OR for emergency presentation increased to 76.68 (95%CI 61.40–96.00), p < 0.001.\ud \ud \ud Conclusion\ud \ud This study highlights that higher weekend mortality rates are a consequence of a lower proportion of elective admissions. Extending the working week to seven days might reduce weekend mortality without reducing the total number of deaths.

ObjectiveCD3+CD8+CD28− cells are increased in the periphery and tissues of rheumatoid arthritis (RA) patients. The aim of this study was to characterise CD3+CD8+CD28− cells for the presence of cell surface receptors that regulate immune activation and function and to track their presence in a disease model of RA. Cell surface receptors expressed by CD3+CD8+CD28− cells were then related to serological and clinical disease parameters to establish whether these cells are prognostic of a clinical response to conventional DMARDs.MethodUsing healthy donor peripheral blood mononuclear (PBMC) cell surface expression of > 50 candidate markers were tested using flow cytometry and compared against CD28 expression. The prevalence of cells expressing the most suitable candidate was investigated in the collagen induced arthritis (CIA) and the antigen-induced arthritis (AIA) models. Fifty RA patients were recruited from University Hospital of Wales (UHW) rheumatology outpatient clinic. Clinical and serological markers of inflammation were noted, and PBMC were analysed using flow cytometry +/− in vitro stimulation.ResultsCD3+CD8+CD28− T cells express CD244, CD57, CX3CR1 and KLRG1. The strongest inverse correlate of CD28 expression was KLRG1. CD3+CD8+CD28−KLRG1+ cells were elevated in experimental models of RA. Notably, Il-10-deficiency was linked with exacerbated arthritis and an increase in the number of CD3+CD8+CD28−KLRG1+ cells, suggesting a regulatory role for Il-10 in their development or survival. In RA patients, CD3+CD8+CD28−KLRG1+ cells correlate with ACPA, RF and ESR, and produce more IL-10 than controls. Finally, these cells are higher in early arthritis patients that do not respond to treatment with synthetic DMARDs at six months.ConclusionKLRG1 is a marker for regulatory CD3+CD8+CD28− cells. The presence of CD3+CD8+CD28-KLRG1+ cells increases with certain measures of disease, and is indicative of poor treatment response to DMARDs in early arthritis.Key MessagesKLRG1 is a marker of CD28 negativity on CD8 T cellsCD3+CD8+CD28−KLRG1+ cells are increased in CIA mice and correlate with disease severity.CD3+CD8+CD28−KLRG1+ cells positively correlate with ESR / ACPA and RF in patients.CD3+CD8+CD28−KLRG1+ cells are increased in Il-10-deficient mice with inflammatory arthritis.CD3+CD8+CD28−KLRG1+ cells produce more Il-10 than CD3+CD8+CD28+KLRG1−cells in RA patients.CD3+CD8+CD28−KLRG1+ cells are higher in patients who do not respond to treatment with synthetic DMARDs after six months.

Musculoskeletal disorders represent the third greatest burden in terms of death and disability in the developed world. Osteoarthritis is the single greatest cause of chronic pain, has no cure, and affects 8.5 and 27 million people in the UK and US, respectively. Osteoarthritis is most prevalent in older people, but as it commonly occurs after joint injury, young people with such injuries are also susceptible. Painful joints are often treated with steroid or hyaluronic acid (HA) injections, but treatments to prevent subsequent joint degeneration remain elusive. In animals, joint injury increases glutamate release into the joint, acting on nerves to cause pain, and joint tissues to cause inflammation and degeneration. This study investigated synovial fluid glutamate concentrations and glutamate receptor (GluR) expression in injured human joints and compared the efficacy of GluR antagonists with current treatments in a mouse model of injury-induced osteoarthritis (ACL rupture). GluRs were expressed in the ligaments and meniscus after knee injury, and synovial fluid glutamate concentrations ranged from 19 to 129 μM. Intra-articular injection of NBQX (GluR antagonist) at the time of injury substantially reduced swelling and degeneration in the mouse ACL rupture model. HA had no effect, and Depo-Medrone reduced swelling for 1 day but increased degeneration by 50%. Intra-articular administration of NBQX modified both symptoms and disease to a greater extent than current treatments. There is an opportunity for repurposing related drugs, developed for CNS disorders and with proven safety in humans, to prevent injury-induced osteoarthritis. This could quickly reduce the substantial burden associated with osteoarthritis.

Background The COVID‐19 pandemic poses a critical global public health crisis. Operating room (OR) best practice in this crisis is poorly defined. This systematic review was performed to identify contemporary evidence relating to OR practice in the context of COVID‐19. Methods MEDLINE was searched systematically using PubMed (search date 19 March 2020) for relevant studies in accordance with PRISMA guidelines. Documented practices and guidance were assessed to determine Oxford Centre for Evidence‐Based Medicine (OCEBM) levels of evidence, and recommendations for practice within five domains were extracted: physical OR, personnel, patient, procedure, and other factors. Results Thirty‐five articles were identified, of which 11 met eligibility criteria. Nine articles constituted expert opinion and two were retrospective studies. All articles originated from the Far East (China, 9; Singapore, 2); eight of the articles concerned general surgery. Common themes were identified within each domain, but all recommendations were based on low levels of evidence (median OCEBM level 5 (range 4–5)). The highest number of overlapping recommendations related to physical OR (8 articles) and procedural factors (13). Although few recommendations related to personnel factors, consensus was high in this domain, with all studies mandating the use of personal protective equipment. Conclusion There was little evidence to inform this systematic review, but there was consensus regarding many aspects of OR practice. Within the context of a rapidly evolving pandemic, timely amalgamation of global practice and experiences is needed to inform best practice., This rapid systematic review was performed to identify contemporary evidence relating to operating room (OR) practice during the COVID‐19 pandemic. Evidence underpinning the review was sparse; however, consensus regarding many aspects of OR practice was apparent. High quality evidence needed

Aim/Purpose: Understanding the educational needs of postgraduate research candidates (PGRs) is essential to facilitate development, support attainment, and maintain graduate quality. Background: The production and effective defence of the research thesis are the summative assessment tools used in postgraduate research education. Examiners’ reports provide a rich source of feedback and indicate the gap between the candidate’s level of performance and that expected for the award. This provides a lens through which to view the unmet training needs of PGR cohorts. Methodology: Following a review of all examiner reports for PGR assessments held over a 12 month period, we explored the quantitative and qualitative dimension data in context in order to identify common training needs for our PGR students. Utilising this theoretical framework and standard thematic analysis, we identified recurring themes and were able to determine key areas for future focus. Contribution: This study utilises independent comment from postgraduate research candidate thesis and oral examination assessment to identify unmet core research training needs. Findings: We recognised seven key areas identified by the examiners for improvement: i) quality of scientific writing, ii) general presentation of thesis, iii) statistics /data analysis, iv) understanding / critical appraisal, v) experimental design, vi) English language and vii) supervision. Academic literacy and numeracy stood out as key areas for future training focus. The results highlight areas for future focus in educational provision and targeted training for PGRs undertaking biomedical and life sciences research within our faculty. Recommendations for Practitioners: Evaluation of postgraduate research programmes should include feedback from a variety of sources and not rely solely on employability and completion rates as measures of success. The examination committees are an important source of feedback on the individual and the programme with regard to attainment of core research skills. Recommendation for Researchers: Regular and wide reaching evaluation of postgraduate research programmes and support available is required to ensure the sector can meet the changing needs of our PGR cohorts. Impact on Society: Doctoral graduates are entering increasingly diverse employment fields. Ensuring the quality of graduates and supporting their journey through candidature ensures the greatest value for society once in the work place. Future Research: This study highlights unmet training needs of PGRs as identified by an inde-pendent expert. The impact of engagement with training and the importance of prior experience are not explored in this study, nor is the student perspective on the process. These will reveal additional dimensions to the evaluation process.

T-cell infiltration into tumors represents a critical bottleneck for immune-mediated control of cancer. We previously showed that this bottleneck can be overcome by depleting immunosuppressive Foxp3+ regulatory T cells (Tregs), a process that can increase frequencies of tumor-infiltrating lymphocytes through promoting the development of specialized portals for lymphocyte entry, namely high endothelial venules (HEVs). In this paper, we used a carcinogen-induced tumor model that allows for coevolution of the tumor microenvironment and the immune response to demonstrate that Treg depletion not only results in widespread disruption to HEV networks in lymph nodes (LNs) but also activates CD8+ T cells, which then drive intratumoral HEV development. Formation of these vessels contrasts with ontogenic HEV development in LNs in that the process is dependent on the TNF receptor and independent of lymphotoxin β receptor–mediated signaling. These intratumoral HEVs do not express the chemokine CCL21, revealing a previously undescribed intratumoral blood vessel phenotype. We propose a model where Treg depletion enables a self-amplifying loop of T-cell activation, which promotes HEV development, T-cell infiltration, and ultimately, tumor destruction. The findings point to a need to test for HEV development as part of ongoing clinical studies in patients with cancer. Cancer Immunol Res; 5(11); 1005–15. ©2017 AACR.

Although the historical bases for graduate training in the United Kingdom(UK) and Scandinavia both stem from the original concept developed byvon Humboldt, and both award a ‘PhD degree’, their paths have diverged.There are thus significant differences in the manner in which graduate trainingis organised. To analyse these differences, two UK graduate programmes(School of Medicine, Cardiff University; Institute of Integrative Biology,University of Liverpool) and two Scandinavian graduate schools (Faculty ofMedicine and Dentistry, University of Bergen; Karolinska Institutet, Stockholm)completed a Self-evaluation questionnaire developed by Organisationof PhD Education in Biomedicine and Health Sciences in the European System(ORPHEUS)). Analysis of the completed questionnaires shows differencesconcerning requirements for admission, the training content of PhDprogrammes, the format of the PhD thesis, how the thesis is assessed and thefinancial model. All programmes recognise that PhD training should preparefor employment both inside and outside of academia, with emphasis ontransferable skills training. However, the analysis reveals some fundamentaldifferences in the direction of graduate programmes in the UK and Scandinavia.In the UK, graduate programmes are directed primarily towardsteaching PhD students to do research, with considerable focus on practicaltechniques. In Scandinavia, the focus is on managing projects and publishingpapers. To some extent, the differences lead to a lack of full recognition ofeach other’s theses as a basis for doing a postdoc. This paper describes thebasis for these differences and compares the two approaches and points toareas in which there is, or might be, convergence. Although the historical bases for graduate training in the United Kingdom(UK) and Scandinavia both stem from the original concept developed by von Humboldt, and both award a ‘PhD degree’, their paths have diverged. There are thus significant differences in the manner in which graduate training is organised. To analyse these differences, two UK graduate programmes(School of Medicine, Cardiff University; Institute of Integrative Biology, University of Liverpool) and two Scandinavian graduate schools (Faculty of Medicine and Dentistry, University of Bergen; Karolinska Institutet, Stock-holm) completed a Self-evaluation questionnaire developed by Organisation of PhD Education in Biomedicine and Health Sciences in the European System (ORPHEUS)). Analysis of the completed questionnaires shows differences concerning requirements for admission, the training content of PhD programmes, the format of the PhD thesis, how the thesis is assessed and the financial model. All programmes recognise that PhD training should prepare for employment both inside and outside of academia, with emphasis on transferable skills training. However, the analysis reveals some fundamental differences in the direction of graduate programmes in the UK and Scandinavia. In the UK, graduate programmes are directed primarily towards teaching PhD students to do research, with considerable focus on practical techniques. In Scandinavia, the focus is on managing projects and publishing papers. To some extent, the differences lead to a lack of full recognition of each other’s theses as a basis for doing a postdoc. This paper describes the basis for these differences and compares the two approaches and points to areas in which there is, or might be, convergence.

Approximately 30 members of the tumor necrosis factor receptor superfamily (TNFRSF) have been identified. They are transmembrane proteins with cysteine‐rich motifs in their extracellular domains that bind to their cognate ligands 1. They are categorized into 3 groups: death domain–containing receptors, decoy receptors, and TNFR‐associated factor–binding receptors. Only 8 TNFRSF members contain a death domain (TNFR type I [TNFRI], death receptor 3 [DR‐3], DR‐4, DR‐5, DR‐6, Fas, nerve growth factor receptor, and ectodysplasin A receptor [EDAR]), of which TNFRI and DR‐3 constitute the principal focus of this article. Interactions between TNF superfamily (TNFSF) ligands and TNFRSF receptors help maintain tissue homeostasis by controlling survival, proliferation, differentiation, and effector function of immune cells. We limit our review to recent advances and novel insights into the roles of TNFRI and DR‐3 in bone and joint biology. Bone cells (osteoblasts, osteoclasts, and osteocytes), fibroblast‐like synoviocytes, chondrocytes, and immune cells that infiltrate the arthritic joint will at different times express a wide range of TNFRSF members and TNFSF ligands. An overview of the current status of our knowledge in this regard is provided in Table 1. The impact of TNFRI activation on bone and inflammatory joint diseases has been researched in great depth 2, 3, but little or no data in the field have been reported on other more recently discovered TNFRSF members such as TROY (TNFRSF expressed on the mouse embryo; TNFRSF19), EDAR, and XEDAR (X‐linked ectodysplasin receptor; TNFRSF27). The unexpected interaction between progranulin (PGRN) and both TNFRI and TNFRII is particularly interesting in the context of arthritis‐associated bone pathology. PGRN levels are elevated in the synovial fluid of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and other arthropathies 4, 5, 6, and PGRN has been shown to inhibit TNF‐induced osteoclastogenesis and promote osteoblast differentiation in mice 7. However, PGRN has a higher binding affinity for TNFRII (antiinflammatory with osteoprotective function) than for TNFRI (predominantly proinflammatory with degenerative function), which suggests conflicting actions. The potential overall impact of these divergent PGRN signaling pathways on the architecture of the arthritic joint has been evaluated 8. Table 1 Cellular expression of death domain–containing TNFRSF members and their association with arthritis*

The most widely cited procedures for radiolabeling antibodies with zirconium-89 for immuno-PET require multi-milligram amounts of antibody which can be cost-prohibitive, particularly during the research and development process. We therefore sought to develop a reliable (89)Zr-radiolabeling procedure that provides high radiochemical yields at the microgram scale.

Objective Macrophage inflammatory protein 1-alpha (CCL3) is a chemokine that regulates macrophage trafficking to the inflamed joint. The agonistic effect of CCL3 on osteolytic lesions in patients with multiple myeloma is recognized; however, its role in skeletal damage during inflammatory arthritis has not been established. The aim of the study was to explore the role of osteoclast-associated CCL3 upon bone resorption, and to test its pharmacological blockade for protecting against bone pathology during inflammatory arthritis. Methods CCL3 production was studied during osteoclast differentiation from osteoclast precursor cells: human CD14-positive mononuclear cells. Mice with CIA were treated with an anti-CCL3 antibody. The effect of CCL3 blockade through mAb was studied through osteoclast number, cytokine production and bone resorption on ivory disks, and in vivo through CIA progression (clinical score, paw diameter, synovial inflammation and bone damage). Results Over time, CCL3 increased in parallel with the number of osteoclasts in culture. Anti-CCL3 treatment achieved a concentration-dependent inhibition of osteoclast fusion and reduced pit formation on ivory disks (P ⩽ 0.05). In CIA, anti-CCL3 treatment reduced joint damage and significantly decreased multinucleated tartrate-resistant acid phosphatase-positive osteoclasts and erosions in the wrists (P < 0.05) and elbows (P < 0.05), while also reducing joint erosions in the hind (P < 0.01) and fore paws (P < 0.01) as confirmed by X-ray. Conclusion Inhibition of osteoclast-associated CCL3 reduced osteoclast formation and function whilst attenuating arthritis-associated bone loss and controlling development of erosion in murine joints, thus uncoupling bone damage from inflammation. Our findings may help future innovations for the diagnosis and treatment of inflammatory arthritis.

Introduction Rheumatoid Arthritis (RA) patients have increased cardiovascular risk (CV) due to accelerated atherosclerosis (ATS), which significantly contributes to excess mortality in RA.1 The increased CV risk cannot be fully explained by traditional risk factors and systemic chronic inflammation appears to play a crucial role. Interestingly, IL-7, a proinflammatory cytokine involved in RA pathogenesis, appears to play a role also in ATS2 but its effect on cardiovascular disease (CVD) in RA has not been studied yet. Objectives To examine serum IL-7 levels and expression of IL-7, IL-7R, CD3 and CD20 in aortic adventitia of RA and non-RA patients with coronary artery disease (CAD) and to search for relationships between systemic IL-7 levels and expression of vascular markers, cardiovascular risk factors including metabolic and inflammatory parameters. Methods We examined 19 RA and 20 non-RA patients undergoing coronary artery bypass graft surgery included in the Feiring Heart Biopsy Study. Serum IL-7 levels were measured by chemiluminescence (MSD). Biopsies from the adventitia of thoracic aorta from a subset of patients (12 RA and 14 non-RA) were stained for IL-7, IL-7R, CD3 and CD20 by immunohistochemistry and scored per mm2 of tissue. Results Non-RA patients had lower IL-7 serum levels than RA (3.4±3.3 vs. 6.7±3.5, p Conclusions Among patients with CAD, those with RA had higher serum IL-7 and a greater expression of both IL-7 and IL-7R is aortic adventitia. Systemic levels of IL-7 were related to its vascular expression. Thus, the IL-7/IL-7R axis may play a role in the accelerated ATS observed in RA; further studies are needed to elucidate the precise role of IL-7 in CV risk in RA. References . Kaplan MJ. Curr. Opin. Rheumatol2006;18(3):289–297. . Damas JK, et al. Circulation2003;107(21):2670–2676. Acknowledgements None. Disclosure of interest None declared

Introduction The link between rheumatoid arthritis (RA) and cardiovascular disease (CVD) is well established but not yet fully understood. Anti-TNF treatment, e.g. with Enbrel, is one of most common therapies used in RA patients and has been shown to have beneficial effects on RA-associated cardiovascular pathologies in these patients. 1 Initial studies in the collagen-induced arthritis (CIA) model revealed an increase in cardiac fibrosis marker galectin-3, both locally in perivascular adipose tissue (PVAT) and systemically in blood plasma during inflammatory arthritis and correlated with impaired vascular function. 2 Objectives This study aims to investigate the impact of anti-TNF treatment on galectin-3 expression and vascular function during CIA. Methods Male DBA/1 mice underwent CIA induction and received either Enbrel or phosphate buffered saline (PBS) by intravenous injection. Vascular function was determined by measuring the constriction response of the thoracic aorta at termination using a myograph. Galectin-3 was measured locally in thoracic PVAT by immunohistochemistry and quantitative polymerise chain reaction (qPCR), and systemically by ELISA. Results Anti-TNF treatment during CIA resulted in a decrease in arthritis severity and progression compared to PBS treated mice, and partially restored vascular function. Galectin-3 expression was significantly reduced in thoracic PVAT, but not in blood plasma, in CIA mice treated with Enbrel. Conclusions In conclusion, decreased galectin-3 expression in Enbrel treated mice is associated with an improvement in arthritis-associated disease activity including arthritis clinical score, joint swelling and vascular function. References . Allanore Y, Avouac J. Cardiovascular risk in rheumatoid arthritis: Effects of anti-TNF drugs. Expert Opin Pharmacother2008May;9(7):1121–8. doi:10.1517/14656566.9.7.1121 . Sime K, Choy EH, Williams AS. Alterations to adipose tissue morphology during inflammatory arthritis is indicative of vasculopathology in DBA/1 mice. Adipocyte2017April 3;6(2):87–101. doi:10.1080/21623945.2017.1295174 (Accessed: 2017 February 21). Disclosure of interest None declared

In vivo mouse models of inflammatory arthritis are extensively used to investigate pathogenic mechanisms governing inflammation-driven joint damage. Two commonly utilized models include collagen-induced arthritis (CIA) and methylated bovine serum albumin (mBSA) antigen-induced arthritis (AIA). These offer unique advantages for modeling different aspects of human disease. CIA involves breach of immunological tolerance resulting in systemic autoantibody-driven arthritis, while AIA results in local resolving inflammatory flares and articular T cell-mediated damage. Despite limitations that apply to all animal models of human disease, CIA and AIA have been instrumental in identifying pathogenic mediators, immune cell subsets and stromal cell responses that determine disease onset, progression, and severity. Moreover, these models have enabled investigation of disease phases not easily studied in patients and have served as testing beds for novel biological therapies, including cytokine blockers and small molecule inhibitors of intracellular signaling that have revolutionized rheumatoid arthritis treatment.

Background Our research has shown that patients with RA have higher proportions of peripheral blood CD3+CD8+CD28- Treg cells compared to healthy individuals. CD3+CD8+CD28- Treg cells in patients with RA have lost their ability to suppress lymphocyte proliferation 1 . Thus CD28 negativity may mark senescent T cells. CD57 2 CD45RA 3,4 and killer-cell lectin like receptor G1 (KLRG1) 5 cell surface molecules have been associated with CD8+ T cell activation and senescence. Defining the phenotypic signature of CD8+CD28- Treg cells will help establish their significance in the immunoregulation of RA. Objectives To use immunofluorescence and flow cytometry to define the phenotype of CD3+CD8+CD28+/- cells in relation to early RA progression. Methods The effector characteristics of peripheral blood CD8 T cells were evaluated by flow cytometry. RA patients with established (n=21) and early disease (n=20) were recruited, and compared to twenty four healthy controls. The mean age of the subjects was 59 (SD=12.5), 25/38 (66%) were female, 27/38 (71%) were anti-CCP positive and 25/38 (66%) rheumatoid factor positive. The mean age for the controls was 43 (SD=11.6), 14/20 (70%) were female. Results Confirming our previous work, a significantly higher proportion of CD3+CD8+CD28- cells was observed in RA patients compared to healthy individuals (P=0.03) (Figure 1). Flow cytometric evaluation of peripheral blood demonstrated a significantly higher expression of CD57, CD45Ra and KLRG1 in CD28- compared to CD28+ T cells. A further evaluation of these markers revealed that 69% of the CD8+CD28- cell pool was KLRG1+, in comparison to 66% being CD57+ and 55% KLRG1+CD57+ double positive. This suggests that KLRG1 is a robust and clinically relevant marker of CD28- T cells in RA. Our current research is investigating the significance of KLRG1 and CD8+CD28- cells as prognostic markers in RA. Conclusions CD3+CD8+CD28- cells are enriched in the peripheral blood of RA patients. KLRG1 expression is increased in line with CD57 and CD45Ra in CD8+CD28- cells, and will be used to evaluate the functional significance of these cells in relation to their activation status and potential senescence in the immunoregulation of RA pathogenesis. References Ceeraz S, Hall C, Choy EH, Spencer J, Corrigall VM. Defective CD8+CD28+ regulatory T cell suppressor function in rheumatoid arthritis is restored by tumour necrosis factor inhibitor therapy. Clin Exp Immunol. 2013 Oct;174(1):18–26. Progressive decrease of CD8high+ CD28+ CD57- cells with ageing.Merino J, Martinez-Gonzalez MA, Rubio M, Inoges S, Sanchez-Ibarrola A, Subira ML Clin Exp Immunol. 1998 Apr; 112(1):48–51. CD28(-)CD8(+) T cells do not contain unique clonotypes and are therefore dispensable. Weinberger B, Welzl K, Herndler-Brandstetter D, Parson W, Grubeck-Loebenstein BImmunol Lett. 2009 Dec 2; 127(1):27–32. KLRG1 signaling induces defective Akt (ser473) phosphorylation and proliferative dysfunction of highly differentiated CD8+ T cells. Henson SM, Franzese O, Macaulay R, Libri V, Azevedo RI, Kiani-Alikhan S, Plunkett FJ, Masters JE, Jackson S, Griffiths SJ, Pircher HP, Soares MV, Akbar AN. Blood. 2009 Jun 25; 113(26):6619–28. Disclosure of Interest None declared

Background Cardiovascular (CV) disease is a major cause of mortality in patients with rheumatoid arthritis (RA). CV risk is increased early in the disease course. Subclinical inflammation and dyslipidaemia are often seen in RA before patients become symptomatic, suggesting the presence of subclinical CV disease. Inflammation, as measured by acute phase reactants, is associated with CV disease in RA. Interleukin (IL)-6 is a major driver of the acute phase response in RA. Notably, systemic elevations in inflammatory cytokines including IL-6 correlate with CV risk. Importantly, IL-6 regulates both immune homeostasis and inflammatory processes linked with chronic disease progression. Control of these processes is regulated by two modes of IL-6 signaling; classical IL-6 receptor signaling and IL-6 trans-signaling. Cellular responses controlled by IL-6 trans-signaling are mediated via soluble IL-6 receptor (sIL-6R) and is widely considered to promote deleterious pro-inflammatory outcomes [1]. We hypothesize that atherosclerosis may predate diagnosis of RA, and is accelerated by IL-6 trans-signaling during active arthritis. Here, we investigate this hypothesis using the established ApoE-deficient ( apoE -/- ) mouse model of atherosclerosis. Objectives To examine the effect of IL-6 classical and trans-signaling on atherosclerosis by administering IL-6 or Hyper-IL-6 (a IL-6: sIL-6R fusion protein) to apoE -/- mice. Methods Male apoE -/- mice were fed a high-fat diet for 8 weeks starting at 8 weeks of age. Mice were divided into 4 groups. Group 1 received IL-6 (160 ng twice weekly, delivered i.p.), and Group 2 and 3 received Hyper-IL-6 (500 ng and 1 μg delivered i.p. twice weekly) for 8 weeks. Group 4 received PBS twice weekly for 8 weeks. Serial transverse 7 um brachiocephalic artery cross-sections were cut and stained with haematoxylin and oil red-O. Lesion size was determined by computer-assisted morphometry, using Image J on stained sections. Brachiocephalic plaque size in mice treated with PBS, IL-6 and Hyper-IL6 were compared using ANOVA and post-hoc Tukey t test. Results Mice treated with Hyper-IL-6 1μg had significantly larger brachiocephalic plaques (mean plaque area 0.73±0.04 mm 2 ) than those administered PBS (0.018±0.01 mm 2 , p 2 , p =0.015; Fig.1A). Similarly, mice administered Hyper-IL-6 [1μg] had a significantly higher percentage of the brachiocephalic artery occupied by plaques (45.3±18.1%) compared to those administered PBS (10.38±6.7%, p p =0.002) (Fig.1B). Mice administered Hyper-IL-6 [0.5μg] had significantly higher percentage plaque (27.7±16.2%) than PBS administered mice, p =0.015. There was no significant difference in total cholesterol, HDL, LDL, triglycerides, free fatty acids or cholesterol:HDL ratio between the groups. Conclusions IL-6 trans-signaling leads to accelerated atherosclerosis in disease susceptible animals. This effect is independent of changes in serum lipid profiles. References Rose-John S. IL-6 Trans-Signaling via the Soluble IL-6 Receptor: Importance for the Pro-Inflammatory Activities of IL-6. Int. J. Biol. Sci. 2012;8(9):1237–1247. Acknowledgements This work was funded by Arthritis Research UK, grant number 20760. Disclosure of Interest None declared

Background Cardiovascular (CV) mortality in patients with rheumatoid arthritis (RA) is up to 50% higher than the general population. Whilst traditional CV risk factors such as smoking, diabetes and hypertension contribute to this increased mortality in RA, they do not fully explain the increase in risk. The British Society for Rheumatology, National Institute for Health and Clinical Excellence and European League Against Rheumatism (EULAR) recommend annual assessment of CV risk in RA patients. Furthermore, EULAR recommends multiplying such traditional CV risk scores by 1.5 for RA patients who meet two of three criteria consisting of (1) disease duration >10 years, (2) positive rheumatoid factor or anti-cyclic citrullinated peptide (anti-CCP) serology, (3) presence of severe extra-articular manifestation, to account for the unexplained increased CV risk in RA. Objectives This study assessed CV risk by QRISK2 score and used carotid ultrasound to determine cardiovascular risk and prevalence of subclinical atherosclerosis at the time of diagnosis of RA. Methods Patients ≥18 years-old with early RA, defined by ACR/EULAR 2010 criteria and diagnosis of RA Results 40 patients, 10 males and 30 females with early RA were recruited. Mean age was 55.7±14.8 years. Rheumatoid factor and anti-CCP antibodies were positive in 62% and 80% of patients respectively. Mean BMI was 27.2±5.9. Twenty-nine percent were current smokers. Mean DAS28 was 3.8±1.3. According to the QRISK2, 54% of patients had >10% risk of CV disease over 10 years. Carotid ultrasound was conducted in 35 patients. Mean CIMT was 0.71±0.19 mm. 10 patients (29%) had carotid plaques. Fourteen patients (40%) had either plaque or CIMT >0.9mm, both considered markers of high CV risk. These patients were termed “ultrasound positive” patients. The sensitivity and specificity of the QRISK2 to predict US positive patients was 82% and 56% respectively. The positive predictive value of the QRISK2 was 63% and the negative predictive value of the QRISK2 was 88%. No patients met >1 criteria for EULAR adjustment of risk as all patients had early RA and none had severe extra-articular manifestations. Conclusions Many patients with early RA have significant CV disease at the time of diagnosis. This suggests subclinical CV disease may be developing before patients become symptomatic. More than half of the patients with early RA fulfil current NICE guidelines for starting lipid-lowering therapy, although of these, over one-third had no subclinical disease on carotid ultrasound. There is scope to improve the sensitivity and specificity of the QRISK2 calculation in RA patients, perhaps with a biomarker. Acknowledgements This work was funded by Arthritis Research UK, grant number 20760 Disclosure of Interest None declared

Background The physiological function of adipose tissue is altered by the host’s inflammatory response. The implications for maintaining human health or causing inflammation-associated cardiovascular disease are ill defined. The purpose of this study was to determine morphological and molecular alterations to perivascular adipose tissue (PVAT) that functionally impair normal vascular function during inflammatory arthritis. Materials and methods Aorta (PVAT intact), renal and gonadal white adipose tissue (WAT) and brown adipose tissue (BAT) from the inter-scapular region were harvested from mice with established Collagen-induced arthritis (CIA) and age-matched naive controls. Morphological analyses were undertaken on paraffin-embedded tissue sections. Molecular markers for WAT, BAT and macrophages were measured by qPCR. Results Cell number increased significantly by 1.8 fold in all adipose (CIA versus naive mice). Adipocytes were compressed by CIA and voluminosity was significantly reduced in PVAT (2-fold) and BAT (4-fold) but not WAT. The classical M1 macrophage marker CD11c was significantly increase by CIA together with M2 markers such as Arg1 and CD206 in thoracic PVAT. iNOS expression (M1 marker) was not altered by CIA. Anti-TNF therapy reduced PVAT-associated inflammation and restored impaired constriction responses caused by CIA in excised aortae. Conclusions These data, reveal potentially important arthritis-associated mechanisms and tissue changes in PVAT and BAT that may be important in the initiation and progression of cardiovascular disease during inflammatory arthritis. We identify CIA as a relevant model to study the abnormal body composition phenotype that are overrepresented in patients with RA and RA-associated cardiovascular comorbidities.

Background In patients with rheumatoid arthritis (RA), osteolysis, osteopenia and osteoporosis are predictive of a severe disease phenotype. CCL3 is a chemokine associated with inflammatory osteolytic lesions and bone resorption by osteoclasts in patients with multiple myeloma. CCL3’s role in modulating bone destruction during inflammatory arthritis has not been established. The purpose of this study was to assess pharmacological blockade of CCL3 as a strategy to prevent or inhibit osteoclast-associated bone damage during inflammatory arthritis. Method Fully differentiated osteoclasts were formed from human CD14-positive mononuclear cell precursors cultured with macrophage-colony stimulating factor (m-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Collagen-induced arthritis (CIA) was selected for in vivo studies. Antibodies (anti-CCL3 and isotype control) were tested for their potency to modulate; (i) osteoclast differentiation and resorption of bone substrate in vitro and (ii) arthritis severity, synovial inflammation by histology and bone erosion by x-ray. Results The concentration of CCL3 in supernatants increased in a time-dependent manner over the 14-day osteoclast culture period (p Conclusion Targeted inhibition of CCL3 protect the synovial joint against arthritis-associated pathology by potent inhibition osteoclast formation and bone erosion. Our data may help guide future innovations for the diagnosis and treatment of inflammatory arthritis.

Introduction: Terminal ileum resection in patients with Crohn’s disease results in vitamin B12 deficiency and these patients are routinely given B12 supplementation to prevent anemia and neuropsychiatric disease. The effect of right hemicolectomy on B12 status in cancer patients has not previously been studied. Total serum B12 is an unreliable test. Second-line diagnostic tests measure methylmalonic acid (MMA) and homocysteine (tHcy) are also used. An assay measuring an active form of B12 is available for routine use. This observational study investigated whether B12 deficiency exists in patients after a right hemicolectomy for cancer. Methods: Patients (n=28; age range=52-88 years) who had undergone a right hemicolectomy for cancer and been in remission for two years were identified from a database and recruited to an outpatient clinic along with an age-matched control group (n=27; age range=52-89 years). Vitamin B12 status in both patients and controls was assessed by the measurement of vitamin B12 (total and active), MMA and tHcy. Results: Concentration of tHcy was significantly higher (p 0.05). Discussion: Right hemicolectomy for malignancy is associated with an increase in tHcy concentrations although a larger study is required to determine the B12 status in post-operative right hemicolectomy patients.

Objective: To investigate the role of death receptor 3 (DR-3) and its ligand tumor necrosis factor–like molecule 1A (TL1A) in the early stages of inflammatory arthritis. Methods: Antigen-induced arthritis (AIA) was generated in C57BL/6 mice deficient in the DR-3 gene (DR3−/−) and their DR3+/+ (wild-type) littermates by priming and intraarticular injection of methylated bovine serum albumin. The joints were sectioned and analyzed histochemically for damage to cartilage and expression of DR3, TL1A, Ly-6G (a marker for neutrophils), the gelatinase matrix metalloproteinase 9 (MMP-9), the aggrecanase ADAMTS-5, and the neutrophil chemoattractant CXCL1. In vitro production of MMP-9 was measured in cultures from fibroblasts, macrophages, and neutrophils following the addition of TL1A and other proinflammatory stimuli. Results: DR3 expression was up-regulated in the joints of wild-type mice following generation of AIA. DR3−/− mice were protected against cartilage damage compared with wild-type mice, even at early time points prior to the main accumulation of Teff cells in the joint. Early protection against AIA in vivo correlated with reduced levels of MMP-9. In vitro, neutrophils were major producers of MMP-9, while neutrophil numbers were reduced in the joints of DR3−/− mice. However, TL1A neither induced MMP-9 release nor affected the survival of neutrophils. Instead, reduced levels of CXCL1 were observed in the joints of DR3−/− mice. Conclusion: DR-3 drives early cartilage destruction in the AIA model of inflammatory arthritis through the release of CXCL1, maximizing neutrophil recruitment to the joint and leading to enhanced local production of cartilage-destroying enzymes.

Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza-induced inflammation. Based on the capacity of interleukin-6 (IL-6) to govern both optimal T-cell responses and inflammatory resolution, we hypothesised that IL-6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza-infected wild-type control and IL-6-deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL-6 displayed a profound defect in their ability to mount an anti-viral T-cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro-inflammatory cytokines, IFN-α and TNF-α. These events were associated with severe lung damage, characterised by profound vascular leakage and death. Our data highlight an essential role for IL-6 in orchestrating anti-viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology.

Background: Mortality is increased in RA patients, mainly due to cardiovascular (CV) disease; however, the biologic mechanisms are unknown. Increased CV risk in RA is attributed to both traditional risk factors and systemic inflammation. CV risk scores, even after modification as recommended by EULAR, underestimate risk in RA and thus there is a need to develop a better means of risk stratification. Murine collagen-induced arthritis (mCIA) is associated with vascular dysfunction, characterized by reduced vascular constriction to 5- hydroxytryptamine (5-HT). This study was undertaken to characterize the relationship between vascular cell adhesion molecule 1 (VCAM-1), which is induced in pro-atherosclerotic conditions in the general population and vascular dysfunction in mCIA and CV risk in RA patients. Methods: mCIA was induced in DBA/1 mice. The severity of arthritis was assessed by the arthritis index score. Constriction responses to 5- HT were used to assess vascular function in isolated sections of thoracic aorta. Serum VCAM-1 was measured with ELISA. Serum VCAM-1, IL-6, ESR and CRP were measured with ELISA in RA patients [182 patients, 4 female:1 male, mean age 60 years (range 21–89), mean disease duration 11.4 years (S.D. 11)]. CV risk was calculated using the Framingham risk calculator and the QRISK2 algorithm. The latter includes RA as an independent CV risk factor. Results: In mCIA, VCAM-1 levels [mean 1500 mg/ml range (929–2528)] correlated with the arthritis index score (r¼0.43, P¼0.03) and negatively correlated with maximal aortic contraction (r¼�0.35, P10% over 10 years [1200 ng/ ml (S.D. 547)] compared with those with

Introduction: Although rheumatoid arthritis (RA) is a disease of articular joints, patients often suffer from co-morbid neuropsychiatric changes, such as anxiety, that may reflect links between heightened systemic inflammation and abnormal regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we apply behavioral neuroscience methods to assess the impact of antigeninduced arthritis (AIA) on behavioral performance in wild type (WT) and interleukin-10 deficient (Il10-/-) mice. Our aim was to identify limb-specific motor impairments, as well as neuropsychological responses to inflammatory arthritis. Methods: Behavioral testing was performed longitudinally in WT and Il10-/- mice before and after the induction of arthritic joint pathology. Footprint analysis, beam walking and open field assessment determined a range of motor, exploratory and anxietyrelated parameters. Specific gene changes in HPA axis tissues were analyzed using qPCR. Results: Behavioral assessment revealed transient motor and exploratory impairments in mice receiving AIA, coinciding with joint swelling. Hind limb coordination deficits were independent of joint pathology. Behavioral impairments returned to baseline by 10 days post-AIA in WT mice. Il10-/- mice demonstrated comparable levels of swelling and joint pathology as WT mice up to 15 days post-AIA, but systemic differences were evident in mRNA expression in HPA axis tissues from Il10-/- mice post-AIA. Interestingly, the behavioral profile of Il10-/- mice revealed a significantly longer time post-AIA for activity and anxiety-related behaviors to recover. Conclusions: The novel application of sensitive behavioral tasks has enabled dissociation between behaviors that occur due to transient joint-specific pathology and those generated by more subtle systemic alterations that manifest post-AIA.

SummaryA 44-year-old alcoholic (and therefore immunocompromised) hospital cleaner presented with general malaise, weight loss, and erythematous skin nodules. Computed tomography scanning revealed a neck mass invading the thyroid gland, pulmonary infiltrates, liver lesions, and deposits on the anterior abdominal wall, consistent with disseminated malignancy. However, tissue diagnosis showed a necro-inflammatory process with no evidence of malignancy. Microscopy and culture of samples failed to detect any infectious pathogen, but after an extended incubation period, Finegoldia magna was isolated. This case study illustrates the importance of tissue diagnosis in suspected disseminated malignancy and raises the risk of acquiring the rarer bacteria amongst hospital staff.

NAMPT, also known as pre-B-cell colony-enhancing factor and visfatin, has been proposed to be involved in preventing apoptosis in cancer cells and, as such, has received a great deal of attention in recent years and stimulated the development to specific inhibitors for treating cancer. The role of NAMPT inhibitors as potential therapeutic agents for other diseases has not been studied extensively. Here, we describe their applicability for treating rheumatoid arthritis. We summarize current knowledge of NAMPT expression in healthy and diseased tissues, thereafter, we focus on pathological mechanisms relevant to rheumatoid arthritis that involve the NAMPT pathway and review the current status of NAMPT inhibitors being evaluated in clinical trials.

Death receptor 3 (DR3; TNFRSF25) and its tumor necrosis factor–like ligand TL1A (TNFSF15) control several processes in inflammatory diseases through the expansion of effector T cells and the induction of proinflammatory cytokines from myeloid and innate lymphoid cells. Using wild-type (DR3 +/+ ) and DR3-knockout (DR3 −/− ) mice, we show that the DR3/TL1A pathway triggers the release of multiple chemokines after acute peritoneal inflammation initiated by a single application of Staphylococcus epidermidis supernatant, correlating with the infiltration of multiple leukocyte subsets. In contrast, leukocyte infiltration was not DR3 dependent after viral challenge with murine cytomegalovirus. DR3 expression was recorded on connective tissue stroma, which provided DR3-dependent release of chemokine (C-C motif) ligand (CCL) 2, CCL7, CXCL1, and CXCL13. CCL3, CCL4, and CXCL10 production was also DR3 dependent, but quantitative RT-PCR showed that their derivation was not stromal. In vitro cultures identified resident macrophages as a DR3-dependent source of CCL3. Whether DR3 signaling could contribute to a related peritoneal pathology was then tested using multiple applications of S. epidermidis supernatant, the repetitive inflammatory episodes of which lead to peritoneal membrane thickening and collagen deposition. Unlike their DR3 +/+ counterparts, DR3 −/− mice did not develop fibrosis of the mesothelial layer. Thus, this work describes both a novel function and essential requirement for the DR3/TL1A pathway in acute, resolving, and chronic inflammation in the peritoneal cavity.

IL-6–mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6–mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.

Tumor necrosis factor (TNF)-like cytokine (TL1A) is a T-cell costimulator that bolsters cytokine-induced activation through death receptor 3 (DR3). To explore the relationship between T-cell activation and TL1A responsiveness, flow cytometry profiled DR3 expression in resting and activated T cells. In human CD4+ T cells, DR3 was induced rapidly following activation and expressed prominently by interleukin (IL)-17-secreting T cells (Th17). Splenic T cells from wild-type and DR3-deficient mice showed that TL1A activation of DR3 inhibits Th17 generation (81±2.6% at 100 ng/ml TL1A) from naive T cells. This response was not associated with suppression of T-cell proliferation. Using neutralizing antibodies or T cells derived from genetically modified mice, TL1A inhibition of Th17 development was found to be independent of IL-2, IL-27, γIFN, IFNAR1, and STAT1. Under suboptimal TCR activation, TL1A continued to block IL-17A secretion, however, the reduced threshold of TCR engagement was now linked with an increase in TL1A-driven proliferation. In contrast, fully committed Th17 cells displayed an altered TL1A responsiveness and in the absence of TCR costimulation supported the maintenance of T cell IL-17A expression. Consequently, TL1A orchestrates unique outcomes in naive and effector T-helper cells, which may affect the proliferation, differentiation and maintenance of Th17 cells in peripheral compartments and inflamed tissues.—Jones, G. W., Stumhofer, J. S., Foster, T., Twohig, J.P., Hertzog, P., Topley, N., Williams, A. S., Hunter, C. A., Jenkins, B. J., Wang, E. C. Y., Jones, S. A. Naive and activated T cells display differential responsiveness to TL1A that affects Th17 generation, maintenance, and proliferation.

It is clear that CD4+ CD25+ Foxp3+ regulatory T (Treg) cells inhibit chronic inflammatory responses as well as adaptive immune responses. Among the CD4+ T-cell population in the skin, at least one-fifth express Foxp3. As the skin is constantly exposed to antigenic challenge and is a common site of vaccination, understanding the role of these skin-resident Treg cells is important. Although the suppressive effect of Treg cells on T cells is well documented, less is known about the types of innate immune cells influenced by Treg cells and whether the Treg cells suppress acute innate immune responses in vivo. To address this we used a mouse melanoma cell line expressing Fas ligand (B16FasL), which induces an inflammatory response following subcutaneous injection of mice. We demonstrate that Treg cells limit this response by inhibiting neutrophil accumulation and survival within hours of tumour cell inoculation. This effect, which was associated with decreased expression of the neutrophil chemoattractants CXCL1 and CXCL2, promoted survival of the inoculated tumour cells. Overall, these data imply that Treg cells in the skin are rapidly mobilized and that this activity serves to limit the amplification of inflammatory responses at this site.

Despite its vital role in innate immunity, complement is involved in a number of inflammatory pathologies and has therefore become a therapeutic target. Most agents generated for anti-complement therapy have short half-lives in plasma, or have been of mouse or human origin, thereby limiting their use either to murine models of disease or to short-term therapy. Here we describe the generation of a long-acting rat therapeutic agent based on the rat complement inhibitor, Crry. Characterisation of various soluble forms of Crry demonstrated that the amino-terminal four short-consensus repeat domains were required for full regulatory and C3b-binding activities. Fusion of these domains to rat IgG2a Fc generated an effective complement inhibitor (rCrry-Ig) with a circulating half-life prolonged from 7 min for Crry alone to 53 h for rCrry-Ig. Systemic administration of rCrry-Ig over 5 weeks generated a weak immune response to the recombinant agent, however this was predominantly IgM in nature and did not neutralise Crry function or cause clearance of the agent from plasma. Administration of rCrry-Ig completely abrogated clinical disease in a rat model of myasthenia gravis whereas soluble Crry lacking the immunoglobulin Fc domain caused a partial response. rCrry-Ig not only ablated clinical disease, but also prevented C3 and C9 deposition at the neuromuscular junction and inhibited cellular infiltration at this site. The long half-life and low immunogenicity of this agent will be useful for therapy in chronic models of inflammatory disease in the rat.

Influenza-specific immune activity not only promotes virus clearance but also causes immunopathology, thereby underlining the importance of mounting a measured anti-viral immune response. Since complement bridges both the innate and adaptive immune systems and has been implicated in defence against influenza, the role of the complement regulator CD59a in modulating the response to influenza was explored. For this purpose, immune responses to influenza virus, strain E61-13-H17, in mice deficient in the complement regulator protein CD59a (Cd59a(-/-) mice) were compared to those in wild-type mice. The severity of lung inflammation was significantly enhanced in the lungs of Cd59a(-/-) mice with increased numbers of infiltrating neutrophils and CD4(+) T cells. When complement was inhibited using soluble complement receptor 1, the frequency of lung-infiltrating neutrophils in influenza-infected Cd59a(-/-) mice was much reduced whilst numbers of CD4(+) T cells remained unchanged. These results demonstrate that CD59a, previously defined as a complement regulator, modulates both the innate and adaptive immune response to influenza virus by both complement-dependent and -independent mechanisms.

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

OBJECTIVE: Local interaction between soluble mediators within the inflamed synovium is a key factor that governs the pathologic outcome of inflammatory arthritides. Our aim was to investigate the interplay between the Th1 lymphokine interferon-gamma (IFNgamma) and pivotal cytokines that drive rheumatoid arthritis (RA) pathology (interleukin-1beta [IL-1beta] and tumor necrosis factor alpha [TNFalpha]) in modulating inflammation and arthritis in vitro and in vivo. METHODS: Monarticular antigen-induced arthritis (AIA) was initiated in IFNgamma-deficient (IFNgamma(-/-)) mice and age-matched wild-type (IFNgamma(+/+)) mice. Joint swelling was measured and histologic analysis was performed in order to assess changes in both inflammatory and degenerative parameters in vivo. In vitro, the influence of IFNgamma in regulating IL-1beta- and TNFalpha-driven CXCL8 and CCL2 production was quantified by enzyme-linked immunosorbent assay. RESULTS: In murine AIA, both inflammatory and degenerative arthritis parameters were significantly exacerbated in the absence of IFNgamma. IFNgamma appeared to be a crucial factor in regulating CXCR2+ neutrophil influx in the joint. In in vitro studies using RA fibroblast-like synoviocytes, IFNgamma modulated both IL-1beta- and TNFalpha-driven chemokine synthesis, resulting in the down-regulation of CXCL8 production. CONCLUSION: IFNgamma exerts antiinflammatory, chondroprotective, and antiosteoclastogenic effects in murine AIA through a mechanism that involves the regulation of chemokine synthesis and local neutrophil recruitment. These studies suggest a potential therapeutic role of modulating IFNgamma signaling in the treatment of inflammatory arthritides.

Ectopic lymphoid-like structures (ELSs) reminiscent of secondary lymphoid organs often develop at sites of chronic inflammation where they contribute to immune-mediated pathology. Through evaluation of synovial tissues from rheumatoid arthritis (RA) patients, we now show that low interleukin-27 (IL-27) expression corresponds with an increased incidence of ELS and gene signatures associated with their development and activity. The presence of synovial ELS was also noted in mice deficient in the IL-27 receptor (IL-27R) after the onset of inflammatory arthritis. Here, pathology was associated with increased synovial expression of pro-inflammatory cytokines, homeostatic chemokines, and transcriptional regulators linked with lymphoid neogenesis. In both clinical and experimental RA, synovial ELS coincided with the heightened local expression of cytokines and transcription factors of the Th17 and T follicular helper (Tfh) cell lineages, and included podoplanin-expressing T cells within lymphoid aggregates. IL-27 inhibited the differentiation of podoplanin-expressing Th17 cells, and an increased number of these cells were observed in IL-27R–deficient mice with inflammatory arthritis. Thus, IL-27 appears to negatively regulate ELS development in RA through control of effector T cells. These studies open new opportunities for patient stratification and treatment.

Human HtrA1 belongs to a widely conserved family of serine proteases involved in various aspects of protein quality control and cell fate. Although HtrA1 has been implicated in the pathology of several diseases, its precise biological functions remain to be established. Through identification of potential HtrA1 targets, studies presented herein propose that within the context of arthritis pathology HtrA1 contributes to cartilage degradation. Elevated synovial HtrA1 levels were detected in fluids obtained from rheumatoid and osteoarthritis patients, with synovial fibroblasts identified as a major source of secreted HtrA1. Mass spectrometry analysis of potential HtrA1 substrates within synovial fluids identified fibronectin as a candidate target, and treatment of fibronectin with recombinant HtrA1 led to the generation of fibronectin-degradation products that may be involved in cartilage catabolism. Consistently, treatment of synovial fibroblasts with HtrA1 or HtrA1-generated fibronectin fragments resulted in the specific induction of matrix metalloprotease 1 and matrix metalloprotease 3 expression, suggesting that HtrA1 contributes to the destruction of extracellular matrix through both direct and indirect mechanisms.

ObjectivesSynovial fluid glutamate concentrations increase in arthritis. Activation of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) increase interleukin-6 (IL-6) release and cause arthritic pain, respectively. We hypothesised that AMPA and KA GluRs are expressed in human arthritis, and that intra-articular NBQX (AMPA/KA GluR antagonist) prevents pain and pathology in antigen-induced arthritis (AIA).MethodsGluR immunohistochemistry was related to synovial inflammation and degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). A single intra-articular NBQX injection was given at induction, and knee swelling and gait of AIA and AIA+NBQX rats compared over 21 days, before imaging, RT-qPCR, histology and immunohistochemistry of joints. Effects of NBQX on human primary osteoblast (HOB) activity were determined.ResultsAMPAR2 and KA1 immunolocalised to remodelling bone, cartilage and synovial cells in human OA and RA, and rat AIA. All arthritic tissues showed degradation and synovial inflammation. NBQX reduced GluR abundance, knee swelling (pConclusionsAMPA/KA GluRs are expressed in human OA and RA, and in AIA, where a single intra-articular injection of NBQX reduced swelling by 33%, and inflammation and degeneration scores by 34% and 27%, respectively, exceeding the efficacy of approved drugs in the same model. AMPA/KA GluR antagonists represent a potential treatment for arthritis.

Interleukin (IL)-6 signaling through its soluble receptor (IL-6 transsignaling) directs transition between innate and acquired immune responses by orchestrating the chemokine-directed attraction and apoptotic clearance of leukocytes. Through analysis of mononuclear cell infiltration in WT and IL-6-deficient mice during peritoneal inflammation, we now report that IL-6 selectively governs T cell infiltration by regulating chemokine secretion (CXCL10, CCL4, CCL5, CCL11, and CCL17) and chemokine receptor (CCR3, CCR4, CCR5, and CXCR3) expression on the CD3+infiltrate. Although blockade of IL-6 trans-signaling prevented chemokine release, chemokine receptor expression remained unaltered suggesting that this response is regulated by IL-6 itself. To dissect the signaling events promoting T cell migration, inflammation was established in knock-in mice expressing mutated forms of the universal signal-transducing element for IL-6-related cytokines gp130. In mice (gp130Y757F/Y757F) deficient in SHP2 and SOCS3 binding, but presenting hyperactivation of STAT1/3, T cell recruitment and CCL5 expression was enhanced. Conversely, both of these parameters were suppressed in mice with ablated gp130-mediated STAT1/3 activation (gp130ΔSTAT/ΔSTAT). T cell migration was related to STAT3 activity, because monoallelic deletion ofStat3ingp130Y757F/Y757Fmice (gp130Y757F/Y757F:Stat3+/-) corrected the exaggerated responses observed ingp130Y757F/Y757Fmice. Consequently, STAT3 plays a defining role in IL-6-mediated T cell migration.

Inappropriate activation of complement contributes to pathology in diverse inflammatory diseases. Soluble recombinant forms of the natural cell membrane regulators of complement are effective in animal models and some human diseases. However, their use is limited for reasons related to cost, short half lives, and propensity to cause unwanted systemic effects. Some of these limitations may be overcome by use of bacterial expression systems, specific targeting moieties, and judicious choice of regulator. Here we describe the application of these strategies to the generation of a membrane-targeted form of CD59. A recombinant soluble form of rat CD59, comprising the first 71 residues of the mature protein and missing the membrane-anchoring signal, was expressed in bacteria, purified, and refolded in a fully active form. The protein was coupled through its carboxyl terminus to a short, synthetic address tag that confers membrane binding activity. Attachment of the membrane address tag markedly increased complement-inhibitory activity assessed in vitro in hemolysis assays. Intra-articular administration of the tagged agent markedly suppressed disease in a model of rheumatoid arthritis in Lewis rats. This novel type of agent, termed sCD59-APT542, offers for the first time the prospect of efficient and specific inhibition of membrane attack complex activity in vivo.

Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.

Regulated recruitment and clearance of neutrophils (PMN) is the hallmark of competent host defense and resolution of inflammation. We now report that IFN-gamma controls PMN infiltration and modulates IL-6 signaling through its soluble receptor (sIL-6R) to promote their apoptosis and clearance. Induction of peritoneal inflammation in IFN-gamma-deficient (IFN-gamma-/-) mice emphasized that the initial rate of PMN recruitment was impaired. This defect in PMN recruitment was also associated with the suppressed intraperitoneal expression of IL-1beta and IL-6. Reconstitution of IFN-gamma signaling restored the rate of PMN infiltration and IL-6 levels and was accompanied by normalization of PMN-activating CXC chemokine expression. To test whether local IL-6 signaling modulated PMN recruitment, inflammation was induced in IFN-gamma-/- and IL-6-/- mice and cytokine signaling adapted by intraperitoneal sIL-6R-IL-6 fusion protein (HYPER-IL-6) or IFN-gamma. Although HYPER-IL-6 attenuated PMN influx in IFN-gamma-/- mice, IFN-gamma had no effect on PMN infiltration in IL-6-/- mice. Examination of the leukocyte infiltrate from IFN-gamma-/-, IL-6-/-, and wild-type mice showed that apoptosis was aberrant in the absence of IFN-gamma and IL-6 as a result of impaired sIL-6R signaling. These data emphasize a pivotal role for IFN-gamma in regulating innate immunity through control of both the recruitment and clearance phases of PMN trafficking.

SUMMARY Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function.

Introduction Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology. Methods Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays. Results In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis. Conclusions These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis. Electronic supplementary material The online version of this article (doi:10.1186/s13075-014-0419-y) contains supplementary material, which is available to authorized users.

SUMMARYWe set out to determine whether inhibition of complement using sCR1 could influence the development and progression of collagen arthritis in the Lewis rat. Collagen arthritis was successfully established in the Lewis rat, using a novel immunization schedule. In separate experiments, cobra venom factor (CVF) and sCR1 were used to achieve systemic complement inhibition. Their respective effects on disease onset and on the progression of established disease compared with saline-treated control animals was explored. Arthritis was assessed by measurement of clinical score, paw diameter and paw volume. Complement inhibition using either CVF or sCR1, prior to the onset of clinical signs of inflammation, delayed the development of disease. CVF was ineffective in the treatment of established disease, whereas sCR1 delayed the progression of disease in affected joints and prevented the recruitment of further joints while the animals were complement-depleted. In the control saline-treated groups the disease continued to progress relentlessly. We conclude that complement activation is important in the initiation and maintenance of inflammation in collagen arthritis. The potent disease-modulating effect of sCR1 provides persuasive evidence that specific complement inhibiting agents may be an effective approach to the treatment of inflammatory joint diseases