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ObjectivesIncreasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation.MethodsT cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes.ResultsSignificant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio.ConclusionsInteraction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders.

Background: The use of the vancomycin wrap to pretreat the hamstring graft in anterior cruciate ligament reconstruction (ACLR) has grown in popularity since it was first described in 2012 and has significantly reduced rates of postoperative infection. However, it remains unknown if this antibiotic treatment affects the molecular composition of the graft. Purpose: To establish whether treatment with vancomycin at 5 mg/mL, the most commonly used concentration, alters the molecular function of the hamstring graft in ACLR. Study Design: Controlled laboratory study. Methods: Surplus hamstring tendon collected after routine ACLR surgery was used for in vitro cell culture and ex vivo tissue experiments. Vancomycin was used at 5 mg/mL in RPMI or saline diluent to treat cells and tendon tissue, respectively, with diluent control conditions. Cell viability at 30, 60, and 120 minutes was assessed via colorimetric viability assay. Tendon cells treated with control and experimental conditions for 1 hour was evaluated using semiquantitative reverse transcription analysis, immunohistochemistry staining, and protein quantitation via enzyme-linked immunosorbent assay for changes in apoptotic, matrix, and inflammatory gene and protein expression. Results: Vancomycin treatment at 5 mg/mL significantly reduced tenocyte viability in vitro after 60 minutes of treatment ( P < .05); however, this was not sustained at 120 minutes. Vancomycin-treated tendon tissue showed no significant increase in apoptotic gene expression, or apoptotic protein levels in tissue or supernatant, ex vivo. Vancomycin was associated with a reduction in inflammatory proteins from treated tendon supernatants (IL-6; P < .05). Conclusion: Vancomycin did not significantly alter the molecular structure of the hamstring graft. Reductions in matrix protein and inflammatory cytokine release point to a potential beneficial effect of vancomycin in generating a homeostatic environment. Clinical Relevance: Vancomycin ACL wrap does not alter the molecular structure of the ACL hamstring graft and may improve graft integrity.

Significance Frozen shoulder (FS) is a classic example of a prevalent debilitating pathological inflammatory fibrosis. Using approaches to dissect the molecular pathways subserving FS, we demonstrate the immune cell landscape in FS shifts from primarily macrophages to T cells in disease. We find T cells from FS tissue are able to secrete the inflammatory cytokine IL-17A, and fibroblasts from FS have greater expression of the receptor IL-17RA. Thus, FS fibroblasts produce greater fibrotic and inflammatory responses following IL-17A stimulation, which can be abrogated following NF-κB pathway inhibition or cytokine blockade using an anti–IL-17A antibody. This single-cell dissection of FS highlights a T cell–driven disease with both small molecule and anti-cytokine attenuation of disease-like features., Frozen shoulder is a common fibroproliferative disease characterized by the insidious onset of pain and restricted range of shoulder movement with a significant socioeconomic impact. The pathophysiological mechanisms responsible for chronic inflammation and matrix remodeling in this prevalent fibrotic disorder remain unclear; however, increasing evidence implicates dysregulated immunobiology. IL-17A is a key cytokine associated with inflammation and tissue remodeling in numerous musculoskeletal diseases, and thus, we sought to determine the role of IL-17A in the immunopathogenesis of frozen shoulder. We demonstrate an immune cell landscape that switches from a predominantly macrophage population in nondiseased tissue to a T cell–rich environment in disease. Furthermore, we observed a subpopulation of IL-17A–producing T cells capable of inducing profibrotic and inflammatory responses in diseased fibroblasts through enhanced expression of the signaling receptor IL-17RA, rendering diseased cells more sensitive to IL-17A. We further established that the effects of IL-17A on diseased fibroblasts was TRAF-6/NF-κB dependent and could be inhibited by treatment with an IKKβ inhibitor or anti–IL-17A antibody. Accordingly, targeting of the IL-17A pathway may provide future therapeutic approaches to the management of this common, debilitating disease.

Tendinopathy is a highly prevalent musculoskeletal pathology associated with incremental injury as result of repetitive microtrauma. We sought to explore the physiological significance of stromal “activation” signatures in a human model of tendinopathy. Torn supraspinatus tendon and matched intact subscapularis tendon biopsies were collected from patients undergoing shoulder surgery while healthy tendon was collected from patients undergoing anterior cruciate ligament (ACL) reconstruction. Expression of stromal activation markers was analyzed at transcript/protein level using qRT‐PCR and immunohistochemistry. Gene expression of stromal activation markers was silenced by siRNA‐mediated knockdown or induced by IL‐1β stimulation. Expression of “activation” markers podoplanin, VCAM‐1 and CD248 was identified in human tendon tissue. Podoplanin and VCAM‐1 expression were significantly increased in tendinopathic tissue. Knockdown of podoplanin and VCAM‐1 in normal and tendinopathic tenocytes did not have any significant effect on expression of matrix genes COL1A1, COL3A1, TNC, or DCN. Similarly, no changes in release of inflammatory mediators IL‐6, IL‐8, and CCL2 were observed in podoplanin/VCAM‐1 knockdown cultures. Our data suggest that silencing expression of stromal “activation” markers does not affect the intrinsic inflammatory profile or matrix regulatory behavior of tenocytes. We propose that the term “activation” is more appropriately reflected by alterations in tenocyte behavior that induce changes in the stromal microenvironment and overall tissue architecture rather than identification through potentially arbitrary phenotypic traits.

We previously showed that exposure to a high-sugar and moderate-fat diet (i.e., Western diet [WD]) in mice induces appreciable skin inflammation and enhances the susceptibility to imiquimod-induced psoriasiform dermatitis, suggesting that dietary components may render the skin susceptible to psoriatic inflammation. In this study, utilizing an IL-23 minicircle-based model with features of both psoriasiform dermatitis and psoriatic arthritis, we showed that intake of WD for 10 weeks predisposed mice not only to skin but also to joint inflammation. Both WD-induced skin and joint injuries were associated with an expansion of IL-17A‒producing γδ T cells and increased expression of T helper type 17 cytokines. After IL-23 minicircle delivery, WD-fed mice had reduced microbial diversity and pronounced dysbiosis. Treatment with broad-spectrum antibiotics suppressed IL-23‒mediated skin and joint inflammation in the WD-fed mice. Strikingly, reduced skin and joint inflammation with a partial reversion of the gut microbiota were noted when mice switched from a WD to a standard diet after IL-23 minicircle delivery. These findings reveal that a short-term WD intake‒induced dysbiosis is accompanied by enhanced psoriasis-like skin and joint inflammation. Modifications toward a healthier dietary pattern should be considered in patients with psoriatic skin and/or joint disease.

OBJECTIVE To assess the involvement of the CCR6/CCL20 axis in psoriatic arthritis (PsA) and psoriasis (PsO) and to evaluate its potential as a therapeutic target. METHODS First, we quantified CCL20 levels in peripheral blood and synovial fluid from PsA patients and examined the presence of CCR6+ cells in synovial and tendon tissue. Utilizing an interleukin-23 minicircle DNA (IL-23 MC) mouse model exhibiting key features of both PsO and PsA, we investigated CCR6 and CCL20 expression as well as the preventive and therapeutic effect of CCL20 blockade. Healthy tendon stromal cells were stimulated in vitro with IL-1β to assess the production of CCL20 by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of conditioned media from stimulated tenocytes in inducing T cell migration was interrogated using a Transwell system. RESULTS We observed an up-regulation of both CCR6 and CCL20 in the enthesis of IL-23 MC-treated mice, which was confirmed in human biopsy specimens. Specific targeting of the CCR6/CCL20 axis with a CCL20 locked dimer (CCL20LD) blocked entheseal inflammation, leading to profound reductions in clinical and proinflammatory markers in the joints and skin of IL-23 MC-treated mice. The stromal compartment in the tendon was the main source of CCL20 in this model and, accordingly, in vitro activated human tendon cells were able to produce this chemokine and to induce CCR6+ T cell migration, the latter of which could be blocked by CCL20LD. CONCLUSION Our study highlights the pathogenic role of the CCR6/CCL20 axis in enthesitis and introduces the prospect of a novel therapeutic approach for treating patients with PsO and PsA.

Background Mechanical stress or damage is a well-known inducer of inflammation in both psoriasis and psoriatic arthritis (PsA). The occurrence of microtrauma at enthesial sites, areas subjected to high mechanic stress, could explain the development of local inflammation (enthesitis) that further extends to the synovial tissue through what it is known synovio-entheseal complex. Current treatment strategies mainly target the immune compartment, however there is growing evidence for the role of the stroma in the development of chronic inflammation. Increasing attention has focused on the interaction between the stroma and immune system and its role in the initiation/development of tissue inflammatory chronicity. Our hypothesis is that stromal cells in the tendon or tenocytes, once activated, are able to recruit and activate T cells into the tendon, which in turn may have an effect on the stroma, altogether leading to chronicity. Methods We assessed the effect of damage on healthy tenocytes after stimulation with conditioned media from tendon explants or IL-1β by qPCR and ELISA. A transwell membrane system was used to test the impact of conditioned media from tenocytes on T cell migration. T cells and tenocytes were co-cultured with or without the presence of a transwell membrane to quantify T cell activation (CD69 by FACS and IFN-γ by ELISA). Changes in gene expression on tenocytes after co-culture with activated T cells were analysed by qPCR. Results In the presence of damage, tenocytes upregulated inflammatory cytokines (IL-1β, IL-6), chemokines (IL-8, CCL2, CCL5, CXCL10) and adhesion molecules (ICAM-1). Of interest, we observed an upregulation of CCL20, both at transcript and protein level. Conditioned media from tenocytes induced T cell migration, especially after stimulation. Co-culture of tenocytes and T cells resulted in contact dependant activation of T cells. These activated T cells also had an effect on tenocytes, further upregulating the production of inflammatory mediators. Conclusion These results support the role of the tendon stromal compartment in the recruitment and activation of T cells, creating a feedback loop that could be involved in the maintenance of the inflammatory process and the development of chronicity in the context of PsA. Moreover, the production of CCL20 by tenocytes after damage could explain the preferential recruitment of Th17 or gamma delta T cells into the tendon. We are further investigating the mechanisms that govern this relationship that could be targeted therapeutically in the future. Disclosures E. Garcia-Melchor None. G. Cafaro None. S. Sood None. L.A.N. Crowe None. M. McLean None. I.B. McInnes None. M. Akbar None. N.L. Millar None.

Targeting STAT pathways may offer previously unidentified approaches in the management of fibrotic disorders., Fibrotic disorders represent common complex disease pathologies that are therapeutically challenging. Inflammation is associated with numerous fibrotic pathogeneses; however, its role in the multifaceted mechanisms of fibrosis remains unclear. IL-13 is implicated in aberrant responses involved in fibrotic disease, and we aimed to understand its role in the inflammatory processes of a common fibrotic disorder, Dupuytren’s disease. We demonstrated T-cells produced IFN-g, which induced IL-13 secretion from mast cells and up-regulated IL-13Ra1 on fibroblasts, rendering them more reactive to IL-13. Consequently, diseased myofibroblasts demonstrated enhanced fibroproliferative effects upon IL-13 stimulation. We established IFN-g and IL-13 responses involved STAT dependent pathways, and STAT targeting (tofacitinib) could inhibit IL-13 production from mast cells, IL-13Ra1 up-regulation in fibroblasts and fibroproliferative effects of IL-13 on diseased myofibroblasts. Accordingly, utilizing Dupuytren’s as an accessible human model of fibrosis, we propose targeting STAT pathways may offer previously unidentified therapeutic approaches in the management of fibrotic disease.

CCR6 is important for the trafficking of IL-17A–producing γδ T cells and required for the development of psoriasiform dermatitis in an IL-23 intradermal injection model. The role of CCR6, however, in IL-23–mediated joint inflammation is unclear. We herein hydrodynamically delivered IL-23 minicircle DNA into wild-type and CCR6-deficient (CCR6-knockout) mice to induce overexpression of IL-23 systemically. After IL-23 gene transfer, wild-type mice exhibited concurrent skin and joint changes that recapitulate some features found in human psoriatic skin and joints. CCR6-knockout mice were resistant to IL-23–induced skin inflammation but exhibited no changes in joint inflammation compared with wild-type mice. Depletion of neutrophils protected wild-type mice from skin and joint disease without suppressing T helper type 17 cytokine expression. In contrast, mice lacking γδ T cells showed a partial reduction in neutrophilic recruitment and a significant decrease in IL-17A expression in skin and paw tissue. Thus, in an IL-23–mediated model that allows concurrent assessment of both skin and joint disease, we showed that CCR6 is critical for inflammation in the skin but not in the joint. Furthermore, our data suggest that neutrophils and γδ T cells are key effector cells in IL-23–mediated skin and joint inflammation in mice.

Background Enthesitis is a hallmark of spondyloarthropaties[1], with mechanical stress or damage in the tendon being proposed as a trigger for the development of inflammation at the enthesis that propagates to the synovial compartment through what has been termed “synovio-enthesial complex”[2]. Increasing evidence supports the role that stromal cells play in the shift of the inflammatory process towards chronicity promoting T cell migration, retention and survival[3]. Therefore, we hypothesize that after tendon damage the crosstalk between stromal and immune compartments contributes to the development of chronic inflammation. Objectives We aimed to assess the effect of tendon stromal cells (tenocytes) on T cell migration and activation and the impact of these activated T cells on the stroma. Methods Tenocytes were explanted from tissue obtained from anterior cruciate ligament (ACL) reconstructions. The effect of damage on tenocytes after stimulation with conditioned media from tendon explants or IL-1s was evaluated by qPCR. A transwell membrane system was used to test the impact of conditioned media from tenocytes on T cell migration. T cells and tenocytes were co-cultured with or without the presence of a transwell membrane to quantify T cell activation (CD69 by FACS and IFN-γ by ELISA). Changes in gene expression on tenocytes after co-culture with activated T cells were analysed by qPCR. Results In the presence of damage, tenocytes upregulated inflammatory mediators (IL-6, COX2), chemokines (CCL2, CCL5, CXCL10, CXCL12) and adhesion molecules (ICAM-1). Conditioned media, particularly after stimulation with IL-1s, from tenocytes induced T cell migration. Co-cultures of tenocytes and T cells resulted in activation of T cells that was contact dependant. In turn, these activated T cells upregulated the production of inflammatory mediators in tenocytes and increased the COL3/COL1 ratio. Conclusion Our results support a communication between the stromal and immune compartment within the tendon that could be involved in the progression towards chronicity in the context of spondyloarthritis. Following damage, tendon stromal cells are able to induce the recruitment of T cells, that once enter the tissue interact with the stroma. Stromal cells are then further activated to produce inflammatory cytokines and chemokines that amplify and maintain this inflammatory response. References [1] G. Schett, et al., “Enthesitis: from pathophysiology to treatment,” Nat. Rev. Rheumatol., vol. 13, no. 12, pp. 731–741, 2017. [2] D. McGonagle, S. Z. Aydin, and A. L. Tan, “The synovio-entheseal complex and its role in tendon and capsular associated inflammation,” J. Rheumatol., vol. 39, no. SUPPL. 89, pp. 11–14, 2012. [3] C. D. Buckley, “Why does chronic inflammation persist: An unexpected role for fibroblasts,” Immunol. Lett., vol. 138, no. 1, pp. 12–14, 2011. Disclosure of Interests Emma Garcia-Melchor: None declared, Giacomo Cafaro: None declared, Lindsay AN Crowe: None declared, Michael McLean: None declared, James H Reilly: None declared, Iain McInnes Grant/research support from: AstraZeneca, Celgene, Compugen, Novartis, Roche, UCB Pharma, Consultant for: AbbVie, Celgene, Galvani, Lilly, Novartis, Pfizer, UCB Pharma, Moeed Akbar: None declared, Neal L Millar: None declared

Introduction: \ud Frozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement.\ud \ud Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the fibroblast compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder,\ud \ud Methods: \ud Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Fibroblast activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1β upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR.\ud \ud Results: \ud Immunohistochemistry demonstrated increased expression of fibroblast activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM).\ud \ud Conclusions: \ud These results show that fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease.

IntroductionFrozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement. Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the stromal compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder,MethodsShoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Stromal activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1β upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR.ResultsImmunohistochemistry demonstrated increased expression of stromal activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM).ConclusionsThese results show that stromal fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated stromal fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

Background Although the role of IL-23/Th17 axis in psoriatic arthritis (PsA) is well known1, little data is available on the contribution of stromal cells to this pathway2. Enthesitis is a common feature of PsA and it may represent the site of onset3,4, suggesting tendon stromal cells (tenocytes) may have an initiating role in Th17 driven pathogenesis. Objectives To assess the ability of stromal cells to produce CCL20, a chemokine able to recruit Th17 cells, and to induce T cell migration. Methods Healthy tenocytes cultured from hamstring tendons and fibroblast-like synoviocytes (FLS) from PsA patients were stimulated with human recombinant IL-1β (1 ng/ml) and IL-17A (1, 10 and 100 ng/ml). Expression of CCL20 transcript and protein were assessed by quantitative PCR and ELISA, respectively. T cell migration assays were performed with magnetically enriched CD3+ cells from peripheral blood of PsA patients and healthy controls using a Transwell system. Following incubation with conditioned media from stimulated stromal cells, the migrated cells were harvested and analysed via light microscopy and flow cytometry. Results Both tenocytes and FLS were able to produce CCL20 following stimulation with IL-1β. Furthermore, the addition of IL-17A induced a synergistic effect with IL-1β. Following cytokine stimulation, diseased stromal cells produced greater levels of CCL20 compared to stimulated healthy tenocytes. In addition, conditioned media from stimulated tenocytes promoted T cell migration, compared with supernatants from unstimulated tenocytes. Conclusions We have shown that tendon and PsA synovium stromal cells are able to produce CCL20 and induce T cell recruitment, suggesting a role in the chemotaxis of Th17 cells. The positive feedback observed with IL1β and IL-17A suggests a close relationship between stromal cells and Th17 cells. References [1] Frleta M, Siebert S, McInnes IB. Curr. Rheumatol. Rep. 2014;16:414. [2] Millar NL, Murrell GAC, McInnes IB. Nat. Rev. Rheumatol. 2017;13:110–122. [3] Benjamin M, McGonagle D. Adv. Exp. Med. Biol. 2009;649:57–70. [4] Sherlock JP, et al. Nat. Med. 2012;18:1069–1076. Acknowledgements Giacomo Cafaro was supported by Partner Fellowship and EULAR Fellowship Disclosure of Interest None declared

Background: The pathophysiological mechanisms behind proliferation of fibroblasts and deposition of dense collagen matrix in idiopathic frozen shoulder remain unclear. Alarmins (also known as danger signals) are endogenous molecules that are released into the extracellular milieu after infection or tissue injury and that signal cell and tissue damage. Purpose: To investigate whether the presence of alarmins is higher in patients with idiopathic frozen shoulder than in control subjects. Study Design: Controlled laboratory study. Methods: Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients with unstable shoulders (control). The samples were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry using antibodies against alarmin molecules including high-mobility group protein B1 (HMGB1), interleukin 33, S100A8, S100A9, and the peripheral nerve marker PGP9.5. Immunoreactivities were rated in a blinded fashion from “none” to “strong.” Immunohistochemical distribution within the capsule was noted. Before surgery, patient-ranked pain frequency, severity, stiffness, and the range of passive shoulder motion were recorded and statistically analyzed. Results: Compared with control patients, patients with frozen shoulder had greater frequency and severity of self-reported pain ( P = .02) and more restricted range of motion in all planes ( P < .05). H&E-stained capsular tissue from frozen shoulder showed fibroblastic hypercellularity and increased subsynovial vascularity. Immunoreactivity of alarmins was significantly stronger in frozen shoulder capsules compared with control capsules ( P < .05). Furthermore, the expression of the alarmin molecule HMGB1 significantly correlated ( r > 0.9, P < .05) with the severity of patient-reported pain. Conclusion: This study demonstrates a potential role for key molecular danger signals in frozen shoulder and suggests an association between the expression of danger molecules and the pain experienced by patients.

La gota ha estat classificada com a malaltia autoinflamatòria pel seu caràcter autolimitat i la participació del sistema immune innat i IL-1β en la seva patogènia. Durant els últims anys, l'evidència que els cristalls d'urat monosòdic (UMS) activen l'inflamasoma NLRP3, juntament amb l'eficàcia del tractament bloquejant de IL-1 en pacients gotosos, han reafirmat el concepte de gota com a malaltia autoinflamatòria. Tot i això, algunes preguntes relatives a la seva patogènia encara no tenen resposta. La presència de cristalls d'UMS en articulacions asimptomàtiques o el perquè no tots els pacients amb hiperuricèmia desenvolupen clínica de gota en són alguns exemples. En la present tesi hem centrat la nostra atenció en el sistema mononuclear fagocític amb la hipòtesi que l'estat d'activació o maduració dels monòcits i macròfags modula la resposta inflamatòria als cristalls d'UMS. Pel que fa als macròfags, la nostra hipòtesi és que els macròfags residents, després d'entrar en contacte amb els cristalls, adquireixen un fenotip inflamatori, sent capaços de produir citocines inflamatòries en presència d'un segon estímul. Per a demostrar-ho, hem emprat un model in vitro de macròfags derivats de monòcits humans polaritzats cap a fenotips M1 o M2 en la presència de GM-CSF o M-CSF respectivament, considerant als M2 com a model de macròfags residents a la sinovial. Tant els macròfags M1 com els M2 foren incapaços de produir IL-1β després d'estimulació amb cristalls d'UMS o LPS. Però en presència de cristalls d'UMS, els macròfags M2 van secretar IL-1β, disminuint a més la seva producció de IL-10, en rebre un segon estímul amb LPS. Inesperadament, l'àcid úric soluble va exercir un efecte supressor en ambdues citocines. Els resultats dels nostres experiments demostren que la producció de IL-1β per part dels macròfags M2 s'explica perquè els cristalls d'UMS són capaços d'activar caspasa-1 a través de l'activació de l'inflamasoma, mentre que LPS indueix la producció de la forma inactiva de pro-IL-1β i caspasa-1. La nostra anàlisi de l'inflamasoma en macròfags M1 i M2 ha mostrat trets diferencials que podrien explicar els seus diferents requeriments per a la producció de IL-1β. Per a investigar si una major reactivitat dels monòcits enfront dels cristalls d'UMS podria explicar perquè uns individus desenvolupen gota i d'altres no, vàrem comparar l'activació de l'inflamasoma en monòcits de pacients gotosos amb controls sans. Els monòcits de pacients gotosos presentaren una major activació de l'inflamasoma en presència dels cristalls d'UMS. Per tant, els nostres resultats suggereixen que els pacients amb gota presenten una major reactivitat enfront dels cristalls d'UMS. Per a completar l'estudi vam genotipar l'exó 3 del gen NLRP3, sense trobar associació de cap polimorfisme amb la gota. Finalment, vam analitzar la distribució de diferents subpoblacions de monòcits en sang perifèrica, observant una expansió dels monòcits amb fenotip intermedi (CD14++CD16+) en pacients durant un brot d'artritis gotosa, mentre que no varen existir diferències entre pacients gotosos asimptomàtics i controls sans., Gout has been classified as an autoinflammatory disease because of its self-remitting course and the involvement of the innate immune system and IL-1β in its pathogenesis. During the last years, the evidence of NLRP3 inflammasome activation by monosodium urate (MSU) crystals and the beneficial effect of IL-1 blockade for the treatment of patients with gout, have reaffirmed the concept of gout as an autoinflammatory disease. However, some questions regarding its pathogenesis remain still unanswered. The presence of MSU crystals in asymptomatic joints or why not all individuals with hyperuricemia develop clinical gout, are some examples. In this thesis we centred our attention in the mononuclear phagocyte system with the hypothesis that the state of activation or maturation of monocytes and macrophages modulates the inflammatory response to MSU crystals. Regarding macrophages, our hypothesis is that resident macrophages, after contact with MSU crystals, acquire an inflammatory phenotype, being able to produce inflammatory cytokines in the presence of a second signal or trigger. Therefore, to test it, we used an in vitro model of human monocyte-derived macrophages polarized towards M1 or M2 phenotypes in the presence of GM-CSF and M-CSF respectively, considering M2 macrophages a model of resident synovial macrophages. M1 and M2 macrophages failed to produce IL-1β after stimulation with MSU crystals or LPS. However, in the presence of MSU, M2 macrophages produced IL-1β and decreased IL-10 secretion when LPS was added. Surprisingly, soluble uric acid had a suppressive effect for expression of both cytokines. The production of IL-1β was explained because MSU crystals increased the presence of active caspase-1, reflecting inflammasome activation, whereas LPS induced pro-IL-1β and inactive caspase-1 production. Analysis of inflammasome activation in M1 and M2 macrophages revealed differences in inflammasome proteins, that could account for their different IL-1β production. To investigate if the capacity of monocytes to react to MSU crystals could explain why some individuals develop gout, we compared the inflammasome activation induced by MSU crystals in PBMCs from gouty patients with healthy controls. PBMCs of patients with gout exhibited enhanced inflammasome activation after culture with MSU. Therefore, our results suggest that patients with gout have an increased reactivity to MSU crystals. To provide further insight, we genotyped exon 3 of NLRP3 gene in gout patients, without finding any association of gout with polymorphisms in this gene. Finally, we analyzed the distribution of different monocyte phenotypes or blood subpopulations in patients with gout. We observed an expansion of the intermediate subpopulation (CD14++CD16+) during gout flares, but no differences were observed in any of the subpopulations between asymptomatic patients and healthy controls.

Background Microparticles (MPs) have been described to function as extracellular vehicles that shuttle microRNAs (miR) from platelets to endothelial cells1 and regulate recipient cell gene expression. Crosstalk of synovial monocytes (MC) with synovial fibroblasts (SF) is a key step to the inflammatory process in rheumatoid arthritis (RA). Objectives Herein, we investigated whether delivery of specific miRs by MC-derived MPs affects RASF behaviour. Methods Blood samples of healthy volunteers and RA patients were collected. MPs were obtained by differential centrifugation. CD14+ cells were isolated from PBMCs by using positive selection and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 6 days. Scanning electron microscopy, nanosight trafficking analysis and flow cytometry (FACS) have been applied to characterize MPs from human CD14+MCs and THP-1 cells. Total and smallRNA sequencing were performed on macrophages (n=4) and RASFs (n=4) as well as on macrophage-derived MPs (n=4). (Pre-)miR-223 expression and copy number was quantified in cells or tissues via TLDA, qPCR & in situ hybridisation with specific primers and probes. Targeted pathways were identified using prediction algorithms (TargetScanHuman6.2). Transfer of miR-223 from MCs to SFs was determined by fluorescence microscopy, direct cell co-culture and FACS, as well as transwell co-culture. RASF proliferation assays with FGF-2 as positive control, as well as THP-1 derived MPs, miR-223 mimic, miR-223 inhibitor, control mimic and inhibitor were carried out. Results MPs from MCs & THP-1 cells are about 250nm in size (range 50–800nm). SmallRNA sequencing revealed high levels of miR-223 in macrophages as opposed to a lack of its expression in RASF. If co-cultured with MCs for 3d, RASF acquire miR-223 expression, but not pre-miR-223 to a significant extent (n=6, Ct values Conclusions miR-223 transport from MCs to SFs by MPs represents a novel intercellular mechanism that could stimulate SF proliferation during initiation or chronicity of synovial inflammation in RA. References Laffont, B. et al. Blood (2013) Acknowledgement We wish to thank Derek Baxter, Diane Vaughan, Margaret Mullin, Pawel Herzyk, Julie Galbraith, Russka Shumnalieva for their support and technical assistance. Disclosure of Interest None declared