Translational targeting of inflammation and fibrosis in frozen shoulder: Molecular dissection of the T cell/IL-17A axis.

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 IKKß 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.

Attenuation of Dupuytren's fibrosis via targeting of the STAT1 modulated IL-13Rα1 response.

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.

Fibroblast activation and inflammation in frozen shoulder.

INTRODUCTION: Frozen 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 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. METHODS: 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-1ß upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR. RESULTS: 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). CONCLUSIONS: 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.

S100A8 & S100A9: Alarmin mediated inflammation in tendinopathy.

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.

Targeting danger molecules in tendinopathy: the HMGB1/TLR4 axis.

OBJECTIVES: To seek evidence of the danger molecule, high-mobility group protein B1 (HMGB1) expression in human tendinopathy and thereafter, to explore mechanisms where HMGB1 may regulate inflammatory mediators and matrix regulation in human tendinopathy. METHODS: Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing 'early pathology') biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. Markers of inflammation and HMGB1 were quantified by reverse transcriptase PCR (RT-PCR) and immunohistochemistry. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction and used through passage 3. In vitro effects of recombinant HMGB1 on tenocyte matrix and inflammatory potential were measured using quantitative RT-PCR, ELISA and immunohistochemistry staining. RESULTS: Tendinopathic tissues demonstrated significantly increased levels of the danger molecule HMGB1 compared with control tissues with early tendinopathy tissue showing the greatest expression. The addition of recombinant human HMGB1 to tenocytes led to significant increase in expression of a number of inflammatory mediators, including interleukin 1 beta (IL-1ß), IL-6, IL-33, CCL2 and CXCL12, in vitro. Further analysis demonstrated rhHMGB1 treatment resulted in increased expression of genes involved in matrix remodelling. Significant increases were observed in Col3, Tenascin-C and Decorin. Moreover, blocking HMGB1 signalling via toll-like receptor 4 (TLR4) silencing reversed these key inflammatory and matrix changes. CONCLUSION: HMGB1 is present in human tendinopathy and can regulate inflammatory cytokines and matrix changes. We propose HMGB1 as a mediator driving the inflammatory/matrix crosstalk and manipulation of the HMGB1/TLR4 axis may offer novel therapeutic approaches targeting inflammatory mechanisms in the management of human tendon disorders.

IL-17A mediates inflammatory and tissue remodelling events in early human tendinopathy.

Increasingly, inflammatory mediators are considered crucial to the onset and perpetuation of tendinopathy. We sought evidence of interleukin 17A (IL-17A) expression in early human tendinopathy and thereafter, explored mechanisms whereby IL-17A mediated inflammation and tissue remodeling in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing 'early pathology') along with control biopsies were collected from patients undergoing shoulder surgery. Markers of inflammation and IL-17A were quantified by RT-PCR and immunohistochemistry. Human tendon cells were derived from hamstring tendon obtained during ACL reconstruction. In vitro effects of IL-17A upon tenocytes were measured using RT-PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V FACS staining. Increased expression of IL-17A was detected in 'early tendinopathy' compared to both matched samples and non-matched control samples (p < 0.01) by RT-PCR and immunostaining. Double immunofluoresence staining revealed IL-17A expression in leukocyte subsets including mast cells, macrophages and T cells. IL-17A treated tenocytes exhibited increased production of proinflammatory cytokines (p < 0.001), altered matrix regulation (p < 0.01) with increased Collagen type III and increased expression of several apoptosis related factors. We propose IL-17A as an inflammatory mediator within the early tendinopathy processes thus providing novel therapeutic approaches in the management of tendon disorders.

Analysis of the cell populations composing the mononuclear cell infiltrates in the labial minor salivary glands from patients with rheumatoid arthritis and sicca syndrome.

OBJECTIVES: Sicca symptoms occur in around 30% of rheumatoid arthritis (RA) patients. Herein, we examined the characteristics of RA patients bearing sicca symptomatology (RA-sicca) with a special focus on the immunohistopathological features of their labial minor salivary gland (LMSG) biopsies. METHODS: Our cohort included 100 consecutive RA patients which were interrogated using a sicca symptoms questionnaire. Positive responders were evaluated for ocular and oral dryness and underwent an LMSG biopsy. All samples were immunohistochemically evaluated for the presence and distribution of specific leukocyte subsets using appropriate markers and for the expression of certain immunoregulatory molecules by salivary gland epithelial cells. Positively stained and total mononuclear cells (MNC) were counted in the entire section. Counts were expressed as cell frequency (percentage of cell type number/total infiltrating MNC number). RESULTS: In the majority (86.1%) of the 44 RA-sicca cases, periductal infiltrates were observed in LMSG biopsies. The frequencies of infiltrating cell subtypes and their correlation with lesion severity were different from that previously described in primary Sjögren's syndrome (pSS). Moreover, DCs and ΜΦs frequencies were increased in RA-sicca patients who had a biopsy focus score <1 and absence of anti-Ro/anti-La autoantibodies, in contrast to what was observed for B cells. In about half of the biopsies, salivary gland epithelial cells expressed CD80/B7.1 molecules, most commonly in patients with a positive biopsy or anti-Ro/anti-La autoantibodies. CONCLUSION: LMSG infiltrates composition in RA-sicca patients is distinct from that described in pSS. These differences, further attest to diverse pathophysiologic processes operating in these two entities.

Inflammation and Neovascularization in Hip Impingement: Not Just Wear and Tear.

BACKGROUND: Femoroacetabular impingement (FAI) is a significant cause of osteoarthritis (OA) in young active patients, but the pathophysiology remains unclear. Increasingly, mechanistic studies point toward an inflammatory component in OA. PURPOSE: This study aimed to characterize inflammatory cell subtypes and neovascularization in FAI by exploring the phenotype and quantification of inflammatory cells and neovascularization in FAI versus OA samples. STUDY DESIGN: Descriptive laboratory study. METHODS: Ten samples of the labrum were obtained from patients with FAI (confirmed diagnosis) during open osteochondroplasty or hip arthroscopic surgery. Control samples of the labrum were collected from 10 patients with OA who were undergoing total hip arthroplasty. Labral biopsy specimens were evaluated immunohistochemically by quantifying the presence of macrophages (CD68, CD206, interleukin-13 [IL-13]), T cells (CD3), mast cells (mast cell tryptase), and vascular endothelium (CD34, vascular endothelial growth factor). RESULTS: Labral biopsy specimens obtained from patients with FAI exhibited significantly greater macrophage, mast cell, and vascular endothelium expression compared with control OA labral samples (P < .05). The most significant difference was noted in macrophage (P < .01) and mast cell (P < .05) expression. Further subtyping of macrophages in FAI using the CD206 tissue marker and IL-13 revealed an M2 phenotype, suggesting that these cells are involved in a regenerate versus degenerate process. There was a modest but significant correlation between mast cells and CD34 expression (r = 0.4, P < .01) in FAI samples. CONCLUSION: This study provides evidence for an inflammatory cell infiltrate in FAI along with significant neovascularization. In particular, the significant infiltration of mast cells and macrophages was demonstrated, suggesting a role for innate immune pathways in the events that mediate hip impingement. CLINICAL RELEVANCE: Further mechanistic studies to evaluate the net contribution and hence therapeutic utility of these cellular lineages and their downstream processes may reveal novel therapeutic approaches to the management of early hip impingement.

MicroRNA29a regulates IL-33-mediated tissue remodelling in tendon disease.

MicroRNA (miRNA) has the potential for cross-regulation and functional integration of discrete biological processes during complex physiological events. Utilizing the common human condition tendinopathy as a model system to explore the cross-regulation of immediate inflammation and matrix synthesis by miRNA we observed that elevated IL-33 expression is a characteristic of early tendinopathy. Using in vitro tenocyte cultures and in vivo models of tendon damage, we demonstrate that such IL-33 expression plays a pivotal role in the transition from type 1 to type 3 collagen (Col3) synthesis and thus early tendon remodelling. Both IL-33 effector function, via its decoy receptor sST2, and Col3 synthesis are regulated by miRNA29a. Downregulation of miRNA29a in human tenocytes is sufficient to induce an increase in Col3 expression. These data provide a molecular mechanism of miRNA-mediated integration of the early pathophysiologic events that facilitate tissue remodelling in human tendon after injury.

IL-21 receptor expression in human tendinopathy.

The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1ß) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

The liver X receptor pathway is highly upregulated in rheumatoid arthritis synovial macrophages and potentiates TLR-driven cytokine release.

OBJECTIVES: Macrophages are central to the inflammatory processes driving rheumatoid arthritis (RA) synovitis. The molecular pathways that are induced in synovial macrophages and thereby promote RA disease pathology remain poorly understood. METHODS: We used microarray to characterise the transcriptome of synovial fluid (SF) macrophages compared with matched peripheral blood monocytes from patients with RA (n=8). RESULTS: Using in silico pathway mapping, we found that pathways downstream of the cholesterol activated liver X receptors (LXRs) and those associated with Toll-like receptor (TLR) signalling were upregulated in SF macrophages. Macrophage differentiation and tumour necrosis factor α promoted the expression of LXRα. Furthermore, in functional studies we demonstrated that activation of LXRs significantly augmented TLR-driven cytokine and chemokine secretion. CONCLUSIONS: The LXR pathway is the most upregulated pathway in RA synovial macrophages and activation of LXRs by ligands present within SF augments TLR-driven cytokine secretion. Since the natural agonists of LXRs arise from cholesterol metabolism, this provides a novel mechanism that can promote RA synovitis.

Hypoxia: a critical regulator of early human tendinopathy.

OBJECTIVES: To seek evidence for the role of hypoxia in early human tendinopathy, and thereafter to explore mechanisms whereby tissue hypoxia may regulate apoptosis, inflammatory mediator expression and matrix regulation in human tenocytes. METHODS: Fifteen torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing 'early pathology') biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of the subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Markers of hypoxia were quantified by immunohistochemical methods. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction. The impact of hypoxia upon tenocyte biology ex vivo was measured using quantitative real-time PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V fluorescence-activated cell sorter staining. RESULTS: Increased expression of hypoxia-inducible factor 1α, Bcl-2 and clusterin was detected in subscapularis tendon samples compared with both matched torn samples and non-matched control samples (p<0.01). Hypoxic tenocytes exhibited increased production of proinflammatory cytokines (p<0.001), altered matrix regulation (p<0.01) with increased production of collagen type III operating through a mitogen-activated protein kinase-dependent pathway. Finally, hypoxia increased the expression of several mediators of apoptosis and thereby promoted tenocyte apoptosis. CONCLUSION: Hypoxia promotes the expression of proinflammatory cytokines, key apoptotic mediators and drives matrix component synthesis towards a collagen type III profile by human tenocytes. The authors propose hypoxic cell injury as a critical pathophysiological mechanism in early tendinopathy offering novel therapeutic opportunities in the management of tendon disorders.

IL-33 induces skin inflammation with mast cell and neutrophil activation.

Psoriasis is a common chronic autoimmune condition of the skin characterized by hyperplasia of epidermal keratinocytes associated with pro-inflammatory cytokines. IL-33 is a new member of the IL-1 superfamily that signals through the ST2 receptor and was originally defined as an inducer of T helper 2 (Th2) cytokines. Recently, broader immune activatory potential has been defined for IL-33 particularly via mast cell activation and neutrophil migration. Here, we show that ST2(-/-) mice exhibit reduced cutaneous inflammatory responses compared with WT mice in a phorbol ester-induced model of skin inflammation. Furthermore, injections of IL-33 into the ears of mice induce an inflammatory skin lesion. This inflammatory response was partially dependent on mast cells as mast cell-deficient mice (Kit(W-sh/W-sh) ) showed delayed responses to IL-33. IL-33 also recruited neutrophils to the ear, an effect mediated in part by increased production of the chemokine KC (CXCL1). Finally, we show that IL-33 expression is up-regulated in the epidermis of clinical psoriatic lesions, compared with healthy skin. These results therefore demonstrate that IL-33 may play a role in psoriasis-like plaque inflammation. IL-33 targeting may provide a new treatment strategy for psoriasis.

Phosphodiesterase 4 (PDE4) regulation of proinflammatory cytokine and chemokine release from rheumatoid synovial membrane.

BACKGROUND: The cAMP-metabolising enzyme, phosphodiesterase 4 (PDE4), has been implicated in a number of immune responses, including tumour necrosis factor α (TNFα) production. To date, few data have directly addressed whether synovial cytokine and chemokine production is modified by PDE4. OBJECTIVE: Using specific PDE4 inhibitors, roflumilast plus two novel inhibitors, INH 0061 and INH 0062, the authors studied the effect of PDE4 inhibition on proinflammatory cytokine and chemokine release from primary rheumatoid arthritis (RA) synovial digest suspensions and in a macrophage T cell co-culture assay system. RESULTS: All PDE4 inhibitors dose-dependently reduced the release of TNFα from primary synovial membrane cultures (n=5), half maximal inhibitory concentration (IC(50)) 300-30 nM, p<0.05. Similarly, a significant suppression in the release the proinflammatory chemokines, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1ß (IC(50) 300-30 nM) and regulated upon activation normal T-cell expressed and secreted (RANTES) (IC(50) 3 nM) was also observed, p<0.05. While interleukin 1ß was also reduced, it did not achieve an IC(50). These observations were further confirmed in a macrophage T cell co-culture system, demonstrating the importance of PDE4 pathways in regulating cytokine/chemokine release in a cellular interaction implicated in inflammatory synovitis. Subsequent studies using the human monocytic cell line U937 also demonstrated cytokine regulation with PDE4 knockdown utilising a small interfering RNA approach. CONCLUSION: These data provide direct evidence of PDE4-dependent pathways in human RA synovial inflammatory cytokine and chemokine release and may provide a novel approach in treating chronic autoimmune conditions such as RA.

Inflammation is present in early human tendinopathy.

BACKGROUND: The cellular mechanisms of tendinopathy remain unclear particularly with respect to the role of inflammation in early disease. The authors previously identified increased levels of inflammatory cytokines in an early human model of tendinopathy and sought to extend these studies to the cellular analysis of tissue. PURPOSE: To characterize inflammatory cell subtypes in early human tendinopathy, the authors explored the phenotype and quantification of inflammatory cells in torn and control tendon samples. DESIGN: Controlled laboratory study. METHODS: Torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from 20 patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilization surgery. Tendon biopsy samples were evaluated immunohistochemically by quantifying the presence of macrophages (CD68 and CD206), T cells (CD3), mast cells (mast cell tryptase), and vascular endothelium (CD34). RESULTS: Subscapularis tendon samples obtained from patients with a torn supraspinatus tendon exhibited significantly greater macrophage, mast cell, and T-cell expression compared with either torn supraspinatus samples or control subscapularis-derived tissue (P < .01). Inflammatory cell infiltrate correlated inversely (r = .5; P < .01) with rotator cuff tear size, with larger tears correlating with a marked reduction in all cell lineages. There was a modest but significant correlation between mast cells and CD34 expression (r = .4; P < .01) in matched subscapularis tendons from shoulders with supraspinatus ruptures. CONCLUSION: This study provides evidence for an inflammatory cell infiltrate in early mild/moderate human tendinopathy. In particular, the authors demonstrate significant infiltration of mast cells and macrophages, suggesting a role for innate immune pathways in the events that mediate early tendinopathy. Clinical Relevance Further mechanistic studies to evaluate the net contribution and hence therapeutic utility of these cellular lineages and their downstream processes may reveal novel therapeutic approaches to the management of early tendinopathy.

Poor predictive value of broad-range PCR for the detection of arthroplasty infection in 92 cases.

BACKGROUND: The diagnosis of prosthetic infection remains a challenge, as no test is 100% sensitive and 100% specific. Recent advances in molecular biology have enabled the detection of infection in culture negative cases. PATIENTS AND METHODS: We evaluated the effectiveness of polymerase chain reaction (PCR) in detecting infection in failed joint replacements prospectively in 91 consecutive patients (92 prosthetic joints) undergoing revision total hip or knee arthroplasty. Synovial fluid was collected intraoperatively and examined by broad-range PCR assay for detection of bacterial DNA. The clinical diagnosis of infection was based on the results of blood tests, preoperative joint aspiration, culture and histology of multiple intraoperative tissue samples, as well as the surgeon's assessment. 12 joints (13%) were infected, but the PCR was positive in 32 cases. The sensitivity of the technique was 92%, the specificity 74%, the accuracy 76%, the positive predictive value 34%, and the negative predictive value was 98%. INTERPRETATION: The PCR technique cannot be recommended for the routine detection of prosthetic infection. The large number of false positive results may represent sample contamination, or bacterial presence related to low-virulence organisms, low bacterial load, or a strong host immune response.