Shauna Kerr

Cutting Edge: Mast Cells Express IL-17A in Rheumatoid Arthritis Synovium

The proinflammatory cytokine IL-17A is considered a crucial player in rheumatoid arthritis (RA) pathogenesis. In experimental models of autoimmune arthritis, it has been suggested that the cellular source of IL-17A is CD4+ T cells (Th17 cells). However, little is known about the source of IL-17 in human inflamed RA tissue. We explored the cellular sources of IL-17A in human RA synovium. Surprisingly, only a small proportion of IL-17–expressing cells were T cells, and these were CCR6 negative. Unexpectedly, the majority of IL-17A expression colocalized within mast cells. Furthermore, we demonstrated in vitro that mast cells produced RORC-dependent IL-17A upon stimulation with TNF-α, IgG complexes, C5a, and LPS. These data are consistent with a crucial role for IL-17A in RA pathogenesis but suggest that in addition to T cells innate immune pathways particularly mediated via mast cells may be an important component of the effector IL-17A response.

IL-33 Shifts the Balance from Osteoclast to Alternatively Activated Macrophage Differentiation and Protects from TNF-α–Mediated Bone Loss

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2−/− bone marrow led to more bone loss compared with the chimeras with ST2+/+ bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206+ AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.

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.

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-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.

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-1β) 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.

Simultaneous activation of the liver X receptors (LXRα and LXRβ) drives murine collagen-induced arthritis disease pathology

Background It has previously been shown that dual activation of the Liver X Receptors (LXRα and LXRβ) by the agonist, GW3965, enhances pathology in a murine model of collagen-induced arthritis. Objective To determine whether LXRα or LXRβ have discrete roles in driving articular inflammation. Methods Arthritis was induced in male C57BL/6 wild-type (WT), LXRα−/−, LXRβ−/− and LXRα/β double KO mice by injection with type II collagen and treated with 30 mg/kg of the LXR agonist GW3965 or vehicle control. The mice were monitored for articular inflammation and cartilage degradation by scoring for clinical signs of arthritis and by histological examination of the joints. Results Administration of 30 mg/kg GW3965 significantly increases the severity of arthritis in WT but not LXRα−/−, LXRβ−/− or LXRα/β KO mice as assessed by an increase in the clinical score, paw thickness and articular histological analysis. Conclusion The proinflammatory effects associated with the administration of GW3965 are mediated specifically through LXRs. The absence of increased disease severity in the LXRα−/− and LXRβ−/− GW3965-treated groups shows for the first time that agonism of both LXRα and LXRβ is required to drive proinflammatory pathways in vivo.

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.

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 Sjögrens 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.

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-1β), 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.