Background Increasingly, inflammatory mediators are considered crucial to the onset and perpetuation of tendinopathy.1 We have previously reported increased expression of key cytokines in human and rodent models of tendinopathy.2 IL-33 and its receptor ST2 have become increasingly associated with musculoskeletal pathologies over the past few years and are purported as critical endogenous tissue danger signals.3 There remains a significant unmet clinical need in the understanding of tendon disorders, largely owing to a lack of in-depth interrogation of the biological mechanisms underpinning the disease process.
Objectives To seek evidence of IL-33 expression in early human tendinopathy and thereafter, to explore mechanisms whereby IL-33 may regulate inflammatory mediators and matrix regulation in human tenocytes and a murine tendon model.
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 subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The impact of IL-33 upon tenocyte biology ex vivo was measured using quantitative RT-PCR, collagen I and III ELISAs and luminex cytokine multiplexes. In vivo work composed of experiments utilising a murine patellar tendon injury model in WT and ST2-/-.
Results IL-33, soluble and membrane bound ST2 transcripts were significantly upregulated in early tendinopathy compared to control or torn tendon biopsies. IL-33 induced dose and time dependent upregulation of total collagen protein accounted for by increased expression of type I but particularly type III collagen mRNA and protein. Following array analysis and consistent with reported IL-33.
IL-33 mRNA was elevated on days 1 and 3 post tendon injury in WT mice. This was significantly reduced in injured ST2-/- mice suggesting autocrine regulation. Analysis of collagen synthesis revealed significantly greater expression of collagen 3 at all time points post injury in WT mice compared to uninjured controls or injured ST2-/- mice Importantly injury of WT mice tendons resulted in a significant decrease in biomechanical strength at Day 1 post injury compared to ST2-/- that recovered by days 7 and 21. These data suggest altered collagen matrix synthesis in ST2-/- mice implicating IL-33/ST2 as an early modulator of collagen changes in tendon injury that has biomechanical significance. Administration of rhIL-33 did not affect Collagen 1 synthesis but did significantly increase Collagen 3 synthesis particularly in injured tendons. Moreover, rhIL33 administration significantly reduced ultimate tendon strength at all time points post injection in WT mice suggesting that such changes were of functional impact.
Finally we directly targeted IL-33 in vivo. Neutralising antibodies to IL-33 attenuated the collagen I to III switch at days 1 and 3 post injury in WT injured mice resulting in a significant increase in biomechanical strength at day 1 post injury WT mice tendons.
Conclusion We herein provide new evidence for a role of IL-33 in the initial steps that lead to the important clinical entity of tendinopathy. Our data implicate IL-33 as an alarmin in early tendinopathy, and importantly, our biomechanical data suggest such expression has a pathogenically relevant role.
References 1 Dourte LM, et al. J Orthop Res. 2008;26(10):1297–305
2 Millar NL, et al. J Bone Joint Surg Br. 2009;91(3):417–24
3 Liew FY. Ann Rheum Dis. Apr 2012 Suppl
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