Chronic inflammation is a feature of Achilles tendinopathy and rupture

Abstract

Background: Recent investigation of human tissue and cells from positional tendons such as the rotator cuff has clarified the importance of inflammation in the development and progression of tendon disease. These mechanisms remain poorly understood in disease of energy-storing tendons such as the Achilles. Using tissue biopsies from patients, we investigated if inflammation is a feature of Achilles tendinopathy and rupture.

Methods: We studied Achilles tendon biopsies from symptomatic patients with either mid-portion tendinopathy or rupture for evidence of abnormal inflammatory signatures. Tendon-derived stromal cells from healthy hamstring and diseased Achilles were cultured to determine the effects of cytokine treatment on expression of inflammatory markers.

Results: Tendinopathic and ruptured Achilles highly expressed CD14+ and CD68+ cells and showed a complex inflammation signature, involving NF-κB, interferon and STAT-6 activation pathways. Interferon markers IRF1 and IRF5 were highly expressed in tendinopathic samples. Achilles ruptures showed increased PTGS2 and interleukin-8 expression. Tendinopathic and ruptured Achilles tissues expressed stromal fibroblast activation markers podoplanin and CD106. Tendon cells isolated from diseased Achilles showed increased expression of pro-inflammatory and stromal fibroblast activation markers after cytokine stimulation compared with healthy hamstring tendon cells.

Conclusions: Tissue and cells derived from tendinopathic and ruptured Achilles tendons show evidence of chronic (non-resolving) inflammation. The energy-storing Achilles shares common cellular and molecular inflammatory mechanisms with functionally distinct rotator cuff positional tendons. Differences seen in the profile of ruptured Achilles are likely to be attributable to a superimposed phase of acute inflammation and neo-vascularisation. Strategies that target chronic inflammation are of potential therapeutic benefit for patients with Achilles tendon disease.

Keywords: Achilles tendon; immunology; orthopaedics; tendinopathy; tendon.

Figure 1

Immunohistochemistry showing the phenotypes of myeloid cells and increased vascularity of diseased Achilles tendons. (A) Quantitative analysis of CD14+ and CD68+ myeloid cells in healthy hamstring and diseased (tendinopathic and ruptured) Achilles tendons. Bars represent median values. (B–D) Representative immunofluorescence images of diseased Achilles tendon sections stained for inflammation activation markers including the interferon pathway (IRF1 and IRF5, purple), the glucocorticoid receptor activation pathway (CD163, red) and STAT-6 pathway (CD206, green). CD68 (green) is a marker of tissue resident macrophages. Cyan shows POPO-1 nuclear stain. Scale bar, 20 µm. (E) Representative images of 3,3′-diaminobenzidine immunostaining (brown) for vascular marker CD31 in healthy hamstring, tendinopathic and ruptured Achilles tendons. Nuclear stain is haematoxylin. Scale bar, 50 µm. Graph shows quantitative analysis of immunostaining for CD31 in healthy hamstring and diseased Achilles tendons.

Figure 2

Expression of inflammatory genes in diseased Achilles tendons. Tendinopathic (n=11 donors) and ruptured Achilles (n=13 donors) tissue samples showed a complex inflammation gene signature encompassing activation of NF-κB (IL-8, PTGS2), interferon (IRF1, IRF5 and CXCL10), STAT-6 (CD206) and glucocorticoid receptor activation pathways (CD163). The inflammation signature of diseased Achilles tendons was compared with healthy hamstring tendons (n=6 donors). Gene expression is shown normalised to β-actin; bars represent median values. mRNA, messenger RNA.

Figure 3

Expression of stromal fibroblast activation markers in healthy hamstring and diseased Achilles tendons. (A) PDPN, CD248 and CD106 mRNA expression in healthy hamstring tendons (n=5 donors), tendinopathic (n=11 donors) and ruptured Achilles tendons (n=13 donors). Gene expression is shown normalised to β-actin; bars represent median values. (B–D) Representative immunofluorescence images of diseased Achilles tendons (tendinopathic) (B) and rupture (C) and healthy hamstring tendon sections (D) stained for markers of stromal activation (PDPN, green; CD248 and CD106, purple) and TLR4 (red). Cyan shows POPO-1 nuclear stain. Scale bar, 20 µm. mRNA, messenger RNA; PDPN, podoplanin; TLR4, toll-like receptor 4.

Figure 4

Expression of markers of inflammation in cultured tendon-derived stromal cells after cytokine treatment. Tendon stromal cells were derived from healthy hamstring (n=6 donors), tendinopathic (n=6 donors) or ruptured Achilles (n=6 donors). (A) PDPN expression after IL-1β treatment (10 ng/mL) for 24 hours determined by flow cytometry. (B) Expression of PDPN mRNA after IL-1β treatment (10 ng/mL) for 24 hours. Expression of interferon target genes IRF5 (C) and IRF1 (D) after IFNγ treatment (20 ng/mL) for 24 hours. Gene expression is shown normalised to β-actin; bars represent median values. **P<0.01, *P<0.05. mRNA, messenger RNA; PDPN, podoplanin.

Figure 5

Schematic summarising the cellular and molecular features of chronic inflammation identified from cross-sectional assessments of functionally distinct tendons. Macrophages in diseased tendons show a mixed signature and express pro-inflammatory macrophage markers (IRF1, IRF5) and markers of alternative macrophage activation including CD206 and CD163. After exposure to inflammatory stimuli, diseased tendon cells become ‘primed’ and express markers of stromal fibroblast activation including PDPN and adopt a more rounded morphology, reflecting a phenotypic shift in their inflammatory profile. Other pro-inflammatory molecules expressed by tendon cells include damage-associated molecular pattern TLR4, IRF1 and IRF5. Achilles tendon ruptures show increased vascularity and highly express interleukin-8 and PTGS2. Chronic inflammation and fibrosis develop due to impaired resolution of inflammation and failure of clearance of apoptotic cells. With successful resolution, expression of pro-inflammatory mediators is moderated, although some degree of stromal fibroblast activation persists. This stromal ‘memory’ may sensitise tendon cells and increase susceptibility to further episodes of inflammation and recurrent tendon disease. PDPN, podoplanin; TLR4, toll-like receptor 4.