Chronic Inflammation Is a Feature of Achilles Tendinopathy and Rupture

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 stromal cells isolated from healthy hamstring and diseased Achilles were cultured to investigate the effects of cytokine treatment on expression of inflammatory markers.

 

Results: Tendinopathic and ruptured Achilles showed increased numbers of CD14+ and CD68+ cells and a complex inflammation signature, including activation of NF-B, 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. Stromal fibroblast activation markers Podoplanin and CD106 were increased in tendinopathic and ruptured Achilles. Cells isolated from diseased Achilles showed increased expression of pro-inflammatory and stromal fibroblast activation markers after cytokine stimulation compared to 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.

 

 

 

 

 

What are the new findings?

• Chronic inflammation is a feature of both mid-portion Achilles tendinopathy and rupture
• Pro-inflammatory profiles differ slightly in ruptured tendons; this is likely to be due to acute inflammation and increased vascularisation resulting from recent trauma
• Although the Achilles and rotator cuff tendons are functionally distinct, they share common cellular and molecular inflammatory disease mechanisms

 

How might it impact on clinical practice in the near future?

 

• Therapeutic strategies must address the underlying biology of Achilles tendon disease if treatment is to be successful
• Strategies that target inflammation are of potential therapeutic benefit for patients with chronic Achilles tendon disease
• New therapeutic approaches are required to promote resolution of chronic tendon inflammation

 

 

INTRODUCTION

Achilles tendon disorders including tendinopathy and rupture are a common cause of pain and loss of function in athletes and non-athletic individuals ¹. These injuries are difficult to treat, require prolonged rehabilitation and have a high frequency of recurrence ^{2 3}. The tendon mid-portion is the most frequently injured site ³, entheseal disease at the calcaneal insertion is less common. The aetiology of tendon disease is complex and multifactorial, encompassing effects of exercise, overuse, ageing and genetic factors ^4-7. Whilst evidence supports the contribution of inflammatory mediators to the onset and progression of tendon disease in the shoulder ^8-11, the relative importance and role of inflammation is highly debated in energy storing tendons such as the Achilles, where historically disease was frequently described as ‘degenerative’ ¹².

More recent studies have identified immune competent cells including macrophages, T cells, mast cells and natural killer cells in human biopsy specimens from non-ruptured chronic tendinopathic Achilles ¹³. However, the phenotypes of these cells are yet to be fully characterized. Apoptosis pathways have also been described as an important signalling cascade implicated in the biology of Achilles tendinopathy ⁶. These studies support the contribution of inflammation in the pathogenesis of Achilles tendon disease, although the precise mechanisms are yet to be elucidated. Of importance, the resident stromal fibroblast population which constitutes the major cell type in tendons remains under investigated.

Improved understanding of the cellular and molecular processes orchestrating inflammation in Achilles tendon disease is essential to identify therapeutic targets that address the underlying disease biology. The mechanisms underpinning the development of chronic inflammation which fails to resolve in persistently symptomatic patients is of particular interest. In the present study, we investigated the cellular and molecular features of inflammation in patient biopsy samples of tendinopathic and ruptured Achilles. We sought to identify if disease of energy storing tendons such as the Achilles shared common inflammatory mechanisms to those previously identified in positional tendons such as the rotator cuff. We hypothesized that these functionally distinct tendons would share common inflammatory mechanisms.

 

 

 

 

RESULTS

 

 

Patient demographics for the study group

Tendon biopsies from patients with Achilles tendinopathy were collected from 10 male and 7 female patients aged between 41 and 74 years (mean, 50.4 ± 8.8 years) that presented for High Volume Injection (HVI). VISA-A scores for Achilles tendinopathy patients ranged from 25-77 (mean 39.6 ±15.4). Mean BMI from the Achilles tendinopathy patient group was 29.9 (±6.1).

Tissue biopsy samples were collected from 15 male and 4 female patients that presented to a Trauma Unit for surgical debridement of an Achilles rupture (n=19). Patients with Achilles rupture were aged between 20 and 67 years (mean, 44.6 ± 11.8 years). VISA-A scores were not available for patients with Achilles ruptures. BMI for the Achilles rupture patient group was (28.8 ± 4).

Healthy hamstring (semitendinosis) tendons were collected from 10 male and 5 female patients undergoing surgical reconstruction of their anterior cruciate ligament. All patients were aged between 18 and 48 years (mean, 25.5± 11 years). BMI from the healthy hamstring tendon patient group was 24.9 (± 2.1).

 

Tendinopathic and ruptured Achilles show a complex tissue inflammation signature and increased vascularity

Using immunohistochemistry, we identified increased numbers of CD14+ and CD68+ cells in tendinopathic and ruptured Achilles (collectively grouped as diseased) compared to healthy hamstring tendons (p=0.0015 and 0.0007 respectively) (Figure 1A). There was no significant difference in the numbers of CD14+ and CD68+ cells between tendinopathic and ruptured Achilles tendons. We further characterised the activation status of these immune cells in samples of tendinopathic and ruptured Achilles in situ. We used a previously validated panel of antibodies associated with macrophage activation, including proteins implicated in pro-inflammatory pathways (IRF5 and IRF1) and alternative macrophage activation (CD206 and CD163) ⁸. Diseased Achilles tendons showed a complex macrophage activation protein signature and expressed markers of Interferon (IRF5, IRF1), STAT-6 (CD206) and glucocorticoid receptor (CD163) macrophage activation pathways (Figure 1B-D). In addition to characterising myeloid cells, we also investigated CD31 as a marker of vascularisation in samples of healthy hamstring, tendinopathic and ruptured Achilles (Figure 1E). Quantitative analysis of immunopositive staining showed increased CD31 expression in tendinopathic (p=0.02) and ruptured Achilles (p=0.0002) compared to healthy hamstring tendons. Ruptures showed increased CD31 expression compared to samples of tendinopathic Achilles (p=0.0003) (Figure 1E). Isotype control staining of corresponding tendon tissues is shown in Figure S1.

Having identified inflammatory proteins in tendinopathic and ruptured Achilles, we studied gene expression in these samples using a panel of genes known to be implicated in macrophage activation⁸.  In support of the protein signatures identified in these samples, diseased Achilles showed a complex inflammation gene signature with some differences between tendinopathic and ruptured samples (Figure 2).  CD163 mRNA was highly expressed by tendinopathic and ruptured Achilles (p=0.002 and 0.03 respectively) compared to healthy hamstring tendons. CD206 mRNA was increased in tendinopathic compared to ruptured Achilles (p=0.0002). Tendinopathic Achilles highly expressed Interferon target genes including IRF1, IRF5 and CXCL10 compared to ruptured and healthy tendons. ALOX15 implicated in resolving inflammation was reduced in tendinopathic compared to healthy tendons (p=0.04). Ruptures showed increased IL-8 and PTGS2 compared to tendinopathic Achilles (p=0.0003 and 0.03 respectively).

Diseased Achilles tendon tissues express stromal fibroblast activation markers

Having focused on characterising the phenotypes of myeloid cells populating diseased Achilles, we investigated if resident tendon cells in these samples also possessed a pro-inflammatory phenotype. Markers of stromal fibroblast activation including PDPN, CD106 and CD248 have not been identified in diseased Achilles tendons. We found PDPN mRNA was increased in tendinopathic (p=0.04) and ruptured Achilles (p=0.01) compared to healthy hamstring tendon tissues (Figure 3A). CD106 mRNA was also increased in tendinopathic (p=0.004) and ruptured Achilles (p=0.04) compared to healthy hamstring. CD248 mRNA was increased in tendinopathic Achilles compared to healthy hamstring (p=0.008). Immunostaining supported increased expression of stromal fibroblast activation markers and pro-inflammatory marker toll-like receptor 4 (TLR4) in sections of tendinopathic and ruptured Achilles compared to healthy hamstring tendons (Figures 3B-D). In diseased Achilles tendons PDPN, CD106 and CD248 co-localized with TLR4.

Diseased Achilles tendon-derived stromal cells are primed for inflammation

Markers of inflammatory pathways and stromal fibroblast activation identified in diseased Achilles tissues were further studied in vitro to compare the effects of cytokine treatment on tendon stromal cells derived from healthy hamstring and diseased tendinopathic and ruptured Achilles tendons. PDPN protein was increased in cells isolated from tendinopathic and ruptured Achilles compared to healthy hamstring under baseline unstimulated conditions (p=0.01 and p=0.004 respectively) (Figure 4A). IL-1β treatment of healthy and diseased tendon cells for 24 hours further induced PDPN. Induction of PDPN was more profound in IL-1β treated tendinopathic and ruptured Achilles compared to IL-1β-treated healthy tendon cells (0.04 and 0.004 respectively) (Figures 4A). Similar observations were made when PDPN mRNA expression was determined after IL-1β treatment of healthy and diseased tendon cells (Figure 4B). IRF5 mRNA was increased in cells isolated from tendinopathic Achilles compared to healthy hamstring under baseline unstimulated conditions (p=0.026) (Figure 4C). IFN treatment induced expression of Interferon target genes in tendon cells derived from healthy hamstring and diseased Achilles. IFN treatment markedly induced IRF5 mRNA in cells isolated from tendinopathic and ruptured Achilles compared to healthy hamstring (p= 0.016 and 0.026 respectively) (Figure 4C). The same treatment also induced IRF1 mRNA in cells isolated from tendinopathic and ruptured Achilles compared to healthy hamstring (p= 0.04 respectively) (Figure 4D).

 

DISCUSSION

The role of inflammation in the pathogenesis of tendon disease is a subject of ongoing debate. During the past 20 years, tendon disease has been characterized as a ‘degenerative’ process devoid of inflammation. Recently, this paradigm has been challenged, suggesting it may disregard a complex role for inflammation in tendon disease. Coupled with improved scientific methodologies and collection of well phenotyped patient biopsy material, our understanding of tendon inflammatory processes has advanced in recent years. Recent studies have improved understanding of the phenotypes of immune cells identified in biopsy samples of diseased human tendons ⁸. Other work has also identified that resident stromal fibroblasts from diseased human tendons show a pro-inflammatory phenotype ¹¹, illustrating that non-immune cells are also implicated in tendon inflammatory processes. Understanding the phenotypes of immune cells and tendon stromal populations is critical to advancing knowledge of inflammation in the pathobiology of Achilles tendon disease.

The purpose of the current study was to investigate the cellular and molecular features of inflammation in diseased Achilles tendons. Using tissues derived from well phenotyped patient cohorts, we identified that inflammation is a feature of both mid-portion Achilles tendinopathy and rupture. The pathways underpinning activation of macrophages to be classified as M1 or M2 subtypes have recently been revised to reflect the key signalling pathways and receptors in common and distinct pathways. These include pro-inflammatory Interferon and NF-B pathways, the pro-fibrotic STAT-6 pathway and inflammation resolving pathways involving glucocorticoid receptor (GCR) activation ¹⁴. We investigated expression of target genes and proteins from these activation pathways in samples of healthy hamstring and diseased Achilles tendons. Tissue samples from Achilles patient cohorts showed a complex inflammation signature, expressing target genes and proteins from Interferon, NF-B, STAT-6 and GCR activation pathways. Both tendinopathic and ruptured Achilles tendons expressed CD206 and CD163, suggestive of established (chronic) inflammation. We identified some differences in the inflammatory profiles depending upon the type of disease. Tendinopathic Achilles showed increased expression of Interferon target genes and proteins including IRF1, IRF5 and CXCL10. We previously identified a similar Interferon signature to that found in tendinopathic Achilles tendons was a feature of tissue biopsies from tendinopathic rotator cuff ⁸. In contrast, ruptured Achilles tendons highly expressed NF-B target genes PTGS2 and IL-8 and showed increased vascularity. Genes associated with angiogenesis including vascular endothelial growth factor have been implicated in the aetiology of Achilles tendon disease ¹⁵. Furthermore, IL-8 is known to be a potent promoter of angiogenesis ¹⁶. This NFB signature of Achilles tendon ruptures was not a feature of tissue samples collected from patients with large to massive rotator cuff tendon tears ⁸. Table 1 summarises the comparative features of disease in functionally distinct tendons such as the Achilles and rotator cuff. Whilst patients with Achilles rupture may have had pre-existing tendon disease prior to rupture, the timing of tissue collection relative to an acute event such as tendon rupture is an important factor in the interpretation of these findings. The differences in inflammation signature between torn Achilles and rotator cuff tendons are likely attributable to the consequences of recent trauma and earlier clinical presentation of patients with Achilles rupture. Conversely, symptom duration and subsequent surgical repair is frequently more protracted (months to years) in patients with rotator cuff tendon tears (Table 1). This phase of acute inflammation and neo-angiogenesis may partly explain the higher success rate of repair of Achilles tendon ruptures compared to repair of torn rotator cuff tendons ¹⁷.

Having characterised the phenotypes of myeloid cells in diseased Achilles tissues, we next investigated the phenotype of resident tendon stromal fibroblasts, (tenocytes) in tissues and cells derived from these patient cohorts. We previously identified that tissues and cells isolated from diseased shoulder tendons expressed markers of stromal fibroblast activation, suggestive of a sustained change in their phenotype as a consequence of exposure to inflammation ¹¹. To our knowledge, the phenotype of fibroblasts populating diseased Achilles tendons has not been described. We discovered that tissue samples from patients with Achilles tendinopathy and rupture also showed increased expression of markers of stromal fibroblast activation, including PDPN, CD106 and CD248. Previous work investigating inflammation in cultured stromal cells isolated from torn rotator cuff tendons identified diseased cells were ‘primed’ for inflammation ⁸. In the current study, we investigated if cells isolated from patients with disease of functionally distinct Achilles tendons were similarly ‘primed’. Treatment of healthy and diseased tendon stromal cells with IL-1 or IFN induced the expression of respective target genes compared to untreated control cells. Notably, cytokine treated tendon stromal cells from diseased Achilles showed a more profound induction of stromal fibroblast activation marker PDPN and inflammatory proteins IRF1 and IRF5 compared to healthy hamstring tendon stromal cells. Collectively, these findings support the concept that tissues and cells from diseased Achilles tendons undergo phenotypic change and may become ‘primed’ after exposure to an inflammatory stimulus. The cellular and molecular features of chronic inflammation common to functionally distinct tendons are summarized in Figure 5, whereby impaired resolution of inflammation and failure to clear apoptotic cells sustains chronic inflammation and fibrosis. Conversely, with successful resolution, expression of pro-inflammatory mediators is moderated, although some degree of stromal fibroblast activation persists ^{8 11}. This stromal ‘memory’ may sensitize tendon stromal cells and increase susceptibility to further episodes of inflammation and recurrent tendon disease.

We acknowledge there are potential limitations with the use of hamstring tendon as a comparator to diseased Achilles tendons including tendon type and donor age differences. However, hamstring tendon was taken from live healthy donors without history of tendinopathy. We believe this is a more suitable comparator than cadaveric Achilles tendon tissues where little is known about whether the tendons were healthy or diseased and tendons were not affected by post mortem changes. In addition, as sex distribution was not evenly matched between Achilles tissue cohorts, it was necessary to pool data for males and females.

In summary the findings from this study demonstrate that tissues from patients with tendinopathic and ruptured Achilles tendons both show evidence of chronic inflammation. Moreover, functionally distinct tendons such as the Achilles and rotator cuff share common cellular and molecular features. We identify slight differences in pro-inflammatory profiles between tendinopathic and ruptured Achilles tendons and suggest that this difference is attributable to superimposed acute inflammation and increased vascularisation, occurring after recent Achilles tendon rupture. We propose that treatment strategies that target non-resolving inflammation are of potential therapeutic benefit for patients with Achilles tendon disease.

 

 

MATERIALS AND METHODS

 

Study Design

The objective of this study was to investigate the cellular and molecular features of inflammation in Achilles tendon disease. Tissue samples were collected from well phenotyped patient cohorts with mid-portion Achilles tendinopathy or rupture. We investigated tissue inflammation signatures in these patient cohorts compared to healthy hamstring tendons. Inflammatory pathways were further studied in cultured tendon derived stromal cells from healthy and diseased tendons. Sample size justification was derived from previous studies that were sufficiently powered to study inflammation in diseased tendon tissues and cells ^{8 11}. For histology and immunostaining, a single blinded investigator acquired images. Details of sample size and replicates are outlined in figure legends.

 

Collection of healthy and diseased tendons

Patients with Achilles tendinopathy were recruited from a Sports Medicine clinic (n=17). Patients presenting to the Sports clinic had been symptomatic for months and failed a standard 3-month eccentric training programme for Achilles tendinopathy. Ultrasonography was performed to confirm the diagnosis of Achilles tendinopathy and suitability for HVI for patients who have failed conservative management, as this procedure represents the next step in standard treatment. Patients completed the VISA-A scoring system¹⁸, a validated and widely used clinical outcome measure scoring from 0 (severe disease) to 100 (normal function). Achilles tendon biopsies were collected from patients that presented for HVI.  This procedure involves injecting 10 ml 0.5% bupivicaine and 30 ml normal saline into the pre Achilles space. Achilles tendon biopsies were collected via percutaneous ultrasound-guided biopsy under local anesthesia prior to high volume injection at the site of ultrasonographic abnormality. The biopsy specimen was taken using a 14G trucut needle inserted into the diseased mid-portion of the Achilles tendon. This validated biopsy technique is adapted from a previously described protocol ¹⁹.

Patients with Achilles tendon rupture were recruited from a Trauma Unit (n=19). Tissue biopsy samples of Achilles tendon ruptures were collected between 36-48 hours after tendon rupture. Tendinopathic and ruptured Achilles tendons were collected under research ethics from the Office for Research Ethics Committees Northern Ireland REC reference 14/NI/1063. Full informed consent according to the Declaration of Helsinki was obtained from all patients. Exclusion criteria included previous intra-tendinous corticosteroid / Platelet Rich Plasma / stem cell injection, extracorporeal shockwave therapy or systemic steroid or methotrexate treatments. Diabetic patients and those receiving systemic anticoagulant therapy were also excluded from the study.

Healthy hamstring (semitendinosis) tendons were collected from 15 patients undergoing surgical reconstruction of their anterior cruciate ligament. Hamstring tendons were collected under research ethics from the Oxford Musculoskeletal Biobank (09/H0606/11).

Processing of tendon samples

Immunohistochemistry and immunofluorescence. Healthy and diseased tendons were immersed in 10% buffered formalin. After fixation, tendons were processed using a Leica ASP300S tissue processor and embedded in paraffin wax. Tissues were sectioned at 6m using a rotary RM2135 microtome (Leica Microsystems Ltd.) onto adhesive glass slides.

Gene expression. Samples of healthy hamstring and diseased Achilles tendons were immediately snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction.

Immunohistochemistry and immunofluorescence for macrophage and stromal fibroblast activation markers

For antigen retrieval, slides were baked at 60°C for 60 min, and tissue sections subjected to deparaffinization and target retrieval steps (heat-mediated antigen retrieval at high pH) using an automated PT Link (Dako). For single staining immunohistochemistry for CD14 and CD68, antibody staining was performed using the EnVision FLEX visualization system with an Autostainer Link 48 (Dako). Antibody binding was visualized using FLEX 3,3′-diaminobenzidine (DAB) substrate working solution and hematoxylin counterstain (Dako) using the recommended manufacturer protocols. After staining, slides were taken through graded alcohol and xylene and mounted in Pertex mounting medium (Histolab). For multiple antibody immunofluorescence staining and image acquisition, protocols were adapted from Dakin et al 2015 ⁸ using primary antibodies listed in Supplementary Table 1. Isotype control antibodies were a cocktail of mouse immunoglobulin G (IgG₁), IgG_2a, IgG_2b, IgG₃, and IgM (Dako) and rabbit immunoglobulin fraction of serum from non-immunized rabbits, solid-phase absorbed (Dako) (Figure S1). Immunofluorescence images were acquired on a Zeiss LSM 710 confocal microscope as previously described ⁸.

Cytokine treatment of tendon-derived stromal cells

IL-1 and IFN respectively induce NF-B and IFN target genes known to be highly expressed in diseased shoulder tendons ^{8 11} and were identified in diseased Achilles tendon tissues in the current study. We therefore investigated if treatment of cultured tendon cells with these cytokines induced more profound expression of markers of stromal fibroblast activation and NF-B and IFN target genes in diseased compared to healthy tendon-derived cells. Tendon cells were isolated from healthy hamstring and diseased Achilles tendons using previously described protocols ¹¹. Cells between passages 1 and 3 were used for all experiments. Cells were seeded at a density of 30,000 cells per well in a 12 well plate (mRNA) or 60,000 cells in a 6 well plate (flow cytometry). Cells were allowed to reach 80% confluence prior to stimulation with IL-1 (10ngml^-1, Sigma) or IFN gamma (20 ngml⁻¹;BioLegend) in DMEM F12 medium (Lonza) containing 1% heat-inactivated human serum (Sigma). Non-treated (vehicle only) cells served as controls for each experiment. After cytokine or vehicle treatment, cells were incubated for 24 hours at 37°C and 5% CO₂ until harvest of the lysate for mRNA or flow cytometry.

 

Extraction of RNA from tendons

Protocols for RNA extraction from healthy and diseased tendon tissues and cells, complementary DNA synthesis and quantitative polymerase chain reaction are described elsewhere ⁸. 2 L cDNA was used in a 10 L qPCR volume with Fast SYBR Green Master Mix (Applied Biosystems) and diluted validated human primers listed in Supplementary Table 2. Duplicate reactions for each gene were run on a ViiA7 qPCR machine (Applied Biosystems) and results were calculated using the DDCt method using reference genes for human -actin and GAPDH. Results were consistent using these reference genes and data are shown normalized to -actin.

Flow cytometry

Flow cytometry was performed as previously described ¹¹. Antibody and isotype cocktails were prepared as indicated in Supplementary Table 3. After washing, cells were fixed using Cytofix fixation buffer (BD Biosciences) for 20 mins at RT. Flow cytometry was performed on a BD LSR Fortessa instrument calibrated daily with BD cytometer setup and tracking beads. Analysis of data was carried out using FlowJo software (Treestar). Tendon cell populations were gated on CD45^– and CD34^– cells.

 

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software). Normality was tested using the Shapiro-Wilk normality test. Pairwise Mann-Whitney U tests were used to test for differences in expression of CD14, CD68 in healthy and diseased tendons. Kruskal-Wallis tests followed by pairwise post hoc Mann-Whitney U tests were used to compare mRNA expression of macrophage and stromal fibroblast activation genes in healthy hamstring and in diseased tendinopathic and ruptured Achilles tendons. Pairwise Mann-Whitney U tests were used to test for differences between mRNA expression of macrophage and stromal fibroblast activation target genes in cytokine-treated healthy and diseased tendon cells. P<0.05 was considered statistically significant.

REFERENCES

 

 

1. Kujala UM, Sarna S, Kaprio J. Cumulative incidence of achilles tendon rupture and tendinopathy in male former elite athletes. Clin J Sport Med 2005;15(3):133-5.

2. Lenskjold A, Kongsgaard M, Larsen JO, et al. The influence of physical activity during youth on structural and functional properties of the Achilles tendon. Scand J Med Sci Sports 2015;25(1):25-31.

3. Jarvinen TA, Kannus P, Maffulli N, et al. Achilles tendon disorders: etiology and epidemiology. Foot Ankle Clin 2005;10(2):255-66.

4. Almekinders LC, Temple JD. Etiology, diagnosis, and treatment of tendonitis: an analysis of the literature. Med Sci Sports Exerc 1998;30(8):1183-90.

5. Dudhia J, Scott CM, Draper ER, et al. Aging enhances a mechanically-induced reduction in tendon strength by an active process involving matrix metalloproteinase activity. Aging Cell 2007;6(4):547-56.

6. Nell EM, van der Merwe L, Cook J, et al. The apoptosis pathway and the genetic predisposition to Achilles tendinopathy. J Orthop Res 2012;30(11):1719-24.

7. Gwilym SE, Watkins B, Cooper CD, et al. Genetic influences in the progression of tears of the rotator cuff. J Bone Joint Surg Br 2009;91(7):915-7.

8. Dakin SG, Martinez FO, Yapp C, et al. Inflammation activation and resolution in human tendon disease. Science translational medicine 2015;7(311):311ra173.

9. Millar NL, Hueber AJ, Reilly JH, et al. Inflammation is present in early human tendinopathy. Am J Sports Med 2010;38(10):2085-91.

10. Millar NL, Akbar M, Campbell AL, et al. IL-17A mediates inflammatory and tissue remodelling events in early human tendinopathy. Sci Rep 2016;6:27149.

11. Dakin SG, Buckley CD, Al-Mossawi MH, et al. Persistent stromal fibroblast activation is present in chronic tendinopathy. Arthritis Res Ther 2017;Jan 25(19(1):16).

12. Tallon C, Maffulli N, Ewen SW. Ruptured Achilles tendons are significantly more degenerated than tendinopathic tendons. Med Sci Sports Exerc 2001;33(12):1983-90.

13. Kragsnaes MS, Fredberg U, Stribolt K, et al. Stereological quantification of immune-competent cells in baseline biopsy specimens from achilles tendons: results from patients with chronic tendinopathy followed for more than 4 years. Am J Sports Med 2014;42(10):2435-45.

14. Murray PJ, Allen JE, Biswas SK, et al. Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines. Immunity 2014;41(1):14-20.

15. Rahim M, El Khoury LY, Raleigh SM, et al. Human Genetic Variation, Sport and Exercise Medicine, and Achilles Tendinopathy: Role for Angiogenesis-Associated Genes. OMICS 2016;20(9):520-7.

16. Belperio JA, Keane MP, Arenberg DA, et al. CXC chemokines in angiogenesis. J Leukoc Biol 2000;68(1):1-8.

17. Carr A, Cooper C, Campbell MK, et al. Effectiveness of open and arthroscopic rotator cuff repair (UKUFF): a randomised controlled trial. The bone & joint journal 2017;99-B(1):107-15.

18. Robinson JM, Cook JL, Purdam C, et al. The VISA-A questionnaire: a valid and reliable index of the clinical severity of Achilles tendinopathy. Br J Sports Med 2001;35(5):335-41.

19. Murphy RJ, Floyd Dean BJ, Wheway K, et al. A Novel Minimally Invasive Ultrasound-Guided Technique to Biopsy Supraspinatus Tendon. Oper Tech Orthop 2013;23(2):56-62.

 

FIGURE LEGENDS

 

FIGURES

Figure 1. Immunohistochemistry showing the phenotypes of myeloid cells and increased vascularity of diseased Achilles tendons. (A) Graphs show quantitative analysis of CD14+ and CD68+ myeloid cells in healthy hamstring and diseased (tendinopathic and ruptured) Achilles tendons. Bar shows median values. Statistically significant differences were calculated using pairwise Mann-Whitney U tests. (B-D)Representative immunofluorescence images of sections of diseased Achilles tendons stained for inflammation activation markers including those of the Interferon pathway (IRF1 and IRF5, purple), the glucocorticoid receptor activation pathway (CD163, red) and the STAT-6 pathway (CD206, green). CD68 (green) is a marker of tissue resident macrophages. Cyan represents POPO-1 nuclear counterstain. Scale bar, 20 m. (E) Representative images of 3,3’-diaminobenzidine immunostaining (brown) for vascular marker CD31 in healthy hamstring, tendinopathic and ruptured Achilles tendons. Nuclear counterstain 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 inflammation activation pathway 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-B (IL-8, PTGS2), Interferon (IRF1, IRF5 and CXCL10), STAT-6  (CD206) and glucocorticoid receptor activation pathways (CD163). The mRNA signature of diseased Achilles tendons was compared with healthy hamstring tendons (n=6 donors). Gene expression is normalized to -actin. Statistically significant differences were calculated using pairwise Mann-Whitney U tests. Bars represent median values.

 

 

Figure 3. Expression of stromal fibroblast activation markers in healthy hamstring and diseased Achilles tendons. (A) mRNA expression of stromal fibroblast activation markers Podoplanin (PDPN), CD248 and CD106 in healthy hamstring tendons (n=5 donors), tendinopathic (n=11 donors) and ruptured Achilles tendons (n=13 donors). Gene expression is normalized to -actin. Statistically significant differences were calculated using pairwise Mann-Whitney U tests. Bars represent median values. (B-D) Representative immunofluorescence images of sections of diseased Achilles tendons (tendinopathic, B and rupture C) and healthy hamstring tendons (D) stained for markers of stromal activation (PDPN, green; CD248 and CD106, purple) and toll-like receptor 4 (TLR4) (red). Cyan represents POPO-1 nuclear counterstain. Scale bar 20m.

 

Figure 4. Expression of markers of inflammation in cultured tendon 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) Expression of stromal fibroblast activation marker Podoplanin (PDPN) after IL-1 treatment (10ngml^-1) for 24 hrs determined by flow cytometry. (B) Expression of PDPN mRNA after IL-1 treatment (10ngml^-1) for 24 hrs. Expression of Interferon target genes IRF5 (C) and IRF1 (D) after IFN treatment (20ngml^-1) for 24hrs. Statistically significant differences were calculated using pairwise Mann-Whitney U tests. Gene expression is normalized to -actin; bars represent median values. **p<0.01, * p<0.05.

 

 

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 an inflammatory stimulus, diseased tendon cells become ‘primed’ and express markers of stromal fibroblast activation including Podoplanin (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 sensitize tendon cells and, increase susceptibility to further episodes of inflammation and recurrent tendon disease.

 

TABLES

 

Table 1. Comparative features of disease in functionally distinct tendons

 

Feature	Achilles tendon disease	Shoulder tendon disease
Symptom duration	acute or chronic	frequently chronic
Type of disease	tendinopathic or ruptured	tendinopathic or torn
Typical time to clinical presentation	days-weeks (rupture)  months-years (tendinopathic)	months-years
Inflammation changes with type of disease	yes	yes
Inflammation changes after treatment in asymptomatic patients	unknown	yes

 

 

SUPPLEMENTARY INFORMATION

 

Figure S1 Isotype control staining of diseased Achilles tendons

Table S1 Primary antibodies used for immunohistochemistry and immunofluorescence

Table S2 Primers used for quantitative polymerase chain reaction

Table S3 Antibodies used for flow cytometry