Harnessing The Regulatory Effect Of Immflammatory Cotokines On Tendon Stem Progenitor Cells For Tendon Calcification Treatment

Tendon disease, also called as tendinopathy, is a common and significant health care problem both in athletes and ageing population. Calcifying and degenerative tendinopathy causes loss of function, pain and secondary osteoarthritis with a substantial economic and social burden. Multiple therapies have been applied in patients with rotator cuff and Achilles’ tendon tendinopathy with mixed outcomes, due to our incomplete understanding of the risk factors and mechanisms under pinning tendon pathology. Thus, insights into the cellular and molecular processes of tendon regeneration and calcification may contribute to the design of new approaches for immune modulation of tendinopathy.Inflammation is crucial to mitigate infections, clear damaged cells and initiate tissue repair. However, chronic inflammation perpetuates tissue remodeling and underlies many chronic diseases, such as cancer, fibrosis, rheumatoid arthritis and the spondyloarthropathies. Growing evidences support the role of inflammation during the development of tendon diseases. Anti-inflammatory drug treatment, such as non-steroidal anti-inflammatory drugs (NSAIDs), and glucocorticoids result in pain relief but unsatisfying functional recovery. Recent studies suggest that self-limiting acute inflammation is essential for tissue stem/progenitor activation and differentiation, angiogenesis and proper restore of injured tissue. These studies highlight that new therapies of tendinopathy require fine-tuned approaches that specific blocks the negative effects of inflammation in an environment while the positive effects of in immune response is preserved. Thus, insights of the immune modulation for tendon regeneration and calcification in a subtype and phase dependent manner may shed light on the development of new therapeutic approaches for tendinopathy.In this study, we (1) characterized the expression profile of inflammatory cytokines during acute and chronic tendon injury, selected targeted inflammatory cytokines for each phase based on the expression trends; (2) examined the regulatory effect of each cytokine on proliferation and differentiation of TSPCs, (3) studied the molecular mechanism underlying the effect of cytokines; (4) manipulated the fate of endogenous TSPCs with small molecules targeting related signaling pathways for tendon regeneration.Part I. Expression profiles of inflammatory cytokines in different phases of tendinopathyWe established rat tendinopathy by injection of collagenase type Ⅰ into Achilles’tendons and analyzed the level of inflammatory cytokines during different phase of inflammation response.1 week after injury, massive immune cells infiltrated into tendon tissues, meanwhile expression of pro-inflammatory cytokine IL-1β was significantly up-regulated; 2 weeks after injury, anti-inflammatory cytokines IL-4 and IL-10 were up-regulated and the number of infiltrated immune cells was decreased.8 weeks after injury, immune cells are rare in tendon tissue, but NF-κB signaling pathway was activated in cells around calcific sites. In addition, in clinical human microarray data, it shows that levels of IL-la and IL-1β were mildly increased and levels of IL-4 and IL-10 were decreased in chronic tendinopathy, indicating imbalance between pro-inflammatory and anti-inflammatory cytokines.In summary, after acute injury of tendon, the pro-inflammatory and anti-inflammatory cytokines were sequentially up-regulated, which mediated the activation and resolution of inflammation. In chronic tendinopathy, the imbalance between pro-inflammatory and anti-inflammatory cytokines may contribute to the development of tendinopathy.Part Ⅱ. Reparative and pathogenic effect of inflammatory cytokinesTo investigate the role of each cytokine in tendon regeneration and calcification, we added IL-1β (5ng/ml), IL-4 (lOng/ml,20ng/ml) and conditioned M1/M2 medium into cultured tendon tissues for 3 days. We observed that IL-4 (20ng/ml) and conditioned M1/M2 medium maintained the expression level of tendon specific transcription factor Scleraxis (Scx), but IL-1β (5ng/ml) suppressed Scx level. To further study effect of IL-1β on TSPCs in a time-dependent manner, we 3-D cultured TSPCs for 5days and 8days with IL-10 (5ng/ml) and for another 3days after IL-1β withdrawal. Scx expression suppressed by short time treatment (5days) of IL-lp is temporary which can be re-induced after IL-1β withdrawal, but suppression by long time treatment (5days) was not. In addition, IL-1β treatment for 1 week not only increased proliferation of TSPCs but also elevated the level of chondral-lineage transcription factor Sox9 and alkaline phosphatase (ALP) staining.In summary, we found that acute increase of IL-1β level promoted proliferation of TSPCs and reversibly suppressed Scx expression, while soluble factors secreted by M2 macrophages, such as IL-4, could maintain the expression level of Scx thus may facilitate teno-lineage commitment of TSPCs. However, long period treatment of IL-1β irreversibly suppressed expression level of Scx and promoted osteochondral differentiation, which may contribute to ectopic calcification.Part Ⅲ. HIF-2α mediated pathogenic effect of IL-1β on TSPCs and the underlying mechanismTo further investigate the molecular mechanism, we analyze the gene expression microarray of human tendinopathy and found that transcription factor HIF-2α was up-regulated in chronic tendinopathy. We observed that HIF-2alpha signaling is markedly activated within stem/progenitor cells recruited at calcified sites of diseased human tendons and heart valves. Pro-inflammatory microenvironment, rather than hypoxia, is correlated with HIF-2alpha activation. Overexpression of active HIF-2alph enhanced osteogenesis of TSPCs, while anti-HIF-2alph shRNA reduced osteogenesis under IL-1β treatment. Several conserved potential hypoxia-responsive elements (HRE) were noted in the regulatory regions upstream and downstream of the transcription start site (TSS) of the SCX gene. Scx expression was down-regulated by activation of HIF-2alph, and was significantly derepressed in human TSPCs upon HIF-2alpha inhibition. Overexpression of Scx could partially block the pro-osteogenic effect of HIF-2alpha on TSPCs.In summary, abnormal upregulation of HIF-2alpha by IL-1β served as a key switch to direct TSPCs differentiation into osteochondral-lineage rather than teno-lineage.Part Ⅳ. Manipulation of immune-mediated response of TSPCs for tendon regenerationWe used small molecules targeting NF-κB and HIF-2α signaling pathway to modulate the IL-1β-mediated imbalanced inflammatory microenvironment and the pathogenic response of TSPCs. Digoxin effectively inhibits HIF-2alpha upregulation induced by IL1β and promotes tenogenesis and inhibits calcification of TSPCs in vitro. Treatment of digoxin 3 days after injury could significantly promote Scx expression and teno-lineage fate specification during tendon repair while inhibit calcification progression. These results indicate that digoxin could modulate pro-calcific response of TSPCs in chronic tendinopathy through inhibiting HIF-2a.Curcumin has been reported to inhibit NF-κB signaling in tenocytes. We observed that curcumin treatment could efficiently maintain Scx expression and promote proliferation of TSPCs.6 weeks treatment of curcumin (3 days after injury) upregulated anti-inflammatory cytokines such as IL-4 and IL-10, while down-regulated the level of pro-inflammatory cytokine IL-1α. Also, curcumin treated macrophages secreted soluble factors that could induce Scx expression in TSPCs. Histological analysis shown that curcumin treated tendons secreted more tendon specific extracellular matrix. These results suggest that curcumin could directly act on TSPCs and indirectly regulate inflammatory microenvironment in chronic tendinopathy.In summary, we discovered that after tendon injury, an acute biphasic inflammatory response mediates an initial proliferative phase followed by a differentiation phase of TSPCs, while chronic imbalance between pro-inflammatory and anti-inflammatory cytokines leads to decreased teno-lineage differentiation of TSPCs. Chronic IL-1β elevation also promote osteochondral differentiation of TSPCs which contribute to ectopic calcification. Abnormal upregulation of HIF-2alpha served as a key switch to direct TSPCs differentiation into osteochondral-lineage rather than teno-lineage. And harnessing the inflammatory microenvironment and TSPCs responses toward IL-1β signaling could promote tendon regeneration while decrease ectopic calcification. Therefore, our results provide new cellular and molecular mechanism underlying immune modulated tendon regeneration and calcification.