THE ROLE OF CYTOKINES AND SUBSTANCE P IN REPETITIVE LOADING-INDUCED BEHAVIORAL DECLINES AND TISSUE FIBROSIS

Abstract

Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance. Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance.