Leptin Enhances M1 Macrophage Polarization and Impairs Tendon-Bone Healing in Rotator Cuff Repair: A Rat Model

BACKGROUND: Rotator cuff tears are common, affecting more than 60% of individuals older than 80 years, and they have been implicated in 70% of patients with shoulder pain. M1 polarization-related inflammation has been reported to be associated with poor healing outcomes of rotator cuff injury, and leptin, an adipokine, has been reported to be a potential activator of inflammation. However, whether leptin affects rotator cuff repair remains unknown. QUESTIONS/PURPOSES: Using in vitro cell experiments and an in vivo rat rotator cuff tear model, we therefore asked: (1) Does leptin promote the M1 polarization of macrophages in vitro and in vivo? (2) Does leptin impair biomechanical strength, the histologic structure of the tendon-bone interface, bone mineral density (BMD), or gait in the rotator cuff tear scenario? (3) Does leptin promote M1 polarization by upregulating the tumor necrosis factor (TNF) pathway? METHODS: The impact of leptin on M1 macrophage polarization in vitro was determined by reverse transcription-polymerase chain reaction (RT-PCR), the Western blot test, and immunofluorescence staining. The effect of leptin on tendon-bone healing was assessed in an in vivo rat rotator cuff tear model by comparing a leptin group with a suture group in terms of gait, biomechanical tensile strength, the histologic structure of the tendon-bone interface, and BMD. In the in vivo experiments, 8-week-old male Sprague Dawley rats were used, adapting a previously developed rat rotator cuff tear model. The supraspinatus tendon was resected from the greater tuberosity bilaterally, and then the tendon was secured to its anatomical footprint using the transosseous single-row technique. In total, 30 rats were randomized into two groups (suture, leptin) by drawing lots (15 rats in each group). They were assessed at 2, 4, and 8 weeks after the surgery. In the suture group, 100 µL of normal saline was injected into the subacromial space after the deltoid muscle was restitched to the original position. In the leptin group, 100 µL of leptin solution (200 ng/mL) was injected into the subacromial space after the deltoid muscle was restitched to the original position. Biomechanical properties including maximal failure load, stiffness, and tensile failure stress were determined to assess the biomechanical strength at 4 and 8 weeks after the surgery. Histologic staining was conducted to compare the structure of the tendon-bone interface between treatment groups. Micro-MRI and micro-CT assessments were conducted to compare the overall healing outcome and BMD between treatment groups. Gait analysis was conducted to compare the stride length and strength between treatment groups. M1 macrophage polarization in vivo at the tendon-bone interface was assessed by immunofluorescence staining. Finally, to explore the underlying mechanism of the effects of leptin, Necrostatin-1 (Nec-1) was used to block the TNF signaling pathway in the in vitro macrophage study, and RT-PCR and Western blot were used to explore the underlying mechanism. RESULTS: Leptin enhanced LPS-induced M1 polarization of macrophages in vitro, showing increased gene expression of CD86, Nos2, and TNF-α as well as increased protein expression of CD86, TNF-α, interleukin-6 (IL-6), and inducible NO synthase (iNOS). The in vivo polarization showed that the M1 polarization of macrophages at the tendon-bone interface was promoted. At 2 weeks postoperatively, there were more M1 cells in the leptin group (53 ± 5 versus 77 ± 8, mean difference 24 [95% confidence interval (CI) 11 to 37]; p = 0.002), although the proportion of M1 cells (ratio of the number of M1 cells to the total number of macrophages) was not higher (18.6% ± 2.9% versus 21.5% ± 1.7%, mean difference 2.9% [95% CI -2.8% to 8.7%]; p = 0.36). At 4 weeks postoperatively, the leptin group exhibited more M1 cells (31 ± 4 versus 50 ± 6, mean difference 19 [95% CI 6 to 32]; p = 0.008) and at a higher proportion (16.4% ± 2.6% versus 23.0% ± 3.0%, mean difference 6.6% [95% CI 0.8% to 12.4%]; p = 0.03). The in vivo experiments showed that leptin impaired tendon-bone healing. At 4 weeks postoperatively, the biomechanical properties of both groups were not different in terms of maximal failure load (12.7 ± 1.6 N versus 12.4 ± 1.8 N, mean difference -0.3 N [95% CI -2.6 to 1.8]; p = 0.91), stiffness (5.1 ± 0.7 N/mm versus 4.6 ± 0.8 N/mm, mean difference -0.5 N/mm [95% CI -1.3 to 0.5]; p = 0.44), and tensile failure stress (2.0 ± 0.3 N/mm 2 versus 2.0 ± 0.3 N/mm 2 , mean difference 0.0 N/mm 2 [95% CI -0.4 to 0.4]; p = 0.99). At 8 weeks postoperatively, the leptin group showed worse maximal failure load (17.6 ± 1.4 N versus 14.1 ± 1.4 N, mean difference -3.5 N [95% CI -5.7 to -1.3]; p = 0.002), stiffness (7.0 ± 0.6 N/mm versus 5.2 ± 0.6 N/mm, mean difference -1.8 N/mm [95% CI -2.7 to -0.9]; p < 0.001), and tensile failure stress (3.4 ± 0.3 N/mm 2 versus 2.8 ± 0.4 N/mm 2 , mean difference -0.6 N/mm 2 [95% CI -1.0 to -0.2]; p = 0.007). Results of histologic staining, image assessments, and gait analysis also demonstrated that leptin impaired the healing process. In vitro experiments showed that leptin upregulated the gene expression of molecules in the TNF pathway, including CCL2 and receptor-interacting protein kinase 1 (RIPK1), and M1 markers, such as TNF-α, CD86, and Nos2; the addition of Nec-1 neutralized the effect of leptin on macrophage polarization, reducing the expression of M1 markers, including TNF-α, CD86, and Nos2, and blocking the TNF signaling pathway, including CCL2 and RIPK. The protein expression exhibited similar trends. CONCLUSION: Based on the results of this study, leptin appears to impair tendon-bone healing in a rat model of rotator cuff tear, promote M1 macrophage polarization at the tendon-bone interface, and upregulate the TNF signaling pathway in macrophages to promote M1 polarization. CLINICAL RELEVANCE: Obesity and fatty infiltration of the rotator cuff muscle are associated with poor healing of rotator cuff tears. In this study, the effect of leptin, an adipose factor, on tendon-bone healing and the underlying mechanism were explored. Future studies might focus on developing novel approaches to improve the tendon-bone healing in patients with obesity by targeting leptin or the TNF signaling pathway with the aid of biomaterials.