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IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways.

ABSTRACT: Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by excessive hypercellularity and extracellular matrix (ECM) component deposition. Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Previous studies show that IL10 has potential therapeutic benefits in terms of preventing and reducing HS formation. However, no studies have examined IL10-mediated autophagy during the pathological process of HS formation. Here, we examined the effect of IL10 on starvation-induced autophagy and investigated the molecular mechanism underlying IL10-mediated inhibition of autophagy in HS-derived fibroblasts (HSFs) under starvation conditions. Immunostaining and PCR analysis revealed that a specific component of the IL10 receptor, IL10 alpha-chain (IL10Rα), is expressed in HSFs. Transmission electron microscopy and western blot analysis revealed that IL10 inhibited starvation-induced autophagy and induced the expression of p-AKT and p-STAT3 in HSFs in a dose-dependent manner. Blocking IL10R, p-AKT, p-mTOR, and p-STAT3 using specific inhibitors (IL10RB, LY294002, rapamycin, and cryptotanshinone, respectively) showed that IL10 inhibited autophagy via IL10Rα-mediated activation of STAT3 (the IL10R-STAT3 pathway) and by directly activating the AKT-mTOR pathway. Notably, these results suggest that IL10-mediated inhibition of autophagy is facilitated by the cross talk between STAT3, AKT, and mTOR; in other words, the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways. Finally, the results also indicate that mTOR-p70S6K is the molecule upon which these two pathways converge to induce IL10-mediated inhibition of autophagy in starved HSFs. In summary, the findings reported herein shed light on the molecular mechanism underlying IL10-mediated inhibition of autophagy and suggest that IL10 is a potential therapeutic agent for the treatment of HS.

SUBMITTER: Shi J

PROVIDER: S-EPMC4823945 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe hypertrophic scar (HS) is a serious fibrotic skin condition and a major clinical problem. Interleukin-10 (IL-10) has been identified as a prospective scar-improving compound based on preclinical trials. Our previous work showed that IL-10 has anti-fibrotic effects in transforming growth factor (TGF)-β1-stimulated fibroblasts, as well as potential therapeutic benefits for the prevention and reduction of scar formation. However, relatively little is known about the mechanisms underlying IL-10-mediated anti-fibrotic and scar-improvement actions.ObjectiveTo explore the expression of the IL-10 receptor in human HS tissue and primary HS fibroblasts (HSFs), and the molecular mechanisms contributing to the anti-fibrotic and scar-improvement capabilities of IL-10.MethodsExpression of the IL-10 receptor was assessed in HS tissue and HSFs by immunohistochemistry, immunofluorescence microscopy, and polymerase chain reaction analysis. Primary HSFs were treated with IL-10, a specific phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) or a function-blocking antibody against the IL-10 receptor (IL-10RB). Next, Western blot analysis was used to evaluate changes in the phosphorylation status of AKT and signal transducers and activators of transcription (STAT) 3, as well as the expression levels of fibrosis-related proteins.ResultsHS tissue and primary HSFs were characterized by expression of the IL-10 receptor and by high expression of fibrotic markers relative to normal controls. Primary HSFs expressed the IL-10 receptor, while IL-10 induced AKT and STAT3 phosphorylation in these cells. In addition, LY294002 blocked AKT and STAT phosphorylation, and also up-regulated expression levels of type I and type III collagen (Col 1 and Col 3) and alpha-smooth muscle actin (α-SMA) in IL-10-treated cells. Similarly, IL-10RB reduced STAT3/AKT phosphorylation and blocked the IL-10-mediated mitigation of fibrosis in HSFs.ConclusionIL-10 apparently inhibits fibrosis by activating AKT and STAT3 phosphorylation downstream of the IL-10 receptor, and by facilitating crosstalk between the PI3K/AKT and STAT3 signal transduction pathways.

Project description:Background: As a fibrotic disease with a high incidence, the pathogenesis of hypertrophic scarring is still not fully understood, and the treatment of this disease is also challenging. In recent years, human adipose-derived mesenchymal stem cells (AD-MSCs) have been considered an effective treatment for hypertrophic scars. This study mainly explored whether the therapeutic effect of AD-MSCs on hypertrophic scars is associated with oxidative-stress-related proteins. Methods: AD-MSCs were isolated from adipose tissues and characterized through flow cytometry and a differentiation test. Afterwards, coculture, cell proliferation, apoptosis, and migration were detected. Western blotting and a quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect oxidative stress-related genes and protein expression in hypertrophic scar fibroblasts (HSFs). Flow cytometry was used to detect reactive oxygen species (ROS). A nude mouse animal model was established; the effect of AD-MSCs on hypertrophic scars was observed; and hematoxylin and eosin staining, Masson's staining, and immunofluorescence staining were performed. Furthermore, the content of oxidative-stress-related proteins, including nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), B-cell lymphoma 2(Bcl2), Bcl2-associated X(BAX) and caspase 3, was detected. Results: Our results showed that AD-MSCs inhibited HSFs' proliferation and migration and promoted apoptosis. Moreover, after coculture, the expression of antioxidant enzymes, including HO-1, in HSFs decreased; the content of reactive oxygen species increased; and the expression of Nrf2 decreased significantly. In animal experiments, we found that, at 14 days after injection of AD-MSCs into human hypertrophic scar tissue blocks that were transplanted onto the dorsum of nude mice, the weight of the tissue blocks decreased significantly. Hematoxylin and eosin staining and Masson's staining demonstrated a rearrangement of collagen fibers. We also found that Nrf2 and antioxidant enzymes decreased significantly, while apoptotic cells increased after AD-MSC treatment. Conclusions: Our results demonstrated that AD-MSCs efficiently cured hypertrophic scars by promoting the apoptosis of HSFs and by inhibiting their proliferation and migration, which may be related to the inhibition of Nrf2 expression in HSFs, suggesting that AD-MSCs may provide an alternative therapeutic approach for the treatment of hypertrophic scars.

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IL10 inhibits starvation-induced autophagy in hypertrophic scar fibroblasts via cross talk between the IL10-IL10R-STAT3 and IL10-AKT-mTOR pathways.

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