Haoran Wang1, Tao Wu1, Feng Hua1, Jinpeng Sun1, Yunfeng Bai1, Weishun Wang2, Jun Liu1, Mingshun Zhang3. 1. Department of Orthopedics, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China. 2. Department of Orthopedics, Sir Run Run Hospital, Nanjing Medical University, Nanjing, China. 3. NHC Key Laboratory of Antibody Technique, Jiangsu Province Engineering Research Center of Antibody Drug, Department of Immunology, Nanjing Medical University, Nanjing, China. 13776698080@139.com.
Abstract
BACKGROUND: Recent evidence suggests that IL-33, a novel member of the IL-1β family, is involved in organ fibrosis. However, the roles of IL-33 and its receptor ST2 in epidural fibrosis post spine operation remain elusive. METHODS: A mouse model of epidural fibrosis was established after laminectomy. IL-33 in the wound tissues post laminectomy was measured with Western blotting, ELISA and immunoflurosence imaging. The fibroblast cell line NIH-3T3 and primary fibroblasts were treated with IL-33 and the mechanisms of maturation of fibroblasts into myofibroblasts were analyzed. To explore roles of IL-33 and its receptor ST2 in vivo, IL-33 knockout (KO) and ST2 KO mice were employed to construct the model of laminectomy. The epidural fibrosis was evaluated using H&E and Masson staining, western-blotting, ELISA and immunohistochemistry. RESULTS: As demonstrated in western blotting and ELISA, IL-33 was increased in epidural wound tissues post laminectomy. The immunoflurosence imaging revealed that endothelial cells (CD31+) and fibroblasts (α-SAM+) were major producers of IL-33 in the epidural wound tissues. In vitro, IL-33 promoted fibroblast maturation, which was blocked by ST2 neutralization antibody, suggesting that IL-33-promoted-fibroblasts maturation was ST2 dependent. Further, IL-33/ST2 activated MAPK p38 and TGF-β pathways. Either p38 inhibitor or TGF-β inhibitor decreased fibronectin and α-SAM production from IL-33-treated fibroblasts, suggesting that p38 and TGF-β were involved with IL-33/ST2 signal pathways in the fibroblasts maturation. In vivo, IL-33 KO or ST2 KO decreased fibronectin, α-SMA and collagen deposition in the wound tissues of mice that underwent spine surgery. In addition, TGF-β1 was decreased in IL-33 KO or ST2 KO epidural wound tissues. CONCLUSION: In summary, IL-33/ST2 promoted fibroblast differentiation into myofibroblasts via MAPK p38 and TGF-β in a mouse model of epidural fibrosis after laminectomy.
BACKGROUND: Recent evidence suggests that IL-33, a novel member of the IL-1β family, is involved in organ fibrosis. However, the roles of IL-33 and its receptor ST2 in epidural fibrosis post spine operation remain elusive. METHODS: A mouse model of epidural fibrosis was established after laminectomy. IL-33 in the wound tissues post laminectomy was measured with Western blotting, ELISA and immunoflurosence imaging. The fibroblast cell line NIH-3T3 and primary fibroblasts were treated with IL-33 and the mechanisms of maturation of fibroblasts into myofibroblasts were analyzed. To explore roles of IL-33 and its receptor ST2 in vivo, IL-33 knockout (KO) and ST2 KO mice were employed to construct the model of laminectomy. The epidural fibrosis was evaluated using H&E and Masson staining, western-blotting, ELISA and immunohistochemistry. RESULTS: As demonstrated in western blotting and ELISA, IL-33 was increased in epidural wound tissues post laminectomy. The immunoflurosence imaging revealed that endothelial cells (CD31+) and fibroblasts (α-SAM+) were major producers of IL-33 in the epidural wound tissues. In vitro, IL-33 promoted fibroblast maturation, which was blocked by ST2 neutralization antibody, suggesting that IL-33-promoted-fibroblasts maturation was ST2 dependent. Further, IL-33/ST2 activated MAPK p38 and TGF-β pathways. Either p38 inhibitor or TGF-β inhibitor decreased fibronectin and α-SAM production from IL-33-treated fibroblasts, suggesting that p38 and TGF-β were involved with IL-33/ST2 signal pathways in the fibroblasts maturation. In vivo, IL-33 KO or ST2 KO decreased fibronectin, α-SMA and collagen deposition in the wound tissues of mice that underwent spine surgery. In addition, TGF-β1 was decreased in IL-33 KO or ST2 KO epidural wound tissues. CONCLUSION: In summary, IL-33/ST2 promoted fibroblast differentiation into myofibroblasts via MAPK p38 and TGF-β in a mouse model of epidural fibrosis after laminectomy.