The antiprotease Spink7 promotes inflammation resolution by modulating multiple proteases activities during wound healing

BACKGROUND: Effective control of inflammation is crucial for the healing of cutaneous wounds, but the molecular mechanisms governing inflammation resolution during wound closure are still not yet clear. Here, we describe a homeostatic mechanism that facilitates the inflammation resolution by timely regulating the targeted proteases activities through antiprotease Spink7 (serine peptidase inhibitor, kazal type 7). METHODS: The expression pattern of Spink7 was investigated by quantitative RT-PCR, immunohistochemistry (IHC) and in situ hybridization. In both Spink7 knockdown and knockout models, quantitative comparisons were made between the healing rate of wounds and histopathological morphometric analysis. Microarrays, multiple chemokine assays, IHC, immunofluorescence, protease activity measurement were performed to explore the underlying mechanisms of Spink7 knockout in impaired wound healing. Radiation-wound combined injury (R-W-CI) model was employed to evaluate the therapeutic effects of Spink7 manipulation. RESULTS: Our study demonstrates that Spink7 is significantly upregulated in the differentiated epidermal granular keratinocytes of proliferative phase during murine wound closure. Both local knockdown of Spink7 levels in wounds using siRNA gel and systemic knockout of Spink7 using KO mice resulted in delayed wound closure with sustained neutrophil infiltration. Loss of Spink7 leads to augmented inflammatory responses, increased production of multiple chemokines/cytokines, and impaired M2 polarization of macrophages in wound healing. Furthermore, loss of Spink7 results in elevated proteolytic activities of uPA, MMP2/9 and KLK5/7 in proliferative phase. However, inhibiting KLK5/7 downstream PAR2 activation exacerbates the phenotype of KO mice. In R-W-CI model, further significant induction of Spink7 is observed in wounds with insufficient inflammatory response. Local suppression of Spink7 promotes wound healing in the R-W-CI model by augmenting inflammation. CONCLUSIONS: Maintaining an endogenous balance between Spink7 and its target proteases is a crucial checkpoint for regulating inflammation resolution during healing. Therefore, manipulating levels of Spink7 might be an effective treatment for impaired wounds caused by inflammatory dysregulation.