Regulation Of Inflammation Resolution By Serine Protease Inhibitor Spink7 And The Underlying Mechanism

Inflammation is a crucial part of immune responses towards invading pathogens or tissue damage.To prevent inefficient removal of the threat but also excessive damage to the host,inflammation processes must be tightly controlled.Thus,it is important that the inflammation processes are terminated in a coordinate manner to prevent tissue damage due to the highly reactive inflammatory environment.Initially,tissue resident macrophages of the innate immune system detect the damaging insult and alarm circulating neutrophils to the site of injury,and then a large number of inflammatory monocytes rapidly infiltrate inflamed site and differentiate into macrophages in response to the action of inflammatory microenvironment.Once activated,classically macrophages exhibit a pro-inflammatory phenotype(M1 type)and secrete large amounts of pro-inflammatory cytokines.With the development of inflammation,neutrophils undergo apoptosis after phagocyte pathogens or necrotic tissues.Subsequently macrophages phagocytize these apoptotic cells and then transit to alternatively activated type 2(M2 type),which has anti-inflammatory features and facilitates tissue repair.Thus,the microenvironment turns to be favorable for inflammation resolution and homeostasis re-establishment at site of inflammation.Inflammation resolution involves complex interactions between immune cells and microenvironment.Meanwhile,dysregulation of key links often leads to impaired inflammation resolution,which is an important cause for a variety of chronic inflammation diseases.Therefore,the in-depth study on the cellular and molecular mechanisms involved in the inflammation resolution and exploration new regulatory strategies are of great significance for the prevention and treatment of inflammatory resolution disorders diseases,such as chronic wounds and inflammatory bowel disease.Serine protease inhibitor kazal type 7(Serine Peptidase Inhibitor,SPINK7),also known as esophageal cancer-related gene 2(Esophagus Cancer-Related Gene 2,ECRG2),is located on human chromosome 5(mouse chromosome 18),and the coding region contains 258 bases,which is translated into 9.23 k D protein products containing 85 amino acids.SPINK7,which is initially recognized as markedly decreased in esophageal cancer by screening of differentially expressed genes,can be secreted into the extracellular,and is constitutively highly expressed in the esophageal epithelium and oral mucosa.Earlier studies have found that SPINK7,as a tumor suppressor,plays an important role in regulating cell proliferation,apoptosis,migration and invasion via two different pathways,namely,extracellular serine protease inhibitor activity and intracellular non-serine protease inhibitor activity.Recent studies reported that SPINK7 as an inhibitory checkpoint for inflammatory responses in esophageal epithelial cell,involved in the development of eosinophilic esophagitis(Eosinophilic Esophagitis,Eo E).It has been reported that SPINK7 expression was significantly reduced in Eo E patients by up to 15-fold compared to normal esophageal epithelium.Knockout or knockdown SPINK7 in normal esophageal epithelial cells can inhibit epithelial differentiation and thus impair its barrier function.More importantly,further investigation indicated that SPINK7 can promote the activation of eosinophils by inhibiting urokinase Plasminogen-type Ativator(u PA)and its specific receptor(urokinase Plasminogen Activator Receptor,u PAR),and also regulate the expression of many chemokines/cytokines by targeting down-regulation Kallikrein 5 protease(KLK5)activity to inhibit the activation of Proteinase-activated receptor 2(PAR-2),thus playing the role of inhibitory checkpoint in inflammatory response.However,the current studies on SPINK7’s role as an inhibitory checkpoint in the regulation of inflammatory response are limited to esophageal epithelium,whether it has broad-spectrum anti-inflammatory effects in other systems remain to be further studied.In the current study,the role of Spink7 in regulating inflammation resolution were systematically studied on both skin wound healing model and experimental colitis model in mouse respectively,and its possible mechanism was preliminarily discussed.In a murine model of acute wound healing,to address the role of Spink7 on the resolution of wound inflammation,we analyzes the effect of loss of Spink7 in the rate of wound closure,re-epithelialization level,infiltration of inflammatory cells,pro-inflammatory cytokines and chemokine level,protease activity level and macrophage polarization ratio by using histological staining,immunohistochemical staining,q RT-PCR,tissue RNA chips,multiple fluorescent enzyme-linked immunosorbent assay,gelatin zymography and the activity of urokinase detection technology.We investigate the effect of impaired inflammation resolution,delayed healing and other phenotypes by inhibiting PAR2 pathway on Spink7 knockout mice by using PAR2 inhibitor through in vivo interventions.To evaluate the effect of Spink7 on LPS-stimulated inflammatory response,and to preliminarily probe the anti-inflammatory mechanism of this gene,we conduct in vitro experiment to over-express Spink7 in Raw264.7 cells by using stable transfection of lentivirus,cell culture conditioned supernatant stimulation,and purified protein stimulation methods.In addition,the feasibility of promoting wound healing by Spink7 small interfering RNA(si RNA)knockdown was assessed by utilizing combined radiation and wound injury model in mouse with delayed wound healing due to insufficient inflammatory response.The primary results and conclusions of this part are summarized as follows:1.The expression of Spink7 was significantly up-regulated in the proliferative stage during cutaneous wound healing in mice,and the inhibition of Spink7 expression by specific si RNAs on the wounds resulted in delayed wound healing characterized by delayed re-epithelialization and impaired inflammation resolution.2.Spink7 knockout mice showed similar results with impaired inflammation resolution during wound healing.Further investigation showed that Spink7 knockout leads to multiple phenotypes including over-activation of inflammation related signaling pathway,high levels of proinflammatory cytokines and chemokines,hyperactivity of proteases including u PA and MMP2/9,significant impairment of M2 polarization of macrophages during the proliferative phase of wound healing(7 days after injury).3.Results of inhibiting PAR2 activation with specific inhibitor in vivo during wound healing indicated that the chronic refractory phenotype caused by knockout of Spink7 was not dependent on the activation of PAR2 pathway.4.Up-regulation of Spink7 levels in macrophage cell line by stable transfection of lentivirus,cell culture conditioned medium and purified protein could inhibit the expression of LPS-induced inflammatory cytokines and other genes.Moreover,we preliminarily found that Spink7 can negatively regulated LPS-induced NF-κB and p38 MAPK signaling pathways activation.5.In mouse combined radiation and wound injury model,the expression of Spink7 was significantly upregulated in 7-d wounds compared with those of acute wounds.Application of Spink7 specific si RNA could increase wound inflammation of this model and enhanced wound closure.To determine the possible role of Spink7 in the inflammation of experimental colitis,we evaluated the effects of Spink7 deletion on clinical symptoms of ulcer in DSS-induced experimental colitis in mouse,by using histological staining,immunohistochemical staining,q RT-PCR,multiple fluorescent enzyme-linked immunosorbent assay,laser confocal microscopic observation,construction of chimeric mice with bone marrow transplantation and bioinformatics analysis.The mainly results and conclusions of this part are summarized as follows:1.In DSS-treated mice,the expression of Spink7 was markedly increased.Loss of Spink7 led to more severe colitis,characterized by a significant loss of body weight,higher disease activity index,more shorted colon length,and higher histological score compared with those in WT mice after DSS exposure.In line with these results,expression and secretion of a series of pro-inflammatory cytokines and chemokines were also significantly higher than those of controls.2.The construction of bone marrow transplanted chimeric mice demonstrated that Spink7 derived from bone marrow hematopoietic cells played a major protective role against experimental colitis.Further results showed that Spink7 was mainly expressed in neutrophils of experimental colitis tissues.3.The expression of SPINK7 in human Inflammatory Bowel Disease(IBD)was evaluated by histochemical staining of SPINK7 and public database analysis.SPINK7 was constitutively expressed in human colonic epithelium,and tends to be downregulated in IBD.In conclusion,our results indicate that Spink7,as an immune inhibitory checkpoint,is a key factor in promoting inflammation resolution in both skin wound healing and experimental colitis murine models.In skin wound healing model,we show that Spink7 absence leads to inflammatory disorder characterized by impairment inflammation resolution phenotype.Our preliminary results indicate that Spink7 can promote inflammation resolution independent of PAR2 activation.Further investigation showed that Spink7 could antagonize LPS induces macrophage polarization to the M1 phenotype through inhibit NF-κB and other signaling pathways.Spink7 knock-down in wounds of combined radiation and wound injury model could increase wound inflammation and enhanced its healing.In experimental colitis,we demonstrate that Spink7 deficiency leads to a significant increase in inflammatory injury sensitivity and neutrophils-derived Spink7 plays important protective role during experimental IBD.These results enrich the theoretical understanding of the cellular and molecular mechanisms of inflammation resolution and provide a novel target on preventing and treating strategies for inflammatory resolution disorders such as chronic wounds and inflammatory bowel disease.