Role Of Chemokine CXCL14 In The Pathogenesis Of Necrotizing Enterocolitis(NEC)

Neonatal necrotizing enterocolitis(NEC)is a severe intestinal infections disease,which is common in newborns and leads to disastrous results.The imbalance of pro-inflammatory response is an important mechanism for NEC,and macrophages are the key to the regulation of intestinal inflammatory response and are related to immune injury in a variety of tissues,just like intestinal epithelial immune injury in NEC.In addition,studies have shown that intestinal macrophages can regulate Paneth cells to maintain the function of intestinal stem cells and promote the renewal and repair of intestinal epithelial cells.These two different conclusions reveal that the main function of intestinal macrophages may have changed in NEC intestine,but the cause of this phenomenon is not clear yet.In this paper,we found that intestinal epithelial cells negatively regulate macrophage TNF-α expression and inhibit inflammatory damage,which together with macrophages promote intestinal epithelial renewal and repair mechanisms,constitute the macrophage-intestinal epithelial cell homeostasis;and the chemokine CXCL14 expressive from intestinal epithelial is a key molecule to maintain this homeostasis;CXCL14 expression in the intestinal epithelium is suppressed during NEC and leads to uncontrolled pro-inflammatory effects of macrophages and suppression of intestinal stem cell function resulting in homeostatic imbalance.The specific studies are as follows.Part Ⅰ The homeostasis between macrophages and intestinal epithelium is broken during NECObjective:The role of intestinal macrophages during NEC was explored through clinical sample studies,animal models and organoid cell experiments.Methods:Immunofluorescence staining was used to label macrophages,intestinal stem cells,and inflammatory factors in clinical samples to observe their changes at NEC;Western blot experiments were performed on tissues of clinical samples to verify the intestinal stem cell-associated WNT/β-catenin pathway and to determine whether the pathway was inhibited at NEC;to clarify the correlation between macrophages in the intestine of NEC and TNF-α and In order to clarify the correlation between macrophages and TNF-α and IFN-γ expression in the intestine of NEC,Clophosome-A-Clodronate Liposomes(Anionic)was used to remove intestinal macrophages in C57/BL mice and perform NEC modeling,and to compare the expression of inflammatory factors in the intestine;small intestine organoid was used as a cell model that simulates the homeostatic environment of intestinal epithelium,human peripheral blood mononuclear cells were collected and sorted to obtain human macrophages after stimulation with CSF1,and mouse macrophages can be obtained by harvesting bone marrow cells after induction of differentiation with CSF1 too;After co-cultured with intestine organoid and stimulated relevant cytokines and Lipopolysaccharide(LPS),and the results were compared by the number of organoid,Western blot and ELISA experiments on key protein molecules,and qRT-PCR experiments on key mRNA molecules,and then explore the mechanism of intestinal macrophages on small intestinal stem cells and intestinal epithelial homeostasis in children with NEC.Results:Immunofluorescence staining of clinical intestinal samples confirmed that macrophages were significantly increased in the NEC intestine,with a disturbed distribution and high expression of NF-κB,while macrophages were regularly distributed around the intestinal crypts in the control group.TNF-α and IFN-γ expression was significantly increased in the NEC intestine,with TNF-α being abundantly expressed in the intestinal stroma of NEC children and co-localized with macrophages.After depletion of mouse intestinal macrophages,TNF-α and IFN-γ expression was downregulated in the intestine of NEC mice,but did not improve the survival rate of NEC mice;TNF-α reduced the proliferation of intestinal organoid by inhibiting the WNT/β-catenin pathway,which is an important inflammatory factor regulating intestinal stem cells,and the effect was dose-dependent;IFN-γ had no inhibitory effect on this pathway;in vitro In vitro co-culture experiments confirmed that macrophages could promote the proliferation of intestinal organoid and protect intestinal organoid against LPS stimulation within a certain range;however,if the concentration of LPS was too high,TNF-α expression in the co-culture system increased rapidly,leading to an obvious limitation of organoid proliferation.The tendency was weaker,suggesting that the intestinal organoid also had a regulatory effect on macrophages.Conclusion:Macrophages and intestinal organoids regulate each other and maintain homeostasis and resist the pro-inflammatory effect in a certain range;However,when the pro-inflammatory effect exceeds a certain limit,the excessive expression of TNF-α by macrophages can affect the function of intestinal stem cells by inhibiting WNT/β-catenin pathway,resulting in the imbalance of intestinal epithelial homeostasis.Part Ⅱ CXCL14 and macrophages are involved in the regulation of intestinal inflammatory injury and repair in NECObjective:To explore the regulatory mechanism between the intestinal inflammatory response and tissue repair related pathways in NEC.Methods:The results of NEC-related experiments were analyzed by searching the GEO database to identify differentially expressed genes between children with NEC and the normal group,and after enrichment analysis,the inflammatory regulatory pathways and epithelial homeostasis-related pathways were clarified,and whether there was an association between the pathways by reciprocal network analysis.The key molecules identified by the raw signal analysis were then validated in the intestinal tissues of clinical children using immunofluorescence and Western blot experiments.Results:By analyzing GSE46619 and GSE178088 data sets,it was found that there is a link between inflammatory factor regulation and epithelial growth repair pathways in the intestine of children with NEC.CXCL14 is involved in inflammatory factors and growth repair-related pathways,mainly expressed in intestinal non-immune cells,and its expression is decreased in NEC;immunofluorescence staining experiments of clinical intestinal samples free of CXCL14 confirmed that CXCL14 expression is down-regulated in the intestine of NEC.Conclusion:CXCL14 is involved in regulatory pathways related to inflammatory factors and growth repair in intestinal tissues,and its expression is reduced in NEC intestine;the trend of CXCL14 expression changes in NEC intestine is opposite to the trend of pro-inflammatory effect of macrophages.Part Ⅲ CXCL14 regulates the expression of TNF-α in macrophages and protects intestinal epitheliumObjective:To clarify the role of CXCL14 in the intestine and its ability to regulate macrophages.Methods:Intraperitoneal injection of CXCL14 was used to intervene in the NEC molds of C57/BL mice,and to clarify whether it has a protective effect on the intestine based on the survival rate of mice,HE staining of intestinal tissues and immunofluorescence staining;LPS was used to stimulate the intestinal organoid to clarify the effect on CXCL14 expression in the intestinal epithelium;in vitro cell experiments were used to verify whether the intestinal organoid could express CXCL14 and to analyze the effect of CXCL14 on TNF-α expression by macrophages.Results:There was no statistically significant difference in the effect of CXCL14 on the survival rate of NEC-molded mice,but HE staining confirmed that it could reduce inflammatory damage in the intestine,and immunofluorescence staining showed that intestinal TNF-α expression was downregulated;ELISA assay confirmed that CXCL14 expression was higher in intestinal organoid medium relative to Caco2 cell line;LPS stimulation reduced CXCL14 concentration in intestinal organoid medium and also inhibited its mRNA expression;CXCL14 reduced TNF-α expression in the co-culture system.Conclusion:CXCL14 is expressed in the intestinal epithelium and is an important cytokine for maintaining intestinal epithelial homeostasis by negatively regulating the expression of TNF-α by intestinal macrophages and antagonizing the pro-inflammatory effect,but excessive LPS can inhibit CXCL14 expression and cause the pro-inflammatory effect of intestinal macrophages to become uncontrolled,leading to the development of NEC.