Investigation Of The Role And Mechanism Of PAD4-mediated NETosis In The Development Of Ulcerative Colitis

BackgroundUlcerative colitis（UC）is a chronic idiopathic intestinal inflammatory disease that is common in Western countries,and in recent years,its incidence has been increasing in China.UC is characterized by repeated disease episodes,resulting in frequent hospitalizations,a significant decline in quality of life,and interruptions to education and work,leading to a heavy medical burden and a negative impact on social development.Although the specific pathogenesis of UC is not yet clear,clinical and basic studies have suggested that disruption of intestinal homeostasis and intestinal mucosal barrier dysfunction are important factors contributing to its occurrence and development.Neutrophils are the most abundant immune effector cells in the innate immune system and act as the first line of defense against pathogen invasion.Recent research has shown that neutrophil activation can lead to the release of neutrophil extracellular traps（NETs）,which are DNA-based scaffolds consisting of condensed chromatin and various proteins,including neutrophil elastase（NE）,myeloperoxidase（MPO）,histone,particularly citrullinated-H3（Cit H3）,that help capture and kill invading microorganisms.While the specific mechanism and functional regulation of NETosis in neutrophils are still unclear,it is generally believed that citrullination mediated by peptidyl arginine deiminase 4（PAD4）is a crucial step in NETosis formation.Many studies have confirmed that PAD4-dependent NETosis is involved in the pathogenesis of various diseases,such as arthritis,sepsis,and thromboembolism.In UC,some scholars have observed an increase in NETosis,but its role in the pathogenesis of UC remains unclear.In this study,we intend to explore the role of NETosis mediated by PAD4 in the occurrence and development of UC by using PAD4knockout mice at the histological level,cellular level and citrullination modification level based on existing studies,in order to find a new direction for the diagnosis and treatment of UC.Contents and Methods1.Detection of NETosis level in colon tissue of DSS mice and UC patients（1）C57/BL6 mice colitis model was established by DSS.DAI score and colon length were used to evaluate the disease activity.HE-staining was used to evaluate the degree of inflammation;the levels of colonic inflammatory factors,intestinal barrier function and NETosis indicators were detected at gene expression level and protein expression level.（2）According to Mayo endoscopic score,human tissue biopsy samples and endoscopic images（healthy control,mild to moderate UC,severe UC）were collected.HE-staining was performed to evaluate the degree of inflammation in each group,and Western blot was used to detect NETosis indicators.（3）The correlation analysis between Mayo endoscopic score and NETosis indicators in patients with UC.2.Detection of NETosis levels and inflammatory barrier indicators in the colon tissue of PAD4 knockout mice（1）PAD4 knockout mice were generated and genotyped.（2）DSS-induced colitis models were established in wild-type and PAD4 knockout mice.DAI score and colon length were used to evaluate the disease activity.HE-staining was used to evaluate the degree of colonic inflammation.Colonic inflammatory factor levels,intestinal barrier function and NETosis indicators were detected at gene expression level and protein expression level.（3）Immunohistochemistry and Western blot were used to evaluate the level of citrullination in colon tissue of PAD4 knockout mice.3.Citrullination sequencing and single-cell RNA sequencing analysis of colon tissue of PAD4 knockout mice（1）Proteomic and citrullination sequencing analysis of DSS-induced colitis in wild-type and PAD4 knockout mice.Western blot was used to verify the citrullinated differential proteins in colon tissue of mice.（2）Single-cell sequencing analysis,cell subtype analysis and functional enrichment analysis of colon tissue from DSS-induced wild-type and PAD4 knockout mice.（3）The high-expression cell types were evaluated by single-cell sequencing and citrullination sequencing,and their colocalization was verified by immunofluorescence in mouse colon tissue.4.Exploration of epithelial cell barrier and apoptosis levels by NETosis-mediated citrullination（1）Immunofluorescence,Western blot and ELISA were used to detect the related indicators of NETosis in peripheral blood neutrophils of mice stimulated by ionomycin.（2）293T cells overexpressing CKMT1 and mutant-CKMT1 were treated with supernatants of neutrophils from Ionomycin-induced wild-type and PAD4 knockout mice.The levels of CKMT1 and citrullinated CKMT1 were detected by immunoprecipitation and Western blot.（3）The expression of CKMT1 in 293T cells was detected after the neutrophil of wild-type mice induced by Ionomycin was treated with protease inhibitor and autophagy inhibitor respectively.（4）Extracellular vesicles（EVs）were extracted from the supernatants of neutrophils stimulated by Ionomycin and detected by transmission electron microscopy and nanoparticle tracking particle analysis.The expression levels of PAD4 and EVs-related markers were detected by Western blot.The expression level of PAD4 in NCM460 cells was detected by Western blot to explore whether PAD4 could enter intestinal epithelial cells through the extracellular vesicle pathway.（5）After neutrophils from wild-type and PAD4-knockout mice were stimulated by Ionomycin,the supernatants of neutrophils stimulated colon cancer cell line Caco-2 and normal colon epithelial cell line NCM460.Western blot was used to detect the total citrullinated protein,epithelial barrier markers and apoptosis-related markers.（6）NCM460 transformation strain overexpressing CKMT1 and mutant-CKMT1 were established.After being treated with supernatants of neutrophils from wild-type and PAD4knockout mice induced by Ionomycin,the levels of CKMT1 and citrullinated CKMT1were detected by immunoprecipitation and Western blot.The epithelial barrier markers and apoptosis-related markers were detected by Western blot.Results1.Significant increase in NETosis levels in colon tissue of DSS mice and UC patients（1）DSS-induced intestinal barrier damage,increased inflammatory markers and increased levels of NETosis in wild-type mice.（2）DSS-induced increase in colonic protein citrullination in wild-type mice was positively correlated with PAD4 expression.（3）The levels of NETosis and protein citrullination in colon tissue of UC patients were increased,which were positively correlated with the endoscopic score.2.The level of NETosis in PAD4 knockout mice with colitis was significantly lower than that in wild-type mice.（1）The inflammation of PAD4 knockout mice with colitis was significantly reduced compared with wild-type mice,and the expression of proinflammatory factors were decreased.（2）The colonic epithelial barrier injury of PAD4 knockout mice was significantly reduced compared with wild-type mice,and the expression of barrier-related molecules was increased.（3）The level of NETosis in the colon of PAD4 knockout mice was significantly lower than that of wild-type mice.3.The level of citrullination in PAD4 knockout colitis mice was significantly reduced.（1）The colon tissue of DSS mice after PAD 4 knockout changed significantly at the proteomic level,and the differentially up-regulated proteins were mainly enriched in the energy metabolism pathway related to mitochondria.The citrullination level of CKMT1with a significant difference was increased in wild-type DSS mice and significantly decreased in PAD4 knockout DSS mice.（2）Two subsets of neutrophils,Padi4^highand Padi4^low,were defined in single-cell sequencing.The former is associated with oxidative phosphorylation,apoptosis,lysosome and other pathways;Eight epithelial cell subtypes were defined,among which immature distal epithelial cells were significantly increased in PAD4 knockout mice,mainly related to oxidative phosphorylation,tricarboxylic acid cycle and intestinal infection.（3）Single-cell sequencing showed that Padi4 gene was highly expressed in neutrophils while low in epithelial cells.Ckmt1 is highly expressed in the epithelium while low in neutrophils.Colon immunofluorescence staining confirmed that CKMT1colocalized with epithelial marker EPCAM,but not with neutrophil marker Ly6G.4.NETosis-mediated CKMT1 citrullination affects epithelial cell barrier and apoptosis.（1）Ionomycin is capable of inducing NETosis in peripheral blood neutrophils of wild-type mice,while such a process is suppressed in PAD4^-/-mice,leading to a significant reduction in NETosis-related protein levels in neutrophils.（2）The increase of CKMT1 citrullination induced by NETosis is mainly mediated by the 242 site,and the decrease in protein level may be associated with the autophagy-lysosome pathway.（3）The NETs supernatant derived from PAD4^-/-mice exhibit a substantial decrease in citrullination level,barrier injury,and apoptosis of Caco-2 and NCM460 cells.（4）PAD4 can enter intestinal epithelial cells through the extracellular vesicle pathway and impact its epithelial barrier and apoptosis by citrullinating 242 site in CKMT1.ConclusionIn this study,we initially observed an increase in NETosis levels in both DSS mice and UC patients.Through PAD4 knockout mice,we discovered that the elimination of PAD4 could significantly alleviate colitis and NETosis levels.Citrullination sequencing analysis demonstrated that the degree of citrullination at 243 site in CKMT1 was significantly higher in wild-type colitis mice,and significantly lower in PAD4 knockout mice.Single-cell RNA sequencing and immunofluorescence co-localization confirmed that CKMT1 was predominantly expressed in the epithelium.At the cellular level,we induced NETosis in peripheral blood neutrophils of mice through ionomycin.We discovered that the supernatant of neutrophils promoted an increase in CKMT1 citrullination,epithelial barrier damage,and apoptosis.Mutation at 242 site in CKMT1 resulted in a reduction in its citrullination,significantly lessened epithelial barrier damage,and significantly decreased levels of apoptosis.Our study revealed that PAD4 could potentially penetrate intestinal epithelial cells through an extracellular vesicle pathway after NETosis,affecting the intestinal epithelial barrier and apoptosis by mediating the citrullination of 242 site in CKMT1（corresponding to 243 in mice）.These findings provide a new avenue for exploring the mechanisms of NETosis in UC.