The Interaction Of ANXA1 Released From Intestinal Epithelial Cells Between Natural Killer Cells To Ulcerative Colitis Mice And The Self-healing Mechanism Research

Part Ⅰ:The mechanism research of ANXA1 secreted by intestinal epithelial cells and NK cells on ulcerative colitis in miceObjective:To explore the expression variation of ANXA1 and whether NK cells gathered under the UC circumstance.Methods:6-8-week-old SPF C57BL/6 female mice were used to construct the ulcerative colitis model by DSS.Collected colon mucosa tissue were used to test ANXA1 gene and protein expression by Quantitative real-time PCR and Western blotting analysis.Same methods were applied to exam ANXA1 gene and protein expression from isolated IECs or EVs extracted from IECs.The percentage of NK cells were detected in MLN and LPMCs from normal mice and UC mice by flow cytometry.NK cells isolated from MLN were stained with CFSE and injected into the tail vein of mice that treated with DSS for 5 days,two days later the colon tissue were harvested to produce frozen section,which were observed under fluorescence microscope to access the CFSE+NK cells’ recruitment in colonic mucosal.Detecting the expression of FPR-1 from NK cells by Western blotting analysis,which is the receptor of ANXA1.Results:In the DSS-induced-UC mice model,ANXA1 increased in colonic mucosal tissue,IECs and EVs;More NK cells were recruited to colonic mucosal compared with normal mice and expressed FPR-1.Conclusions:IECs secreted excessive ANXA1 during UC mice model,and maybe wrapped in EVs to influence the NK cells.Over expressed ANXA1 might combine with NK cells through FPR-1,then the interaction affected the pathology of UC.Part Ⅱ:The interaction of ANXA1 released from intestinal epithelial cells and NKG2A expressed by NK cells on ulcerative colitispathology development and self-healing mechanism researchObjective:The interaction of ANXA1 released from intestinal epithelial cells and NK cells expressed NKG2A on UC mice,and the mechanism of this interaction on UC development.And to explore the self-healing mechanism of UC.Methods:Mice received normal saline or anti-ASGM1 Ab alone or/and in the present of anti-ANXA1 Ab by intraperitoneal injection.Then record the change of weight and colon length.Colon tissue were harvested and prepared for hematoxylin and eosin(HE)staining and pathological score.The percentage of neutrophils were tested in separated LPMCs by flow cytometry.A part of isolated NK cells were treated with ANXA1 or EVs;another part were co-cultured with IECs adding anti-ANXA1 Ab in transwell or not.To test IFN-y,TNF-a,NKp46,KLRG1,NKG2D and NKG2A genes expression from NK cells by Quantitative real-time PCR analysis,and to detect NKG2A protein expression by Western blotting analysis.Pretreated and untreated NK cells were transferred by tail vein injection respectively 4 days after anti-ASGM1 Ab injection to UC mice.Recording the change of weight and colon length and colon tissue were prepared for hematoxylin and eosin(HE)staining and histology score.Mice received anti-NKG2A Ab or IgG Ab,which repeated every 2 days to maintain NKG2A blocking.Recording the change of weight and colon length of mice and colon tissue were prepared for HE staining and histology score.The percentage of neutrophil in LPMCs were detected by flow cytomery.And to detect the expression of CD69,ROS,IL-6,IL-17A genes from isolated neutrophils by Real-time PCR analysis.Results:Depleting NK cells or blocking ANXA1 exacerbates inflammation of mice accompany with more neutrophil recruitment.Transferation of NK cells stimulated by ANXA1 alleviated inflammation of UC.Blocking NKG2A exacerbated inflammation by affecting recruitment and activation of neutrophils.Conclusions:Confirming the immune mechanism of IECs with NK cells in UC pathology development that IECs secreting excessive ANXA1 through EVs to influence the expression of NKG2A receptor in NK cells,alleviating the pathology of UC by affecting recruitment and activation of neutrophils,which might take part into the whole self-healing mechanism.