CD47 Mediated Efferocytosis Protect Against Acinar Cell Necrosis Of Acute Pancreatitis

Acute pancreatitis(AP)is a disease of the digestive system that originates from damage to pancreatic acinar cells and affects adjacent tissues and distal organs to varying degrees.When associated with extensive pancreatic necrosis,it is unpredictable and potentially fatal.Excessive or repeated damage to acinar cells can lead to pancreatic necrosis and simultaneously release a large number of proinflammatory mediators and inflammatory chemokines that can lead to pancreatic necrosis and infection,systemic inflammatory responses,and organ failure.It can progress to severe acute pancreatitis(SAP),which involves a dangerous condition,a high mortality rate,and great difficulty in clinical management,resulting in a heavy burden on medical resources and socioeconomic pressure.It is well known that AP is a disease related to cell death,and acinar cell damage and death caused by pancreatic enzyme activation are typical pathological features of AP.Once the damage to acinar cells triggers cell death,it may lead to rupture of the cell membrane and release of contents(protease,oxide,etc.),stimulate the release of inflammatory factors,and aggravate local pancreatic injury and even cause systemic inflammatory responses.Early apoptotic cells cooperate with some harmful secretory substances,including oxidative metabolites,zymogen particles,and inflammatory factors,to attract activated macrophages,which rapidly move to the site of inflammatory damage,a process known as "efferocytosis."As a key weapon in the regression of inflammation,cell burial may effectively protect against secondary cell necrosis and inflammatory responses and help regulate the progression of atherosclerosis,autoimmune diseases and other conditions.However,it is not yet fully understood how apoptotic acinar cells are degraded by the body in the AP state and what intervention options are available.Recent studies have shown that CD47,a transmembrane protein designated as a "do not eat me" signaling molecule on the cell surface,is overexpressed in vascular cells with atherosclerotic lesions,which is associated with irreversible death of apoptotic cells,impairs macrophage function with its clearance ability,produces an effect of "anti-efferocytosis",exacerbates the inflammatory response,and significantly increases the mortality rate in severe disease.In this study,we investigated for the first time the relationship between early damaged acinar cells(apoptotic cells)and the "cell burial effect" of macrophages and examined their molecular regulatory mechanisms in AP.Here,we confirm for the first time that pancreatic necrosis is closely related to the upregulation of CD47 expression,and that suppression of CD47 expression(by genetic intervention or pharmacological methods)has a potential therapeutic effect on AP as it represents an important "do not eat me" signal.We further elucidate the function,mechanism,and clinical relevance of efferocytosis and pancreatic necrosis as follows:① In human and mouse AP pancreatic tissues,we observed changes in CD47 expression by immunofluorescence staining,WB and qPCR,and confirmed the presence of cell burial at AP by transmission electron microscopy and confocal laser scanning microscopy;② We constructed CD47 knockout mice,used anti-CD47 neutralizing antibodies,miRNA agonists and pancreas-specific AAV-pan to regulate CD47 expression and promote the cellular spilling effect of macrophages on apoptotic acinar cells to protect hydatin-induced pancreatic necrosis in AP mice.In addition,we constructed a mouse model of severe acute pancreatitis by bile duct ligation(BDL)to simulate clinical acute biliary pancreatitis in humans.Similarly,we also observed upregulation of CD47 expression and enhanced cell burial in the mouse model induced by BDL AP.Inhibition of CD47(CD47 knockout mice and neutralizing antibodies against CD47)can improve pancreatic tissue necrosis and inflammatory responses and increase mouse survival.The expression of CD47 is regulated by TRIM28/miR133a,and the effect of regulating the TRIM28/miR133a/CD47 molecular axis in promoting cell shedding in cae-AP was verified;④ We established a co-culture system to simulate a phagocytic state of intercellular interaction in vitro,further confirming that TRIM28/miR133a strictly regulates the expression of CD47.All research results indicate that CD47-mediated cell burial is critical for preventing pancreatic necrosis,suggesting that targeting CD47 regulation has clinical significance for the treatment of AP.The research content is divided into the following five parts:Chapter 1CD47 was significantly over expressed in pancreatic tissue in acute pancreatitis.Objective:To study the expression of CD47 in mouse and human pancreatic tissue in acute pancreatitis.Methods:We established a mouse model of acute pancreatitis by intraperitoneal injection of hydantoin.Blood and pancreatic tissues were collected from mice at 6,12,and 24 hours after the first hydantoin injection,and blood and pancreatic tissues were also collected from control mice.Pancreatic tissues from mice with acute pancreatitis(6 hours)and control mice were selected for RNA-seq sequencing,and qPCR assays were then performed based on the sequencing results.Pancreatic tissue from mice with acute pancreatitis was stained with H&E at all time points,and serum amylase and lipase levels were measured.Simultaneously,protein and RNA were extracted from pancreatic tissues,and CD47 protein and mRNA levels were detected by WB and qPCR.Phagocytosis of macrophages was observed by transmission electron microscopy,and efferocytosis was observed by confocal laser scanning microscopy(TUNEL staining of apoptosis of acinar cells and Mac3 staining of macrophages).Pancreatic tissue from mice with acute pancreatitis and control mice was stained with CD47 and amylase immunofluorescence.In addition,we purchased a chip of human pancreatitis tissue through Biomax in the United States,and the adjacent tissue was taken from patients with pancreatic cancer in our hospital as a normal control.The human pancreatic tissue was also subjected to H&E staining,CD47 immunohistochemical staining,and Mac3/TUNEL immunofluorescence staining.The correlation between the expression of CD47 in pancreatic tissue and pancreatic necrosis was analyzed.Results:RNA-seq sequencing of mouse pancreatic tissue revealed a total of 10493 gene alterations,of which 5360 genes were increased and 5133 genes were downregulated.The GO component analysis of all differential genes revealed significant changes in the"phagocytosis" pathway or"phagocytosis" pathway(NES+2.0922,P=0.0012).GSEA analysis showed that in mice with acute pancreatitis,there were significant changes in the signaling pathways associated with the inflammatory immune response and cell death,where"phagocytosis" is located.Volcanic map and thermal map analysis based on the differential basis of the "phagocytosis" pathway showed that cell burial-related molecules(mainly molecules involved in the clearance of apoptotic cells by macrophages)CD47 and the ligand SIRP a were significantly upregulated in the pancreatic tissue of mice with acute pancreatitis.However,mRNA levels of four known "do not eat me" signaling molecules associated with cell burial showed an increase in CD47 and CD31 expression and an increase in CD47 by more than 20-fold.Immunofluorescence and WB also confirmed that CD47 protein levels are highly expressed,especially in damaged acinar cells,and correlate positively with disease severity.In pancreatic tissue from mice with acute pancreatitis,we observed macrophage activation and phagocytosis of necrotic contents(dead cells and debris,damaged mitochondria,lysosomes,etc.).Mac3/TUNEL immunofluorescence staining showed an increase in macrophage infiltration and cell burial in mice in the acute phase.In patients with acute pancreatitis,we also observed high expression of CD47 in injured acinar cells,and there was also a phenomenon of cell burial.Conclusion:The above results suggest that cell spillage and high expression of CD47 in injured acinar cells are hallmarks of acute pancreatitis,and CD47 may serve as a new target for clinical prevention and treatment of acute pancreatitis in the future.Chapter 2Targeted inhibition of CD47 mediated efferocytosis and protected acinar necrosis in mice of acute pancreatitisObjective:The protective effect of inhibiting CD47 in acute pancreatitis in vitro and in vivo experiments.Methods:We used CRISPER/Cas9 technology to construct CD47 knockout mice,then introduced anti-CD47 antibodies that have entered clinical research.Used the above two methods to target inhibited CD47.Cae and BDL were used to construct murine model of acute pancreatitis.Serum and pancreatic tissue were collected and serum enzyme(amylase,lipase)and inflammatory factor levels were measured.Efferocytosis was observed by TEM,and CLSM(Mac3/TUNEL staining staining).In addition,we isolated immune cells in the pancreatic tissue of AP mice induced by Cae and detected the activation of macrophages by flow cytometry.In the in vitro experiment,we performed experiments with both PACs and 266-6 acinar cells.First,the effect of inhibition of CD47 on necrosis of acinar cells was tested.Bone marrow-derived macrophages were extracted and stimulated to mature with MCSF to establish a co-culture system with CCK-induced acinar cells.Macrophages were collected for flow cytometry to observe the effect of targeted inhibition of CD47 on efferocytosis(extraction of CD47 KO acinar cells or application of antiCD47 and siCD47 gene intervention).Results:Both methods of inhibiting CD47 expression(CD47 knockout mice,anti-CD47 antibody)could promote efferocytosis(significant increase in co-localization of apoptosis in acinar cells by TUNEL/Mac3 staining)and protect acinar necrosis in mice with acute pancreatitis.Flow cytometry results suggest that inhibition of CD47 could also reduce macrophage infiltration and alter the immunological microenvironments.In vitro coculture experiments,targeted inhibition of CD47 increased macrophage efferocytosis rate without affecting acinar death.Similarly,we observed that inhibition of CD47 altered the activation state of macrophages,consistent with experimental results in vivo.Conclusion:Inhibition of CD47 promoted efferocytosis,protected acinar necrosis,and reduce the inflammatory response in mice with acute pancreatitis.Chapter 3Mechanism of targeted inhibiting CD47 mediated efferocytosis in acinar necrosis of acute pancreatitisPurpose:To clarify the upstream regulatory molecules of CD47 mediated efferocytosis in acute pancreatitis.Methods:TargetScan 8.0 was used to calculate and predict the upstream regulatory molecule miRNA and its regulatory sites of CD47.Simulated peptides(Agomir in vivo,Mimic,and inhibitor in vitro)were used to up regulate or inhibit miRNA expression,and miRNA expression was verified by FISH staining,qPCR.The effects of upstream regulatory molecules on acute pancreatitis were observed by H&E staining,serum enzymology,and serum inflammatory factors.The phenomenon of efferocytosis in pancreatic tissue was observed by TEM and CLSM.Subsequently,mimic-RNA was transfected into 266-6 cells and cholecystokinin was used to establish an AP model in vitro.Results:TargetScan 8.0 calculated and simulated that the upstream regulatory molecule of CD47 was miR133a.The application of agomir-miR133a significantly increased the expression of miR133a in pancreatic tissue(both FISH staining and qPCR observed a 5-fold increase in miR133a expression),and downregulated the level of CD47 protein and mRNA.Upregulation of miR133a expression promoted efferocytosis in mice with acute pancreatitis,and reduced serum enzyme levels.In vitro and vivo,upregulation of miR133a promoted the level of macrophage efferocytosis,while under the condition of simultaneous application of anti CD47 antibodies,the spilling effect was canceled.miRWalk showed that the upstream regulatory molecule of miR133a was TRIM28.TRIM28 and CD47 protein levels in pancreatic tissues of TRIM28 knockout mice(shTRIM28)were significantly decreased,while miR133a mRNA levels were upregulated.TRIM28 knockout(shTRIM28)promoted efferocytosis in vitro and in vivo.In vitro experiments,the upregulation effect of cellular burial was counteracted by using anti CD47 or the simulated peptide inhibitor miR133a.Conclusion:The above results highlight the molecular signaling axis that regulates cellular burial of damaged acinar cells,namely the TRIM28/miR133a/CD47 signaling axis.