| Background and aims:Acute pancreatitis（AP）is characterized by numerous etiologies,and an intractable course of treatment,and its pathogenesis has not been fully elucidated.Pancreatic acinar cells（PACs）injury is a key initiating event of AP,in which it plays an important role.Classical studies have suggested that receptor interacting protein kinase 3（RIPK3）and its downstream molecule mixed lineage kinase domain like protein（MLKL）are involved in the pathogenesis of inflammatory diseases by inducing necroptosis,but their specific roles in AP are inconclusive,especially the molecular mechanisms by which they regulate PACs injury.Macrophages are prone to polarization induced by injured cells at the site of inflammation,and there are studies showing that RIPK3-and MLKL-mediated cellular injury is associated with polarization of infiltrating macrophages.Macrophages are dominated by the proinflammatory M1 subtype in AP,but whether and how macrophage polarization is regulated by injured PACs remains unclear.Based on this,we aimed to explore whether and how RIPK3 and MLKL are involved in AP,explore the mechanisms by which they regulate pancreatic acinar cell injury,and investigate their association with macrophage polarization in the hope of providing a theoretical basis for further scientific research and clinical translation.Methods:1.In the first part of the study,we constructed AP by cerulein combined with lipopolysaccharide in C57BL/6J mice,and serum amylase and HE staining of the pancreas were used to clarify the presence of AP.Immunohistochemistry,western blotting,and immunofluorescence were used to detect the expression of RIPK3,p-RIPK3,MLKL,p-MLKL,and p-Ca MKII in the pancreas.For cerulein-stimulated PACs isolated from C57BL/6J mice,the expression of RIPK3 and MLKL was assessed using quantitative real-time PCR（q PCR）,and the expression of RIPK3,p-RIPK3,MLKL,and p-MLKL was detected by western blotting.To explore the regulatory relationship between MLKL and RIPK3,we constructed AP in Ripk3-/-mice and detected the expression of MLKL and p-MLKL in the pancreas using western blotting and immunohistochemistry.In addition,we isolated PACs from Ripk3-/-mice and treated them with cerulein.The expression of MLKL was detected using q PCR,and the expression of MLKL and p-MLKL was measured by western blotting.2.In the second part of the study,we constructed AP and their controls in C57BL/6J（WT）,Mlkl-/-and Ripk3-/-mice.Pancreatic HE staining,immunohistochemical staining of TNF-αand IL-6,serum amylase,and the ratio of pancreas weight to body weight were used to compare the severity of AP between groups.Immunohistochemical staining of p-p65,p-p38 and cleaved-caspase11 was used to determine whether knockout of MLKL or RIPK3 affected the activation of the NF-κB,MAPK and caspase11pathways.Immunofluorescence staining of Amylase-Tunel and Amylase-PI was used to detect PACs death.Pancreatic apoptosis was assessed by immunohistochemical staining of Bax,Bcl-2 and cleaved-caspase3.LDH release assays were used to examine the cell death rate of cerulein-treated,WT and Mlkl-/-mouse-derived PACs.Immunofluorescence staining of F4/80-i NOS and F4/80-CD206 was used to detect the infiltration of pancreatic M1 and M2 macrophages,respectively.To define the effect of PACs injury on macrophage polarization,we isolated PACs from WT and Mlkl-/-mice and collected conditioned medium,which was used to culture primary peritoneal macrophages.Peritoneal macrophage polarization was detected by immunofluorescence staining and q PCR.3.In the third part of the study,we isolated PACs from WT and Mlkl-/-mice,collected cells after cerulein treatment,applied transcriptome sequencing to identify their differential gene expression profiles,and identified the most significantly differentially expressed molecule,CXCL10.To verify the sequencing results,we constructed AP in WT and Mlkl-/-mice,isolated PACs from WT and Mlkl-/-mice and treated them with cerulein.Pancreatic CXCL10 was detected by immunofluorescence staining,CXCL10 in mouse serum and PACs supernatant was detected by ELISA,and cellular CXCL10 was detected by q PCR.To define the effect of CXCL10 in conditioned medium on peritoneal macrophage polarization,we neutralized CXCL10 in conditioned medium and then cultured macrophages with it.Peritoneal macrophage polarization was detected by immunofluorescence staining and q PCR.We also collected serum samples from patients with AP and healthy controls to detect CXCL10 levels by ELISA to evaluate its clinical relevance.4.In the fourth part of the study,we constructed AP in C57BL/6J mice treated with CXCL10 neutralizing antibodies.Pancreatic HE staining,serum amylase,serum inflammatory mediator TNF-αand IL-6 were performed to clarify whether in vivo neutralization of CXCL10 can alleviate AP.In addition,we detected pancreatic M1 and M2 macrophages by immunofluorescence staining of F4/80-i NOS and F4/80-CD206 to clarify whether in vivo neutralization of CXCL10 affects the polarization of pancreatic macrophages in mice with AP.Results:1.Significant upregulation of p-MLKL,but not RIPK3 and p-RIPK3,was observed in the pancreas of AP mice and in cerulein-stimulated PACs.In the pancreas of Ripk3-/-AP mice and cerulein-treated PACs derived from Ripk3-/-mice,significant upregulation of p-MLKL was also observed,suggesting that the phosphorylation of MLKL was independent of RIPK3,which is considered its canonical upstream molecule.2.The significant upregulation of p-Ca MKII and its substantial colocalization with p-MLKL in the pancreas of AP mice suggest a possible interaction between them.3.Compared with WT-AP mice,Mlkl-/-AP mice had lower pancreatic pathological scores of HE staining,serum amylase,ratio of pancreatic weight to body weight,and the expression of pancreatic TNF-αand IL-6.However,pancreatic injury was not alleviated in Ripk3-/-AP mice.4.The expression of p-p65,p-p38,and cleaved-caspase11 was significantly upregulated in the pancreas of WT-AP,Mlkl-/-AP,and Ripk3-/-AP mice compared with controls but was not significantly different among the three groups,suggesting that knockout of MLKL or RIPK3 does not alter the activation of these pathways.In addition,there were no significant differences in the cell counts of Amylase+Tunel+and Amylase+PI+cells in the pancreas among the three groups.The expression of the apoptotic proteins Bax,Bcl-2,and cleaved-caspase3 was also not significantly different between groups.The cell death rates of cerulein-treated PACs derived from WT and Mlkl-/-mice were also similar,suggesting that the protective effect of MLKL deletion was independent of PACs death.5.The cell count and proportion of M1 macrophages in the pancreas of WT-AP and Ripk3-/-AP mice were significantly elevated and were not significantly different between the two groups.Interestingly,the cell count and proportion of pancreatic M1 macrophages in Mlkl-/-AP mice were significantly reduced compared with those in WT-AP and Ripk3-/-AP mice,accompanied by an elevated cell count and proportion of M2 macrophages.The results suggested that knockout of RIPK3 did not affect macrophage polarization in the pancreas of AP mice,but knockout of MLKL significantly reduced M1 polarization of pancreatic macrophages.6.Conditioned medium from cerulein-treated,WT mouse-derived PACs exhibited a strong M1 polarizing effect on peritoneal macrophages,whereas conditioned medium from Mlkl-/-PACs showed a much weaker M1polarizing effect.7.Transcriptome sequencing of cerulein-treated PACs derived from WT and Mlkl-/-mice identified a large number of differentially expressed genes,with CXCL10 being the most significantly downregulated gene following MLKL deletion.CXCL10 was significantly upregulated in the pancreas and serum of WT-AP mice,as well as in cerulein-treated PACs and their supernatants,whereas knockout of MLKL significantly reduced the expression of CXCL10.8.Neutralization of CXCL10 in the conditioned medium of cerulein-treated,WT mouse-derived PACs significantly attenuated their ability to promote M1 polarization of peritoneal macrophages.9.We collected serum samples from 22 patients with AP and 6 healthy controls.The results showed a significant upregulation of serum CXCL10in patients with AP compared with controls.10.The pancreatic pathological scores of HE staining,serum amylase,and serum inflammatory mediators TNF-αand IL-6 in WT-AP mice were significantly reduced after in vivo neutralization of CXCL10.In addition,the proportion of pancreatic M1 macrophages was significantly decreased,while the proportion of M2 macrophages was significantly increased.Conclusions:1.MLKL was phosphorylated in AP in a RIPK3-independent manner and abundantly localized to PACs.Ca MKII might be the upstream regulator of MLKL.2.Knockout of MLKL alleviated AP in mice,whereas knockout of RIPK3 had no protective effect.The protective effect of MLKL deletion was associated with a reduction in pancreatic M1 macrophage infiltration.CXCL10 was significantly upregulated during PACs injury,and it is a key molecule in promoting M1 polarization of macrophages.Knockout of MLKL in PACs significantly reduced their expression of CXCL10 and impaired their pro-M1 ability.3.CXCL10 was significantly upregulated in the serum of patients with AP.In vivo neutralization of CXCL10 alleviated AP in mice,and its protective effect correlated with a reduction in pancreatic M1 macrophage infiltration.Figures:34;Tables:41;References:118. |
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