Specificity protein 1/ZIP8/zinc axis reduced macrophage extracellular traps to alleviate severe acute pancreatitis in mice

Figure 1 Macrophage extracellular traps are present in severe acute pancreatitis. A: Hematoxylin-eosin staining revealed pathological changes in the pancreatic and lung tissues in acute pancreatitis models induced by caerulein, and in severe acute pancreatitis (SAP) models induced by either caerulein or duct ligation; B: Levels of plasma amylase and lipase (n = 4); C: Immunofluorescence staining revealed the protein expression levels of citrullinated histone H3 (CitH3) (red fluorescence) and F4/80 (green fluorescence) in SAP; D: Levels of double-stranded DNA (dsDNA) in plasma (n = 4); E and F: Neutrophils were depleted from mice using an anti-Ly6G antibody, followed by induction of the SAP model. Cluster of differentiation (CD) 11b and Ly6G antibodies were subsequently used for staining, and flow cytometry was performed to determine the purity of CD11b and Ly6G in blood and pancreatic tissues; G: Levels of dsDNA in plasma (n = 5); H: After pretreatment with an anti-Ly6G antibody, the expression of F4/80 and CitH3 in pancreatic tissue was assessed by immunofluorescence. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; Nc: Negative control; AP: Acute pancreatitis; SAP: Severe acute pancreatitis; CitH3: Citrullinated histone H3; DAPI: 4’,6-diamidino-2-phenylindole; dsDNA: Double-stranded DNA.

Figure 2 Degradation of macrophage extracellular traps alleviates the severity of severe acute pancreatitis. A: Experimental flowchart: C57 mice were pretreated with anti-Ly6G antibody, followed by induction of severe acute pancreatitis and treatment with DNase I; B: Representative hematoxylin-eosin-stained images of pancreatic and lung tissues; C and D: Expression levels of amylase and lipase in plasma (n = 7); E and F: Representative immunohistochemical images of cleaved caspase 3 (brown granules) in pancreatic tissue, accompanied by statistical analysis (n = 7); G: Expression level of 16S rDNA in plasma (n = 4); H: Plasma fluorescein isothiocyanate (FITC) signal in each group of mice following oral gavage with FITC-dextran (n = 4). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; Ctrl: Control; DNase I: Deoxyribonuclease I; Casp: Caspase; IHC: Immunohistochemical; 16S rDNA: 16S ribosomal DNA; FITC: Fluorescein isothiocyanate.

Figure 3 Macrophage extracellular trap formation depends on the peptidylarginine deiminase 4 pathway and reactive oxygen species, and M1 macrophages exhibit a strong ability to form macrophage extracellular traps. A and B: Primary macrophages were cocultured with phorbol 12-myristate 13-acetate and pancreatic acinar cells (following injury induced by caerulein), and the expression levels of double-stranded DNA (dsDNA) were subsequently measured in each group (n = 3); C and D: Representative immunofluorescence images of citrullinated histone H3 (CitH3) and F4/80 in macrophages from each group (C), as well as statistical analysis of the positivity rate of CitH3 (D), a macrophage extracellular trap marker (n = 5); E-H: After the administration of the peptidylarginine deiminase 4 inhibitor and anti-Ly6G, either alone or in combination, in the severe acute pancreatitis model, the levels of dsDNA in mouse plasma were measured (E), hematoxylin-eosin staining of pancreatic and lung tissues was performed (F), and the activities of amylase and lipase in plasma were determined (G and H) (n = 7); I and J: Representative immunofluorescence images of CitH3 and F4/80 in macrophages from each group, along with statistical analysis of CitH3 (n = 4). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; MET: Macrophage extracellular trap; PMA: Phorbol 12-myristate 13-acetate; BMDMs: Bone marrow-derived macrophages; Nc: Negative control; dsDNA: Double-stranded DNA; CitH3: Citrullinated histone H3; Cl-amidine: BB-Cl-amidine; Ctrl: Control; DAPI: 4’,6-diamidino-2-phenylindole; NAC: N-Acetylcysteine; MFI: Mean fluorescence intensity.

Figure 4 Zinc supplementation can inhibit the formation of macrophage extracellular traps. A and B: Representative immunofluorescence images of citrullinated histone H3 (CitH3) (red) and F4/80 (green) in macrophages, along with statistical analysis of the relative expression levels of CitH3 (n = 4); C: Double-stranded DNA (dsDNA) levels in the cell supernatant (n = 4); D: Macrophages were cocultured with pancreatic acinar cells treated with caerulein in the presence of phorbol 12-myristate 13-acetate. The intracellular zinc levels in the macrophages were measured using a zinc ion probe and statistically analyzed (n = 4); E: Zinc concentration in serum (n = 4); F-J: After supplementing severe acute pancreatitis model mice were supplemented with different concentrations of zinc, pathological changes in pancreatic and lung tissues were assessed by hematoxylin-eosin staining (F), plasma levels of amylase and lipase were measured (G and H), immunofluorescence staining for CitH3 and F4/80 in pancreatic tissue was performed (I), and the concentration of dsDNA in plasma was determined (J) (n = 8). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; PMA: Phorbol 12-myristate 13-acetate; Nc: Negative control; CitH3: Citrullinated histone H3; DAPI: 4’,6-diamidino-2-phenylindole; dsDNA: Double-stranded DNA; AP: Acute pancreatitis; SAP: Severe acute pancreatitis; MFI: Mean fluorescence intensity.

Figure 5 Reduced ZIP8 expression in macrophages reduces zinc levels and is linked to enhanced macrophage extracellular trap formation; zinc supplementation can lower reactive oxygen species levels and suppress macrophage extracellular trap formation. A: A transcriptomic analysis was conducted to identify differentially expressed genes in the macrophages of the macrophage extracellular trap (MET)-induced group and negative control groups; B: The expression level of ZIP8 messenger RNA (mRNA) in bone marrow-derived macrophages (BMDMs) was measured using quantitative real-time polymerase chain reaction (q-PCR) (n = 3); C: Representative immunofluorescence images showing ZIP8 expression in BMDMs; D: After ZIP8 was overexpressed, ZIP8 mRNA expression in BMDMs was measured using q-PCR (n = 3); E: Zinc levels in BMDMs were assessed using a fluorescent zinc probe (n = 4); F: Representative fluorescence images of citrullinated histone H3 (CitH3) and F4/80 in macrophages, together with quantitative analysis of CitH3 fluorescence intensity (n = 4); G: Changes in the mRNA expression levels of peptidylarginine deiminase 4, nicotinamide adenine dinucleotide phosphate hydrogen oxidase 2, and toll-like receptor 2 in macrophages within the MET induction system, with or without ZIP8 overexpression, were measured by q-PCR (n = 4); H: Intracellular reactive oxygen species levels were measured using a 2’,7’-dichlorodihydrofluorescein diacetate fluorescent probe assay (n = 4). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; PMA: Phorbol 12-myristate 13-acetate; Nc: Negative control; CitH3: Citrullinated histone H3; BMDMs: Bone marrow-derived macrophages; DAPI: 4’,6-diamidino-2-phenylindole; mRNA: Messenger RNA; Oe: Overexpression; MFI: Mean fluorescence intensity; PAD4: Peptidylarginine deiminase 4; TLR2: Toll-like receptor 2; NOX2: Nicotinamide adenine dinucleotide phosphate hydrogen oxidase 2; ROS: Reactive oxygen species; DCFH-DA: 2’,7’-dichlorodihydrofluorescein diacetate.

Figure 6 The overexpression of ZIP8 mitigates the severity of severe acute pancreatitis by decreasing macrophage extracellular trap formation. A: Schematic diagram of the experimental procedure; B: Western blot analysis of ZIP8 protein expression levels in peritoneal macrophages (n = 4); C: Quantitative real-time polymerase chain reaction analysis of ZIP8 messenger RNA expression levels in peritoneal macrophages (n = 7); D: Representative immunofluorescence images showing ZIP8 (green) and cluster of differentiation 68 (red) expression; E and F: Hematoxylin-eosin staining demonstrating the pathological effects of ZIP8 overexpression on pancreatic and lung tissues; G and H: Plasma levels of amylase and lipase (n = 7); I: Plasma levels of double-stranded DNA (n = 7); J: Representative fluorescence images of citrullinated histone H3 (CitH3) and F4/80 in the pancreas, together with quantitative analysis of CitH3 fluorescence intensity (n = 7). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. AAV: Adeno-associated virus; Ctrl: Control; DAPI: 4’,6-diamidino-2-phenylindole; mRNA: Messenger RNA; CD: Cluster of differentiation; CitH3: Citrullinated histone H3; dsDNA: Double-stranded DNA; MFI: Mean fluorescence intensity.