High expression of P-selectin induces neutrophil extracellular traps via the PSGL-1/Syk/Ca2+/PAD4 pathway to exacerbate acute pancreatitis

Abstract

Background: Excessive neutrophil extracellular traps (NETs) is involved in the progression of acute pancreatitis (AP) but the mechanisms controlling NETs formation in AP are not fully understood. Therefore, our study sought to investigate the mechanism of the highly expressed P-selectin stimulating the formation of NETs in AP.

Methods: NETs formation was detected by flow cytometry, immunofluorescence staining, and cf-DNA and MPO-DNA complexes were measured as biomarkers of NETs formation. Neutrophils treated with P-selectin and pharmacological inhibitors were examined by western blot, immunofluorescence staining and flow cytometry. Mouse model of AP was established by caerulein and the effect of inhibiting P-selectin by PSI-697 on the level of NETs and PAD4 in pancreatic tissue was observed. The severity of AP was evaluated by histopathological score and the detection of serum amylase and lipase.

Results: Patients with AP had elevated levels of NETs and P-selectin compared with healthy volunteers. Stimulation of P-selectin up-regulated the expression of PSGL-1, increased the phosphorylation of Syk, mediated intracellular calcium signal and led to the activation and expression of PAD4, which modulated NETs formation in neutrophils. Pretreament with PSI-697 blunted NETs formation and PAD4 expression in the pancreatic tissue, and ameliorated the severity of AP in mice.

Conclusion: Taken together, these results suggest that P-selectin induces NETs through PSGL-1 and its downstream Syk/Ca²⁺/PAD4 signaling pathway, and that targeting this pathway might be a promising strategy for the treatment of AP.

Keywords: P-selectin; P-selectin glycoprotein ligand-1; acute pancreatitis; neutrophil extracellular traps; peptidylarginine deiminase 4.

Figure 1

Elevated levels of NETs and P-selectin in AP patients. (A) Peripheral blood was collected from healthy individuals and AP patients. Neutrophils were isolated, and NETs formation was detected by flow cytometry. Representative flow data showing CitH3-positive and MPO-positive cells defined as NETs. (B) Quantification of circulating NETs in healthy controls and AP patients. (n=5, **P < 0.01) (C) Representative images of neutrophils stained with MPO (red), CitH3 (Green) and DAPI (blue). Scale bar: 50 μm. The white arrows indicated NET-forming cells. (D) The proportion of NET-forming cells in total neutrophils were quantified. (n=6, ****P < 0.0001) The serum levels of cf-DNA (E) and MPO-DNA complexes (F) were determined in serums of healthy individuals and AP patients. (n=12, ****P < 0.0001) (G) Serum levels of P-selectin in the control group and AP patients determined by ELISA. (n=12, ****P < 0.0001).

Figure 2

High level of P-selectin leads to increased PSGL-1 expression and NETs formation in human neutrophils. Human peripheral blood neutrophils were isolated and treated with P-selectin recombinant protein at concentrations of 10, 20 and 50 nM. (A) Representative images of neutrophil stained with PSGL-1 (yellow) and DAPI (blue). The inset box from each group is magnified. Scale bar: 5 μm and 20 μm, respectively. (B) Cell lysates were collected and subjected to Western blot analysis for PSGL-1. GAPDH was used as a loading control. Data from one representative experiment are shown. (C) Relative intensities of PSGL-1 against GAPDH. (n=3, **P < 0.01) (D) Representative images of neutrophils stained with MPO (red), CitH3 (Green) and DAPI (blue). The inset box from each group is magnified. Scale bar: 50 μm and 20 μm, respectively. The arrows indicate NETs. (E) NET-forming cells per field are quantified. (n=6, ****P < 0.0001) The neutrophil supernatants were assessed for cf-DNA (F) and MPO-DNA complexes (G). (n=3, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

High level of P-selectin-induced NETs are regulated by Syk and PAD4. Neutrophils were pre-treated with Syk inhibitor (Syki, 4 μM of PRT-060318) or PAD4 inhibitor (PAD4i, 10 μM of GSK484) for 1 h before treated with P-selectin for 4 h. (A) Neutrophils were immune-stained with Abs to MPO, CitH3, and DAPI nuclear stain. Scale bar: 50 μm. The arrows indicate NETs. (B) NET-forming cells per field are quantified. (n=6, **P < 0.01, ***P < 0.001) The neutrophil supernatants were assessed for cf-DNA (C) and MPO-DNA complexes (D). (n=3, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

High level of P-selectin induces NETs formation through Syk/Ca²⁺/PAD4 signaling pathway. Neutrophils were pretreated with Syk inhibitor (Syki, 4 μM of PRT-060318) or Ca2+ chelator (3 μM of BAPTA-AM) for 1h and then were stimulated with P-selectin recombinant protein. Neutrophils were loaded with Ca²⁺ fluorescence probe Fluo-4 AM and then were analyzed by fluorescence microscopy and flow cytometry. (A) Representative images of neutrophils labeled by Fluo-4 AM under fluorescence microscope. Scale bar: 100 μm. (B, C) Quantitative analysis of the proportion of Fluo-4 AM⁺ neutrophils and fluorescence density of Fluo-4 AM under fluorescence microscope. (n=3, *P < 0.05, **P < 0.01) (D) Flow cytometry observation and quantification of Fluo-4 AM in neutrophils in different groups. Neutrophils treated with ionomycin were used as positive control. (E, F) Quantitative analysis of the Fluo-4 AM⁺ neutrophils ratio and the mean fluorescence intensity (MFI) detected by flow cytometry. (n=3, *P < 0.05, **P < 0.01) Neutrophils were pretreated with Syk inhibitor (G) or Ca²⁺ chelator (K) for 1h and then were stimulated with P-selectin recombinant protein. Cell lysates were collected and subject to Western blot analysis for p-Syk, PAD4 and CitH3. GAPDH was used as loading control. (H–J, L–N) Relative intensities are quantified by ImageJ. (n=3, N.S. represents no significant difference, *P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 5

Inhibition of P-selectin binding to PSGL-1 reduces the level of NETs in pancreas of AP mice. (A) Drug administration and construction of AP mice. (B) Colocalization of MPO (red) with CitH3 (green)in the pancreas of AP mice. The inset box from each group is magnified. Scale bar: 50 μm and 20 μm, respectively. The arrows indicate NETs in pancreas. (C) The pancreatic tissue was harvested for lysis and lysates were immunoblotted for CitH3 and β-actin. (D) The relative expression levels of CitH3 were quantified. (n=3, **P < 0.01, ***P < 0.001) The serum levels of cf-DNA (E) and MPO-DNA complexes (F) were determined in mice of different groups. (n=6, *P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 6

Inhibition of P-selectin binding to PSGL-1 by PSI-697 alleviated pancreatic histopathological injury and reduced serum enzyme levels in AP. (A) Representative H&E staining of pancreatic tissues. The inset box from each group is magnified. Scale bar: 100 μm and 50μm, respectively. (B–E) Histopathological scores of pancreatic tissues. (n=6, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000) (F, G) Serum amylase and lipase levels in mice. (n=6, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 7

Schematic diagram (created with BioRender.com) depicting the mechanism of high level of P-selectin-induced NETs formation in AP. Upon stimulation of P-selectin, PSGL-1 and its downstream Syk/Ca²⁺/PAD4 signaling pathway induce neutrophils to form NETs and aggravate the deterioration of AP.