Introduction

Acute pancreatitis (AP) is a common clinical emergency, with 20%–30% of cases progressing to severe acute pancreatitis (SAP), which has a mortality rate of up to 40%. Gut microbiota dysbiosis is closely associated with AP severity, yet its underlying mechanisms remain unclear.

Aims & Methods

This study aimed to elucidate how microbiota dysbiosis exacerbates AP.AP was induced in Specific Pathogen-Free (SPF) and Germ-Free (GF) C57BL/6 male mice to investigate the effects of microbiota dysbiosis on neutrophil recruitment, neutrophil extracellular trap (NET) formation, pancreatic injury, and systemic inflammation. Single-cell sequencing of neutrophils from SPF and GF AP mice was conducted to assess microbiota-driven changes in neutrophil distribution, gene expression, and signaling pathways. Serum cfDNA levels were measured in 50 AP patients and 30 healthy controls. Key findings were validated in vivo and in vitro.

Results

Serum cfDNA (NETs) levels were significantly elevated in AP patients compared to healthy controls and decreased during recovery. GF-treated mice exhibited reduced pancreatic edema, inflammatory infiltration, and acinar cell necrosis, with lower pathology scores. Immunohistochemistry confirmed decreased MPO and F4/80-positive cells in pancreatic tissues, while ELISA showed reduced levels of amylase, lipase, IL-6, and TNF-α in GF-treated mice. Single-cell sequencing revealed a lower proportion of pancreatic neutrophils and an increase in acinar cells in GF-treated mice, whereas the NET formation pathway was activated in SPF-treated mice. The TLR2/MYD88/NLRP3/Caspase-1 signaling cascade, which promotes NET formation, was also activated in SPF mice. Consistent with pathway analysis, CXCL1, CCL9, and CXCR2 expression was upregulated, while Reg3β expression was downregulated in SPF_AP mice. Dual immunofluorescence demonstrated reduced MPO/CitH3 (NETs) formation in GF-treated mice. Neutrophils from GF-treated mice released significantly less cfDNA upon PMA stimulation. Serum cfDNA levels were elevated in both AP-induced mice and AP patients.

Conclusion

Gut microbiota dysbiosis exacerbates AP severity by promoting neutrophil recruitment, activating the TLR2/NLRP3/MYD88/Caspase-1 signaling cascade, and enhancing NET formation, leading to aggravated pancreatic injury and systemic inflammation. These findings highlight microbiota modulation as a potential therapeutic strategy to prevent AP progression.

Disclosure

The authors declare that they have no conflict of interests.