Single-cell RNA-seq of neutrophils from germ-free and specific-pathogen-free mice reveals gut-microbiota-driven heterogeneity and NETosis in acute pancreatitis

We performed single-cell RNA sequencing (scRNA-seq) using the 10x Genomics platform to characterize pancreatic immune cell populations in germ-free (GF) and specific-pathogen-free (SPF) mice with cerulein-induced acute pancreatitis (AP). scRNA-seq analysis identified distinct neutrophil subpopulations influenced by gut microbiota presence. SPF mice showed enrichment of pro-inflammatory neutrophil clusters associated with increased neutrophil extracellular trap (NET) formation, whereas GF mice exhibited neutrophil subsets expressing reparative and protective signatures. Our data highlight the role of gut microbiota in shaping immune responses during AP. Following induction of AP by repeated intraperitoneal injection of cerulein (100 µg/kg hourly for 10 doses), pancreatic tissues were harvested from GF and SPF male C57BL/6 mice (7–8 weeks old). Single-cell suspensions of pancreatic immune cells were obtained by enzymatic digestion, and libraries were prepared using the 10x Genomics Chromium Single Cell 3′ v3.1 protocol. Sequencing was performed on an Illumina NovaSeq 6000 (paired-end, 2×150 bp). Raw FASTQ files and processed data (gene-cell count matrices) were deposited in GEO