Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in infection and inflammatory diseases by facilitating the formation of neutrophil extracellular traps (NETs). However, the substrates of PAD4 and its exact role in inflammatory bowel disease (IBD) remain unclear. In this study, we employed single-cell RNA sequencing (scRNA-seq) and substrate citrullination mapping to decipher the role of PAD4 in intestinal inflammation associated with IBD. Our results demonstrated that PAD4 deficiency alleviated colonic inflammation and restored intestinal barrier function in a dextran sulfate sodium (DSS)-induced colitis mouse model. scRNA-seq analysis revealed significant alterations in intestinal cell populations, with reduced neutrophil numbers and changes in epithelial subsets upon PAD4 deletion. Gene expression analysis highlighted pathways related to inflammation and epithelial cell function. Furthermore, we found that neutrophil-derived extracellular vesicles (EVs) carrying PAD4 were secreted into intestinal epithelial cells (IECs). Within IECs, PAD4 citrullinates mitochondrial creatine kinase 1 (CKMT1) at the R242 site, leading to reduced CKMT1 protein stability via the autophagy pathway. This action compromises mitochondrial homeostasis, impairs intestinal barrier integrity, and induces IECs apoptosis. IEC-specific depletion of CKMT1 exacerbated intestinal inflammation and apoptosis in mice with colitis. Clinical analysis of IBD patients revealed elevated levels of PAD4, increased CKMT1 citrullination, and decreased CKMT1 expression. In summary, our findings highlight the crucial role of PAD4 in IBD, where it modulates IECs plasticity via CKMT1 citrullination, suggesting that PAD4 may be a potential therapeutic target for IBD.

肽基精氨酸脱亚胺酶 4 (PAD4) 通过促进中性粒细胞胞外陷阱 (NET) 的形成，在感染和炎症性疾病中发挥关键作用。然而，PAD4 的底物及其在炎症性肠病 (IBD) 中的确切作用仍不清楚。在这项研究中，我们采用单细胞 RNA 测序 (scRNA-seq) 和底物瓜氨酸图谱来破译 PAD4 在 IBD 相关肠道炎症中的作用。我们的结果表明，PAD4 缺陷可减轻葡聚糖硫酸钠 (DSS) 诱导的结肠炎小鼠模型中的结肠炎症并恢复肠道屏障功能。 scRNA-seq 分析揭示了 PAD4 缺失后肠道细胞群发生显着变化，中性粒细胞数量减少，上皮亚群发生变化。基因表达分析强调了与炎症和上皮细胞功能相关的途径。此外，我们发现携带 PAD4 的中性粒细胞来源的细胞外囊泡 (EV) 被分泌到肠上皮细胞 (IEC) 中。在 IEC 内，PAD4 在 R242 位点瓜氨酸线粒体肌酸激酶 1 (CKMT1)，通过自噬途径导致 CKMT1 蛋白稳定性降低。这种作用会损害线粒体稳态，损害肠道屏障完整性，并诱导 IEC 凋亡。 IEC 特异性去除 CKMT1 会加剧结肠炎小鼠的肠道炎症和细胞凋亡。 IBD 患者的临床分析显示 PAD4 水平升高、CKMT1 瓜氨酸化增加以及 CKMT1 表达降低。总之，我们的研究结果强调了 PAD4 在 IBD 中的关键作用，它通过 CKMT1 瓜氨酸化调节 IEC 可塑性，表明 PAD4 可能是 IBD 的潜在治疗靶点。