Dragoni Gabriele, Ke Bo-Jun, Picariello Lucia, Abdurahiman Saeed, Ceni Elisabetta, Biscu Francesca, Mello Tommaso, Polvani Simone, Innocenti Tommaso, Spalart Valérie, Milani Stefano, D'Hoore André, Bislenghi Gabriele, Scaringi Stefano, Verstockt Bram, De Hertogh Gert, Martinod Kimberly, Galli Andrea, Matteoli Gianluca, Vermeire Séverine
Department of Chronic Diseases and Metabolism, Translational Research Center for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium.
Gastroenterology Research Unit, Department of Experimental and Clinical Biochemical Sciences "Mario Serio", University of Florence, Florence, Italy.
J Crohns Colitis. 2025 Jan 11;19(1). doi: 10.1093/ecco-jcc/jjae121.
During early phases of inflammation, activated neutrophils extrude neutrophil extracellular traps (NETs) in a peptidyl arginine deiminase 4 (PAD4)-dependent manner, aggravating tissue injury and remodeling. In this study, we investigated the potential pro-fibrotic properties and signaling of NETs in Crohn's disease (CD).
NETs and activated fibroblasts were labeled on resected ileum from CD patients by multiplex immunofluorescence staining. NETs-treated human primary intestinal fibroblasts were analyzed by bulk RNA sequencing to uncover cell signaling pathways, and by high-throughput imaging to assess collagen production and migratory activity. Consequentially, TLR2/NF-κB pathway was evaluated by transfection of CCD-18Co fibroblasts with an NF-κB-luciferase reporter plasmid, incorporating C29 to block TLR2 signaling. A chronic dextran sulfate sodium (DSS) mouse model was used to define the specific role of PAD4 deletion in neutrophils (MRP8-Cre, Pad4fl/fl).
Immunofluorescence showed spatial colocalization of NETs and activated fibroblasts in ileal ulcerations of CD patients. Transcriptomic analysis revealed upregulation of pro-fibrotic genes and activation of Toll-like receptor signaling pathways in NETs-treated fibroblasts. NETs treatment induced fibroblast proliferation, diminished migratory capability, and increased collagen release. Transfection experiments indicated a substantial increase in an NF-κB expression with NETs, whereas C29 led to decreased expression and release of collagen. In line, a significant reduction in collagen content was observed in the colon of MRP8-Cre, Pad4fl/fl mice subjected to chronic DSS colitis.
NETs potentially serve as an initial stimulus for pathological activation of fibroblasts within the intestine via the TLR2/NF-κB pathway. Given their early involvement in inflammation, inhibition of PAD4 might offer a strategy to modulate both inflammation and fibrogenesis in CD.
在炎症早期阶段，活化的中性粒细胞以肽基精氨酸脱亚氨酶4（PAD4）依赖的方式释放中性粒细胞胞外诱捕网（NETs），加重组织损伤和重塑。在本研究中，我们调查了NETs在克罗恩病（CD）中的潜在促纤维化特性及信号传导。
通过多重免疫荧光染色对CD患者切除的回肠中的NETs和活化的成纤维细胞进行标记。对经NETs处理的人原代肠道成纤维细胞进行批量RNA测序以揭示细胞信号通路，并通过高通量成像评估胶原蛋白产生和迁移活性。随后，通过用NF-κB荧光素酶报告质粒转染CCD-18Co成纤维细胞并加入C29阻断TLR2信号传导来评估TLR2/NF-κB通路。使用慢性葡聚糖硫酸钠（DSS）小鼠模型来确定中性粒细胞中PAD4缺失（MRP8-Cre，Pad4fl/fl）的具体作用。
免疫荧光显示NETs与活化的成纤维细胞在CD患者回肠溃疡中存在空间共定位。转录组分析显示，经NETs处理的成纤维细胞中促纤维化基因上调且Toll样受体信号通路激活。NETs处理诱导成纤维细胞增殖、迁移能力减弱并增加胶原蛋白释放。转染实验表明，NETs使NF-κB表达显著增加，而C29导致胶原蛋白表达和释放减少。同样，在患有慢性DSS结肠炎的MRP8-Cre，Pad4fl/fl小鼠的结肠中观察到胶原蛋白含量显著降低。
NETs可能通过TLR2/NF-κB通路作为肠道内成纤维细胞病理活化的初始刺激因素。鉴于它们早期参与炎症反应，抑制PAD4可能为调节CD中的炎症和纤维化提供一种策略。
