Background: The aim of this study was to investigate the association of the formation of neutrophil extracellular traps (NETs) with gut leakage in type 1 (T1D) and type 2 diabetes (T2D).
Methods: In all, 105 subjects (56 T1D, 49 T2D) were included in the study. Eight biomarkers of NET formation and gut leakage (ie, protein arginine deiminase type 4 [PAD4], neutrophil elastase [NE], proteinase 3 [PR3], complement 5a [C5a], α1 -antitrypsin [AAT], DNase I, zonulin, and lipopolysaccharide [LPS]) were measured in serum samples by ELISA. Neutrophils were isolated and stimulated by phorbol myristate acetate to form NETs in vitro. Neutrophil intracellular contents were then collected and used as antigens to detect anti-neutrophil cytoplasmic antibodies (ANCA) in the serum.
Results: There was an increase in NET-associated proteins (PAD4, NE, PR3, C5a, AAT and DNase I) in new-onset T1D patients but not in those with T2D. Of PAD4, NE, and PR3, PAD4 was found to be the most sensitive biomarker for the diagnosis of T1D. Furthermore, circulating levels of zonulin and LPS were not only increased, but were also strongly correlated with NET formation and ANCA generation in T1D patients.
Conclusions: This study provides evidence that increased formation of NETs, particularly PAD4, is closely associated with gut leakage in T1D but not T2D, and suggests that microorganisms and the release of neutrophil cytoplasmic antigen during the formation of NETs may be involved in the pathogenesis of T1D.
背景: 本研究旨在探索1型(T1D)和2型(T2D)糖尿病中中性粒细胞胞外诱捕网(neutrophil extracellular traps，NETs)的形成与肠道渗漏性是否具有相关性。 方法: 本研究对象共计有105例糖尿病患者，其中包括56例T1D患者与49例T2D患者。通过酶联免疫吸附法来测定患者血清中8种与NETs形成及肠道渗漏性相关的生物标志物含量，包括精氨酸脱亚氨酶4[PAD4]、中性细胞弹性蛋白酶[NE]、蛋白酶3[PR3]、补体5a[C5a]、α1-抗胰蛋白酶[AAT]、脱氧核糖核酸酶I[DNase I]、连蛋白[zonulin]及脂多糖[LPS]。此外，通过体外刺激中性粒细胞形成NETs并收集其形成过程中释放的细胞内容物，将其作为中性粒细胞胞质抗原来检测血清中抗中性粒细胞胞质抗体(anti-neutrophil cytoplasmic antibodies，ANCA)的含量。 结果: 与健康人相比，我们检测到在新发的T1D患者血清中，NETs相关蛋白(PAD4、NE、PR3、C5a、AAT和DNase I)的含量显著升高，但其在T2D患者血清中变化不明显。此外，在可能作为T1D诊断的生物标志物中，PAD4与NE和PR3相比，表现出更高的灵敏度。在T1D患者体内，我们不仅检测到高水平的zonulin和LPS的含量，还发现其与NETs及ANCA的形成具有极显著的相关性。 结论: 本研究表明在T1D而非T2D患者中，NETs的形成，特别是血清中PAD4含量的变化与肠道渗漏密切相关，并提示微生物入侵及NETs形成过程中中性粒细胞胞质抗原的释放可能是T1D的重要致病因素。.
Keywords: 1型糖尿病; 2型糖尿病; gut leakage; neutrophil extracellular traps; type 1 diabetes; type 2 diabetes; 中性粒细胞胞外诱捕网; 肠道渗漏.
© 2018 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.