Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Inflamm Bowel Dis. 2010 May;16(5):736-42. doi: 10.1002/ibd.21144.
Human neutrophil peptide 1 (HNP-1) is a defensin with antibacterial activity secreted by various cells as a component of the innate immune host defense. NOD2 is a cytoplasmic protein that recognizes bacterial derived muramyl dipeptide, and is involved in bacterial clearance. The aim of the present study was to investigate the relationship between antibacterial activity of NOD2 and HNP-1 expression in epithelial cell lines.
Gentamicin protection assay using Salmonella typhimurium was performed in Caco-2 cells. The mRNA level was determined by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and defensin expression was assessed by Western blot and enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappaB activation was assessed using pIV luciferase and Renilla plasmids. A NOD2 mutant was generated by site-directed mutagenesis.
Among the defensins tested, only HNP-1 expression is induced in colonic epithelial model HCT116 cells after MDP-LD stimulation. HNP-1 secretion is significantly increased after MDP-LD stimulation in the cell supernatant of intestinal epithelial cells expressing endogenous NOD2, but not in cells that lack endogenous NOD2 expression. HNP-1 is required for NOD2-dependent NF-kappaB activation after MDP-LD stimulation since hnp-1 siRNA transfection abrogated the response to MDP-LD stimulation. The antibacterial function of NOD2 against S. typhimurium was impaired when expression of HNP-1 was blocked by siRNA.
HNP-1 secretion depends on NOD2 stimulation by MDP-LD and contributes to antibacterial activity in intestinal epithelial cells expressing endogenous NOD2, but not NOD2 3020insC mutant associated with increased susceptibility to Crohn's disease.
人中性粒细胞肽 1（HNP-1）是一种具有抗菌活性的防御素，由各种细胞分泌，作为先天免疫宿主防御的组成部分。NOD2 是一种细胞质蛋白，可识别细菌衍生的 muramyl dipeptide，并参与细菌清除。本研究旨在探讨上皮细胞系中 NOD2 的抗菌活性与 HNP-1 表达之间的关系。
使用鼠伤寒沙门氏菌进行 Caco-2 细胞的庆大霉素保护试验。通过定量逆转录聚合酶链反应（RT-PCR）测定 mRNA 水平，并通过 Western blot 和酶联免疫吸附试验（ELISA）评估防御素表达。通过 pIV 荧光素酶和 Renilla 质粒评估核因子-kappaB 激活。通过定点突变生成 NOD2 突变体。
在所测试的防御素中，仅在 MDP-LD 刺激后结肠上皮模型 HCT116 细胞中诱导 HNP-1 表达。在表达内源性 NOD2 的肠上皮细胞的细胞上清液中，MDP-LD 刺激后 HNP-1 分泌显著增加，但在缺乏内源性 NOD2 表达的细胞中则不然。MDP-LD 刺激后，hnp-1 siRNA 转染可阻断对 MDP-LD 刺激的反应，表明 HNP-1 是 NOD2 依赖性 NF-kappaB 激活所必需的。当通过 siRNA 阻断 HNP-1 的表达时，NOD2 对鼠伤寒沙门氏菌的抗菌功能受损。
HNP-1 的分泌依赖于 MDP-LD 对 NOD2 的刺激，并有助于表达内源性 NOD2 的肠上皮细胞中的抗菌活性，但对与克罗恩病易感性增加相关的 NOD2 3020insC 突变体则不然。
