La Ferla Katia, Seegert Dirk, Schreiber Stefan
Department of Pediatrics, University of Ulm, Prittwitzstrasse 43, 89075 Ulm, Germany.
Int J Colorectal Dis. 2004 Jul;19(4):334-42. doi: 10.1007/s00384-004-0583-7. Epub 2004 Apr 22.
The involvement of bacteria in the pathogenesis of inflammatory bowel disease has been discussed for several years. In this study we evaluated the ability of E. coli isolates from inflamed and noninflamed colonic mucosa to activate NF-kappaB.
Fifteen bacterial strains from inflamed and six from noninflamed colonic tissues from IBD patients. Their ability to induce NF-kappaB activation was examined in vitro by gel-shift assays. The activation of the TNF-alpha promoter was determined by reporter gene assays. Bacterial isolates were characterized by invasion assays, electron microscopy, and PCR.
Four of 15 E. coli bacterial isolates from inflamed IBD tissues induced NF-kappaB activity in intestinal epithelial cells as determined by gel-shift assays. NF-kappaB activation was only seen with living bacteria but not with heat-inactivated cells. Isolates from noninflamed tissues and a wild-type E. coli control strain induced a weaker or no activation. Reporter gene assays with a construct comprising a luciferase gene driven by the TNF-alpha promoter revealed that isolates from Crohn's disease patients induced a stronger activation of the TNF-alpha gene than isolates from ulcerative colitis patients. The isolated bacteria invaded HT-29 cells, although typical virulence genes for enteropathogenic, enterhemorrhagic, or enteroinvasive E. coli, i.e., eae, tir, EspA, Per (A-C), ipaC, were not detected in these cells. Bacterial invasion was additionally confirmed by electron microscopy examination.
Our results indicate that E. coli strains can be found in the mucosa of some IBD patients which are able to activate NF-kappaB similar to known pathogenic strains. The absence of several virulence genes in these cells suggests that they are members of the luminal flora which acquire as yet unidentified virulence determinants and are therefore involved in the pathophysiology of IBD.
细菌在炎症性肠病发病机制中的作用已被讨论多年。在本研究中，我们评估了从发炎和未发炎的结肠黏膜分离出的大肠杆菌激活核因子κB（NF-κB）的能力。
从炎症性肠病（IBD）患者的发炎结肠组织中分离出15株细菌菌株，从未发炎结肠组织中分离出6株。通过凝胶迁移试验在体外检测它们诱导NF-κB激活的能力。通过报告基因试验确定肿瘤坏死因子-α（TNF-α）启动子的激活情况。通过侵袭试验、电子显微镜检查和聚合酶链反应（PCR）对细菌分离株进行鉴定。
凝胶迁移试验表明，从发炎的IBD组织中分离出的15株大肠杆菌菌株中有4株可诱导肠上皮细胞中的NF-κB活性。仅活细菌能激活NF-κB，热灭活的细菌则不能。从未发炎组织中分离出的菌株以及野生型大肠杆菌对照菌株诱导的激活作用较弱或无激活作用。用包含由TNF-α启动子驱动的荧光素酶基因的构建体进行报告基因试验表明，与来自溃疡性结肠炎患者的分离株相比，来自克罗恩病患者的分离株对TNF-α基因的激活作用更强。分离出的细菌可侵袭HT-29细胞，尽管在这些细胞中未检测到致病性、出血性或侵袭性大肠杆菌的典型毒力基因，即eae、tir、EspA、Per（A-C）、ipaC。电子显微镜检查进一步证实了细菌的侵袭。
我们的结果表明，在一些IBD患者的黏膜中可发现能激活NF-κB的大肠杆菌菌株，类似于已知的致病菌株。这些细胞中缺乏几种毒力基因表明它们是管腔菌群的成员，获得了尚未确定的毒力决定因素，因此参与了IBD的病理生理过程。
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