Basler Tina, Jeckstadt Sabine, Valentin-Weigand Peter, Goethe Ralph
Institut fuer Mikrobiologie, Zentrum fuer Infektionsmedizin, Stiftung Tieraerztliche Hochschule Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany.
J Leukoc Biol. 2006 Mar;79(3):628-38. doi: 10.1189/jlb.0905520. Epub 2006 Jan 13.
Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic enteritis in ruminants. In addition, MAP is presently the most favored pathogen linked to Crohn's disease. In this study, we were interested in dissecting the molecular mechanisms of macrophage activation or deactivation after infection with MAP. By subtractive hybridization of cDNAs, we identified the immune-responsive gene 1 (IRG1), which was expressed substantially higher in lipopolysaccharide (LPS)-stimulated than in MAP-infected murine macrophage cell lines. A nuclear run-on transcription assay revealed that the IRG1 gene was activated transcriptionally in LPS-stimulated and MAP-infected macrophages with higher expression in LPS-stimulated cells. Analysis of post-transcriptional regulation demonstrated that IRG1 mRNA stability was increased in LPS-stimulated but not in MAP-infected macrophages. Furthermore, IRG1 gene expression of macrophages infected with the nonpathogenic Mycobacterium smegmatis differed from those of LPS-stimulated and MAP-infected macrophages. At 2 h postinfection, M. smegmatis-induced IRG1 gene expression was as low as in MAP-infected, and 8 h postinfection, it increased nearly to the level in LPS-stimulated macrophages. Transient transfection experiments revealed similar IRG1 promoter activities in MAP- and M. smegmatis-infected cells. Northern analysis demonstrated increased IRG1 mRNA stability in M. smegmatis-infected macrophages. IRG1 mRNA stabilization was p38 mitogen-activated protein kinase-independent. Inhibition of protein synthesis revealed that constitutively expressed factors seemed to be responsible for IRG1 mRNA destabilization. Thus, our data demonstrate that transcriptional and post-transcriptional mechanisms are responsible for a differential IRG1 gene expression in murine macrophages treated with LPS, MAP, and M. smegmatis.
副结核分枝杆菌（MAP）可引起反刍动物的慢性肠炎。此外，目前MAP是与克罗恩病关联度最高的病原体。在本研究中，我们旨在剖析巨噬细胞感染MAP后激活或失活的分子机制。通过对cDNA进行消减杂交，我们鉴定出免疫反应基因1（IRG1），其在脂多糖（LPS）刺激的小鼠巨噬细胞系中的表达显著高于MAP感染的细胞系。一项细胞核转录活性分析显示，IRG1基因在LPS刺激的巨噬细胞和MAP感染的巨噬细胞中均被转录激活，且在LPS刺激的细胞中表达更高。转录后调控分析表明，IRG1 mRNA稳定性在LPS刺激的巨噬细胞中增加，而在MAP感染的巨噬细胞中未增加。此外，感染非致病性耻垢分枝杆菌的巨噬细胞的IRG1基因表达与LPS刺激的巨噬细胞和MAP感染的巨噬细胞不同。感染后2小时，耻垢分枝杆菌诱导的IRG1基因表达与MAP感染的情况一样低，而在感染后8小时，其表达几乎增加到LPS刺激的巨噬细胞中的水平。瞬时转染实验显示，MAP和耻垢分枝杆菌感染的细胞中IRG1启动子活性相似。Northern分析表明，耻垢分枝杆菌感染的巨噬细胞中IRG1 mRNA稳定性增加。IRG1 mRNA的稳定化不依赖p38丝裂原活化蛋白激酶。蛋白质合成抑制实验表明，组成性表达的因子似乎是导致IRG1 mRNA不稳定的原因。因此，我们的数据表明，转录和转录后机制导致了LPS、MAP和耻垢分枝杆菌处理的小鼠巨噬细胞中IRG1基因的差异表达。
