Department of Clinical and Experimental Medicine, TARGID, KU Leuven, Leuven, Belgium.
Clermont Université, Inserm U1071, Université d'Auvergne, INRA USC 2018, Clermont-Ferrand, France Centre Hospitalier Universitaire, Clermont-Ferrand, France.
Gut. 2014 Aug;63(8):1265-74. doi: 10.1136/gutjnl-2012-303205. Epub 2013 Oct 3.
Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up.
We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis.
Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (p(FDR)=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly.
Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors.
在 IBD 中观察到微生物群落组成改变、免疫反应变化和肠道屏障功能受损。这些特征大多受蛋白酶及其抑制剂控制，以维持肠道内稳态。蛋白酶不受控制或过度水解会导致胃肠道发生病理改变。本研究旨在通过遗传关联研究和功能随访，验证之前系统评价中确定的 IBD 候选蛋白酶。
我们在来自五个欧洲 IBD 转诊中心的 2320 例 CD 患者、2112 例 UC 患者和 1796 例健康对照者的大型多中心队列中进行了遗传关联研究。随后，我们对候选基因进行了广泛的功能评估，以探索其在 IBD 发病机制中的因果关系。
在四个基因中的 10 个单核苷酸多态性（SNP）与 CD 显著相关：CYLD、USP40、APEH 和 USP3。CYLD 是最显著的基因，内含子中 rs12324931 是最强的相关 SNP（p（FDR）=1.74e-17，OR=2.24（1.83 至 2.74））。在四个基因中的五个 SNP 与 UC 显著相关：USP40、APEH、DAG1 和 USP3。CYLD 以及其他一些相关基因，是泛素蛋白酶体系统（UPS）的一部分。因此，我们确定 IBD 相关的黏附侵袭性大肠杆菌（AIEC）是否可以调节 UPS 功能。用 AIEC LF82 参考株感染肠上皮细胞，通过减少多泛素化蛋白的积累、增加 26S 蛋白酶体活性和降低 NF-κB 调节剂 CYLD 的蛋白水平来调节 UPS 周转率。这导致 IκB-α降解和 NF-κB 激活。这种活性对于 AIEC 的致病性非常重要，因为降低 CYLD 会导致 AIEC LF82 增加在细胞内复制的能力。
我们的研究结果揭示了 UPS，特别是 CYLD，是 IBD 发病机制的一个重要因素，这一因素受到遗传和微生物因素的共同影响。
