Division of Clinical Medicine, National Institute of Cholera and Enteric Diseases, Kolkata, India.
Division of Clinical Medicine, National Institute of Cholera and Enteric Diseases, Kolkata, India.
Biochim Biophys Acta Gen Subj. 2017 Jan;1861(1 Pt A):3365-3377. doi: 10.1016/j.bbagen.2016.08.019. Epub 2016 Aug 31.
Chemokines play key roles in immune homeostasis and inflammatory response. Considering the role of Ccl20 and Toll-like receptor 9 (TLR9) in gut homeostasis and inflammatory bowel disease (IBD), regulation of Ccl20 by bacterial DNA, the TLR9 ligand, merits in-depth studies.
We analyzed Ccl20 expression in various epithelial cell (EC) lines by q-PCR and ELISA. In-vivo expression was investigated in isolated murine colonocytes by immunoblotting. Transcriptional regulation of Ccl20 was studied by reporter assays, gene knock-down, electrophoretic mobility shift assay and chromatin immunoprecipitation. Activation of upstream kinases was checked by immunoblotting.
We showed low levels of Ccl20 expression in mouse colonic ECs, but marked induction by in vivo treatment with bacterial DNA. This corroborated with persistent Ccl20 induction in different EC lines. We found involvement of MAP-kinases during the early hours after stimulation, and a novel AP-1site (-252bp) regulated the expression in colonic ECs. More importantly, mutually exclusive transcriptional regulation by AP-1 (cjun/cfos) and non-canonical NF-κB (RelB/p52) downstream of MEK-ERK and NIK-IKK-α-NF-κB2 (p100) phosphorylation, respectively was responsible for persistent Ccl20 expression in the colonic cells, while canonical NF-κB isoforms played no role.
Persistent Ccl20 induction by TLR9 in colonic ECs involves early and delayed activation of two independent signaling pathways. This is the first report of non-canonical NF-κB activation and Ccl20 expression in the colonic ECs by TLR9.
Our study will help to better understand immune regulation by Ccl20 in the intestine and may be exploited for future development of novel therapeutics against IBD.
趋化因子在免疫稳态和炎症反应中发挥关键作用。鉴于 Ccl20 和 Toll 样受体 9（TLR9）在肠道稳态和炎症性肠病（IBD）中的作用，细菌 DNA（TLR9 配体）对 Ccl20 的调节值得深入研究。
我们通过 q-PCR 和 ELISA 分析了各种上皮细胞（EC）系中的 Ccl20 表达。通过免疫印迹法研究了分离的鼠结肠细胞中的体内表达。通过报告基因检测、基因敲低、电泳迁移率变动分析和染色质免疫沉淀研究 Ccl20 的转录调节。通过免疫印迹法检查上游激酶的激活。
我们显示，在鼠结肠 EC 中 Ccl20 的表达水平较低，但体内用细菌 DNA 处理后明显诱导。这与不同 EC 系中持续的 Ccl20 诱导相符。我们发现刺激后早期涉及 MAP 激酶，并且一个新的 AP-1 位点（-252bp）调节结肠 EC 中的表达。更重要的是，MEK-ERK 和 NIK-IKK-α-NF-κB2（p100）磷酸化下游的 AP-1（cjun/cfos）和非典型 NF-κB（RelB/p52）的转录调节是相互排斥的，分别负责结肠细胞中持续的 Ccl20 表达，而经典 NF-κB 同工型则没有作用。
TLR9 在结肠 EC 中持续诱导 Ccl20 涉及两条独立信号通路的早期和延迟激活。这是 TLR9 在结肠 EC 中激活非典型 NF-κB 和表达 Ccl20 的第一个报道。
我们的研究将有助于更好地理解 Ccl20 在肠道中的免疫调节，并可能为未来开发针对 IBD 的新型治疗方法提供帮助。
