Biphasic Ccl20 regulation by Toll-like receptor 9 through the activation of ERK-AP-1 and non-canonical NF-κB signaling pathways

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 1; pp. 3365 - 3377
• Main Authors: Dutta, Pujarini, Ta, Atri, Thakur, Bhupesh Kumar, Dasgupta, Nirmalya, Das, Santasabuj
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017
• Subjects: Animals; AP-1; Bacterial DNA; Ccl20; Cell Line; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Chemokine CCL20 - genetics; Chemokine CCL20 - metabolism; chemokines; Chemotaxis - drug effects; chromatin; Dendritic Cells - cytology; Dendritic Cells - drug effects; Dendritic Cells - metabolism; DNA; DNA, Bacterial - pharmacology; Enterocytes - drug effects; Enterocytes - metabolism; enzyme-linked immunosorbent assay; epithelial cells; Epithelium - drug effects; Epithelium - metabolism; Extracellular Signal-Regulated MAP Kinases - metabolism; gel electrophoresis; Gene Expression Regulation - drug effects; genes; Hematopoietic Stem Cells - drug effects; Hematopoietic Stem Cells - metabolism; homeostasis; Humans; IEC; immunoblotting; inflammation; inflammatory bowel disease; Intestine, Small - drug effects; Intestine, Small - metabolism; intestines; ligands; Mice; Models, Biological; Myeloid Differentiation Factor 88 - metabolism; NF-kappa B - metabolism; Persistent induction; phosphorylation; phosphotransferases (kinases); precipitin tests; Protein Subunits - metabolism; Protein Transport - drug effects; RelB/P52; Signal Transduction; therapeutics; Time Factors; TLR9; TNF Receptor-Associated Factor 6 - metabolism; Toll-like receptor 9; Toll-Like Receptor 9 - metabolism; transcription (genetics); Transcription Factor AP-1 - metabolism; transcription factor NF-kappa B; Transcription, Genetic - drug effects; Transcriptional Activation - drug effects; Transcriptional Activation - genetics; PP; ILF; Bacterial DNA; Persistent induction; IEC; iDC; IBD; FAE; LTβR; ActD; PBMC; AP-1; DAI (DLM-1/ZBP-1); PRR; PAMP; Ccl20; TLR9; TLR; EC; RelB/P52; DC; MAPKs
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.08.019

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. •Bacterial DNA induces Ccl20 in colonic epithelial cells.•In the epithelium, Ccl20 induction is persistent, but is transient in hematopoietic cells.•Biphasic Ccl20 regulation during early and late phases by distinct signaling pathways•Late phase Ccl20 induction requires non-canonical NF-κB pathways and Ccl20 promoter shows RelB/p52 binding.