A20 is an early responding negative regulator of Toll‐like receptor 5 signalling in intestinal epithelial cells during inflammation

• Published in: Clinical and experimental immunology Vol. 159; no. 2; pp. 185 - 198
• Main Authors: Oshima, N., Ishihara, S., Rumi, M. A. K., Aziz, M. M., Mishima, Y., Kadota, C., Moriyama, I., Ishimura, N., Amano, Y., Kinoshita, Y.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.02.2010; Blackwell; Oxford University Press; Blackwell Science Inc
• Subjects: A20; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cell Line, Tumor; Cells, Cultured; Chemokine CXCL2 - genetics; Chemokine CXCL2 - metabolism; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; DNA-Binding Proteins; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Epithelial Cells - pathology; Flagellin - administration & dosage; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Gene Expression - drug effects; HT29 Cells; Humans; Immune system; Immunohistochemistry; Inflammation - metabolism; Inflammation - pathology; intestinal inflammation; Intestines - pathology; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Kinases; Lipopolysaccharides - administration & dosage; Medical research; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; negative regulator; NF‐κB; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Rodents; Signal Transduction - drug effects; TLR; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - metabolism; Toll-Like Receptor 5 - genetics; Toll-Like Receptor 5 - metabolism; Translational Studies; Tumor Necrosis Factor alpha-Induced Protein 3; Toll like receptor 5; Digestive system; Gut; Toll like receptor; Biochemistry; Inflammation; Regulator; intestinal inflammation; Signal transduction; NF-κB; negative regulator; Epithelial cell; Early; Transcription factor NFκB; TLR; A20
• ISSN: 0009-9104; 1365-2249
• DOI: 10.1111/j.1365-2249.2009.04048.x

Summary Several negative regulatory mechanisms control Toll‐like receptor (TLR)‐mediated inflammatory responses and restore immune system balance, including the zinc‐finger protein A20, a negative regulator of TLR signalling that inhibits nuclear factor kappa B (NF‐κB) activity. In the present study, we investigated TLR‐5‐mediated A20 expression and its role in intestinal epithelial cells (IECs) during inflammation. HCT‐15 and HT‐29 cells were stimulated with flagellin, then the expressions of A20, interleukin‐1 receptor‐associated kinase (IRAK‐M) and Tollip were evaluated using RNase protection assay. Furthermore, experimental colitis was induced in tlr4‐deficient CH3/HeJ mice by administration of dextran sodium sulphate (DSS), then flagellin was injected anally, and the colonic expression of A20 was examined by real‐time polymerase chain reaction (PCR) and immunohistochemistry. To confirm flagellin‐induced expression of A20, we employed an organ culture system. The role of A20 in flagellin‐induced tolerance induction was evaluated in vitro, using a gene knock‐down method targeting A20. A20 expression increased rapidly and peaked at 1 h after flagellin stimulation in cultured IECs, then declined gradually to the basal level. In vivo, anal injection of flagellin induced epithelial expression of A20 in injured colonic tissue, whereas flagellin did not cause a significant increase in A20 expression in non‐injured normal tissue, which was also confirmed in vitro using the organ culture system. Gene knock‐down using A20 siRNA did not influence tolerance induced by restimulation with flagellin. A20 is an early response negative regulator of TLR‐5 signalling in IECs that functions during intestinal inflammation. Our results provide new insights into the negative feedback regulation of TLR‐5 signalling that maintains the innate immune system in the gut.