title: A20 is an early responding negative regulator of Toll-like receptor 5 signalling in intestinal epithelial cells during inflammation.
abstract: 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-kappaB) 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.
authors.affiliation: Department of Internal Medicine II, Shimane University School of Medicine, Izumo, Shimane, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Oshima
authors.forename: S
authors.initials: S
authors.lastname: Ishihara
authors.forename: M A K
authors.initials: MA
authors.lastname: Rumi
authors.forename: M M
authors.initials: MM
authors.lastname: Aziz
authors.forename: Y
authors.initials: Y
authors.lastname: Mishima
authors.forename: C
authors.initials: C
authors.lastname: Kadota
authors.forename: I
authors.initials: I
authors.lastname: Moriyama
authors.forename: N
authors.initials: N
authors.lastname: Ishimura
authors.forename: Y
authors.initials: Y
authors.lastname: Amano
authors.forename: Y
authors.initials: Y
authors.lastname: Kinoshita
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D054426:Chemokine CXCL2 / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005408:Flagellin / Q000008:administration & dosage; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology; D007422:Intestines / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000008:administration & dosage; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000235:genetics / Q000378:metabolism*; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3
chemical_list: D054426:Chemokine CXCL2; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D005408:Flagellin; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D003546:Cysteine Endopeptidases; C513261:Tnfaip3 protein, mouse