Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-kappaB-independent pathway.
Recognition of bacterial products by the innate immune system is dependent on pattern-recognition receptors: toll-like receptor 9 (TLR-9) in the case of bacterial DNA. We hypothesized that bacterial DNA can directly affect enteric epithelial cells. RT-PCR revealed constitutive TLR-9 mRNA expression in three human colonic epithelial cell lines (T84, HT-29, Caco-2) and THP-1 monocytes. Epithelial cells, in six-well culture plates or on filter supports, were exposed to E. coli DNA (1-50 microg/ml), synthetic CpG-rich oligonucleotides, or calf thymus DNA for 6-48 h. Exposure to E. coli DNA resulted in an increase in IL-8 mRNA, and a time- and dose-dependent increase in IL-8 secretion. Also, CpG oligonucleotides induced epithelial IL-8 production, whereas calf thymus DNA did not. Exposure to E. coli DNA resulted in phosphorylation of ERK 1/2 MAPK and inhibitors of ERK activity (PD98059, UO126) significantly reduced the evoked IL-8 production. In contrast, inhibitors of NFkappaB activity (PDTC, SN50) did not block E. coli DNA-induced IL-8 production. Electrophoretic mobility shift assays revealed that E. coli DNA stimulated epithelial AP-1 but not NFkappaB activation. The barrier (i.e., transepithelial resistance) and ion transport parameters of epithelial monolayers (assessed in Ussing chambers) were unaltered following E. coli DNA exposure. Thus model gut epithelia express TLR-9 mRNA and, while maintaining their barrier function, can respond to E. coli DNA by increased IL-8 production.
Akhtar
Mahmood
M
Intestinal Disease Research Programme, McMaster University, HSC-3N5C, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.
Watson
James L
JL
Nazli
Aisha
A
McKay
Derek M
DM
D018938:Caco-2 Cells; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D003106:Colon / Q000166:cytology; D051546:Cyclooxygenase 2; D004269:DNA, Bacterial / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D015232:Dinoprostone / Q000096:biosynthesis; D020895:Enterocytes / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D019073:HT29 Cells; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis*; D017136:Ion Transport / Q000187:drug effects; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics; D051217:Toll-Like Receptor 9
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D016209:Interleukin-8; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D020928:Mitogen-Activated Protein Kinases; D015232:Dinoprostone