Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium: role of iNOS and COX-2.
OBJECTIVE
Various invasive pathogens cause diarrhea, but the mechanism(s) are poorly understood. We hypothesized that nitric oxide and prostaglandins might modulate chloride secretory and barrier properties of the infected intestinal epithelium and that diarrhea is caused, in part, by altered expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2).


METHODS
Studies were conducted in human intestinal epithelial cell lines (HT29/cl.19A, Caco-2, and T84). Cells were infected with enteroinvasive Escherichia coli (EIEC 029:NM) or Salmonella dublin (SD), or nonpathogenic, noninvasive bacteria (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]). Infected cells and controls were tested for transepithelial resistance, chloride secretion, prostaglandin E2, guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate, and protein expression.


RESULTS
Cells infected with EIEC or SD, but not uninfected controls or ST/LA-exposed monolayers, showed a progressive reduction in transepithelial resistance starting at 6-12 hours. Infected HT29/cl.19A and Caco-2 cells, but not T84 cells, also showed an increase in total nitrite. Expression of iNOS, and consequently COX-2, was also increased, followed by increased production of prostaglandins and cyclic nucleotides. Furthermore, basal and stimulated chloride secretory responses to various agonists were enhanced in HT29/cl.19A and Caco-2 cells after infection with enteroinvasive bacteria, and this effect was reversed for some agonists by iNOS or COX-2 inhibitors. Increased expression of cystic fibrosis transmembrane conductance regulator and NKCC1 was also observed in EIEC or SD-infected cells vs. controls, secondary to NO synthase activity.


CONCLUSIONS
Up-regulation of iNOS and COX-2 by enteroinvasive bacteria can modulate chloride secretion and barrier function in intestinal epithelial cells. Thus, these enzymes represent possible therapeutic targets in infectious diarrhea.
Resta-Lenert
Silvia
S
Department of Medicine, and Biomedical Sciences Ph.D. Program, University of California, San Diego, School of Medicine, San Diego, California, USA. srestalenert@ucsd.edu
Barrett
Kim E
KE
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D018938:Caco-2 Cells; D002712:Chlorides / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D003967:Diarrhea / Q000201:enzymology / Q000382:microbiology; D015232:Dinoprostone / Q000378:metabolism; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000201:enzymology / Q000382:microbiology; D004927:Escherichia coli Infections / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000382:microbiology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009578:Nitrobenzenes / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D012480:Salmonella Infections / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D064506:Solute Carrier Family 12, Member 2; D013449:Sulfonamides / Q000494:pharmacology; D015854:Up-Regulation / Q000502:physiology; D014882:Water-Electrolyte Balance / Q000502:physiology
D000067956:Adenylyl Cyclase Inhibitors; C505032:CFTR protein, human; D002712:Chlorides; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D009578:Nitrobenzenes; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D000242:Cyclic AMP; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D015232:Dinoprostone; D019331:NG-Nitroarginine Methyl Ester