Identification of specific targets for the gut mucosal defense factor intestinal alkaline phosphatase.
Intestinal alkaline phosphatase (IAP) is a small intestinal brush border enzyme that has been shown to function as a gut mucosal defense factor, but its precise mechanism of action remains unclear. We investigated the effects of IAP on specific bacteria and bacterial components to determine its molecular targets. Purulent fluid from a cecal ligation and puncture model, specific live and heat-killed bacteria (Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes), and a variety of proinflammatory ligands (LPS, CpG DNA, Pam-3-Cys, flagellin, and TNF) were incubated with or without calf IAP (cIAP). Phosphate release was determined by using a malachite green assay. The various fluids were applied to target cells (THP-1, parent HT-29, and IAP-expressing HT-29 cells) and IL-8 secretion measured by ELISA. cIAP inhibited IL-8 induction by purulent fluid in THP-1 cells by >35% (P < 0.005). HT29-IAP cells had a reduced IL-8 response specifically to gram-negative bacteria; >90% reduction compared with parent cells (P < 0.005). cIAP had no effect on live bacteria but attenuated IL-8 induction by heat-killed bacteria by >40% (P < 0.005). cIAP exposure to LPS and CpG DNA caused phosphate release and reduced IL-8 in cell culture by >50% (P < 0.005). Flagellin exposure to cIAP also resulted in reduced IL-8 secretion by >40% (P < 0.005). In contrast, cIAP had no effect on TNF or Pam-3-Cys. The mechanism of IAP action appears to be through dephosphorylation of specific bacterial components, including LPS, CpG DNA, and flagellin, and not on live bacteria themselves. IAP likely targets these bacterially derived molecules in its role as a gut mucosal defense factor.
Chen
Kathryn T
KT
Dept. of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
Malo
Madhu S
MS
Moss
Angela K
AK
Zeller
Skye
S
Johnson
Paul
P
Ebrahimi
Farzad
F
Mostafa
Golam
G
Alam
Sayeda N
SN
Ramasamy
Sundaram
S
Warren
H Shaw
HS
Hohmann
Elizabeth L
EL
Hodin
Richard A
RA
D000005:Abdomen / Q000382:microbiology; D000038:Abscess / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001419:Bacteria / Q000187:drug effects; D001424:Bacterial Infections / Q000378:metabolism / Q000517:prevention & control; D018407:Bacterial Physiological Phenomena; D002417:Cattle; D002460:Cell Line; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology; D005408:Flagellin / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D016209:Interleukin-8 / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000382:microbiology; D008070:Lipopolysaccharides / Q000378:metabolism*; D008089:Listeria monocytogenes / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D012486:Salmonella typhimurium / Q000502:physiology; D051193:Toll-Like Receptors / Q000378:metabolism
C408982:CPG-oligonucleotide; D004269:DNA, Bacterial; D016209:Interleukin-8; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; D051193:Toll-Like Receptors; D005408:Flagellin; D000469:Alkaline Phosphatase