Identification of specific targets for the gut mucosal defense factor intestinal alkaline phosphatase

Abstract

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.

Fig. 1.

Exogenous intestinal alkaline phosphatase (IAP) inhibits IL-8 induction by necrotic cecal contents incubated with HT29 cells. Serially diluted cecal contents from a cecal ligation and puncture (CLP) model were incubated with varying concentrations of calf IAP (cIAP) for 2 h, then applied to THP-1 cell cultures. At 33 units/well, cIAP incubation caused >35% inhibition in IL-8 induction. *P < 0.05.

Fig. 2.

Cellular IAP expression inhibits IL-8 induction in cell culture by gram-negative bacteria. A: Escherichia coli 1569 and Salmonella typhimurium were applied to HT29 cells [multiplicity of infection (MOI) 1,000:1, incubation time 2 h], with a resultant 10- and 14-fold increase, respectively, in IL-8 induction. This induction was blocked in HT29-IAP cells. B: incubation with E. coli 1554, 1555, and 1557 (MOI 100:1, incubation time 2 h) resulted in modest 1.3-, 1.4-, and 1.7-increases in IL-8 induction, which were blocked in HT29-IAP cells. *P < 0.05; **P < 0.005. C: Listeria monocytogenes (MOI 1,000:1, incubation time 2 h) caused a 38-fold increase in IL-8 induction in both parent and HT29-IAP cells (P < 0.05 vs. control).

Fig. 3.

Exogenous IAP dephosphorylates heat-killed bacteria and inhibits their ability to induce IL-8 in cell culture. A: live E. coli 1569, S. typhimurium, and L. monocytogenes (MOI 1,000:1) were incubated with cIAP (100 units/cell culture well, 2 h), and free phosphate release was quantified by the malachite green assay. There was no change in phosphate release with or without (+/−) cIAP incubation. When the bacteria were first heat killed, and then incubated with cIAP for 2 h, there was a resultant 1.18- to 1.36-fold increase in free phosphate. B–D: live, intact bacteria incubated +/−cIAP were then applied to HT29 cell cultures, and there was no difference in IL-8 secretion. Heat-killed bacteria incubated with cIAP, when applied to HT29 cells, resulted in a >45% inhibition in IL-8 induction in all cases. *P < 0.05; **P < 0.005; ***P < 0.0005.

Fig. 4.

Exogenous IAP dephosphorylates the bacterial ligands LPS and CpG DNA, but not Pam-3-Cys or TNF-α. A and B: cIAP (100 units) was incubated with LPS (1 μg/ml) or CpG DNA (10 μM) for 30 min. Two- and 5-fold increases (P < 0.05) in free phosphate were observed. C and D: there were no differences in free phosphate when Pam-3-Cys (50 μg/ml) or TNF (30 ng/ml) were incubated with cIAP. **P < 0.005.

Fig. 5.

Exogenous IAP dephosphorylates LPS and CpG DNA over time. A and B: as cIAP incubation times with LPS and CpG DNA were increased to 24 h, the absolute change in free phosphate continued to increase. C and D: conversely, there were no increases in free phosphate with cIAP incubated with Pam-3-Cys or TNF.

Fig. 6.

Exogenous IAP inhibits the ability of LPS, CpG DNA, and flagellin to induce IL-8 in cell culture, but not Pam-3-Cys or TNF-α. All ligands induced IL-8 in cell cultures over a varying range of concentrations. A and B: when LPS and CpG DNA were incubated with cIAP (100 units/well, 2 h), then applied to HT29 cells for 24 h, IL-8 induction was reduced by >50% (P < 0.005). C: flagellin incubation with cIAP (100 units/well for 16 h), then applied to THP-1 cells for 24 h, resulted in reduced IL-8 induction by >40% (P < 0.005). D and E: Pam-3-Cys and TNF incubation with cIAP did not inhibit IL-8 induction.

Fig. 7.

Cellular IAP expression inhibits induction of IL-8 by LPS and CpG DNA but not by TNF-α. LPS (1 μg/ml) and CpG DNA (2 μM) were applied to HT29 cells, with IL-8 induction; the magnitude of IL-8 induction was reduced in HT29-IAP cells by >20% (P < 0.005). With TNF (15 ng/ml), there was no difference in the magnitude of IL-8 induction between HT29 and HT29-IAP cells. **P < 0.005.