A novel approach to maintain gut mucosal integrity using an oral enzyme supplement

Abstract

Objective: To determine the role of intestinal alkaline phosphatase (IAP) in enteral starvation-induced gut barrier dysfunction and to study its therapeutic effect as a supplement to prevent gut-derived sepsis.

Background: Critically ill patients are at increased risk for systemic sepsis and, in some cases, multiorgan failure leading to death. Years ago, the gut was identified as a major source for this systemic sepsis syndrome. Previously, we have shown that IAP detoxifies bacterial toxins, prevents endotoxemia, and preserves intestinal microbiotal homeostasis.

Methods: WT and IAP-KO mice were used to examine gut barrier function and tight junction protein levels during 48-hour starvation and fed states. Human ileal fluid samples were collected from 20 patients postileostomy and IAP levels were compared between fasted and fed states. To study the effect of IAP supplementation on starvation-induced gut barrier dysfunction, WT mice were fasted for 48 hours +/- IAP supplementation in the drinking water.

Results: The loss of IAP expression is associated with decreased expression of intestinal junctional proteins and impaired barrier function. For the first time, we demonstrate that IAP expression is also decreased in humans who are deprived of enteral feeding. Finally, our data demonstrate that IAP supplementation reverses the gut barrier dysfunction and tight junction protein losses due to a lack of enteral feeding.

Conclusions: IAP is a major regulator of gut mucosal permeability and is able to ameliorate starvation-induced gut barrier dysfunction. Enteral IAP supplementation may represent a novel approach to maintain bowel integrity in critically ill patients.

Figure 1

Lower level of endogenous IAP disrupts gut barrier function. WT and IAP-KO mice (n=5) were orally gavaged with FITC-dextran to assess intestinal permeability. Ninety minutes later, mice were euthanized, blood was collected via heart puncture and serum assayed for FITC-dextran concentration. Serum levels of FITC-dextran of IAP-KO mice were compared to those of WT: (A) Serum 4-kDa FITC-Dextran (μg/ml). (B) Serum 10-kDa FITC-Dextran (μg/ml). Upon euthanasia in the same mice groups (n=5), terminal ileum was harvested to extract tissue RNA. qRT-PCR was performed to determine the levels of tight junction protein expression. (C) Junctional protein mRNA expression in the terminal ileum of IAP-KO mice compared to WT. Values are expressed as mean ± SEM. Statistics: Statistical significance between two groups was tested using the two-tailed Student’s t test.; *p < 0.05, **p < 0.01.

Figure 2

Lower level of endogenous IAP in intestinal loops results in higher inflammatory response of mice macrophages to bacterial proinflammatory mediators. Laparotomy was performed on IAP-KO and WT mice (n = 5 per group) under general anesthesia and a 5-cm jejunal loop was constructed. 100 μl of either endotoxin-free water, LPS (100 ng/ml), Flagellin (100 ng/ml) or CpG-DNA (10 μg/ml) were instilled by injection into the loop. After 2 hours, while still under anesthesia, the loop was harvested, and the supernatants of the luminal contents were added to murine RAW 264.7 cells, incubated overnight, and TNF-α levels in the media quantified by ELISA. Values are expressed as mean ± SEM. Statistics: Statistical significance between two groups was tested using the two-tailed Student’s t test.; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Starvation disrupts gut barrier function. Groups of mice (n=5) were food starved for 48h with free access to drinking water. The control groups (n=5) were housed with free access to regular chow diet and drinking water. Mice were orally gavaged with FITC-dextran to assess intestinal permeability. Ninety minutes later, mice were euthanized, blood was collected via heart puncture and serum assayed for FITC-dextran concentration. Serum levels of FITC-dextran of starved mice were compared to those of well-fed mice: (A) Serum 4-kDa FITC-Dextran (μg/ml). (B) Serum 10-kDa FITC-Dextran (μg/ml). Upon euthanasia in the same mice groups (n=5), terminal ileum was harvested to extract tissue RNA. qRT-PCR was performed to determine the levels of tight junction protein expression. (C) Junctional protein mRNA expression in the terminal ileum of starved mice compared to well-fed mice. Values are expressed as mean ± SEM. Statistics: Statistical significance between two groups was tested using the two-tailed Student’s t test.; *p < 0.05.

Figure 4

Starvation diminishes the endogenous anti-inflammatory factors in the gut. Groups of mice (n=5) were fed or food starved for 48 hours with free access to drinking water, followed by construction of the loop in the proximal jejunum. 100 μl of either endotoxin-free water or LPS (100 ng/ml) were instilled into the loops using a 28-gauge needle. After 2 hours, while still under anesthesia, the loop was harvested, and the supernatants of the luminal contents were added to murine RAW 264.7 cells, incubated overnight, and TNF-α levels in the media quantified by ELISA. Values are expressed as mean ± SEM. Statistics: Statistical significance between two groups was tested using the two-tailed Student’s t test.; *p < 0.05.

Figure 5

Enteral fasting reduces IAP levels in human. Ileal fluid were collected from 20 patients undergoing ileostomy construction. IAP levels were compared from the first postoperative sample of enteric contents (fasted state) with their last postoperative sample collected (fed state). Values are expressed as mean ± SEM. Statistics: Statistical significance between fast and fed status was tested using the Wilcoxon signed rank statistical test.; ***p < 0.001.

Figure 6

Exogenous IAP supplementation improves tight junction protein abundance. WT mice (n=5) were housed in MGH animal facility with free access to normal chew diet and drinking water. In one of the groups (n=5), IAP was added to drinking water at a concentration of 300 U/ml. After 10 days of treatment, the mice were euthanized and intestinal tissues were collected for RNA extraction. Values are expressed as mean ± SEM. Statistics: Statistical significance between IAP treated and no treatment groups was tested using the two-tailed Student’s t test.; *p < 0.05.

Figure 7

Exogenous IAP supplementation reverses the effect of starvation on gut barrier function. WT mice (n = 5 per group) were housed with no chow diet for 48 h and had free access to drinking water supplemented with IAP 150 U/ml, IAP 300 U/ml or IAP vehicle as a control group. After 48 h of no food, were orally gavaged with FITC-dextran to assess intestinal permeability. Ninety minutes later, mice were euthanized, blood was collected and serum assayed for FITC-dextran concentration. Serum levels of FITC-dextran of IAP treated mice were compared to those of vehicle treated group: (A) Serum 4-kDa FITC-Dextran (μg/ml). (B) Serum 10-kDa FITC-Dextran (μg/ml). Upon euthanasia in the same mice groups (n=5), terminal ileum was harvested to extract tissue RNA. qRT-PCR was performed to determine the levels of tight junction protein expression. (C) Junctional protein mRNA expression in the terminal ileum of IAP treated mice compared to no treatment. Values are expressed as mean ± SEM. Statistics: Statistical significance between the groups was tested using one-way analysis of variance with Tukey’s multiple comparison posttests.; *p < 0.05, **p < 0.01.

Figure 8

Gut homeostasis during enteral nutrition. The brush border enzyme IAP appears to play a crucial role in regulating the gut barrier function through a mechanism that involves the dephosphorylation of bacterial proinflammatory mediators in the intestinal lumen. By dephosphorylating bacterial toxins, IAP blocks their binding to TLR receptors, preventing inflammation in the gut. Inflammatory cytokines exerts an inhibitory effect on the expression of tight junction proteins, resulting in disrupted gut barrier function. Lack of enteral feeding diminishes IAP levels in the intestine, resulting in hypersensitivity to bacterial toxins and elevated levels of inflammatory cytokines. Depletion of IAP leads to increased intestinal permeability.