Angela K. Moss

Intestinal alkaline phosphatase prevents metabolic syndrome in mice

Metabolic syndrome comprises a cluster of related disorders that includes obesity, glucose intolerance, insulin resistance, dyslipidemia, and fatty liver. Recently, gut-derived chronic endotoxemia has been identified as a primary mediator for triggering the low-grade inflammation responsible for the development of metabolic syndrome. In the present study we examined the role of the small intestinal brush-border enzyme, intestinal alkaline phosphatase (IAP), in preventing a high-fat-diet–induced metabolic syndrome in mice. We found that both endogenous and orally supplemented IAP inhibits absorption of endotoxin (lipopolysaccharides) that occurs with dietary fat, and oral IAP supplementation prevents as well as reverses metabolic syndrome. Furthermore, IAP supplementation improves the lipid profile in mice fed a standard, low-fat chow diet. These results point to a potentially unique therapy against metabolic syndrome in at-risk humans.

Intestinal alkaline phosphatase preserves the normal homeostasis of gut microbiota

Background and aims The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated. Methods Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria. Results IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium). Conclusions IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

Intestinal Alkaline Phosphatase Has Beneficial Effects in Mouse Models of Chronic Colitis

Background: The brush border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and is protective against dextran sulfate sodium (DSS)‐induced acute injury in rats. The present study evaluated the potential therapeutic role for orally administered calf IAP (cIAP) in two independent mouse models of chronic colitis: 1) DSS‐induced chronic colitis, and 2) chronic spontaneous colitis in Wiskott‐Aldrich Syndrome protein (WASP)‐deficient (knockout) mice that is accelerated by irradiation. Methods: The wildtype (WT) and IAP knockout (IAP‐KO) mice received four cycles of 2% DSS ad libitum for 7 days. Each cycle was followed by a 7‐day DSS‐free interval during which mice received either cIAP or vehicle in the drinking water. The WASP‐KO mice received either vehicle or cIAP for 6 weeks beginning on the day of irradiation. Results: Microscopic colitis scores of DSS‐treated IAP‐KO mice were higher than DSS‐treated WT mice (52 ± 3.8 versus 28.8 ± 6.6, respectively, P < 0.0001). cIAP treatment attenuated the disease in both groups (KO = 30.7 ± 6.01, WT = 18.7 ± 5.0, P < 0.05). In irradiated WASP‐KO mice cIAP also attenuated colitis compared to control groups (3.3 ± 0.52 versus 6.2 ± 0.34, respectively, P < 0.001). Tissue myeloperoxidase activity and proinflammatory cytokines were significantly decreased by cIAP treatment. Conclusions: Endogenous IAP appears to play a role in protecting the host against chronic colitis. Orally administered cIAP exerts a protective effect in two independent mouse models of chronic colitis and may represent a novel therapy for human IBD. (Inflamm Bowel Dis 2011)

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.

Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.

Intestinal Alkaline Phosphatase Prevents Antibiotic-Induced Susceptibility to Enteric Pathogens

Objective:To determine the efficacy of oral supplementation of the gut enzyme intestinal alkaline phosphatase (IAP) in preventing antibiotic-associated infections from Salmonella enterica serovar Typhimurium (S. Typhimurium) and Clostridium difficile. Background:The intestinal microbiota plays a pivotal role in human health and well-being. Antibiotics inherently cause dysbiosis, an imbalance in the number and composition of intestinal commensal bacteria, which leads to susceptibility to opportunistic bacterial infections. Previously, we have shown that IAP preserves the normal homeostasis of intestinal microbiota and that oral supplementation with calf IAP (cIAP) rapidly restores the normal gut flora. We hypothesized that oral IAP supplementation would protect against antibiotic-associated bacterial infections. Methods:C57BL/6 mice were treated with antibiotic(s) ± cIAP in the drinking water, followed by oral gavage of S. Typhimurium or C. difficile. Mice were observed for clinical conditions and mortality. After a defined period of time, mice were killed and investigated for hematological, inflammatory, and histological changes. Results:We observed that oral supplementation with cIAP during antibiotic treatment protects mice from infections with S. Typhimurium as well as with C. difficile. Animals given IAP maintained their weight, had reduced clinical severity and gut inflammation, and showed improved survival. Conclusions:Oral IAP supplementation protected mice from antibiotic-associated bacterial infections. We postulate that oral IAP supplementation could represent a novel therapy to protect against antibiotic-associated diarrhea (AAD), C. difficile-associated disease (CDAD), and other enteric infections in humans.

Tu1620 Intestinal Alkaline Phosphatase Is an Endogenous Anti-Inflammatory Factor

Introduction: Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme known to have the ability to detoxify in-vitro many pro-inflammatory bacterial components, including lipopolysaccharides (LPS), lipoteichoic acid (LTA), flagellin, CpG-DNA and uridine diphosphate (UDP). Gastrointestinal tract inflammation and endotoxemia due to elevated bacterial toxic components in the gut and disruption of intestinal permeability play a crucial role in the development and progression of a wide spectrum of diseases. In this study we sought to determine whether the endogenous IAP enzyme functions as an anti-inflammatory factor. Methods: We established a novel intestinal loop model to study the impact of endogenous IAP on the inflammatory activity of different bacterial components within a physiologic in vivo environment. The model was set up in wild type (WT) vs. IAP knockout (KO) mice of approximately 25 grams (n=5 for all groups). In another setting, we applied a fast (48 hours) vs. fed mouse model. Under general anesthesia, a 5 cm segment of proximal jejunum was carefully tied off at the proximal and distal ends, to isolate the loop. Different concentrations of LPS (100 ng/ml), LTA (5 μg/ml), flagellin (100 ng/ml), CpG-DNA (100 μg/ml) or UDP (1 mM) were injected into the loop and the luminal content was collected 2 hours later. Then, the supernatants were applied to RAW264.7 murine macrophage cells in triplicate and incubated overnight. LPS, LTA, Flagellin, CpG-DNA or UDP were directly applied to the cells as positive controls and endotoxin-free water was applied as a negative control. Tumor necrosis factor-alpha (TNF-α) levels were subsequently measured by sandwich ELISA. Results: All studied bacterial components induced a marked increase in TNFα levels from the RAW264.7 cells, whereas little TNF-α was seen in the case of endotoxinfree water alone. The luminal contents from the WT mice resulted in significantly lower TNF-α levels compared to the luminal contents from the KO mice for all studied bacterial toxins: LPS (600.9±75.47 vs. 946.2±55.99 pg/ml, p= 0.006), LTA (223.1±62.85 vs. 536.2±54.64 pg/ml, p= 0.005), flagellin (679.9±60.05 vs. 1008.8±61.15 pg/ml, p= 0.005), CpG-DNA (638.7±61.81 vs. 949.6±57.36 pg/ml , p= 0.006) and UDP (212.5±15.77 vs. 312.6±26.60 pg/ml, p= 0.012). Luminal contents from fed mice resulted in lower TNF-a levels compared to fasted mice for LPS (585.4±76.35 vs. 900.2±63.62 pg/ml, p=0.013). Conclusions: IAP detoxifies and prevents the inflammatory effects of LPS, LTA, flagellin, CpG-DNA and UDP in the gut. The loss of IAP expression that occurs with fasting could be responsible for the systemic sepsis syndrome seen in critically ill patients.

W1866 Local Peritoneal Irrigation With Intestinal Alkaline Phosphatase is Protective Against Peritonitis in Mice

Background The brush-border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and detoxifies different toll-like receptor ligands. This study aimed to determine the therapeutic effects of locally administered calf IAP (cIAP) in a cecal ligation and puncture (CLP) model of polymicrobial sepsis.