Tu1620 Intestinal Alkaline Phosphatase Is an Endogenous Anti-Inflammatory Factor

Abstract

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.