F. M. Kuehn1, S. R. Hamarneh1, J. M. Ramirez1, A. R. Munoz1, F. Adiliaghdam1, S. A. Morrison1, R. A. Hodin1 1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA
Introduction: The inability of epithelial cells to cope with various stresses and cellular damage plays a crucial role in the development of many inflammatory diseases in the gut. The exact functions of the brush border enzyme intestinal alkaline phosphatases (IAP) in various cellular pathways are not well understood. We propose that IAP functions as an important modulator of intracellular homeostasis and stress responses in the gut.
Methods: Functional analysis and identification of IAP-binding proteins were performed using Liquid chromatography-tandem mass spectrometry (LC-MS) after immunoprecipitation of IAP complexes from Caco-2 cells in vitro. Furthermore, IAP knockout and overexpressing Caco-2 cells were developed using CRISPR/Cas9 gene editing technique and cell transfection, respectively. Subsequently, the effect of IAP activation or inhibition on inflammatory cytokine levels was measured using qPCR in the IAP-knockout and overexpressing Caco-2 cells. Additionally, the role of IAP in Caco-2 cell survival was assayed after incubation with high doses of TNF-α.
Results: Analysis of IAP protein complexes showed that IAP binds to key modulators of the NFκB, TNF-α and TLR-4 pathways. Overexpression of human IAP significantly reduced mRNA-levels of inflammatory cytokines in Caco-2 cells: TNF-α (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.08 Vs. 0.19 ± 0.1 Relative Expression, p= 0.01), IL-1β (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.4 Vs. 0.47 ± 0.06 Relative Expression, p= 0.001) and IL-8 (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.26 vs. 0.46 ± 0.16 Relative Expression, p= 0.046). IAP deletion increased the expression of TNF-α (IAP WT vs. IAP-KO Cells, 1.0 ± 0.2 Vs. 7.9 ± 0.7 Relative Expression, p= 0.007) and IL-1β (IAP WT vs. IAP-KO Cells, 1.0 ± 0.6 Vs. 11.4 ± 0.1.1 Relative Expression, p= 0.004) and IL-8 (IAP WT vs. IAP-KO Cells, 1.0 ± 0.12 Vs. 15.9 ± 0.8 Relative Expression, p= 0.0013). Furthermore, overexpression of IAP resulted in significantly less inflammation and cytokine production in Caco-2 cells when incubated with inflammatory mediators such as TNF-α (IAP WT vs. IAP-overexpressing Cells, 9.7 ± 1.8 Vs. 1.2 ± 0.46 Relative Expression, p= 0.004), LPS and bacterial contents. Additionally, higher IAP levels significantly increased Caco-2 survival after incubation with TNF-α 20ng/mL for 24 hours (IAP WT vs. IAP-overexpressing Cells, 30.5 ± 7.2 Vs. 90.3 ± 11.1 % Survival, p= 0.018).
Conclusion: Endogenous IAP functions as a modulator of the stress response and inflammatory pathways in intestinal epithelial cells. Intracellular IAP pathways in the gut may represent an important therapeutic target to prevent or treat a variety of gut inflammatory conditions.