Nutrition and Health Sciences, Department of
First Advisor
Dr Regis Moreau
Date of this Version
7-2023
Abstract
Mechanistic target of rapamycin complex 1 (mTORC1) signaling impacts intestinal inflammation by modulating the production of cytokines in both intestinal epithelial cells and macrophages. Recent evidence revealed that THP-1-derived macrophages with elevated mTORC1 activity (THP-1shTSC2 macrophages), exhibited increased proinflammatory cytokine production when stimulated with lipopolysaccharide (LPS) compared to counterpart macrophages with repressed or baseline mTORC1 activity. Furthermore, it has been shown that mTORC1 governs intestinal barrier function as evidenced by elevated transepithelial electrical resistance (TEER) and para/transcellular permeability in Raptor-depleted Caco-2 cells (cells with repressed mTORC1 activity) compared to Caco-2 cells with elevated or baseline mTORC1 activity. The present study investigated the barrier function characteristics of Caco-2 cell monolayers upon exposure to secretagogues produced by activated THP-1-derived macrophages. Conditioned media from LPS-treated (LP) or LPS+curcumin-treated (LC) THP-1shTSC2 macrophages were applied basolaterally to Raptor-depleted Caco-2 cells (R Caco-2 cells) and Caco-2 cells with baseline mTORC1 activity (S Caco-2 cells) grown and differentiated in Transwells. TEER and paracellular transport of FITC-dextran 4 kDa were measured to assess the integrity of the epithelial barrier. Results showed that upon LP exposure, the initially elevated TEER of R Caco-2 cells collapsed and exhibited increased permeability to FITC-dextran 4 kDa (P<0.0001) while S Caco-2 cells experienced a temporary decrease in TEER and mild leakiness to FITC-dextran followed by the recovery of their pre-treatment TEER. These results suggested that inflammatory factors secreted by macrophages in LP disrupted epithelial Caco-2 cell transport, particularly when mTORC1 activity was downregulated. LP dose-dependently disrupted the TEER of R Caco-2 cells (P<0.001), whereas LC alleviated the disruption. The study identified TNF-α as one of the macrophage-derived secretagogues causing TEER disruption since TNF-α blocking antibody effectively prevented the decrease of R Caco-2 cells TEER caused by basolaterally supplied recombinant TNF-α or LP (P<0.001). Additionally, results showed that the p38 MAPK, JNK, and IKKβ signaling pathways were involved in the collapse of TEER since specific pharmacological inhibitors provided protection (P<0.001). In conclusion, these studies provided new insights into how mTORC1, p38 MAPK, JNK, and IKKβ signaling pathways impact intestinal epithelial barrier function in Caco-2 cells upon macrophage and TNF-α challenges and inferred potential therapeutic targets for enhancing intestinal barrier integrity.
Advisor: Regis Moreau
Comments
A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Nutrition and Health Sciences, Under the Supervision of Professor Regis Moreau. Lincoln, Nebraska: July 2023
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