Stimulation of Naïve Adipose-Derived Stem Cells with Mesenteric Lymph from Chronic Alcohol-Fed Rats Induces IL-33 Release Associated with Cellular Necrosis

Abstract

Our studies show that chronic alcohol feeding in rodents induced mesenteric lymphatic leakage into surrounding perilymphatic adipose tissue (PLAT) that was associated with increased fat regulatory T cells (fTregs), that were not increased in the circulation, suggesting local expansion of fTregs in PLAT. Increased visceral fTregs are implicated in age-associated insulin resistance. The IL-33 receptor, ST2, has been implicated in fTreg cell development and is critical for their accumulation in PLAT. IL-33 is released upon cell death by visceral adipose tissue stromal or adipose derived stem cells (ADSCs) and acts as an alarmin to signal tissue damage. Whether lymphatic leakage in alcohol-fed animals results in death of ADSCs, release of IL-33, and activation of the IL-33/ST2 signaling pathway leading to PLAT fTreg accumulation has yet to be explored. Here, we tested the hypothesis that stimulation of naïve ADSCs with lymph from chronic alcohol-fed animals would lead to increased IL-33 via cell death. Male Fischer 344 rats were randomized to a Lieber-DeCarli liquid diet containing 36% of calories from alcohol or pair-fed an isocaloric diet for 10 weeks (N=10/group). Mesenteric lymph was collected from anesthetized alcohol- and pair-fed animals via the lymph fistula technique. PLAT ADSCs, isolated from age-matched, naïve, chow-fed rats were cultured until adherent and stimulated with 5% v/v lymph from alcohol- or pair-fed animals for 24 hrs. Supernatant was collected, and IL-33 concentration was measured by commercially available ELISA. Adherent ADSCs were stained with 7-AAD and CytoCalcein fluorescent dyes to assess cell viability using fluorescence microscopy. Viable and necrotic cells were counted using QuPath software. Statistical differences were assessed using one-way ANOVA in GraphPad Prism. Results show a 2.5-fold increase in the concentration of IL-33 in supernatant of ADSCs stimulated with lymph from alcohol-fed animals compared to ADSCs stimulated with lymph from pair-fed animals. In addition, ADSC necrosis was 4-fold greater in wells stimulated with lymph from alcohol-fed animals than in those stimulated with control lymph or vehicle. In contrast, lymph stimulation of ADSCs from control-fed animals did not show a significant increase in IL-33 release as compared to vehicle treated controls. These data indicate presence of mediators that induce IL-33 release in lymph from alcohol-fed animals and support the hypothesized involvement of the IL-33/ST2 pathway in chronic alcohol associated ADSC cell death. Supported by K01AA026640 (FSS), 1F30AA030909-01 (KW), 5T32AA007577-23 (PM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.