Abstract
Although tumor necrosis factor-α (TNF-α) is a known major inflammatory mediator in inflammatory bowel disease (IBD) and has various effects on intestinal epithelial cell (IEC) homeostasis, the changes in IECs in the early inflammatory state induced during short-time treatment (24 h) with TNF-α remain unclear. In this study, we investigated TNF-α-induced alterations in IECs in the early inflammatory state using mouse jejunal organoids (enteroids). Of the inflammatory cytokines, i.e., TNF-α, IL-1β, IL-6, and IL-17, only TNF-α markedly increased the mRNA level of macrophage inflammatory protein 2 (MIP-2; the mouse homologue of interleukin-8), which is induced in the early stages of inflammation. TNF-α stimulation (3 h and 6 h) decreased the mRNA level of the stem cell markers leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) and polycomb group ring finger 4 and the progenitor cell marker prominin-1, which is also known as CD133. In addition, TNF-α treatment (24 h) decreased the number of Lgr5-positive cells and enteroid proliferation. TNF-α stimulation at 3 h and 6 h also decreased the mRNA level of chromogranin A and mucin 2, which are respective markers of enteroendocrine and goblet cells. Moreover, enteroids treated with TNF-α (24 h) not only decreased the integrity of tight junctions and cytoskeletal components but also increased intercellular permeability in an influx test with fluorescent dextran, indicating disrupted intestinal barrier function. Taken together, our findings indicate that short-time treatment with TNF-α promotes the inflammatory response and decreases intestinal stem cell activity and barrier function.
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taken from b. The bar represents mean ± SE (n = 3). Different lowercase letters indicate significant differences (p < 0.05). d Time course of MIP-2 level secreted by enteroids after stimulation of 15 ng/mL TNF-α. Enteroids were cultured in enteroid culture medium with TNF-α (15 ng/mL) for 0–24 h. MIP-2 secretion was measured using ELISA. The data represents mean ± SE (n = 3). Different lowercase letters indicate significant differences (p < 0.05)
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The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We express our gratitude to Dr. Hans Clevers (Hubrecht Institute) for the kind gift of the Noggin-secreting cell line, Dr. Eitaro Aihara (Cincinnati University) for kindly donating the R-spondin-secreting cell line, and Dr. Tokiyoshi Ayabe (Hokkaido University) for the kind gift of anti-lysozyme antibody.
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This work was supported by Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for JSPS fellows Grant Number JP18J22466.
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YS, MS, KI, MT, YSK, and KKH designed the experiments. YS and HH performed the experiments. YS, MS, and KKH wrote the paper.
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Animal experiments were conducted in accordance with the guidelines for the maintenance and handling of experimental animals established by the Tokyo University of Agriculture Ethics Committee (Approval No: No. 2020037).
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Saito, Y., Shimizu, M., Iwatsuki, K. et al. Effect of short-time treatment with TNF-α on stem cell activity and barrier function in enteroids. Cytotechnology 73, 669–682 (2021). https://doi.org/10.1007/s10616-021-00487-y
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DOI: https://doi.org/10.1007/s10616-021-00487-y