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Cholinergic Activation of Primary Human Derived Intestinal Epithelium Does Not Ameliorate TNF-α Induced Injury

Abstract

Introduction

The intestinal epithelium contains specialized cells including enterocytes, goblet, Paneth, enteroendocrine, and stem cells. Impaired barrier integrity in Inflammatory Bowel Disease is characterized by elevated levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Prior studies in immortalized lines such as Caco-2, without native epithelial heterogeneity, demonstrate the amelioration of TNF-α compromised barrier integrity via nicotinic (nAChR) or muscarinic (mAChR) acetylcholine receptor activation.

Methods

A tissue-engineered model of primary human small intestinal epithelium was derived from dissociated organoids cultured on collagen-coated Transwells. Differentiation was accomplished with serum-containing media and compared to Caco-2 and HT-29 regarding alkaline phosphatase expression, transepithelial electrical resistance (TEER), and IL-8 secretion. Inflammation was modeled via basal stimulation with TNF-α (25 ng/mL) with or without nicotine (nAChR agonist) or bethanechol (mAChR agonist). Apoptosis, density (cells/cm2), TEER, lucifer yellow permeability, 70 kDa dextran transport, cell morphology, and IL-8 secretion were characterized.

Results

Primary intestinal epithelium demonstrates significant functional differences compared to immortalized cells, including increased barrier integrity, IL-8 expression, mucus production, and the presence of absorptive and secretory cells. Exposure to TNF-α impaired barrier integrity, increased apoptosis, altered morphology, and increased secretion of IL-8. Stimulation of nAChR with nicotine did not ameliorate TNF-α induced permeability nor alter 70 kDa dextran transport. However, stimulation of mAChR with bethanechol decreased transport of 70 kDa dextran but did not ameliorate TNF-α induced paracellular permeability.

Conclusions

A primary model of intestinal inflammation was evaluated, demonstrating nAChR or mAChR activation does not have the same protective effects compared to immortalized epithelium. Inclusion of other native stromal support cells are underway.

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Acknowledgments

S.H., and A.K. conceived the study. S.H., A.K., and S.M. provided experimental design input. S.H., and W.L. conducted the experiments. S.H. analyzed the data and was the primary manuscript author. S.H., W.L., and E.S., prepared and maintained human organoid cultures. E.S., and D.B. supplied human intestinal tissue for organoid establishment and WRN conditioned medium. All authors provided input towards the manuscript. The authors thank funding support from the National Institute of Health award numbers R21EB025395 Trailblazer (AK and RK), R01EB021908 BRP (AK and DB), R01DK084056, P30HD18655 and P30DK034854 (DTB) and the Department of Chemical Engineering at Northeastern University for start-up funding (AK and RK).

Conflict of interest

The authors Sanjin Hosic, Will Lake, Eric Stas, Ryan Koppes, David T. Breault, Shashi K. Murthy, Abigail N. Koppes do not declare any competing interests or conflicts.

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Hosic, S., Lake, W., Stas, E. et al. Cholinergic Activation of Primary Human Derived Intestinal Epithelium Does Not Ameliorate TNF-α Induced Injury. Cel. Mol. Bioeng. 13, 487–505 (2020). https://doi.org/10.1007/s12195-020-00633-0

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Keywords

  • Muscarinic
  • Nicotinic
  • mAChR
  • nAChR
  • Organoid
  • Inflammation
  • Tumor necrosis factor