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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster U18 - Effect of bile salts/phosphatidylcholine micelles on intestinal epithelium inflammation and barrier integrity during bacterial co-culture

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Bile is a key modulator of intestinal epithelial-immune-bacteria cross-talk with important effects in health and disease. Currently, experimental models used to study the relationships between bile salts (BS) and the intestinal mucosa are constrained by the limitations of xenogeneic animal models that do not recapitulate the human response, or low physiological relevance of in vitro models using cancerous cell lines, resulting in an unclear ability to accurately depict the cellular responses to bile exposure in the human body. Intestinal organoids, while highly useful tools for studying intestinal physiology, present challenges associated with apical exposure and permeability, as differentiated organoids generally have apical cell surfaces facing an enclosed lumen compartment. Perhaps most importantly, most in vitro systems lack key features of the intestinal environment that mediate interactions between bile salts and epithelial cells, most notably commensal microbes and immune cells. Bacteria-host cell co-cultures present challenges related to achieving stable bacteria density and requirements for low oxygen environment for some gut bacteria.

In this study, we established an intestinal in vitro model comprised of a simplified BS pool, human duodenal epithelial monolayers, dendritic cells, and a simplified microbial consortium consisting of commensal strains to capture key physiological features for analyzing bile-bacteria-host cell crosstalk. We utilized this model to demonstrate that immune-competent intestinal response to BS is dependent on the presence of bacteria and oxygen levels, while response to bacterial consortia and the bacterial consortia composition are in turn modulated by BS.

Materials and

Methods:  Aerobic BS/PC micelles exposure: Human intestinal organoids were broken into cells and cultured on Type I collagen coated Transwell for 4-5 days of expansion and 2 days of conventional or air liquid interface differentiation. Upon the differentiation, apical medium was changed to 10% modified M9 in 1X DPBS (+/+) containing model bile micelles (BS/PC) comprised of Sodium Taurocholate (Sigma-Aldrich, 86339), Sodium Glycochenodeoxycholate (Sigma-Aldrich, 16564-43-5), Sodium Tauroursodeoxycholate (EMD Millipore, 580549) and Phosphatidylcholine (Avanti, 840051P). The total bile salt concentrations used matched physiological concentrations of 4 mM (fasted state) and 12 mM (fed state) in human duodenal luminal fluid. Cultures were exposed to bile salt preparations for 24 hours.

Anaerobic BS/PC micelles exposure: 48 hrs. before bacterial seeding, Bifidobacterium longum ECE2100, Prevotella menlaninogenica ATCC 25845, C. bifermentans ATCC 638 and S. mitis NCIMB 13770 were revived and expanded, respectively. Upon initiation of bile salt exposure, bacterial mixes were prepared in an anaerobic chamber using chemically defined M9 medium supplemented with or without 4 mM/1 mM, 8 mM/1 mM, 12 mM/3 mM BS/PC at 2 x 105 units/ml (6 x 104 units/well, 1.5 x 104 units/strain). Bacterial mixes were collected by 1 ml BD syringe with 28-gauge needle for seeding into Transwell inserts with apical chambers sealed by rubber stoppers. After seeding, the stopper injection holes were sealed by Vaseline.

After exposure, RT-qPCR, ELISA, immunofluorescent staining, and Lucifer Yellow assay were conducted to measure bacterial density or epithelial cell gene expression, host cell inflammatory responses, monolayer morphology or viability, and monolayer permeability.

Results, Conclusions, and Discussions: BS/PC exposure under normoxia did not impact monolayer viability or barrier integrity at conditions mimicking the fasted state. However, at fed state concentration, both viability and permeability of monolayer were adversely affected. In addition, we noticed the viability of epithelial cells was increased in ALI model after 8/2 mM and 12/3 mM BS/PC exposure in compared to conventional model.

Bacterial co-cultures including fasted state BS/PC maintained barrier function and a relative confluent monolayer. In the absence of bile, incorporating a bacterial consortium into the model and co-culturing under apical hypoxic conditions resulted in loss of barrier function by the 24-hour endpoint. RT-qPCR data revealed lower bacterial numbers in systems incorporating BS/PC, suggesting BS/PC created a more bacteriostatic environment in vitro compared to culture in the absence of bile. Moreover, the inclusion of bile modulated the host immune response in bacterial co-cultures, resulted in markedly decreased levels of TNF- α and IL-10 secretion.

Surprisingly, fasted state BS/PC, while compatible during exposure in normoxia, was toxic to intestinal models in hypoxia, leading to cell death and elevated permeability of monolayers after 24-hour exposure. However, the simplified consortium alleviated the toxicity of fasted state bile and increased the cell viability and barrier function of epithelial cells under hypoxia.

Our results support the utility of a human physiologically relevant small intestinal model that can recapitulate key features of the native mucosal environment for capturing in vivo observations pertaining to intestinal host-microbiome interactions. Using this model, we revealed the sensitivity of human intestinal epithelial monolayers to varying concentrations of bile. The exposure of bile in the model regulated bacterial communities and created a more bacteriostatic environment, potentially aiding in stabilizing the co-culture system via this mechanism. Oxygen was also found to be a critical factor affecting bile toxicity to cells. The model commensal consortium aided in sustaining intestinal monolayer integrity upon exposure to normal concentrations of bile mimicking fasted state under hypoxia. The mucus layer was also shown bile toxicity buffering property to intestinal cells, indicating the importance of physiological mucus structures to reproduce in vivo interactions from bile-intestinal-microbial in vitro studies.

Acknowledgements (Optional):  BS/PC exposure under normoxia did not impact monolayer viability or barrier integrity at conditions mimicking the fasted state. However, at fed state concentration, both viability and permeability of monolayer were adversely affected. In addition, the viability of epithelial cells was increased in ALI model after 8/2 mM and 12/3 mM BS/PC exposure compared to conventional model, suggesting the enhanced mucus production in ALI culture had a protective effect.

Bacterial co-cultures including fasted state BS/PC maintained barrier function and a relatively confluent monolayer. In the absence of bile, however, incorporating a bacterial consortium into the model and co-culturing under apical hypoxic conditions resulted in loss of barrier function by the 24-hour endpoint. RT-qPCR data revealed lower bacterial numbers in systems incorporating BS/PC, suggesting BS/PC created a more bacteriostatic environment in vitro compared to culture in the absence of bile. Moreover, the inclusion of bile modulated the host immune response in bacterial co-cultures, resulted in markedly decreased levels of TNF- α and IL-10 secretion.

Surprisingly, fasted state BS/PC, while compatible during exposure in normoxia, was toxic to intestinal models in hypoxia, leading to cell death and elevated permeability of monolayers after 24-hour exposure. However, the simplified consortium alleviated the toxicity of fasted state bile and increased the cell viability and barrier function of epithelial cells under hypoxia.

Our results support the utility of a human physiologically relevant small intestinal model that can recapitulate key features of the native mucosal environment for capturing host cell-microbiome cross-talk and its modulation by bile. Using this model, we revealed the sensitivity of human intestinal epithelial monolayers to varying concentrations of bile. The exposure of bile in the model regulated bacterial communities and created a more bacteriostatic environment, potentially aiding in stabilizing the co-culture system via this mechanism. Oxygen was also found to be a critical factor affecting bile toxicity to cells. The model commensal consortium aided in sustaining intestinal monolayer integrity upon exposure to normal concentrations of bile mimicking fasted state under hypoxia. The mucus layer was also shown to be important in buffering bile toxicity to intestinal cells.