Response of human enteroids treated with microbial metabolites derived from a multistage in vitro fermentation system

Abstract

Intestinal epithelial cell interactions with bacteria and bacterial metabolites are critical for homeostatic maintenance of the gastrointestinal tract and understanding how bacterial metabolites may help improve intestinal function is of great interest. Current host‐integration models are limited, therefore, a continuous multi‐stage in vitro fermentation system has been designed to simulate microbial activity across different regions of the large intestine, ascending, transverse, and descending colon (AC, TC, DC, respectively) using pooled human fecal samples. This system allows for examination of bacterial function specific to a given region. Outputs include filtered fecal supernatants from each region that can be used for metabolite identification and host‐metabolite interactions. Three‐dimensional human duodenal enteroids were treated with 12.5% of AC, TC, DC, or none for 24 h. Expression of stem cell marker LGR5 decreased 73% with AC (P < 0.05) compared to control (CON) with numerical decreases in TC and DC. Expression of goblet cell marker MUC2 was significantly increased (>100‐fold) in all treatments compared to CON. Adherens and tight junction (TJ) markers CDH1 and OCLN were increased by four and two‐fold, respectively, for TC and DC treatments (P < 0.05). Enteroids were then grown on Transwell inserts and treated with 25% AC, TC, and DC for three days. Transepithelial resistance peaked after two days and increased by 3.5, 2.1, and 2.9‐fold for AC, TC, and DC supernatants, respectively, compared to CON samples (P < 0.05). Interestingly, after three days, LGR5 expression was increased by 4.5‐fold with TC (P < 0.05). Expression of Paneth cell marker LYZ, as well as mucin markers MUC3a and TFF3 were markedly decreased in all treatments compared to CON (P < 0.05). However, MUC2 was increased over 30‐fold with TC and DC compared to CON (P < 0.05). Expression of TJ marker TJP1 increased (>3.5‐fold; P < 0.05) in all treatments, although no differences were found in CDH1. Expression of OCLN and CLDN3 were only increased in DC and TC respectively compared to AC (P < 0.05), which had the lowest expression overall. Expression of pattern‐recognition receptor TLR4 was increased in TC (41‐fold) and DC (53‐fold; P < 0.05) compared to CON. Short‐chain fatty acid (SCFA) analysis of AC, TC, and DC showed a progressive increase in total SCFA production (138, 185, and 203 mM) with individual SCFA increasing with the exception of butyrate (77–79 mM). Overall, fecal supernatants, regardless of fermentation site, altered cell proliferation, differentiation and physiological responses. Abundance of SCFA's and other metabolites, based on fermentation site may be a primary driver of specific cellular responses. This model system provides a relevant integration model to study bacterial metabolite‐host interactions.Support or Funding InformationDepartment of Defense Combat Feeding Research and Engineering Program project “Gut Microbiome and Performance Nutrition” and The Applied Research for the Advancement of Science and Technology Priorities (ARAP) Program on Synthetic Biology for Military Environments (SBME), funded by the Office of the Secretary of Defense.