Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation

Janneke Elzinga,Clara Belzer,Benthe Van Der Lugt,Wilma T Steegenga,Pradeep Dudeja

doi:10.1371/journal.pone.0261191

Abstract

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.

Highlights

The surface of the human gastro-intestinal (GI) tract is covered by a layer of mucus, protecting the host from pathogens, harmful chemical or biological substances and physical damage [1,2,3,4]
To investigate Semi-Wet interface with Mechanical Stimulation (SWMS)-specific effects leading to higher mucus production of the SWMS culturing method, HT29-MTX-E12 cells were grown for 14 days under static and SWMS conditions, and microarray analysis was performed on mRNA of the cells
We investigated the expression of genes encoding for mucins in HT29-MTX-E12 cells, since we know from previous studies that culturing this cell type under SWMS conditions led to increased mucin type 2 (MUC2) production [27]

Summary

Introduction

The surface of the human gastro-intestinal (GI) tract is covered by a layer of mucus, protecting the host from pathogens, harmful chemical or biological substances and physical damage [1,2,3,4]. Defects in this mucus layer have been implicated in several intestinal pathologies. The mucus layer does protect the underlying epithelium from these high bacterial numbers, and provides a binding site and nutrition-rich niche for residing intestinal bacteria, such as mucin-degrading bacterial species which liberate short-chain fatty acids for cross-feeding bacteria and the host [9,10,11]. Mucin has been shown to be a major driver of intestinal microbiota composition in vitro [12]

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