Background

The interface of the body with the external environment is formed by mucosal surfaces. These mucosal epithelial tissues can be found for example in the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye. Due to their exposure to the external environment many microorganisms populate these tissues. Therefore, these epithelia have evolved multiple mechanisms of defense in response to their vulnerability to microbial attack. Many defensive compounds are secreted into the mucosal fluid, including mucins, antibodies, defensins, protegrins, collectins, cathelicidins, lysozyme, histatins, and nitric oxide (Kagnoff and Eckmann, 1997; Lu et al., 2002; Raj and Dentino, 2002).

To date, more than 20 genes encoding mucins have been identified in humans (Corfield, 2015). The human mucin (MUC) family comprises membrane-bound (MUC1, MUC3A/B, MUC4, MUC12, MUC13, MUC15 – 17, MUC20 and MUC21) and secreted mucins (MUC2, MUC5AC, MUC5B, MUC6 – 9, MUC19) (Moran et al., 2011; Tailford et al., 2015).

The mucus layer of the intestinal epithelial surface is mainly composed of the secreted mucin MUC2, but the membrane-bound mucins MUC1, MUC3 and MUC4 are also expressed (Kim and Ho, 2010). In addition, the intestinal mucus layer differs in terms of composition, organization and thickness along the gastrointestinal tract (Tailford et al., 2015). The secreted mucins form a gel-like structure adherent to the apical cell surface that constitutes a physical and chemical barrier between the luminal contents and the underlying epithelium (Allan, 2011). Inflammasome activity controls the secretion of mucus in goblet cells and increased secretion of mucus is observed as the microbiota becomes more diverse (Jakobsson et al., 2015). It is becoming apparent that mucus plays a crucial role in maintaining a homeostatic balance between microbiota and its host. Even small deviations from this dynamic interaction can have significant health effects, among which are colitis, colorectal cancer and susceptibility to infection (McGuckin et al., 2011; Hansson, 2012; Chen and Stappenbeck, 2014).

In this protocol, we describe the culture of in vitro models producing different amounts of mucus depending on their culture condition (static vs. semi-wet with mechanical stimulation (Navabi et al., 2013). These models are based on the little to no mucus-producing HT29 cell line, a human colon adenocarcinoma cell line, and its high mucus-producing derivative HT29-MTX-E12 (E12). Furthermore, it is described how to quantify the mucus produced in the different models by ELISA. In addition, the mucolytic compound N-acetyl-L-cysteine (NAC) is used to remove adherent mucus in order to quantify the amount of secreted adherent mucus. A schematic overview of the workflow described in this protocol is provided in Figure 1.

The method described in this protocol is suitable to study the protective effect of mucus against bacterial toxins (Reuter et al., 2018), to test the effect of different culture conditions on mucus production (Navabi et al., 2013) or to analyze diffusion of molecules through the mucus layer. Since the ELISA used in this protocol is available for different species and mucus proteins, other cell types can also be used.