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Interactions between epithelial and immune cells with the gut microbiota have wide-ranging effects on many aspects of human health. Therefore, there is value in developing in vitro models capable of performing highly controlled studies of such interactions. However, several critical factors that enable long term homeostasis between bacterial and mammalian cultures have yet to be established. In this study, we explored a model consisting of epithelial and immune cells, as well as four different bacterial species (Bacteroides fragilis KLE1958, Escherichia coli MG1655, Lactobacillus rhamnosus KLE2101, or Ruminococcus gnavus KLE1940), over a 50 hour culture period. Interestingly, both obligate and facultative anaerobes grew to similar extents in aerobic culture environments during the co-culture period, likely due to measured microaerobic oxygen levels near the apical surface of the epithelia. It was demonstrated that bacteria elicited reactive oxygen species (ROS) production, and that the resulting oxidative damage heavily contributed to observed epithelial barrier damage in these static cultures. Introduction of a ROS scavenger significantly mitigated oxidative damage, improving cell monolayer integrity and reducing lipid peroxidation, although not to control (bacteria-free culture) levels. These results indicate that monitoring and mitigating ROS accumulation and oxidative damage can enable longer term bacteria-intestinal epithelial cultures, while also highlighting the significance of additional factors that impact homeostasis in mammalian cell-bacteria systems.

Intestinal homeostasis is tightly regulated by the orchestrated actions of a multitude of cell types, including enterocytes, goblet cells and immune cells. Disruption of intestinal barrier function can increase susceptibility to pathogen invasion and destabilize commensal microbial-epithelial-immune interaction, manifesting in various intestinal and systemic pathologies. However, a quantitative understanding of how these cell types communicate and collectively contribute to tissue function in health and disease is lacking. Here, we utilized a human intestinal epithelial-dendritic cell model and multivariate analysis of secreted factors to investigate the cellular crosstalk in response to physiological and/or pathological cues (e.g., endotoxin, non-steroidal anti-inflammation drug (NSAID)). Specifically, we demonstrated that treatment with diclofenac (DCF), an NSAID commonly used to treat inflammation associated with acute infection and other conditions, globally suppressed cytokine secretion when dosed in isolation. However, the disruption of barrier function induced by DCF allowed for luminal lipopolysaccharide (LPS) translocation and activation of resident immune cells that overrode the anti-inflammatory influence of DCF. DCF-facilitated inflammation in the presence of LPS was in part mediated by upregulation of macrophage migration inhibitory factor (MIF), an important regulator of innate immunity. However, while neutralization of MIF activity normalized inflammation, it did not lead to intestinal healing. Our data suggest that systems-wide suppression of inflammation alone is insufficient to achieve mucosal healing, especially in the presence of DCF, the target of which, the COX-prostaglandin pathway, is central to mucosal homeostasis. Indeed, DCF removal post-injury enabled partial recovery of intestinal epithelium functions, and this recovery phase was associated with upregulation of a subset of cytokines and chemokines, implicating their potential contribution to intestinal healing. The results highlight the utility of an intestinal model capturing immune function, coupled with multivariate analysis, in understanding molecular mechanisms governing response to microbial factors, supporting application in studying host-pathogen interactions.

The gut microbiome and its interactions with epithelial and immune cells have wide-ranging effects on many aspects of human health. Thus, in vitro models that enable highly controlled studies of these interactions are of value, yet critical parameters enabling long term homeostasis between bacteria and mammalian cultures have not been established. In this study, we developed a model incorporating epithelial and immune cells as well as different bacterial species (B. fragilis, E. coli, L. rhamnosus, or R. gnavus) over a 50 hour culture period. Interestingly, both obligate and facultative anaerobes grew to similar extents in aerobic culture environments in co-culture with epithelial and immune cells, potentially due to measured microaerobic oxygen levels near the epithelial apical surface. It was demonstrated that bacteria elicited reactive oxygen species (ROS) production, and that these species heavily contribute to observed epithelial barrier damage in these static cultures. Introduction of a ROS scavenger significantly mitigated ROS-mediated damage, improving cell monolayer integrity and reducing lipid peroxidation, although not to control (bacteria-free culture) levels. These results indicate that monitoring and mitigating ROS concentrations can enable longer term bacteria-intestinal epithelial cultures, but also highlight the significance of additional factors that impact homeostasis in mammalian cell-bacteria systems.

The consumption of generally regarded as safe emulsifiers has increased, and has been associated with an increased prevalence of inflammatory bowel and metabolic diseases, as well as an altered microbiome. The mucus barrier, which selectively controls the transport of particulates and microorganisms to the underlying epithelial layer, has been previously shown to be altered by dietary salts and lipids. However, the potential impact of emulsifiers on the protective mucus barrier, its permeability, and associated structural changes are not clear. In this study, we analyzed changes in the mucus barrier to both passively diffusing nanoparticles and actively swimming E. coli upon exposure to two emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (Tween). When exposed to CMC, mucus pore size decreased, which resulted in significantly slower E. coli speed and particle diffusion rates through mucus. Tween exposure minimally impacted mucus microstructure and particle diffusion, but increased E. coli speed in mucus. Moreover, both emulsifiers appeared to alter mucus amount and thickness in rat intestinal tissue and mucus-producing cell cultures. These results indicate that acute exposure to emulsifiers impacts barrier and structural properties of intestinal mucus, modulating interactions between intestinal lumen contents, microbes, and underlying tissue, which may contribute to development of intestinal inflammation.

Intestinal mucus provides a significant barrier to transport of orally delivered drug carriers, as well as other particulates (e.g. food, microbes). The relative significance of particle size, surface chemistry, and dosing medium to mucus barrier properties is not well characterized, but important in designing delivery systems targeted to the intestinal mucosa. In this study, multiple particle tracking (MPT) was used to study diffusion of 20–500 nm diameter carboxylate- and polyethylene glycol-(PEG-)functionalized polystyrene model carriers through intestinal mucus. The impact of exposure to mucus in buffer versus a partially digested triglyceride mixture was explored. Effective diffusivity of particles in intestinal mucus decreased with an increasing particle size less than and more than theoretically (Stokes–Einstein) expected in a homogenous medium when dosed in buffer and model-fed state intestinal contents, respectively. For example, effective diffusivity decreased 2.9- versus 20-fold with increase in the particle size from 100 to 500 nm when dosed to mucus in buffer versus lipid-containing medium. Functionalization with PEG dramatically decreased sensitivity to lipids in a dosing medium. The results indicate that reduction of particle size may increase particle transport through intestinal mucus barriers, but these effects are strongly dependent on intestinal contents and particle surface chemistry.

Mucus forms a protective hydrogel layer over the intestinal epithelium, presenting a selective and robust barrier to the uptake of particulates and microbe invasion. Disease can alter mucus production and composition, thus potentially modifying mucosal barrier properties. Hirschsprung’s disease (HD) is a developmental abnormality of the nervous system often complicated by intestinal infection. An investigation of colonic mucus barrier properties in an HD animal model, endothelin receptor B mutant mice, revealed significantly reduced microsphere (passive) and microbe (active) transport rates (7-fold and 3.6-fold, respectively, in proximal colonic mucus) relative to wild-type. Transport differences were evident in both the ganglionic and aganglionic colon segments, in agreement with the risk of Hirschsprung’s disease-associated enterocolitis after surgery to remove aganglionic colon segments. The development of therapies aimed at altering colonic mucus barrier properties could be explored towards preventing the onset of enterocolitis in Hirschsprung’s disease. Keywords: mucus, Hirschsprung’s disease, nanoparticle diffusion, microbe transport, intestine 1. Introduction Mucus, a highly complex hydrogel composed of mucin glycoproteins with a pore size reported to be approximately 10–500 nm,[1] provides a barrier against particulate materials and biological pathogens. The barrier properties of mucus are a combined result of physical effects, such as size exclusion and hydrodynamic drag, and intermolecular interactions between diffusing entities and mucus components.[1–5] The significance of the mucus barrier in controlling access to the underlying epithelium raises the question of whether mucosal structural and/or chemical changes occur in disease states, potentially contributing to pathogen invasion. Hirschsprung’s disease (HD), also known as intestinal aganglionosis, is a developmental abnormality of the enteric nervous system that affects 1 in 5,000 live births.[6, 7] HD is characterized by the lack of ganglion cells along a variable length of the distal intestine, resulting in the absence of peristalsis in the aganglionic segment and dilation of the colon proximally.[6, 7] HD is associated with the development of Hirschsprung’s-associated enterocolitis (HAEC), a severe inflammation of the intestinal mucosa that presents with abdominal distension, diarrhea, and fever and can progress to sepsis and death.[6, 7] HAEC, the leading cause of death in HD, occurs in up to 50% of untreated HD cases, and the risk persists even after surgical removal of the aganglionic intestine, suggesting that the abnormality associated with HAEC extends beyond the region of aganglionosis.[6] It is thought that HD leads to changes in colonic microbiota, innate immunity, and epithelial barrier properties,[8] any of which may contribute to the risk of developing enterocolitis. Although these theories are being investigated, little attention has been focused on the potential contributory role of mucus barrier properties in this disease. Recent insight into changes in HD mucosal properties has been gained by investigating mucus turnover rate, mucin gene expression, and histological structure.[8–13] These studies have shown that HD is associated with lowered mucus turnover rate, lowered mucin concentrations, abnormal mucin ratios (neutral mucins:acidic-sulphomucins),[13] and differences in mucin producing goblet cell size and proliferation compared to the healthy state.[14] These results therefore support differences in mucus quantity and composition in HD, but do not specifically test potential changes in mucus barrier properties that may play a role in microbe invasion and in the development of enterocolitis. Given the important role of mucus in protecting underlying epithelia from microbial invasion, we hypothesize that the inherent barrier properties of colonic mucus are altered in HD. To test this hypothesis, transport of passively diffusing entities (polystyrene microspheres) and actively moving microbes (Escherichia coli) through colonic mucus in an HD animal model were investigated. In this work, live imaging and multiple particle tracking (MPT),[15–21] a powerful technique enabling non-disruptive investigation of transport across the mucosal layer, was employed. Particle and microbe trajectories were used to calculate time-averaged mean squared displacements (MSDs) and average velocities to quantitatively characterize transport across mucus layers. Results supported differences in colonic mucus barriers between HD and wild-type (WT) animals. A deeper understanding of the mechanisms underlying mucus barrier differences could lead to therapeutic treatments to modulate mucus barrier properties and potentially prevent the onset of HAEC.

The basement membrane of small intestinal epithelium possesses complex topography at multiple scales ranging from the mesoscale to nanoscale. Specifically, intestinal crypt-villus units are comprised of hundred-micron-scale well-like invaginations and finger-like projections; intestinal cell phenotype is related to location on this crypt-villus unit. A biomimetic intestinal cell culture system composed of type I collagen based permeable cell culture membranes incorporating both micron-scale intestinal crypt-like topography and nanometer scale topography was fabricated. Membranes were pre-incubated with either laminin (Ln) or fibronectin (Fn), inoculated with intestinal epithelial Caco-2 cells and cultured for 1-21 days to study the relative significance of influence of crypt-like topography and biomimetic substrate chemistry on cell phenotype. Crypt-like topography inhibited Caco-2 differentiation during early culture, as evidenced by slower cell spreading and lower brush border enzyme activity. For example, alanine aminopeptidase activity was lower on Ln-coated patterned collagen ( approximately 3.4+/-0.24mU/mg) compared to flat collagen (10.84+/-0.55mU/mg) at day 7. Caco-2 cultured on Fn-coated collagen started to spread earlier (1 day vs 3 days) and formed longer protrusions than on Ln-coated collagen. Pre-coating of Ln enhanced cell differentiation, as the maximum activity of a cell differentiation marker (alkaline phosphatase) was 2-3 times higher than on Fn-coated collagen, and maintained differentiated phenotype in long term (up to 21 days) culture. In general, compared to substrate topography, coating with ECM protein had more prominent and longer effect on cell behavior. Crypt-like topography affected Caco-2 spreading and differentiation during early culture, however the effect diminished as culture progressed. This information will benefit intestinal tissue engineering scaffold design, and modification of in vitro intestinal cell models.

Lipid based drug delivery systems, and in particular self-emulsifying drug delivery systems (SEDDS), show great potential for enhancing oral bioavailability but have not been broadly applied, largely due to lack of general formulation guidance. To help understand how formulation design influences physicochemical emulsion properties and associated function in the gastrointestinal environment, a range of twenty-seven representative self-emulsifying formulations were investigated. Two key functions of emulsion-based drug delivery systems, permeability enhancement and drug release, were studied and statistically related to three formulation properties - oil structure, surfactant hydrophilic liphophilic balance (HLB) values, and surfactant-to-oil ratio. Three surfactants with HLB values ranging from 10 to 15 and three structurally different oils (long chain triglyceride, medium chain triglyceride, and propylene glycol dicaprylate/dicaprate) were combined at three different weight ratios (1:1, 5:1, 9:1). Unstable formulations of low HLB surfactant (HLB=10) had a toxic effect on cells at high (1:1) surfactant concentrations, indicating the importance of formulation stability for minimizing toxicity. Results also indicate that high HLB surfactant (Tween 80) loosens tight junction at high (1:1) surfactant concentrations. Release coefficients for each emulsion system were calculated. Incorporation of a long chain triglyceride (Soybean oil) as the oil phase increased the drug release rate constant. These results help establish an initial foundation for relating emulsion function to formulation design and enabling bioavailability optimization across a broad, representative range of SEDDS formulations.

A microfabrication approach was utilized to create topographic analogs of intestinal crypts on a polymer substrate. It was hypothesized that biomimetic crypt-like micro-architecture may induce changes in small intestinal cell (i.e. Caco-2 cell) phenotype. A test pattern of micro-well features with similar dimensions (50, 100, and 500 microm diameter, 50 microm spacing, 120 microm in depth) to the crypt structures found in native basal lamina was produced in the surface of a poly(dimethylsiloxane) (PDMS) substrate. PDMS surfaces were coated with fibronectin, seeded with intestinal-epithelial-cell-like Caco-2 cells, and cultured up to fourteen days. The cells were able to crawl along the steep side walls and migrated from the bottom to the top of the well structures, completely covering the surface by 4-5 days in culture. The topography of the PDMS substrates influenced cell spreading after seeding; cells spread faster and in a more uniform fashion on flat surfaces than on those with micro-well structures, where cell protrusions extending to micro-well side walls was evident. Substrate topography also affected cell metabolic activity and differentiation; cells had higher mitochondrial activity but lower alkaline phosphatase activity at early time points in culture (2-3 days post-seeding) when seeded on micro-well patterned PDMS substrates compared to flat substrates. These results emphasize the importance of topographical design properties of a scaffolds used for tissue engineered intestine.

Orally delivered drugs and nutrients must diffuse through mucus to enter the circulatory system, but the barrier properties of mucus and their modulation by physiological factors are generally poorly characterized. The main objective of this study was to examine the impact of physicochemical changes occurring upon food ingestion on gastrointestinal (GI) mucus barrier properties. Lipids representative of postprandial intestinal contents enhanced mucus barriers, as indicated by a 10–142-fold reduction in the transport rate of 200 nm microspheres through mucus, depending on surface chemistry. Physiologically relevant increases in [Ca 2+ ] resulted in a 2–4-fold reduction of transport rates, likely due to enhanced cross-linking of the mucus gel network. Reduction of pH from 6.5 to 3.5 also affected mucus viscoelasticity, reducing particle transport rates approximately 5–10-fold. Macroscopic visual observation and micro-scale lectin staining revealed mucus gel structural changes, including clumping into regions into which particles did not penetrate. Histological examination indicated food ingestion can prevent microsphere contact with and endocytosis by intestinal epithelium. Taken together, these results demonstrate that GI mucus barriers are significantly altered by stimuli associated with eating and potentially dosing of lipid-based delivery systems; these stimuli represent broadly relevant variables to consider upon designing oral therapies.

It is recognized that topographical features such as ridges and grooves can dramatically influence cell phenotype, motivating the development of substrates with precisely biomimetic topography for study of the influence on cultured cells. Intestinal basement membrane topography has been precisely replicated using plasma enhanced chemical vapor deposition (CVD) of poly(2-hydroxyethyl methacrylate) (pHEMA) on native tissue. The ability for CVD pHEMA to coat and retain the complex architecture of the intestinal basement membrane at the micrometer scale was demonstrated using electron microscopy and surface chemical analysis (XPS). The suitability of CVD pHEMA as a cell culture substrate was assessed. Caco-2 cells maintained a high (>85%) viability on CVD pHEMA. Cell attachment and proliferation on CVD pHEMA were similar to those observed on materials traditionally used for cell culture and microfabrication purposes. Results indicate that CVD pHEMA is useful for development of precise (micrometer-scale) topographically biomimetic substrates for cell culture.

Gastrointestinal mucus, a complex network of highly branched glycoproteins and macromolecules, is the first barrier through which orally delivered drug and gene vectors must traverse. The diffusion of such vectors can be restricted by the high adhesivity and viscoelasticity of mucus. In this investigation, the barrier properties of gastrointestinal mucus to particle transport were explored using real-time multiple particle tracking. The influence of surface chemistry on particle transport rates was examined using amine-, carboxylate-, and sulfate-modified polystyrene nanoparticles. A strong dependence of particle mobility in gastrointestinal mucus on surface charge was observed, with anionic particles diffusing 20-30 times faster than cationic particles. Comparison of diffusion coefficients calculated for gastrointestinal mucus with significantly varying values previously reported in the literature for other mucus sources, including cervicovaginal mucus and cystic fibrosis sputum, highlight the dependence of mucus barrier properties on the anatomical source. A significant degree of transport rate heterogeneity was also observed in native gastrointestinal mucus, suggesting a highly heterogeneous distribution of pore sizes. Furthermore, the suitability of purified mucin as a model system for transport studies was assessed by comparing particle transport rates between native intestinal mucus and purified porcine gastric mucin. Particle transport rates were approximately threefold lower in native mucus compared to purified mucin for anionic particles, yet comparable for cationic particles. Differences between barrier properties of the purified mucin preparation and native mucus depended on specific carrier properties, indicating that the purified mucin preparation does not provide an accurate model system for native mucus.