Highly Biomimetic Ectodermal Epithelial Organoids for Epithelial Barrier Stimulation Assays.

The epithelial barrier theory has been proposed to link the onset of many chronic inflammatory diseases to the damaged epithelial barrier induced by numerous environmental factors, including food emulsifiers. This study aimed to investigate the effects of food emulsifiers kappa-, iota- and lambda-carrageenan on intestinal epithelial barrier integrity and the underlying mechanisms. The cytotoxicity and apoptosis of carrageenans were determined using Hoechst/propidium iodide staining and FITC annexin V/propidium iodide staining, respectively. The gut-on-a-chip was established and transepithelial electrical resistance measurement, paracellular flux assay, immunofluorescence staining of tight junction proteins, RNA-sequencing and targeted proteomics were performed. Kappa-, iota- and lambda-carrageenans induced dose-dependent cytotoxicity, with lambda-carrageenan showing a more severe cytotoxic effect in relatively lower doses. All three carrageenans elicited a dose- and time-dependent decrease of gut epithelial barrier strength, a significant increase in the paracellular flux and irregular and heterogeneous staining of occludin and ZO-1 compared to the untreated group, suggesting the disruption of the intestinal epithelial barrier integrity. Transcriptomics data revealed that iota- and lambda-carrageenan induced more severe pro-inflammatory responses, which were associated with the upregulation of genes involved in TNF signalling, IL-17 signalling cellular response to chemokines, cholesterol metabolism and NF-kappa B signalling pathways. Targeted proteomics data from exposed gut-on-a-chips indicated an upregulation of inflammation- and immune response-related proteins for all three carrageenans. The present study provides direct evidence for the detrimental effects of kappa-, iota- and lambda-carrageenans on gut epithelial cell activation and barrier integrity. The underlying mechanism of epithelial barrier disruption was largely attributed to the activation of innate immune responses by carrageenans, resulting in a pro-inflammatory response.

Previous studies have shown that high concentrations of ethanol (>/=40%) cause functional damage of the gastrointestinal epithelial barrier by direct cytotoxic effect on the epithelial cells. The effects of lower noncytotoxic doses of ethanol on epithelial barrier function are unknown. A major function of gastrointestinal epithelial cells is to provide a barrier against the hostile substances in the gastrointestinal lumen. The apicolaterally located tight junctions (TJs) form a paracellular seal between the lateral membranes of adjacent cells and act as a paracellular barrier. In this study, we investigated the effects of lower doses of ethanol on intestinal epithelial TJ barrier function using filter-grown Caco-2 intestinal epithelial monolayers. The Caco-2 TJ barrier function was assessed by measuring epithelial resistance or paracellular permeability of the filter-grown monolayers. Ethanol (0, 1, 2.5, 5, 7.5, and 10%) produced a dose-related drop in Caco-2 epithelial resistance and increase in paracellular permeability. Ethanol also produced a progressive disruption of TJ protein (ZO-1) with separation of ZO-1 proteins from the cellular junctions and formation of large gaps between the adjacent cells. Ethanol, at the doses used (</=10%), did not cause cytotoxicity (lactate dehydrogenase release) to the Caco-2 cells. Ethanol produced a disassembly and displacement of perijunctional actin and myosin filaments from the perijunctional areas. On ethanol removal, actin and myosin filaments rapidly reassembled at the cellular borders. Ethanol stimulated the Caco-2 myosin light chain kinase (MLCK) activity but did not affect the MLCK protein levels. Specific MLCK inhibitor ML-7 inhibited both ethanol increases in MLCK activity and TJ permeability without affecting the MLCK protein levels. Consistent with these findings, metabolic inhibitors sodium azide and 2,4-dinitrophenol significantly prevented ethanol-induced increase in Caco-2 TJ permeability, whereas cycloheximide or actinomycin D had no effect. The results of this study indicate that ethanol at low noncytotoxic doses causes a functional and structural opening of the Caco-2 intestinal epithelial TJ barrier by activating MLCK.

Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis

The epithelial barrier is a critical component of innate immunity and provides protection against microbial invasion. Claudin-1, a tight junction protein, is known to contribute to the epithelial cell barrier. An experimentally induced rat periodontal disease model was used to study the effects of lipopolysaccharide (LPS) on the expression of tight junction-associated molecule genes in the junctional epithelium. LPS was applied for 8 wk in the gingival sulcus, and junctional epithelium was collected by laser-capture microdissection and subjected to microarray analysis. Microarray analysis identified that expression of the claudin-1 gene was decreased in the epithelium by chronic LPS challenge. Immunohistochemical analysis confirmed the expression of claudin-1 protein in junctional epithelium and that 8 wk of chronic LPS topical application significantly reduced claudin-1 expression. The effect of LPS on claudin-1 protein expression was validated using a porcine junctional epithelial cell culture Transwell model. The epithelial barrier, as measured using transmembrane resistance, was significantly reduced after 3 wk of LPS challenge and this was associated with a decreased level of expression of claudin-1 protein. These results confirm that the initiation of experimental periodontal disease is associated with reduction in the expression of claudin-1 gene and protein. This decreased level of a critical tight junction protein may result in the disruption of barrier function and may play an important role in the initiation of periodontal disease.

Transient opening of tricellular vertices controls paracellular transport through the follicle epithelium during Drosophila oogenesis.

Metallothionein 2 activation by pravastatin reinforces epithelial integrity and ameliorates radiation-induced enteropathy.

The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight. Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction. To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties.

Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn2+-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax.

Vocal folds are a viscoelastic multilayered structure responsible for voice production. Vocal fold epithelial damage may weaken the protection of deeper layers of lamina propria and thyroarytenoid muscle and impair voice production. Systemic dehydration can adversely affect vocal function by creating suboptimal biomechanical conditions for vocal fold vibration. However, the molecular pathobiology of systemically dehydrated vocal folds is poorly understood. We used an in vivo rabbit model to investigate the complete gene expression profile of systemically dehydrated vocal folds. The RNA-Seq based transcriptome revealed 203 differentially expressed (DE) vocal fold genes due to systemic dehydration. Interestingly, function enrichment analysis showed downregulation of genes involved in cell adhesion, cell junction, inflammation, and upregulation of genes involved in cell proliferation. RT-qPCR validation was performed for a subset of DE genes and confirmed the downregulation of DSG1, CDH3, NECTIN1, SDC1, S100A9, SPINK5, ECM1, IL1A, and IL36A genes. In addition, the upregulation of the transcription factor NR4A3 gene involved in epithelial cell proliferation was validated. Taken together, these results suggest an alteration of the vocal fold epithelial barrier independent of inflammation, which could indicate a disruption and remodeling of the epithelial barrier integrity. This transcriptome provides a first global picture of the molecular changes in vocal fold tissue in response to systemic dehydration. The alterations observed at the transcriptional level help to understand the pathobiology of dehydration in voice function and highlight the benefits of hydration in voice therapy.

The epithelium functions as a barrier to the external environment, which is maintained in large part by cell junctions. Age‐related and chronic inflammatory conditions of the skin result in a loss of epithelial barrier integrity through the degradation of cell adhesion proteins. Granzyme B (GzmB) is a serine protease that accumulates in the extracellular milieu in a number of autoimmune skin conditions. As such, GzmB may contribute to the progressive loss of epithelial barrier function. We hypothesized that GzmB disrupts epithelial barrier function through the proteolytic cleavage of cell junction proteins. Human keratinocytes were treated with exogenous GzmB, in the presence or absence of a GzmB inhibitor. Subsequently, epithelial barrier function was assessed by Electric Cell‐substrate Impedance Sensing (ECIS) and immunofluorescence (confocal) microscopy was used to visualize E‐cadherin. GzmB treatment resulted in a loss of E‐cadherin staining on the cell membrane which was further supported by western blot analysis of cell supernatants and biochemical cleavage assays, where we observed a dose‐dependent increase in E‐cadherin fragmentation. E‐cadherin cleavage was markedly decreased when treated with a GzmB inhibitor. Moreover, ECIS analysis showed a noticeable decline in barrier function after GzmB treatment. In summary, GzmB contributes to a decline in epithelial barrier function in part through the proteolytic cleavage of E‐cadherin.Support or Funding InformationBC Lung Association

The environmental and occupational risk we confront from agricultural chemicals increases as their presence in natural habitats rises to hazardous levels, building a major part of the exposome. This is of particular concern in low- and middle-income countries, such as Brazil, known as a leading producer of agricultural commodities and consumer of pesticides. As long as public policies continue to encourage the indiscriminate use of pesticides and governments continue to support this strategy instead of endorsing sustainable agricultural alternatives, the environmental burden that damages epithelial barriers will continue to grow. Chronic exposure to environmental contaminants in early life can affect crucial barrier tissue, such as skin epithelium, airways, and intestine, causing increased permeability, leaking, dysbiosis, and inflammation, with serious implications for metabolism and homeostasis. This vicious cycle of exposure to environmental factors and the consequent damage to the epithelial barrier has been associated with an increase in immune-mediated chronic inflammatory diseases. Understanding how the harmful effects of pesticides on the epithelial barrier impact cellular interactions mediated by endogenous sensors that coordinate a successful immune system represents a crucial challenge. In line with the epithelial barrier hypothesis, this narrative review reports the available evidence on the effects of pesticides on epithelial barrier integrity, dysbiosis, AhR signaling, and the consequent development of immune-mediated inflammatory diseases.

Genetically induced disruption of the intestinal epithelial barrier leads to development of intestinal inflammation. In the interleukin-10 gene-deficient inflammatory bowel disease (IBD) mouse model, for instance, a primary defect in intestinal epithelial integrity occurs before the development of enterocolitis. In humans, a causal role for epithelial barrier disruption is still controversial. Although studies with first-degree relatives of IBD patients suggests an underlying role of impaired barrier function, a primary epithelial barrier defect in IBD patients has not been confirmed. The purpose of this article is to examine whether a primary epithelial barrier disruption is a prerequisite for the development of intestinal inflammation or whether intestinal inflammation can develop in the absence of epithelial disruption. We examined the intestinal epithelial integrity of the T cell receptor (TCR)-alpha gene-deficient mouse model of IBD. In vivo colonic permeability, determined by mannitol transmural flux, was assessed in 6-week-, 12-week-, and 25-week-old TCR-alpha gene-deficient and wild-type control mice using a single-pass perfusion technique. Mice were scored for intestinal histological injury and intestinal cytokine levels measured in organ cultures. Systemic responses to bacterial antigens were determined through 48-h spleen cell cultures stimulated with sonicate derived from endogenous bacterial strains. In contrast with previous findings in the interleukin-10 gene-deficient IBD model, TCR-alpha gene-deficient mice did not demonstrate evidence of primary intestinal epithelial barrier disruption at any age, despite developing a moderate to severe colitis within 12 weeks. A rise in intestinal interferon (IFN)-gamma levels preceded the onset of mucosal inflammation and then correlated closely with the degree of intestinal inflammation and injury. Spleen cells from TCR-alpha gene-deficient mice released IFN-gamma in response to stimulation with endogenous luminal bacterial antigens, a finding that suggests that the systemic response to bacterial antigens occurred independently of epithelial barrier disruption. Intestinal inflammation and a systemic response to bacterial antigens can develop in the absence of a measurable disruption of intestinal permeability.

How does global warming contribute to disorders originating from an impaired epithelial barrier?

To understand developmental processes it is essential to consider both, molecular / genetic data and structural aspects of morphogenesis and differentiation. Integration of both approaches is beneficial to interpret the data obtained and unravel tissue and organism formation. Sequential morphological changes during animal development can be followed by different microscopic methods that enable us to combine imaging of intact embryos, elucidation of the tissue and cell architecture at different size scales, localization of tissue constituents in their original location and correlative imaging, supplemented by analytical microscopy at the specific stages of development. Here we present a combined use of light microscopy, histology, transmission (TEM) and scanning electron microscopy (SEM), labelling with specific ligands and analytical microscopy to unravel the differentiation of epidermis and gut epithelium in a crustacean model. Our study addresses the interrelation of tissue morphogenesis, epithelial cell differentiation at the ultrastructural level and differentiation of the corresponding apical extracellular matrix, the cuticle, examined in the embryos and marsupial larvae of Porcellio scaber . Cuticle is a chitin‐based matrix, secreted apically by ectodermal epithelia during development and renewal in arthropods. We integrate structural information at different scale levels with data on matrix elemental and molecular composition in the selected developmental stages. Developmental stages and gross morphological changes in epidermal and gut tissues were identified by imaging the intact embryos and larvae (Fig. 1A), fluorescence labelling of nuclei and imaging of histological sections (Fig. 1B). Architecture of tissues, cells and matrix was characterized at the levels of light and electron microscopy. To determine the organic scaffold composition of the matrix, we performed localization of macromolecules containing N ‐acetyl‐glucosamine (chitin) by labelling with lectin wheat germ agglutinin. The epidermal cuticle in crustaceans is in addition mineralized, forming a stiff exoskeleton. Analytical microscopy was applied to examine calcification of the forming cuticular matrix in marsupial larvae, performed by energy dispersive X‐ray spectroscopy (EDS) to analyse elemental composition and Raman spectroscopy to determine mineral forms in the cuticle. The results show that several apical matrices are produced sequentially during epidermis and gut morphogenesis and that matrix renewal is coupled with major morphological modifications such as growth of appendages, elongation or bending of embryo body and hatching from egg envelopes. The early stages of apical matrix formation are similar in the epidermis and gut. Later in marsupial larval stages, specialization of both cuticles is evident, implying the establishment of their specific functions. Exoskeletal cuticle with elaborate surface structures (Fig. 2A) differentiates in several layers displaying chitin‐protein fibres patterns (Fig. 2B) and is already prominently calcified. Hindgut cuticle displays differentiation in a homogenous electron lucent procuticle and prominent electron dense epicuticle (Fig. 3A).In concert with the stages of apical matrix formation, a gradual formation of the subapical cell junctions, one of the key factors that determines cell polarity, was evidenced (Fig. 3B). We consider our model system as valuable system to study tissue and cell differentiation in their native environment, with applying the integrated structure‐function perspective.

Cigarette smoke affects differentiation and function of primary human bronchial epithelial cells