Epithelial Health Research Articles

BEST4+ cells in the intestinal epithelium.

The recent development of single-cell transcriptomics highlighted the existence of a new lineage of mature absorptive cells in the human intestinal epithelium. This subpopulation is characterized by the specific expression of Bestrophin 4 (BEST4) and of other marker genes including OTOP2, CA7, GUCA2A, GUCA2B, and SPIB. BEST4+ cells appear early in development and are present in all regions of the small and large intestine at a low abundance (<5% of all epithelial cells). Location-specific gene expression profiles in BEST4+ cells suggest their functional specialization in each gut region, as exemplified by the small intestine-specific expression of the ion channel CFTR. The putative roles of BEST4+ cells include sensing and regulation of luminal pH, tuning of guanylyl cyclase-C signaling, transport of electrolytes, hydration of mucus, and secretion of antimicrobial peptides. However, most of these hypotheses lack functional validation, notably because BEST4+ cells are absent in mice. The presence of BEST4+ cells in human intestinal organoids indicates that this in vitro model should be suitable to study their role. Recent studies showed that BEST4+ cells are also present in the intestinal epithelium of macaque, pig, and zebrafish and, here, we report their presence in rabbits, which suggests that these species could be appropriate animal models to study BEST4+ cells during the development of diseases and their interactions with environmental factors such as diet or the microbiota. In this review, we summarize the existing literature regarding BEST4+ cells and emphasize the description of their predicted roles in the intestinal epithelium in health and disease.NEW & NOTEWORTHY BEST4+ cells are a novel subtype of mature absorptive cells in the human intestinal epithelium highlighted by single-cell transcriptomics. The gene expression profile of BEST4+ cells suggests their role in pH regulation, electrolyte secretion, mucus hydration, and innate immune defense. The absence of BEST4+ cells in mice requires the use of alternative animal models or organoids to decipher the role of this novel type of intestinal epithelial cells.

Inflammation regulation in airway epithelium in health and disease

Inflammation regulation in airway epithelium in health and disease

Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey.

Many whey proteins, peptides and protein-derived amino acids have been suggested to improve gut health through their anti-oxidant, anti-microbial, barrier-protective and immune-modulating effects. Interestingly, although the degree of hydrolysis influences peptide composition and, thereby, biological function, this important aspect is often overlooked. In the current study, we aimed to investigate the effects of whey protein fractions with different degrees of enzymatic hydrolysis on the intestinal epithelium in health and disease with a novel 2D human intestinal organoid (HIO) monolayer model. In addition, we aimed to assess the anti-microbial activity and immune effects of the whey protein fractions. Human intestinal organoids were cultured from adult small intestines, and a model enabling apical administration of nutritional components during hypoxia-induced intestinal inflammation and normoxia (control) in crypt-like and villus-like HIO was established. Subsequently, the potential beneficial effects of whey protein isolate (WPI) and two whey protein hydrolysates with a 27.7% degree of hydrolysis (DH28) and a 50.9% degree of hydrolysis (DH51) were assessed. In addition, possible immune modulatory effects on human peripheral immune cells and anti-microbial activity on four microbial strains of the whey protein fractions were investigated. Exposure to DH28 prevented paracellular barrier loss of crypt-like HIO following hypoxia-induced intestinal inflammation with a concomitant decrease in hypoxia inducible factor 1 alpha (HIF1α) mRNA expression. WPI increased Treg numbers and Treg expression of cluster of differentiation 25 (CD25) and CD69 and reduced CD4+ T cell proliferation, whereas no anti-microbial effects were observed. The observed biological effects were differentially mediated by diverse whey protein fractions, indicating that (degree of) hydrolysis influences their biological effects. Moreover, these new insights may provide opportunities to improve immune tolerance and promote intestinal health.

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research. Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells. Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-ß and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects. Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research. Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells. Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-ß and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects. Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Bronchial epithelium epithelial-mesenchymal plasticity forms aberrant basaloid-like cells in vitro.

Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid cells were identified in fibrotic lung tissue as a novel epithelial cell type displaying a partial EMT phenotype. The developmental origin of these cells remains unknown. To elucidate the role of EMT in the development of aberrant basaloid cells from the bronchial epithelium, we mapped EMT-induced transcriptional changes at the population and single-cell levels. Human bronchial epithelial cells grown as submerged or air-liquid interface (ALI) cultures with or without EMT induction were analyzed by bulk and single-cell RNA-Sequencing. Comparison of submerged and ALI cultures revealed differential expression of 8,247 protein coding (PC) and 1,621 long noncoding RNA (lncRNA) genes and revealed epithelial cell-type-specific lncRNAs. Similarly, EMT induction in ALI cultures resulted in robust transcriptional reprogramming of 6,020 PC and 907 lncRNA genes. Although there was no evidence for fibroblast/myofibroblast conversion following EMT induction, cells displayed a partial EMT gene signature and an aberrant basaloid-like cell phenotype. The substantial transcriptional differences between submerged and ALI cultures highlight that care must be taken when interpreting data from submerged cultures. This work supports that lung epithelial EMT does not generate fibroblasts/myofibroblasts and confirms ALI cultures provide a physiologically relevant system to study aberrant basaloid-like cells and mechanisms of EMT. We provide a catalog of PC and lncRNA genes and an interactive browser (https://bronc-epi-in-vitro.cells.ucsc.edu/) of single-cell RNA-Seq data for further exploration of potential roles in the lung epithelium in health and lung disease.

Epithelial Heat Shock Proteins Mediate the Protective Effects of Limosilactobacillus reuteri in Dextran Sulfate Sodium-Induced Colitis.

Defects in gut barrier function are implicated in gastrointestinal (GI) disorders like inflammatory bowel disease (IBD), as well as in systemic inflammation. With the increasing incidence of IBD worldwide, more attention should be paid to dietary interventions and therapeutics with the potential to boost the natural defense mechanisms of gut epithelial cells. The current study aimed to investigate the protective effects of Limosilactobacillus reuteri ATCC PTA 4659 in a colitis mouse model and delineate the mechanisms behind it. Wild-type mice were allocated to the control group; or given 3% dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis; or administered L. reuteri for 7 days as pretreatment; or for 14 days starting 7 days before subjecting to the DSS. Peroral treatment with L. reuteri improved colitis severity clinically and morphologically and reduced the colonic levels of Tumor necrosis factor-α (TNF-α) (Tnf), Interleukin 1-β (Il1β), and nterferon-γ (Ifng), the crucial pro-inflammatory cytokines in colitis onset. It also prevented the CD11b+Ly6G+ neutrophil recruitment and the skewed immune responses in mesenteric lymph nodes (MLNs) of CD11b+CD11c+ dendritic cell (DC) expansion and Foxp3+CD4+ T-cell reduction. Using 16S rRNA gene amplicon sequencing and RT-qPCR, we demonstrated a colitis-driven bacterial translocation to MLNs and gut microbiota dysbiosis that were in part counterbalanced by L. reuteri treatment. Moreover, the expression of barrier-preserving tight junction (TJ) proteins and cytoprotective heat shock protein (HSP) 70 and HSP25 was reduced by colitis but boosted by L. reuteri treatment. A shift in expression pattern was also observed with HSP70 in response to the pretreatment and with HSP25 in response to L. reuteri-DSS. In addition, the changes of HSPs were found to be correlated to bacterial load and epithelial cell proliferation. In conclusion, our results demonstrate that the human-derived L. reuteri strain 4659 confers protection in experimental colitis in young mice, while intestinal HSPs may mediate the probiotic effects by providing a supportive protein–protein network for the epithelium in health and colitis.

Identifying key regulators of the intestinal stem cell niche.

The intestinal tract is lined by a single layer of epithelium that is one of the fastest regenerating tissues in the body and which therefore requires a very active and exquisitely controlled stem cell population. Rapid renewal of the epithelium is necessary to provide a continuous physical barrier from the intestinal luminal microenvironment that contains abundant microorganisms, whilst also ensuring an efficient surface for the absorption of dietary components. Specialised epithelial cell populations are important for the maintenance of intestinal homeostasis and are derived from adult intestinal stem cells (ISCs). Actively cycling ISCs divide by a neutral drift mechanism yielding either ISCs or transit-amplifying epithelial cells, the latter of which differentiate to become either absorptive lineages or to produce secretory factors that contribute further to intestinal barrier maintenance or signal to other cellular compartments. The mechanisms controlling ISC abundance, longevity and activity are regulated by several different cell populations and signalling pathways in the intestinal lamina propria which together form the ISC niche. However, the complexity of the ISC niche and communication mechanisms between its different components are only now starting to be unravelled with the assistance of intestinal organoid/enteroid/colonoid and single-cell imaging and sequencing technologies. This review explores the interaction between well-established and emerging ISC niche components, their impact on the intestinal epithelium in health and in the context of intestinal injury and highlights future directions and implications for this rapidly developing field.

Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.

SummaryRecent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy.

The Airway Epithelium-A Central Player in Asthma Pathogenesis.

Asthma is a chronic inflammatory airway disease characterized by variable airflow obstruction in response to a wide range of exogenous stimuli. The airway epithelium is the first line of defense and plays an important role in initiating host defense and controlling immune responses. Indeed, increasing evidence indicates a range of abnormalities in various aspects of epithelial barrier function in asthma. A central part of this impairment is a disruption of the airway epithelial layer, allowing inhaled substances to pass more easily into the submucosa where they may interact with immune cells. Furthermore, many of the identified susceptibility genes for asthma are expressed in the airway epithelium. This review focuses on the biology of the airway epithelium in health and its pathobiology in asthma. We will specifically discuss external triggers such as allergens, viruses and alarmins and the effect of type 2 inflammatory responses on airway epithelial function in asthma. We will also discuss epigenetic mechanisms responding to external stimuli on the level of transcriptional and posttranscriptional regulation of gene expression, as well the airway epithelium as a potential treatment target in asthma.

Insulin-like growth factor binding protein-7: A marker of conjunctivalization in an animal model of limbal stem cell deficiency

PurposeLimbal stem cell deficiency (LSCD) is characterized by the loss of limbal epithelial stem cells, resulting in a pathological process termed ‘conjunctivalization’ which compromises corneal transparency, leading to blindness. Current diagnosis for LSCD is limited because reliable conjunctiva-specific biomarkers are lacking. This study sought to address this shortcoming through the serendipitous discovery of insulin-like growth factor binding protein (IGFBP)-7. MethodsIGFBP-7 expression was determined in normal (n=83) and conjunctivalized (n=52) mouse corneas with experimentally-induced LSCD, and in cadaveric normal human corneas (n=7) and human pterygia (n=15); a disease characterized by the invasion of a conjunctivalized, fibrovascular pannus. Clinical assessments including slit-lamp microscopy, fluorescein staining and impression cytology, and biochemical, molecular and immunological assays were also conducted. ResultsMass spectrometry of conditioned media from mouse limbal explant-derived cells revealed the presence of IGFBP-7. This factor was expressed in normal limbal and conjunctival epithelium and conjunctivalized corneas from mice with LSCD, and in human pterygium epithelium but not in normal mouse or human corneal epithelium. Four weeks after inducing LSCD, IGFBP-7 staining was increased by 2.9-fold in mouse corneas compared to steady-state, and by 1.6-fold in impression cytology specimens derived from the same mice. Notably, IGFBP-7 was detected approximately 2-weeks earlier than Muc5AC. ConclusionsThis study provides novel insights into the specificity of IGFBP-7 for the mammalian conjunctival epithelium in health and disease. A point-of-care test for IGFBP-7 could be developed to assist clinicians in early diagnosis, and in monitoring disease progression, severity and therapeutic outcomes in patients with LSCD.

Anti-Aspergillus Activities of the Respiratory Epithelium in Health and Disease.

Respiratory epithelia fulfil multiple roles beyond that of gaseous exchange, also acting as primary custodians of lung sterility and inflammatory homeostasis. Inhaled fungal spores pose a continual antigenic, and potentially pathogenic, challenge to lung integrity against which the human respiratory mucosa has developed various tolerance and defence strategies. However, respiratory disease and immune dysfunction frequently render the human lung susceptible to fungal diseases, the most common of which are the aspergilloses, a group of syndromes caused by inhaled spores of Aspergillus fumigatus. Inhaled Aspergillus spores enter into a multiplicity of interactions with respiratory epithelia, the mechanistic bases of which are only just becoming recognized as important drivers of disease, as well as possible therapeutic targets. In this mini-review we examine current understanding of Aspergillus-epithelial interactions and, based upon the very latest developments in the field, we explore two apparently opposing schools of thought which view epithelial uptake of Aspergillus spores as either a curative or disease-exacerbating event.

Endogenous and exogenous control of gastrointestinal epithelial function: building on the legacy of Bayliss and Starling.

Transport of fluid and electrolytes in the intestine allows for appropriate adjustments in luminal fluidity while reclaiming water used in digesting and absorbing a meal, and is closely regulated. This article discusses various endogenous and exogenous mechanisms whereby transport is controlled in the gut, placing these in the context of the ideas about the neurohumoral control of alimentary physiology that were promulgated by William Bayliss and Ernest Starling. The article considers three themes. First, mechanisms that intrinsically regulate chloride secretion, centred on the epidermal growth factor receptor (EGFr), are discussed. These may be important in ensuring that excessive chloride secretion, with the accompanying loss of fluid, is not normally stimulated by intestinal distension as the meal passes through the gastrointestinal tract. Second, mechanisms whereby probiotic microorganisms can impart beneficial effects on the gut are described, with a focus on targets at the level of the epithelium. These findings imply that the commensal microbiota exert important influences on the epithelium in health and disease. Finally, mechanisms that lead to diarrhoea in patients infected with an invasive pathogen, Salmonella, are considered, based on recent studies in a novel mouse model. Diarrhoea is most likely attributable to reduced expression of absorptive transporters and may not require the influx of neutrophils that accompanies infection. Overall, the goal of the article is to highlight the many ways in which critical functions of the intestinal epithelium are regulated under physiological and pathophysiological conditions, and to suggest possible targets for new therapies for digestive disease states.

Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model.

SummaryHuman embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

Cell death at the intestinal epithelial front line.

The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family.

Bitter and sweet taste receptors in the respiratory epithelium in health and disease.

Taste receptors on the tongue communicate information to the brain about the nutrient content or potential toxicity of ingested foods. However, recent research has now shown that taste receptors are also expressed far beyond the tongue, from the airway and gastrointestinal epithelia to the pancreas and brain. The functions of many of these so-called extraoral taste receptors remain unknown, but emerging basic science and clinical evidence suggests that bitter and sweet taste receptors in the airway are important in sensing bacteria and regulating innate immunity. This review focuses on the role of bitter and sweet taste receptors in human airway innate immunity and the potential clinical relevance to airway infections. The T2R38 bitter taste receptor in sinonasal cilia detects bitter bacterial quorum-sensing molecules and activates nitric oxide-dependent innate immune responses. Polymorphisms that underlie T2R38 functionality also appear to be involved in susceptibility to upper respiratory infection and chronic rhinosinusitis (CRS). Bitter and sweet receptors in specialized sinonasal solitary chemosensory cells control antimicrobial peptide secretion, which may have important implications for airway infections in CRS patients as well as patients with diabetes mellitus. Future research on taste receptors in the airway has tremendous potential to identify immune mechanisms involved in host-pathogen interactions and thus reveal novel therapeutic targets.

Regenerative inflammation: lessons from Drosophila intestinal epithelium in health and disease.

Intestinal inflammation is widely recognized as a pivotal player in health and disease. Defined cytologically as the infiltration of leukocytes in the lamina propria layer of the intestine, it can damage the epithelium and, on a chronic basis, induce inflammatory bowel disease and potentially cancer. The current view thus dictates that blood cell infiltration is the instigator of intestinal inflammation and tumor-promoting inflammation. This is based partially on work in humans and mice showing that intestinal damage during microbially mediated inflammation activates phagocytic cells and lymphocytes that secrete inflammatory signals promoting tissue damage and tumorigenesis. Nevertheless, extensive parallel work in the Drosophila midgut shows that intestinal epithelium damage induces inflammatory signals and growth factors acting mainly in a paracrine manner to induce intestinal stem cell proliferation and tumor formation when genetically predisposed. This is accomplished without any apparent need to involve Drosophila hemocytes. Therefore, recent work on Drosophila host defense to infection by expanding its main focus on systemic immunity signaling pathways to include the study of organ homeostasis in health and disease shapes a new notion that epithelially emanating cytokines and growth factors can directly act on the intestinal stem cell niche to promote “regenerative inflammation” and potentially cancer.

The Laryngeal Epithelium in Reflux

The epithelium plays a critical role in the maintenance of laryngeal health. This is evident in that laryngeal disease may result when the integrity of the epithelium is compromised by insults such as laryngopharyngeal reflux. In this article, we will review the structure and function of the laryngeal epithelium and summarize the impact of laryngopharyngeal reflux on the epithelium. Research investigating the ramifications of reflux on the epithelium has improved our understanding of laryngeal disease associated with laryngopharyngeal reflux. It further highlights the need for continued research on the laryngeal epithelium in health and disease.

The Retinal Pigment Epithelium in Health and Disease

Retinal pigment epithelial cells (RPE) constitute a simple layer of cuboidal cells that are strategically situated behind the photoreceptor (PR) cells. The inconspicuousness of this monolayer contrasts sharply with its importance [1]. The relationship between the RPE and PR cells is crucial to sight; this is evident from basic and clinical studies demonstrating that primary dysfunctioning of the RPE can result in visual cell death and blindness. RPE cells carry out many functions including the conversion and storage of retinoid, the phagocytosis of shed PR outer segment membrane, the absorption of scattered light, ion and fluid transport and RPE-PR apposition. The magnitude of the demands imposed on this single layer of cells in order to execute these tasks, will become apparent to the reader of this review as will the number of clinical disorders that take origin from these cells.

The airway epithelium in health and disease: “calm on the surface, paddling furiously underneath”

The airway epithelium, which is exposed to antigens and microbes throughout respiration, provides an enormous and delicate interface with the external environment. In major conducting airways the epithelium is covered...