A cafeteria diet triggers intestinal inflammation and oxidative stress in obese rats.

The gastrointestinal alterations associated with the consumption of an obesogenic diet, such as inflammation, permeability impairment and oxidative stress, have been poorly explored in both diet-induced obesity (DIO) and genetic obesity. The aim of the present study was to examine the impact of an obesogenic diet on the gut health status of DIO rats in comparison with the Zucker (fa/fa) rat leptin receptor-deficient model of genetic obesity over time. For this purpose, female Wistar rats (n 48) were administered a standard or a cafeteria diet (CAF diet) for 12, 14·5 or 17 weeks and were compared with fa/fa Zucker rats fed a standard diet for 10 weeks. Morphometric variables, plasma biochemical parameters, myeloperoxidase (MPO) activity and reactive oxygen species (ROS) levels in the ileum were assessed, as well as the expressions of proinflammatory genes (TNF-α and inducible nitric oxide synthase (iNOS)) and intestinal permeability genes (zonula occludens-1, claudin-1 and occludin). Both the nutritional model and the genetic obesity model showed increased body weight and metabolic alterations at the final time point. An increase in intestinal ROS production and MPO activity was observed in the gastrointestinal tracts of rats fed a CAF diet but not in the genetic obesity model. TNF-α was overexpressed in the ileum of both CAF diet and fa/fa groups, and ileal inflammation was associated with the degree of obesity and metabolic alterations. Interestingly, the 17-week CAF group and the fa/fa rats exhibited alterations in the expressions of permeability genes. Relevantly, in the hyperlipidic refined sugar diet model of obesity, the responses to chronic energy overload led to time-dependent increases in gut inflammation and oxidative stress.

Supplemental psyllium fibre regulates the intestinal barrier and inflammation in normal and colitic mice.

Our previous study demonstrated that supplemental psyllium fibre increased cytoprotective heat-shock protein (Hsp) 25 levels in the intestinal cells of mice. Here, we examined the effect of psyllium fibre on colonic gene and protein expression and faecal microbiota in normal and colitic mice to improve the understanding of the preventive role of the supplement. DNA microarray analysis revealed that a 10 % psyllium fibre diet administered for 5 d up-regulated eleven extracellular matrix (ECM)-associated genes, including collagens and fibronectins, in normal mice. Acute colitis was induced using dextran sodium sulphate (DSS) in mice that were administered a pre-feeding 5 to 10 % psyllium fibre diet for 5 d. Psyllium fibre partially ameliorated or resolved the DSS-induced colon damage and inflammation characterised by body weight loss, colon shortening, increased levels of pro-inflammatory cytokines and decreased tight junction protein expression in the colon. Analysis of faecal microbiota using denaturing gradient gel electrophoresis of the PCR-amplified 16S rRNA gene demonstrated that psyllium fibre affected the colonic microbiota. Intestinal permeability was evaluated by growing intestinal Caco-2 cell monolayers on membrane filter supports coated with or without fibronectin and collagen. Cells grown on collagen and fibronectin coating showed higher transepithelial electrical resistance, indicating a strengthening of barrier integrity. Therefore, increased Hsp25 levels and modification of colonic ECM contribute to the observed psyllium-mediated protection against DSS-induced colitis. Furthermore, ECM modification appears to play a role in the strengthening of the colon barrier. In conclusion, psyllium fibre may be useful in the prevention of intestinal inflammatory diseases.

The neurogliovascular unit in hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neurological complication of hepatic dysfunction and portosystemic shunting. It is highly prevalent in patients with cirrhosis and is associated with poor outcomes. New insights into the role of peripheral origins in HE have led to the development of innovative treatment strategies like faecal microbiota transplantation. However, this broadening of view has not been applied fully to perturbations in the central nervous system. The old paradigm that HE is the clinical manifestation of ammonia-induced astrocyte dysfunction and its secondary neuronal consequences requires updating. In this review, we will use the holistic concept of the neurogliovascular unit to describe central nervous system disturbances in HE, an approach that has proven instrumental in other neurological disorders. We will describe HE as a global dysfunction of the neurogliovascular unit, where blood flow and nutrient supply to the brain, as well as the function of the blood-brain barrier, are impaired. This leads to an accumulation of neurotoxic substances, chief among them ammonia and inflammatory mediators, causing dysfunction of astrocytes and microglia. Finally, glymphatic dysfunction impairs the clearance of these neurotoxins, further aggravating their effect on the brain. Taking a broader view of central nervous system alterations in liver disease could serve as the basis for further research into the specific brain pathophysiology of HE, as well as the development of therapeutic strategies specifically aimed at counteracting the often irreversible central nervous system damage seen in these patients.

Hypoxia Inducible Factor (HIF) Hydroxylases as Regulators of Intestinal Epithelial Barrier Function.

Human health is dependent on the ability of the body to extract nutrients, fluids, and oxygen from the external environment while at the same time maintaining a state of internal sterility. Therefore, the cell layers that cover the surface areas of the body such as the lung, skin, and gastrointestinal mucosa provide vital semipermeable barriers that allow the transport of essential nutrients, fluid, and waste products, while at the same time keeping the internal compartments free of microbial organisms. These epithelial surfaces are highly specialized and differ in their anatomic structure depending on their location to provide appropriate and effective site-specific barrier function. Given this important role, it is not surprising that significant disease often is associated with alterations in epithelial barrier function. Examples of such diseases include inflammatory bowel disease, chronic obstructive pulmonary disease, and atopic dermatitis. These chronic inflammatory disorders often are characterized by diminished tissue oxygen levels (hypoxia). Hypoxia triggers an adaptive transcriptional response governed by hypoxia-inducible factors (HIFs), which are repressed by a family of oxygen-sensing HIF hydroxylases. Here, we review recent evidence suggesting that pharmacologic hydroxylase inhibition may be of therapeutic benefit in inflammatory bowel disease through the promotion of intestinal epithelial barrier function through both HIF-dependent and HIF-independent mechanisms.

Celiac Disease: Role of the Epithelial Barrier.

In celiac disease (CD) a T-cell-mediated response to gluten is mounted in genetically predisposed individuals, resulting in a malabsorptive enteropathy histologically highlighted by villous atrophy and crypt hyperplasia. Recent data point to the epithelial layer as an under-rated hot spot in celiac pathophysiology to date. This overview summarizes current functional and genetic evidence on the role of the epithelial barrier in CD, consisting of the cell membranes and the apical junctional complex comprising sealing as well as ion and water channel-forming tight junction proteins and the adherens junction. Moreover, the underlying mechanisms are discussed, including apoptosis of intestinal epithelial cells, biology of intestinal stem cells, alterations in the apical junctional complex, transcytotic uptake of gluten peptides, and possible implications of a defective epithelial polarity. Current research is directed toward new treatment options for CD that are alternatives or complementary therapeutics to a gluten-free diet. Thus, strategies to target an altered epithelial barrier therapeutically also are discussed.

Mechanisms of Intestinal Epithelial Barrier Dysfunction by Adherent-Invasive Escherichia coli.

Pathobiont expansion, such as that of adherent-invasive Escherichia coli (AIEC), is an emerging factor associated with inflammatory bowel disease. The intestinal epithelial barrier is the first line of defense against these pathogens. Inflammation plays a critical role in altering the epithelial barrier and is a major factor involved in promoting the expansion and pathogenesis of AIEC. AIEC in turn can exacerbate intestinal epithelial barrier dysfunction by targeting multiple elements of the barrier. One critical element of the epithelial barrier is the tight junction. Increasing evidence suggests that AIEC may selectively target protein components of tight junctions, leading to increased barrier permeability. This may represent one mechanism by which AIEC could contribute to the development of inflammatory bowel disease. This review article discusses potential mechanisms by which AIEC can disrupt epithelial tight junction function and intestinal barrier function.

Causality of small and large intestinal microbiota in weight regulation and insulin resistance.

OBJECTIVE: The twin pandemics of obesity and Type 2 diabetes (T2D) are a global challenge for health care systems. Changes in the environment, behavior, diet, and lifestyle during the last decades are considered the major causes. A Western diet, which is rich in saturated fat and simple sugars, may lead to changes in gut microbial composition and physiology, which have recently been linked to the development of metabolic diseases. METHODS: We will discuss evidence that demonstrates the influence of the small and large intestinal microbiota on weight regulation and the development of insulin resistance, based on literature search. RESULTS: Altered large intestinal microbial composition may promote obesity by increasing energy harvest through specialized gut microbes. In both large and small intestine, microbial alterations may increase gut permeability that facilitates the translocation of whole bacteria or endotoxic bacterial components into metabolic active tissues. Moreover, changed microbial communities may affect the production of satiety-inducing signals. Finally, bacterial metabolic products, such as short chain fatty acids (SCFAs) and their relative ratios, may be causal in disturbed immune and metabolic signaling, notably in the small intestine where the surface is large. The function of these organs (adipose tissue, brain, liver, muscle, pancreas) may be disturbed by the induction of low-grade inflammation, contributing to insulin resistance. CONCLUSIONS: Interventions aimed to restoring gut microbial homeostasis, such as ingestion of specific fibers or therapeutic microbes, are promising strategies to reduce insulin resistance and the related metabolic abnormalities in obesity, metabolic syndrome, and type 2 diabetes. This article is part of a special issue on microbiota.

NF-κB inhibitors impair lung epithelial tight junctions in the absence of inflammation.

NF-κB (p50/p65) is the best characterized transcription factor known to regulate cell responses to inflammation. However, NF-κB is also constitutively expressed. We used inhibitors of the classical NF-κB signaling pathway to determine whether this transcription factor has a role in regulating alveolar epithelial tight junctions. Primary rat type II alveolar epithelial cells were isolated and cultured on Transwell permeable supports coated with collagen for 5 d to generate a model type I cell monolayer. Treatment of alveolar epithelial monolayers overnight with one of 2 different IκB kinase inhibitors (BAY 11-7082 or BMS-345541) resulted in a dose-dependent decrease in TER at concentrations that did not affect cell viability. In response to BMS-345541 treatment there was an increase in total claudin-4 and claudin-5 along with a decrease in claudin-18, as determined by immunoblot. However, there was little effect on the total amount of cell-associated claudin-7, occludin, junctional adhesion molecule A (JAM-A), zonula occludens (ZO)-1 or ZO-2. Moreover, treatment with BMS-345541 resulted in altered tight junction morphology as assessed by immunofluorescence microscopy. Cells treated with BMS-345541 had an increase in claudin-18 containing projections emanating from tight junctions ("spikes") that were less prominent in control cells. There also were several areas of cell-cell contact which lacked ZO-1 and ZO-2 localization as well as rearrangements to the actin cytoskeleton in response to BMS-345541. Consistent with an anti-inflammatory effect, BMS-345541 antagonized the deleterious effects of lipopolysaccharide (LPS) on alveolar epithelial barrier function. However, BMS-345541 also inhibited the ability of GM-CSF to increase alveolar epithelial TER. These data suggest a dual role for NF-κB in regulating alveolar barrier function and that constitutive NF-κB function is required for the integrity of alveolar epithelial tight junctions.

Bacterial protein AvrA stabilizes intestinal epithelial tight junctions via blockage of the C-Jun N-terminal kinase pathway.

The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junctions (TJ) and epithelial functions in intestinal infection and inflammation. In a previous study, we have demonstrated that a bacterial effector AvrA plays a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. However, the molecular mechanisms by which AvrA-modulates TJ protein expression remain unknown. AvrA possesses acetyltransferase activity toward specific mitogen-activated protein kinase kinases (MAPKKs) and potently inhibits the c-Jun N-terminal kinase (JNK) pathway in inflammation. Inhibition of the JNK pathway is known to inhibit the TJ protein disassemble. Therefore, we hypothesize that AvrA stabilizes intestinal epithelial TJs via c-Jun and JNK pathway blockage. Using both in vitro and in vivo models, we showed that AvrA targets the c-Jun and JNK pathway that in turn stabilizes TJ protein ZO-1. Inhibition of JNK abolished the effect of AvrA on ZO-1. We further determined that AvrA suppressed the transcription factor activator protein-1, which was regulated by activated JNK. Moreover, we identified the functional domain of AvrA that directly regulated TJs using a series of AvrA mutants. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampens the inflammatory response of the host. Our findings have uncovered a novel role of the bacterial protein AvrA in suppressing the inflammatory response of the host through JNK-regulated blockage of epithelial cell barrier function.

Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation.

The epithelial tight junction determines the paracellular water and ion movement in the intestine and also prevents uptake of larger molecules, including antigens, in an uncontrolled manner. Claudin-2, one of the 27 mammalian claudins regulating that barrier function, forms a paracellular channel for small cations and water. It is typically expressed in leaky epithelia like proximal nephron and small intestine and provides a major pathway for the paracellular transport of sodium, potassium, and fluid. In intestinal inflammation (Crohn's disease, ulcerative colitis), immune-mediated diseases (celiac disease), and infections (HIV enteropathy), claudin-2 is upregulated in small and large intestine and contributes to diarrhea via a leak flux mechanism. In parallel to that upregulation, other epithelial and tight junctional features are altered and the luminal uptake of antigenic macromolecules is enhanced, for which claudin-2 may be partially responsible through induction of tight junction strand discontinuities.

Blood cells and endothelial barrier function.

The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction.

Arp2/3 complex function in the epidermis.

An exciting frontier in biology is understanding the functions of basic cell biological machinery in complex tissues. This approach is expected to uncover novel modes of regulation as well as reveal how core machinery is repurposed by different tissues to accomplish different physiological outputs. F-actin plays roles in cell shape, adhesion, migration and signaling - diverse functions that require a specific organization established by a myriad of regulators. Here, we discuss the role of the actin nucleating Arp2/3 complex and the unexpected roles that it plays in a stratified epithelial tissue, the epidermis. While many expected phenotypes such as defects in architecture and cell adhesion were lacking, loss of the Arp2/3 complex activity resulted in epidermal barrier and differentiation defects. This teaches us that, while informative, cell culture approaches are limiting and that studies of the Arp2/3 complex in diverse tissues are expected to yield many more surprises.

Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion.

Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCDcl1). Suppression of either ZO-1 or ZO-2 resulted in increased G0/G1 retention in mCCDcl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered ß1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDcl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.

The interdependence of the Rho GTPases and apicobasal cell polarity.

Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease.

Epithelial junctions and Rho family GTPases: the zonular signalosome.

The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of "zonular signalosome" is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors.