Correction: Chronic Alcohol Ingestion Increases Mortality and Organ Injury in a Murine Model of Septic Peritonitis.

[This corrects the article DOI: 10.1371/journal.pone.0062792.].

Claudin-based barrier differentiation in the colonic epithelial crypt niche involves Hopx/Klf4 and Tcf7l2/Hnf4-α cascades.

Colonic enterocytes form a rapidly renewing epithelium and barrier to luminal antigens. During renewal, coordinated expression of the claudin family of genes is vital to maintain the epithelial barrier. Disruption of this process contributes to barrier compromise and mucosal inflammatory diseases. However, little is known about the regulation of this critical aspect of epithelial cell differentiation. In order to identify claudin regulatory factors we utilized high-throughput gene microarrays and correlation analyses. We identified complex expression gradients for the transcription factors Hopx, Hnf4a, Klf4 and Tcf7l2, as well as 12 claudins, during differentiation. In vitro confirmatory methods identified 2 pathways that stimulate claudin expression; Hopx/Klf4 activation of Cldn4, 7 and 15, and Tcf7l2/Hnf4a up-regulation of Cldn23. Chromatin immunoprecipitation confirmed a Tcf7l2/Hnf4a/Claudin23 cascade. Furthermore, Hnf4a conditional knockout mice fail to induce Cldn23 during colonocyte differentiation. In conclusion, we report a comprehensive screen of colonic claudin gene expression and discover spatiotemporal Hopx/Klf4 and Tcf7l2/Hnf4a signaling as stimulators of colonic epithelial barrier differentiation.

Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions.

Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability.

The relative balance of GM-CSF and TGF-ß1 regulates lung epithelial barrier function.

Lung barrier dysfunction is a cardinal feature of the acute respiratory distress syndrome (ARDS). Alcohol abuse, which increases the risk of ARDS two- to fourfold, induces transforming growth factor (TGF)-ß1, which increases epithelial permeability and impairs granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent barrier integrity in experimental models. We hypothesized that the relative balance of GM-CSF and TGF-ß1 signaling regulates lung epithelial barrier function. GM-CSF and TGF-ß1 were tested separately and simultaneously for their effects on lung epithelial cell barrier function in vitro. TGF-ß1 alone caused an ∼ 25% decrease in transepithelial resistance (TER), increased paracellular flux, and was associated with projections perpendicular to tight junctions ("spikes") containing claudin-18 that colocalized with F-actin. In contrast, GM-CSF treatment induced an ∼ 20% increase in TER, decreased paracellular flux, and showed decreased colocalization of spike-associated claudin-18 with F-actin. When simultaneously administered to lung epithelial cells, GM-CSF antagonized the effects of TGF-ß1 on epithelial barrier function in cultured cells. Given this, GM-CSF and TGF-ß1 levels were measured in bronchoalveolar lavage (BAL) fluid from patients with ventilator-associated pneumonia and correlated with markers for pulmonary edema and patient outcome. In patient BAL fluid, protein markers of lung barrier dysfunction, serum α2-macroglobulin, and IgM levels were increased at lower ratios of GM-CSF/TGF-ß1. Critically, patients who survived had significantly higher GM-CSF/TGF-ß1 ratios than nonsurviving patients. This study provides experimental and clinical evidence that the relative balance between GM-CSF and TGF-ß1 signaling is a key regulator of lung epithelial barrier function. The GM-CSF/TGF-ß1 ratio in BAL fluid may provide a concentration-independent biomarker that can predict patient outcomes in ARDS.

Chronic alcohol ingestion increases mortality and organ injury in a murine model of septic peritonitis.

BACKGROUND: Patients admitted to the intensive care unit with alcohol use disorders have increased morbidity and mortality. The purpose of this study was to determine how chronic alcohol ingestion alters the host response to sepsis in mice. METHODS: Mice were randomized to receive either alcohol or water for 12 weeks and then subjected to cecal ligation and puncture. Mice were sacrificed 24 hours post-operatively or followed seven days for survival. RESULTS: Septic alcohol-fed mice had a significantly higher mortality than septic water-fed mice (74% vs. 41%, p = 0.01). This was associated with worsened gut integrity in alcohol-fed mice with elevated intestinal epithelial apoptosis, decreased crypt proliferation and shortened villus length. Further, alcohol-fed mice had higher intestinal permeability with decreased ZO-1 and occludin protein expression in the intestinal tight junction. The frequency of splenic and bone marrow CD4+ T cells was similar between groups; however, splenic CD4+ T cells in septic alcohol-fed mice had a marked increase in both TNF and IFN-γ production following ex vivo stimulation. Neither the frequency nor function of CD8+ T cells differed between alcohol-fed and water-fed septic mice. NK cells were decreased in both the spleen and bone marrow of alcohol-fed septic mice. Pulmonary myeloperoxidase levels and BAL levels of G-CSF and TFG-ß were higher in alcohol-fed mice. Pancreatic metabolomics demonstrated increased acetate, adenosine, xanthine, acetoacetate, 3-hydroxybutyrate and betaine in alcohol-fed mice and decreased cytidine, uracil, fumarate, creatine phosphate, creatine, and choline. Serum and peritoneal cytokines were generally similar between alcohol-fed and water-fed mice, and there were no differences in bacteremia, lung wet to dry weight, or pulmonary, liver or splenic histology. CONCLUSIONS: When subjected to the same septic insult, mice with chronic alcohol ingestion have increased mortality. Alterations in intestinal integrity, the host immune response, and pancreatic metabolomics may help explain this differential response.

Roles for claudins in alveolar epithelial barrier function.

Terminal airspaces of the lung, alveoli, are sites of gas exchange that are sensitive to disrupted fluid balance. The alveolar epithelium is a heterogeneous monolayer of cells interconnected by tight junctions at sites of cell-cell contact. Paracellular permeability depends on claudin (cldn)-family tight junction proteins. Of over a dozen alveolar cldns, cldn-3, cldn-4, and cldn-18 are the most highly expressed; other prominent alveolar claudins include cldn-5 and cldn-7. Cldn-3 is primarily expressed by type II alveolar epithelial cells, whereas cldn-4 and cldn-18 are expressed throughout the alveolar epithelium. Lung diseases associated with pulmonary edema, such as alcoholic lung syndrome and acute lung injury, affect alveolar claudin expression, which is frequently associated with impaired fluid clearance due to increased alveolar leak. However, recent studies have identified a role for increased cldn-4 in protecting alveolar barrier function following injury. Thus, alveolar claudins are dynamically regulated, tailoring lung barrier function to control the air-liquid interface.

Differential effects of claudin-3 and claudin-4 on alveolar epithelial barrier function.

Alveolar barrier function depends critically on the claudin family tight junction proteins. Of the major claudins expressed by alveolar epithelial cells, claudin (Cldn)-3 and Cldn-4 are the most closely related by amino acid homology, yet they differ dramatically in the pattern of expression. Previously published reports have shown that Cldn-3 is predominantly expressed by type II alveolar epithelial cells; Cldn-4 is expressed throughout the alveolar epithelium and is specifically upregulated in response to acute lung injury. Using primary rat alveolar epithelial cells transduced with yellow fluorescent protein-tagged claudin constructs, we have identified roles for Cldn-3 and Cldn-4 in alveolar epithelial barrier function. Surprisingly, increasing expression of Cldn-3 decreased alveolar epithelial barrier function, as assessed by transepithelial resistance and dye flux measurements. Conversely, increasing Cldn-4 expression improved alveolar epithelial transepithelial resistance compared with control cells. Other alveolar epithelial tight junction proteins were largely unaffected by increased expression of Cldn-3 and Cldn-4. Taken together, these results demonstrate that, in the context of the alveolar epithelium, Cldn-3 and Cldn-4 have different effects on paracellular permeability, despite significant homology in their extracellular loop domains.

Claudins: control of barrier function and regulation in response to oxidant stress.

Claudins are a family of nearly two dozen transmembrane proteins that are a key part of the tight junction barrier that regulates solute movement across polarized epithelia. Claudin family members interact with each other, as well as with other transmembrane tight junction proteins (such as occludin) and cytosolic scaffolding proteins (such as zonula occludens-1 (ZO-1)). Although the interplay between all of these different classes of proteins is critical for tight junction formation and function, claudin family proteins are directly responsible for forming the equivalent of paracellular ion selective channels (or pores) with specific permeability and thus are essential for barrier function. In this review, we summarize current progress in identifying structural elements of claudins that regulate their transport, assembly, and function. The effects of oxidant stress on claudins are also examined, with particular emphasis on lung epithelial barrier function and oxidant stress induced by chronic alcohol abuse.

Deciliation is associated with dramatic remodeling of epithelial cell junctions and surface domains.

Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and likely represents a conserved cellular reaction. However, little is known about whether primary cilia are shed from mammalian epithelial cells and what impact deciliation has on polarized cellular organization. We show that several chemically distinct agents trigger autotomy in epithelial cells. Surprisingly, deciliation is associated with a significant, but reversible increase in transepithelial resistance. This reflects substantial reductions in tight junction proteins associated with "leaky" nephron segments (e.g., claudin-2). At the same time, apical trafficking of gp80/clusterin and gp114/CEACAM becomes randomized, basal-lateral delivery of Na,K-ATPase is reduced, and expression of the nonciliary apical protein gp135/podocalyxin is greatly decreased. However, ciliogenesis-impaired MDCK cells do not undergo continual junction remodeling, and mature cilia are not required for autotomy-associated remodeling events. Deciliation and epithelial remodeling may be mechanistically linked processes, because RNAi-mediated reduction of Exocyst subunit Sec6 inhibits ciliary shedding and specifically blocks deciliation-associated down-regulation of claudin-2 and gp135. We propose that ciliary autotomy represents a signaling pathway that impacts the organization and function of polarized epithelial cells.