Tissue barrier function is directly mediated by tight junction transmembrane proteins known as claudins. Cells that form tight junctions typically express multiple claudin isoforms which suggests that heterotypic (head-to-head) binding between different claudin isoforms may play a role in regulating paracellular permeability. However, little is known about motifs that control heterotypic claudin compatibility. We found that although claudin-3 and claudin-4 were heteromerically compatible when expressed in the same cell, they did not heterotypically interact despite having extracellular loop (EL) domains that are highly conserved at the amino acid level. Claudin-1 and -5, which were heterotypically compatible with claudin-3, did not heterotypically bind to claudin-4. In contrast, claudin-4 chimeras containing either the first EL domain or the second EL domain of claudin-3 were able to heterotypically bind to claudin-1, claudin-3, and claudin-5. Moreover, a single point mutation in the first extracellular loop domain of claudin-3 to convert Asn 44 to the corresponding amino acid in claudin-4 (Thr) produced a claudin capable of heterotypic binding to claudin-4 while still retaining the ability to bind to claudin-1 and -5. Thus, control of heterotypic claudin-claudin interactions is sensitive to small changes in the EL domains.
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.
The lung is dynamically remodeled in response to injury, which alters extracellular matrix composition, and can lead to either healthy or impaired lung regeneration. To determine how changes in extracellular matrix can influence alveolar epithelial barrier function, we examined the expression and function of tight junction proteins by rat alveolar epithelial type II cells cultured on one of three different matrix components: type I collagen or fibronectin, matrix glycoproteins which are highly expressed in injured lungs, or laminin, a basement membrane matrix component. Of note, alveolar epithelial cells cultured for 2 days on fibronectin formed high-resistance barriers and showed continuous claudin-3 and claudin-18 localization to the plasma membrane, as opposed to cells cultured on either type I collagen or laminin, which had low resistance monolayers and had areas of cell-cell contact that were claudin deficient. The barrier formed by cells cultured on fibronectin also had preferential permeability to chloride as compared with sodium. Regardless of the initial matrix composition, alveolar epithelial cells cultured for 5 days formed high-resistance barriers, which correlated with increased claudin-18 localization to the plasma membrane and an increase in zonula occludens-1. Day 5 cells on laminin had significantly higher resistance than cells on either fibronectin or type I collagen. Thus, although alveolar epithelial cells on fibronectin formed rapid barriers, it was at the expense of producing an optimized barrier.
BackgroundPatients 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.MethodsMice 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.ResultsSeptic 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-β 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.ConclusionsWhen 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.
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