Lesions and neurologic disability in inflammatory CNS diseases such as multiple sclerosis (MS) result from the translocation of leukocytes and humoral factors from the vasculature, first across the endothelial blood-brain barrier (BBB) and then across the astrocytic glia limitans (GL). Factors secreted by reactive astrocytes open the BBB by disrupting endothelial tight junctions (TJs), but the mechanisms that control access across the GL are unknown. Here, we report that in inflammatory lesions, a second barrier composed of reactive astrocyte TJs of claudin 1 (CLDN1), CLDN4, and junctional adhesion molecule A (JAM-A) subunits is induced at the GL. In a human coculture model, CLDN4-deficient astrocytes were unable to control lymphocyte segregation. In models of CNS inflammation and MS, mice with astrocyte-specific Cldn4 deletion displayed exacerbated leukocyte and humoral infiltration, neuropathology, motor disability, and mortality. These findings identify a second inducible barrier to CNS entry at the GL. This barrier may be therapeutically targetable in inflammatory CNS disease.

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18^–/– AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18^–/– mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 20 and 100 nm, carboxylate (negatively charged) or amidine (positively charged)-modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy, we investigated PNP colocalization with early endosome antigen–1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of 22Na and 14C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20–40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2–3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen–1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type–specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells’ roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription–polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell–specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.

Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na+ gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type–specific mouse knockout lines in which the β1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na+ absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump β3 subunit expression and β2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump β1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell–specific roles in the lung.