Hyperoxia induces alveolar epithelial-to-mesenchymal cell transition.

Myofibroblast accumulation is a pathological feature of lung diseases requiring oxygen therapy. One possible source for myofibroblasts is through the epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AEC). To study the effects of oxygen on alveolar EMT, we used RLE-6TN and ex vivo lung slices and found that hyperoxia (85% O2, H85) decreased epithelial proteins, presurfactant protein B (pre-SpB), pro-SpC, and lamellar protein by 50% and increased myofibroblast proteins, α-smooth muscle actin (α-SMA), and vimentin by over 200% (P < 0.05). In AEC freshly isolated from H85-treated rats, mRNA for pre-SpB and pro-SpC was diminished by ∼50% and α-SMA was increased by 100% (P < 0.05). Additionally, H85 increased H2O2 content, and H2O2 (25-50 µM) activated endogenous transforming growth factor-ß1 (TGF-ß1), as evident by H2DCFDA immunofluorescence and ELISA (P < 0.05). Both hyperoxia and H2O2 increased SMAD3 phosphorylation (260% of control, P < 0.05). Treating cultured cells with TGF-ß1 inhibitors did not prevent H85-induced H2O2 production but did prevent H85-mediated α-SMA increases and E-cadherin downregulation. Finally, to determine the role of TGF-ß1 in hyperoxia-induced EMT in vivo, we evaluated AEC from H85-treated rats and found that vimentin increased ∼10-fold (P < 0.05) and that this effect was prevented by intraperitoneal TGF-ß1 inhibitor SB-431542. Additionally, SB-431542 treatment attenuated changes in alveolar histology caused by hyperoxia. Our studies indicate that hyperoxia promotes alveolar EMT through a mechanism that is dependent on activation of TGF-ß1 signaling.

Enterobacterial common antigen mutants of Salmonella enterica serovar Typhimurium establish a persistent infection and provide protection against subsequent lethal challenge.

Infection with Salmonella spp. is a significant source of disease globally. A substantial proportion of these infections are caused by Salmonella enterica serovar Typhimurium. Here, we characterize the role of the enterobacterial common antigen (ECA), a surface glycolipid ubiquitous among enteric bacteria, in S. Typhimurium pathogenesis. Construction of a defined mutation in the UDP-N-acetylglucosamine-1-phosphate transferase gene, wecA, in two clinically relevant strains of S. Typhimurium, TML and SL1344, resulted in strains that were unable to produce ECA. Loss of ECA did not affect the gross cell surface ultrastructure, production of lipopolysaccharide (LPS), flagella, or motility. However, the wecA mutant strains were attenuated in both oral and intraperitoneal mouse models of infection (P<0.001 for both routes of infection; log rank test), and virulence could be restored by complementation of the wecA gene in trans. Despite the avirulence of the ECA-deficient strains, the wecA mutant strains were able to persistently colonize systemic sites (spleen and liver) at moderate levels for up to 70 days postinfection. Moreover, immunization with the wecA mutant strains provided protection against a subsequent lethal oral or intraperitoneal challenge with wild-type S. Typhimurium. Thus, wecA mutant (ECA-negative) strains of Salmonella may be useful as live attenuated vaccine strains or as vehicles for heterologous antigen expression.

Hyperoxia induces paracellular leak and alters claudin expression by neonatal alveolar epithelial cells.

BACKGROUND: Premature neonates frequently require oxygen supplementation as a therapeutic intervention that, while necessary, also exposes the lung to significant oxidant stress. We hypothesized that hyperoxia has a deleterious effect on alveolar epithelial barrier function rendering the neonatal lung susceptible to injury and/or bronchopulmonary dysplasia (BPD). MATERIALS AND METHODS: We examined the effects of exposure to 85% oxygen on neonatal rat alveolar barrier function in vitro and in vivo. Whole lung was measured using wet-to-dry weight ratios and bronchoalveolar lavage protein content and cultured primary neonatal alveolar epithelial cells (AECs) were measured using transepithelial electrical resistance (TEER) and paracellular flux measurements. Expression of claudin-family tight junction proteins, E-cadherin and the Snail transcription factor SNAI1 were measured by Q-PCR, immunoblot and confocal immunofluorescence microscopy. RESULTS: Cultured neonatal AECs exposed to 85% oxygen showed impaired barrier function. This oxygen-induced increase in paracellular leak was associated with altered claudin expression, where claudin-3 and -18 were downregulated at both the mRNA and protein level. Claudin-4 and -5 mRNA were also decreased, although protein expression of these claudins was largely maintained. Lung alveolarization and barrier function in vivo were impaired in response to hyperoxia. Oxygen exposure also significantly decreased E-cadherin expression and induced expression of the SNAI1 transcription factor in vivo and in vitro. CONCLUSIONS: These data support a model in which hyperoxia has a direct impact on alveolar tight and adherens junctions to impair barrier function. Strategies to antagonize the effects of high oxygen on alveolar junctions may potentially reverse this deleterious effect.