Postnatal Ozone Exposure Disrupts Alveolar Development, Exaggerates Mucoinflammatory Responses, and Suppresses Bacterial Clearance in Developing Scnn1b-Tg+ Mice Lungs.

Increased levels of ambient ozone, one of the six criteria air pollutants, result in respiratory tract injury and worsening of ongoing lung diseases. However, the effect of ozone exposure on the respiratory tract undergoing active lung development and simultaneously experiencing mucoinflammatory lung diseases, such as cystic fibrosis, remains unclear. To address these questions, we exposed Scnn1b transgenic (Scnn1b-Tg+) mice, a mouse model of cystic fibrosis-like lung disease, and littermate wild-type (WT) mice to ozone from postnatal days (PND) 3-20 and examined the lung phenotypes at PND21. As compared with filtered air (FA)-exposed WT mice, the ozone-exposed WT mice exhibited marked alveolar space enlargement, in addition to significant eosinophilic infiltration, type 2 inflammation, and mucous cell metaplasia. Ozone-exposed Scnn1b-Tg+ mice also exhibited significantly increased alveolar space enlargement, which was also accompanied by exaggerated granulocytic infiltration, type 2 inflammation, and a greater degree of mucus obstruction. The alveolar space enlargement in ozone-exposed WT, FA-exposed Scnn1b-Tg+, and ozone-exposed Scnn1b-Tg+ mice was accompanied by elevated levels of MMP12 protein in macrophages and Mmp12 mRNA in the lung homogenates. Finally, although bacterial burden was largely resolved by PND21 in FA-exposed Scnn1b-Tg+ mice, ozone-exposed Scnn1b-Tg+ mice exhibited compromised bacterial clearance, which was also associated with increased levels of IL-10, an immunosuppressive cytokine, and marked mucus obstruction. Taken together, our data show that ozone exposure results in alveolar space remodeling during active phases of lung development and markedly exaggerates the mucoinflammatory outcomes of pediatric-onset lung disease, including bacterial infections, granulocytic inflammation, mucus obstruction, and alveolar space enlargement.

Dexmedetomidine alleviates pulmonary edema through the epithelial sodium channel (ENaC) via the PI3K/Akt/Nedd4-2 pathway in LPS-induced acute lung injury.

Dexmedetomidine (Dex), a highly selective α2-adrenergic receptor (α2AR) agonist, has an anti-inflammatory property and can alleviate pulmonary edema in lipopolysaccharide (LPS)-induced acute lung injury (ALI), but the mechanism is still unclear. In this study, we attempted to investigate the effect of Dex on alveolar epithelial sodium channel (ENaC) in the modulation of alveolar fluid clearance (AFC) and the underlying mechanism. Lipopolysaccharide (LPS) was used to induce acute lung injury (ALI) in rats and alveolar epithelial cell injury in A549 cells. In vivo, Dex markedly reduced pulmonary edema induced by LPS through promoting AFC, prevented LPS-induced downregulation of α-, ß-, and γ-ENaC expression, attenuated inflammatory cell infiltration in lung tissue, reduced the concentrations of TNF-α, IL-1ß, and IL-6, and increased concentrations of IL-10 in bronchoalveolar lavage fluid (BALF). In A549 cells stimulated with LPS, Dex attenuated LPS-mediated cell injury and the downregulation of α-, ß-, and γ-ENaC expression. However, all of these effects were blocked by the PI3K inhibitor LY294002, suggesting that the protective role of Dex is PI3K-dependent. Additionally, Dex increased the expression of phosphorylated Akt and reduced the expression of Nedd4-2, while LY294002 reversed the effect of Dex in vivo and in vitro. Furthermore, insulin-like growth factor (IGF)-1, a PI3K agonists, promoted the expression of phosphorylated Akt and reduced the expression of Nedd4-2 in LPS-stimulated A549 cells, indicating that Dex worked through PI3K, and Akt and Nedd4-2 are downstream of PI3K. In conclusion, Dex alleviates pulmonary edema by suppressing inflammatory response in LPS-induced ALI, and the mechanism is partly related to the upregulation of ENaC expression via the PI3K/Akt/Nedd4-2 signaling pathway.

Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease.

BACKGROUND: Neutrophils are key components of the exacerbated inflammation and tissue damage in cystic fibrosis (CF) airways. Neutrophil extracellular traps (NETs) trap and kill extracellular pathogens. While NETs are abundant in the airways of CF patients and have been hypothesized to contribute to lung damage in CF, the in vivo role of NETs remains controversial, partially due to lack of appropriate animal models. The goal of this study was to detect NETs and to further characterize neutrophil-mediated inflammation in the airways of mice overexpressing the epithelial sodium channel (ßENaC-Tg mice on C57BL/6 background) in their lung with CF-like airway disease, in the absence of any apparent bacterial infections. METHODS: Histology scoring of lung tissues, flow cytometry, multiplex ELISA, immunohistochemistry and immunofluorescence were used to characterize NETs and the airway environment in uninfected, ßENaC-Tg mice at 6 and 8 weeks of age, the most chronic time points so far studied in this model. RESULTS: Excessive neutrophilic infiltration characterized the lungs of uninfected, ßENaC-Tg mice at 6 and 8 weeks of age. The bronchoalveolar lavage fluid (BALF) of ßENaC-Tg mice contains increased levels of CF-associated cytokines and chemokines: KC, MIP-1α/ß, MCP-1, G-CSF, IL-5, and IL-6. The BALF of ßENaC-Tg mice contain MPO-DNA complexes, indicative of the presence of NETs. Immunofluorescence and flow cytometry of BALF neutrophils and lung tissues demonstrated increased histone citrullination, a NET-specific marker, in ßENaC-Tg mice. CONCLUSIONS: NETs are detected in the airways of ßENaC-Tg mice, in the absence of bacterial infections. These data demonstrate the usefulness of the ßENaC-Tg mouse to serve as a model for studying the role of NETs in chronic CF airway inflammation.

Scnn1b-Transgenic BALB/c Mice as a Model of Pseudomonas aeruginosa Infections of the Cystic Fibrosis Lung.

The opportunistic pathogen Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF), a condition that predisposes patients to chronic lung infections. P. aeruginosa lung infections are difficult to treat because P. aeruginosa adapts to the CF lung, can develop multidrug resistance, and can form biofilms. Despite the clinical significance of P. aeruginosa, modeling P. aeruginosa infections in CF has been challenging. Here, we characterize Scnn1b-transgenic (Tg) BALB/c mice as P. aeruginosa lung infection models. Scnn1b-Tg mice overexpress the epithelial Na+ channel (ENaC) in their lungs, driving increased sodium absorption that causes lung pathology similar to CF. We intranasally infected Scnn1b-Tg mice and wild-type littermates with the laboratory P. aeruginosa strain PAO1 and CF clinical isolates and then assessed differences in bacterial clearance, cytokine responses, and histological features up to 12 days postinfection. Scnn1b-Tg mice carried higher bacterial burdens when infected with biofilm-grown rather than planktonic PAO1; Scnn1b-Tg mice also cleared infections more slowly than their wild-type littermates. Infection with PAO1 elicited significant increases in proinflammatory and Th17-linked cytokines on day 3. Scnn1b-Tg mice infected with nonmucoid early CF isolates maintained bacterial burdens and mounted immune responses similar to those of PAO1-infected Scnn1b-Tg mice. In contrast, Scnn1b-Tg mice infected with a mucoid CF isolate carried high bacterial burdens, produced significantly more interleukin 1ß (IL-1ß), IL-13, IL-17, IL-22, and KC, and showed severe immune cell infiltration into the bronchioles. Taken together, these results show the promise of Scnn1b-Tg mice as models of early P. aeruginosa colonization in the CF lung.

Myeloid CD11c+ Antigen-Presenting Cells Ablation Prevents Hypertension in Response to Angiotensin II Plus High-Salt Diet.

Increasing evidence shows that antigen-presenting cells (APCs) are involved in the development of inflammation associated to hypertension. However, the potential role of APCs in the modulation of renal sodium transport has not been addressed. We hypothesized that APCs participate in renal sodium transport and, thus, development of high blood pressure in response to angiotensin II plus a high-salt diet. Using transgenic mice that allow the ablation of CD11chigh APCs, we studied renal sodium transport, the intrarenal renin-angiotensin system components, blood pressure, and cardiac/renal tissue damage in response to angiotensin II plus a high-salt diet. Strikingly, we found that APCs are required for the development of hypertension and that the ablation/restitution of APCs produces rapid changes in the blood pressure in mice with angiotensin II plus a high-salt diet. Moreover, APCs were necessary for the induction of intrarenal renin-angiotensin system components and affected the modulation of natriuresis and tubular sodium transporters. Consistent with the prevention of hypertension, the ablation of APCs also prevented cardiac hypertrophy and the induction of several indicators of renal and cardiac damage. Thus, our findings indicate a prominent role of APCs as modulators of blood pressure by mechanisms including renal sodium handling, with kinetics that suggest the involvement of tubular cell functions in addition to the modulation of inflammation and adaptive immune response.

Inflammatory cytokines regulate renal sodium transporters: how, where, and why?

Hypertension is growing in epidemic proportions worldwide and is now the leading preventable cause of premature death. For over a century, we have known that the kidney plays a critical role in blood pressure regulation. Specifically, abnormalities in renal sodium transport appear to be a final common pathway that gives rise to elevated blood pressure regardless of the nature of the initial hypertensive stimulus. However, it is only in the past decade that we have come to realize that inflammatory cytokines secreted by innate and adaptive immune cells, as well as renal epithelial cells, can modulate the expression and activity of sodium transporters all along the nephron, leading to alterations in pressure natriuresis, sodium and water balance, and ultimately hypertension. This mini-review highlights specific cytokines and the transporters that they regulate and discusses why inflammatory cytokines may have evolved to serve this function.

Modulation of ENaC, CFTR, and iNOS expression in bronchial epithelial cells after stimulation with Staphylococcus epidermidis (94B080) and Staphylococcus aureus (90B083).

Bacteria affect the respiratory epithelium, which is covered by airway surface liquid (ASL) and mucus. Ion concentrations in the ASL are determined by the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC). Neonatal sepsis is a major risk factor for subsequent pulmonary disease in preterm newborns. Predominating are coagulase-negative staphylococci (e.g., Staphylococccus epidermidis and Staphylococccus aureus). The aim of this study was to investigate modulation of CFTR, ENaC, mucins, proinflammatory cytokines, and inducible nitric oxide synthase (iNOS) in respiratory epithelial cells after S. epidermidis 94B080 and S. aureus 90B083 exposure. Bronchial epithelial cells were incubated with S. epidermidis 94B080 and S. aureus 90B083 (neonatal blood isolates) for 1-36 h. Expression of CFTR, ENaC, iNOS, and mucins was analyzed by real-time PCR and Western blotting. Release of cytokines was analyzed by ELISA, and production of NO by the Griess assay. Expression of CFTR significantly decreased after 36 h incubation with S. epidermidis and more prominently with S. aureus, whereas S. epidermidis caused a significant increase in the expression of ß- and γ-ENaC. Expression of iNOS increased, but NO was not detected. Both staphylococci caused a decrease in the intracellular Ca(2+) concentration. S. aureus induced increased secretion of IL-6, IL-8, and transforming nuclear factor (TNF)-α in a time-dependent manner as compared with S. epidermidis. In conclusion, expression of ENaC, CFTR, and iNOS is modulated by exposure to S. aureus 90B083 and S. epidermidis 94B080. S. aureus is more potent in causing release of IL-6, IL-8, and TNF-α by bronchial epithelial cells as compared with S. epidermidis. The mRNA expression for the mucus proteins MUC2, MUC5AC, and MUC5B could not be measured, neither in the presence nor in the absence of bacteria.

Glucocorticoid-induced Leucine zipper 1 stimulates the epithelial sodium channel by regulating serum- and glucocorticoid-induced kinase 1 stability and subcellular localization.

Serum- and glucocorticoid-induced kinase 1 (SGK1) is a multifunctional protein kinase that markedly influences various cellular processes such as proliferation, apoptosis, glucose metabolism, and sodium (Na(+)) transport via the epithelial Na(+) channel, ENaC. SGK1 is a short-lived protein, which is predominantly targeted to the endoplasmic reticulum (ER) to undergo rapid proteasome-mediated degradation through the ER-associated degradation (ERAD) system. We show here that the aldosterone-induced chaperone, GILZ1 (glucocorticoid-induced leucine zipper protein-1) selectively decreases SGK1 localization to ER as well as its interaction with ER-associated E3 ubiquitin ligases, HRD1 and CHIP. GILZ1 inhibits SGK1 ubiquitinylation and subsequent proteasome-mediated degradation, thereby prolonging its half-life and increasing its steady-state expression. Furthermore, comparison of the effect of GILZ1 with that of proteasome inhibition (by MG-132) supports the idea that these effects of GILZ1 are secondary to physical interaction of GILZ1 with SGK1 and enhanced recruitment of SGK1 to targets within an "ENaC regulatory complex," thus making less SGK1 available to the ERAD machinery. Finally, effects of GILZ1 knockdown and overexpression strongly support the idea that these effects of GILZ1 are functionally important for ENaC regulation. These data provide new insight into how the manifold activities of SGK1 are selectively deployed and strengthened through modulation of its molecular interactions, subcellular localization, and stability.