Tove Berg

Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-β1) levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (α-SMA) and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease.

Glucocorticoids regulate the CCSP and CYP2B1 promoters via C/EBPβ and δ in lung cells

Glucocorticoids have several important roles in the lung and play a key role in lung development and maturation. However, the specific molecular mechanisms of glucocorticoid action in lung are unclear. In this study, we have investigated two glucocorticoid-regulated genes expressed in the lung epithelium, the secretory protein CCSP, and the P450-enzyme CYP2B1. In transient transfections of lung epithelial cells, glucocorticoids increased expression from the CCSP and CYP2B1 promoters and we demonstrated that induction was dependent on the integrity of C/EBP-binding sites in both promoters. Electrophoretic mobility shift assays revealed increased DNA-binding of C/EBPβ and C/EBPδ after glucocorticoid treatment, which was not correlated to altered protein levels. The results of this study indicate a previously unknown role for C/EBP transcription factors in glucocorticoid signaling in the lung epithelium.

Lung Epithelial CCAAT/Enhancer-binding Protein-β Is Necessary for the Integrity of Inflammatory Responses to Cigarette Smoke

RATIONALE Cigarette smoke is the major cause of chronic obstructive pulmonary disease and lung cancer. The mechanisms by which smoking induces pulmonary dysfunction are complex, involving stress from toxic components and inflammatory responses. Although CCCAAT/enhancer-binding protein (C/EBP)-β is known as a key intracellular regulator of inflammatory signaling, its role in pulmonary inflammation has not been established. OBJECTIVES To characterize the role of C/EBPβ in the airway epithelial response to cigarette smoke. METHODS mRNA expression in the airway epithelium of current, former, and never-smokers, and in in vitro cigarette smoke extract-treated primary human airway epithelial cells, was analyzed by microarray and quantitative real-time polymerase chain reaction, respectively. Mice with lung epithelial-specific inactivation of C/EBPβ were generated and exposed to cigarette smoke for 4 or 11 days. Lung histology, bronchoalveolar lavage cell differentials, and expression of inflammatory and innate immune mediators in the lungs were assessed. MEASUREMENTS AND MAIN RESULTS C/EBPβ was significantly down-regulated in the airway epithelium of both current and former smokers compared with never-smokers, and in cigarette smoke-treated primary human airway epithelial cells in vitro. Cigarette smoke-exposed mice with a lung epithelial-specific inactivation of C/EBPβ displayed blunted respiratory neutrophil influx and compromised induction of neutrophil chemoattractants growth-regulated oncogene-α, macrophage inflammatory protein-1γ, granulocyte colony-stimulating factor, and serum amyloid A 3 and proinflammatory cytokines tumor necrosis factor-α and interleukin-1β, compared with smoke-exposed controls. Inhibition of C/EBPβ in human airway cells in vitro caused a similarly compromised response to smoke. CONCLUSION Our data suggest a previously unknown role for C/EBPβ and the airway epithelium in mediating inflammatory and innate immune responses to cigarette smoke.

Airway epithelial cell differentiation during lung organogenesis requires C/EBPα and C/EBPβ

Background: CCAAT/enhancer‐binding protein (C/EBP)α is crucial for lung development and differentiation of the pulmonary epithelium. Conversely, no lung defects have been observed in C/EBPβ‐deficient mice, although C/EBPβ trans‐activate pulmonary genes by binding to virtually identical DNA‐sequences as C/EBPα. Thus, the pulmonary phenotype of mice lacking C/EBPβ could be explained by functional replacement with C/EBPα. We investigated whether C/EBPα and C/EBPβ have overlapping functions in regulating lung epithelial differentiation during organogenesis. Epithelial differentiation was assessed in mice with a lung epithelial–specific (SFTPC‐Cre‐mediated) deletion of C/EBPα (CebpaΔLE), C/EBPβ (CebpbΔLE), or both genes (CebpaΔLE; CebpbΔLE). Results: Both CebpaΔLE mice and CebpaΔLE; CebpbΔLE mice demonstrated severe pulmonary immaturity compared to wild‐type littermates, while no differences in lung histology or epithelial differentiation were observed in CebpbΔLE mice. In contrast to CebpaΔLE mice, CebpaΔLE; CebpbΔLE mice also displayed undifferentiated Clara cells with markedly impaired protein and mRNA expression of Clara cell secretory protein (SCGB1A1), compared to wild‐type littermates. In addition, ectopic mucus‐producing cells were observed in the conducting airways of CebpaΔLE; CebpbΔLE mice. Conclusions: Our findings demonstrate that C/EBPα and C/EBPβ play pivotal, and partly overlapping roles in determining airway epithelial differentiation, with possible implications for tissue regeneration in lung homeostasis and disease. Developmental Dynamics 241:911–923, 2012.

Gpr116 Receptor Regulates Distinctive Functions in Pneumocytes and Vascular Endothelium

Despite its known expression in both the vascular endothelium and the lung epithelium, until recently the physiological role of the adhesion receptor Gpr116/ADGRF5 has remained elusive. We generated a new mouse model of constitutive Gpr116 inactivation, with a large genetic deletion encompassing exon 4 to exon 21 of the Gpr116 gene. This model allowed us to confirm recent results defining Gpr116 as necessary regulator of surfactant homeostasis. The loss of Gpr116 provokes an early accumulation of surfactant in the lungs, followed by a massive infiltration of macrophages, and eventually progresses into an emphysema-like pathology. Further analysis of this knockout model revealed cerebral vascular leakage, beginning at around 1.5 months of age. Additionally, endothelial-specific deletion of Gpr116 resulted in a significant increase of the brain vascular leakage. Mice devoid of Gpr116 developed an anatomically normal and largely functional vascular network, surprisingly exhibited an attenuated pathological retinal vascular response in a model of oxygen-induced retinopathy. These data suggest that Gpr116 modulates endothelial properties, a previously unappreciated function despite the pan-vascular expression of this receptor. Our results support the key pulmonary function of Gpr116 and describe a new role in the central nervous system vasculature.

Lung epithelial-C/EBPβ contributes to LPS-induced inflammation and its suppression by formoterol.

The inflammatory processes associated with pulmonary disorders remains incompletely understood. CCAAT/enhancer-binding protein (C/EBP)β is implicated in inflammatory lung disorders as well as in β(2)-adrenoceptor signaling. We hypothesized that C/EBPβ in the lung epithelium contributes to lipopolysaccharide (LPS)-induced airway neutrophilia and expression of neutrophil chemoattractant chemokine (C-X-C) motif ligand (CXCL)1, as well as the suppressive effects of long-acting β(2)-agonists (LABAs) and glucocorticoids (GCs). To investigate this, mice with a lung epithelial-specific deletion of C/EBPβ (Cebpb(ΔLE)) and control littermates (Cebpb(fl/fl)) were pre-treated with a LABA, formoterol and/or a GC, budesonide, and challenged with LPS. Inflammatory cell recruitment in bronchoalveolar lavage (BAL) fluid and pulmonary expression of inflammatory mediators were investigated. In addition, the ability of formoterol to increase C/EBP transactivation was assessed in vitro. LPS-challenged Cebpb(ΔLE) mice exhibited fewer BAL neutrophils and lower pulmonary expression of CXCL1 versus Cebpb(fl/fl) mice. Suppression of LPS-induced neutrophilia by formoterol was impaired in Cebpb(ΔLE) mice and Cxcl1 expression was increased. However, suppression of the neutrophilia by budesonide with/without formoterol was preserved. Further studies indicated that C/EBP transactivation was increased by the cAMP elevating agent forskolin and formoterol in a β(2)-adrenoceptor dependent manner. Thus, C/EBPβ in the lung epithelium contributes to LPS-induced CXCL1 expression and airway neutrophilia as well as to the suppressive effects of formoterol. Reduced C/EBPβ activity, observed in smokers with chronic obstructive pulmonary disease, may impair the responsiveness to LABAs when used without GCs.

Human Enterovirus Species B in Ileocecal Crohn's Disease

OBJECTIVES:Advanced ileocecal Crohn’s disease (ICD) is characterized by strictures, inflammation in the enteric nervous system (myenteric plexitis), and a high frequency of NOD2 mutations. Recent findings implicate a role of NOD2 and another CD susceptibility gene, ATG16L1, in the host response against single-stranded RNA (ssRNA) viruses. However, the role of viruses in CD is unknown. We hypothesized that human enterovirus species B (HEV-B), which are ssRNA viruses with dual tropism both for the intestinal epithelium and the nervous system, could play a role in ICD.METHODS:We used immunohistochemistry and in situ hybridization to study the general presence of HEV-B and the presence of the two HEV-B subspecies, Coxsackie B virus (CBV) and Echovirus, in ileocecal resections from 9 children with advanced, stricturing ICD and 6 patients with volvulus, and in intestinal biopsies from 15 CD patients at the time of diagnosis.RESULTS:All patients with ICD had disease-associated polymorphisms in NOD2 or ATG16L1. Positive staining for HEV-B was detected both in the mucosa and in myenteric nerve ganglia in all ICD patients, but in none of the volvulus patients. Expression of the cellular receptor for CBV, CAR, was detected in nerve cell ganglia.CONCLUSIONS:The common presence of HEV-B in the mucosa and enteric nervous system of ICD patients in this small cohort is a novel finding that warrants further investigation to analyze whether HEV-B has a role in disease onset or progress. The presence of CAR in myenteric nerve cell ganglia provides a possible route of entry for CBV into the enteric nervous system.

A Relationship between Epithelial Maturation, Bronchopulmonary Dysplasia, and Chronic Obstructive Pulmonary Disease

Premature infants frequently develop bronchopulmonary dysplasia (BPD). Lung immaturity and impaired epithelial differentiation contribute together with invasive oxygen treatment to BPD onset and disease progression. Substantial evidence suggests that prematurity is associated with long term pulmonary consequences. Moreover, there is increasing concern that lung immaturity at birth may increase the risk of developing chronic obstructive pulmonary disease (COPD). The mechanisms contributing to this phenomenon remains unknown, largely as a consequence of inadequate experimental models and clinical follow-up studies. Recent evidence suggests that defective transcriptional regulation of epithelial differentiation and maturation may contribute to BPD pathogenesis as well as early onset of COPD. The transcriptional regulators CCAAT/enhancer-binding protein (C/EBP)α and C/EBPβ, SMAD family member (Smad)3, GATA binding protein (GATA)6, and NK2 homeobox (NKX)2-1 are reported to be involved in processes contributing to pathogenesis of both BPD and COPD. Increased knowledge of the mechanisms contributing to early onset COPD among BPD survivors could translate into improved treatment strategies and reduced frequency of respiratory disorders among adult survivors of BPD. In this paper, we introduce critical transcriptional regulators in epithelial differentiation and summarize the current knowledge on the contribution of impaired epithelial maturation to the pathogenesis of inflammatory lung disorders.

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.