Intratracheal infection of mice with adenovirus is associated with subsequent pulmonary inflammation and edema. Water movement through the air space-capillary barrier in the distal lung is facilitated by aquaporins (AQPs). To investigate the possibility that distal lung AQPs undergo altered regulation under conditions of aberrant fluid handling in the lung, we analyzed messenger RNA (mRNA) and protein expression of AQPs 1 and 5 in the lungs of mice 7 and 14 d after infection with adenovirus. Here, we demonstrate that AQP1 and AQP5 show decreased expression following adenoviral infection. Northern blot analysis showed significantly decreased mRNA levels of AQP1, which is expressed in the capillary endothelium, and AQP5, which is expressed in alveolar epithelium, in the lungs of mice both 7 and 14 d after infection. Immunoblotting studies demonstrated significantly reduced levels of AQP1 and AQP5 protein after infection as well. In addition, mRNA expression of the alpha subunit of the epithelial sodium channel was reduced in the lungs of mice 7 and 14 d after adenoviral infection. In contrast, mRNA expression of the alpha1 subunit of the Na,K-adenosine triphosphatase in the lung was unaltered. Immunohistochemical analysis demonstrated that the decreases in AQP1 and AQP5 expression were not localized to regions of overt inflammation but were found throughout the lung. Thus, this study provides the first report of AQP gene regulation in an in vivo model of pulmonary inflammation and edema. Decreased AQP1 and AQP5 levels during adenoviral infection suggest a role for AQP1 and AQP5 in the abnormal fluid fluxes detected during pulmonary inflammation.

Although epidemiologic data strongly suggest a role for inhaled environmental pollutants in modulating the susceptibility to respiratory infection in humans, the underlying cellular and molecular mechanisms have not been well studied in experimental systems. The current study assessed the impact of inhaled diesel engine emissions (DEE) on the host response in vivo to a common pediatric respiratory pathogen, respiratory syncytial virus (RSV). Using a relatively resistant mouse model of RSV infection, prior exposure to either 30 μg/m3 particulate matter (PM) or 1,000 μg/m3 PM of inhaled DEE (6 h/d for seven consecutive days) increased lung inflammation to RSV infection as compared with air-exposed RSV-infected C57Bl/6 mice. Inflammatory cells in bronchoalveolar lavage fluid were increased in a dose-dependent manner with regard to the level of DEE exposure, concomitant with increased levels of inflammatory mediators. Lung histology analysis indicated pronounced peribronchial and peribronchiolar inflammation concordant with the level of DEE exposure during infection. Mucous cell metaplasia was markedly increased in the airway epithelium of DEE-exposed mice following RSV infection. Interestingly, both airway and alveolar host defense and immunomodulatory proteins were attenuated during RSV infection by prior DEE exposure. DEE-induced changes in inflammatory and lung epithelial responses to infection were associated with increased RSV gene expression in the lungs following DEE exposure. These findings are consistent with the concept that DEE exposure modulates the lung host defense to respiratory viral infections and may alter the susceptibility to respiratory infections leading to increased lung disease.

Clara cell secretory protein (CCSP) has been shown to have anti-inflammatory and immunomodulatory functions in the lung. Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and young children. RSV usually infects small airways and likely interacts with the Clara cells of bronchioles. To determine a possible role for CCSP during acute RSV infection, CCSP-deficient (CCSP^−/−) and wild-type (WT) mice were intratracheally infected with RSV and the lung inflammatory and immune responses to RSV infection were assessed. RSV-F gene expression was increased in the lungs of CCSP^−/− mice as compared with WT mice following RSV infection, consistent with increased viral persistence. Lung inflammation was significantly increased in CCSP^−/− mice as compared with WT mice after infection. Moreover, although the levels of Th1 cytokines were similar, the levels of Th2 cytokines and neutrophil chemokines were increased in the lungs of CCSP^−/− mice following infection. Physiologic endpoints of exacerbated lung disease, specifically airway reactivity and mucus production, were increased in CCSP^−/− mice after RSV infection. Importantly, restoration of CCSP in the airways of CCSP^−/− mice abrogated the increased viral persistence, lung inflammation, and airway reactivity. These findings suggest a role for CCSP and Clara cells in regulating lung inflammatory and immune responses to RSV infection.

Replication-deficient adenovirus vectors (Avs) have shown high-efficiency gene transfer in a variety of animal models, but demonstrated lower than expected efficiency in the intensely inflammatory milieu of the respiratory tract of individuals with cystic fibrosis (CF). Specific acquired immune responses directed at adenovirus capsid proteins are known to limit the duration of transgene expression and the effectiveness of vector readministration. In these models, however, nonspecific inflammation is also frequently noted to accompany specific immune responses. Because inflammation can occur early after Av administration, we hypothesized that inflammation may block Av-mediated gene transfer in the lung independent of specific immune responses. To evaluate this hypothesis, we measured pulmonary gene transfer and expression in the absence or presence of the potent antiinflammatory agent dexamethasone. To address and eliminate concerns over the potentially confounding effects of systemic, vector-specific acquired immune responses, evaluations were confined to a 3-day period following Av administration and were carried out, in parallel, in normal and immunodeficient (athymic) mice. Dexamethasone significantly reduced Av-associated inflammation in all animals as measured by a significant reduction of blinded, quantitative lung histopathology scores and by reduced proinflammatory cytokine release. Concomitant with reduced inflammation, gene transfer efficiency was significantly increased in both normal and immunodeficient animals as measured by transgene product activity (β-galactosidase) in total lung homogenates 3 days after vector administration. This finding could not be explained by a direct effect of dexamethasone on transgene specific activity. To begin to understand the molecular mechanisms of Av-induced inflammatory responses, lung levels of the chemoattractive chemokines MIP-2, MIP-1α, and MCP-1 were quantified. All were elevated significantly in Av-exposed animals. Dexamethasone reduced levels of MCP-1 and MIP-1α, but not MIP-2, consistent with the observed pattern of inflammatory cell changes. Expression of several proinflammatory cytokines including TNF-α, IL-6, IL-1β, and IFN-γ were also elevated in Av-exposed animals and modulated by dexamethasone. These observations demonstrate that nonspecific inflammation is an important determinant of the efficiency of in vivo pulmonary gene transfer and expression independent of specific immune responses and may have important implications for human gene therapy for diseases of the lung. Adenovirus vectors have important potential for in vivo gene therapy. However, both animal studies and human clinical trials have demonstrated relatively low-efficiency gene transfer to airway epithelium in vivo and have also demonstrated induction of acquired, specific immune responses as well as nonspecific inflammatory host responses. While acquired immune responses to Av have been studied extensively, fewer studies have focused on nonspecific innate host responses. In this study, we evaluated the effect of nonspecific inflammation on the efficiency of pulmonary gene transfer and expression. To eliminate concerns about effects on acquired immune host responses, studies were also carried out in immunodeficient animals. Dexamethasone blocked inflammation in both normal and immunodeficient mice and significantly improved gene transfer and expression in the lung. These results demonstrate an important role for inflammation in reducing the effectiveness of in vivo gene transfer and expression to the lung.

Rationale: Sepsis is a leading cause of morbidity and mortality. Currently, early diagnosis and the progression of the disease are difficult to make. The integration of metabolomic and transcriptomic data in a primate model of sepsis may provide a novel molecular signature of clinical sepsis. Objectives: To develop a biomarker panel to characterize sepsis in primates and ascertain its relevance to early diagnosis and progression of human sepsis. Methods: Intravenous inoculation of Macaca fascicularis with Escherichia coli produced mild to severe sepsis, lung injury, and death. Plasma samples were obtained before and after 1, 3, and 5 days of E. coli challenge and at the time of killing. At necropsy, blood, lung, kidney, and spleen samples were collected. An integrative analysis of the metabolomic and transcriptomic datasets was performed to identify a panel of sepsis biomarkers. Measurements and Main Results: The extent of E. coli invasion, respiratory distress, lethargy, and mortality was dependent on the bacterial dose. Metabolomic and transcriptomic changes characterized severe infections and death, and indicated impaired mitochondrial, peroxisomal, and liver functions. Analysis of the pulmonary transcriptome and plasma metabolome suggested impaired fatty acid catabolism regulated by peroxisome-proliferator activated receptor signaling. A representative four-metabolite model effectively diagnosed sepsis in primates (area under the curve, 0.966) and in two human sepsis cohorts (area under the curve, 0.78 and 0.82). Conclusions: A model of sepsis based on reciprocal metabolomic and transcriptomic data was developed in primates and validated in two human patient cohorts. It is anticipated that the identified parameters will facilitate early diagnosis and management of sepsis.