Famke Aeffner

Double-stranded RNA induces similar pulmonary dysfunction to respiratory syncytial virus in BALB/c mice

Both respiratory syncytial virus (RSV) and influenza A virus induce nucleotide/P2Y purinergic receptor-mediated impairment of alveolar fluid clearance (AFC), which contributes to formation of lung edema. Although genetically dissimilar, both viruses generate double-stranded RNA replication intermediates, which act as Toll-like receptor (TLR)-3 ligands. We hypothesized that double-stranded RNA/TLR-3 signaling underlies nucleotide-mediated inhibition of amiloride-sensitive AFC in both infections. We found that addition of the synthetic double-stranded RNA analog poly-inosinic-cytidylic acid [poly(I:C)] (500 ng/ml) to the AFC instillate resulted in nucleotide/P2Y purinergic receptor-mediated inhibition of amiloride-sensitive AFC in BALB/c mice but had no effect on cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) transport. Poly(I:C) also induced acute keratinocyte cytokine-mediated AFC insensitivity to stimulation by the β-adrenergic agonist terbutaline. Inhibitory effects of poly(I:C) on AFC were absent in TLR-3(-/-) mice and were not replicated by addition to the AFC instillate of ligands for other TLRs except TLR-2. Intranasal poly(I:C) administration (250 μg/mouse) similarly induced nucleotide-dependent AFC inhibition 2-3 days later, together with increased lung water content and neutrophilic inflammation. Intranasal treatment of BALB/c mice with poly(I:C) did not induce airway hyperresponsiveness at day 2 but did result in insensitivity to airway bronchodilation by β-adrenergic agonists. These findings suggest that viral double-stranded RNA replication intermediates induce nucleotide-mediated impairment of amiloride-sensitive AFC in both infections, together with β-adrenergic agonist insensitivity. Both of these effects also occur in RSV infection. However, double-stranded RNA replication intermediates do not appear to be sufficient to induce either adenosine-mediated, CFTR-dependent Cl(-) secretion in the lung or severe, lethal hypoxemia, both of which are features of influenza infection.

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

Background: Cystic fibrosis is characterized by defective autophagy and increased Burkholderia cenocepacia infection. Results: The depletion of SQSTM1/p62 from ΔF508 macrophages improves bacterial clearance via autophagy. Conclusion: p62 expression level determines the fate of B. cepacia infection in ΔF508 macrophages. Significance: Our study reveals the role of p62 in diseases characterized by protein aggregates that compromise autophagy by consuming essential autophagy molecules. Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.

Influenza A H1N1 induces declines in alveolar gas exchange in mice consistent with rapid post-infection progression from acute lung injury to ARDS

Background  Patients with severe seasonal or pandemic influenza pneumonia frequently develop acute respiratory distress syndrome (ARDS). One clinical diagnostic criterion for ARDS is the PaO2:FiO2 ratio, which is an index of alveolar gas exchange. However, effects of H1N1 influenza infection on PaO2:FiO2 ratios and related pathophysiologic readouts of lung function have not been reported in mice.

Mouse Models of Acute Respiratory Distress Syndrome A Review of Analytical Approaches, Pathologic Features, and Common Measurements

Acute respiratory distress syndrome (ARDS) is a severe pulmonary reaction requiring hospitalization, which is incited by many causes, including bacterial and viral pneumonia as well as near drowning, aspiration of gastric contents, pancreatitis, intravenous drug use, and abdominal trauma. In humans, ARDS is very well defined by a list of clinical parameters. However, until recently no consensus was available regarding the criteria of ARDS that should be evident in an experimental animal model. This lack was rectified by a 2011 workshop report by the American Thoracic Society, which defined the main features proposed to delineate the presence of ARDS in laboratory animals. These should include histological changes in parenchymal tissue, altered integrity of the alveolar capillary barrier, inflammation, and abnormal pulmonary function. Murine ARDS models typically are defined by such features as pulmonary edema and leukocyte infiltration in cytological preparations of bronchoalveolar lavage fluid and/or lung sections. Common pathophysiological indicators of ARDS in mice include impaired pulmonary gas exchange and histological evidence of inflammatory infiltrates into the lung. Thus, morphological endpoints remain a vital component of data sets assembled from animal ARDS models.

Activation of A1-Adenosine Receptors Promotes Leukocyte Recruitment to the Lung and Attenuates Acute Lung Injury in Mice Infected with Influenza A/WSN/33 (H1N1) Virus

ABSTRACT We have shown that bronchoalveolar epithelial A1-adenosine receptors (A1-AdoR) are activated in influenza A virus-infected mice. Alveolar macrophages and neutrophils also express A1-AdoRs, and we hypothesized that activation of A1-AdoRs on these cells will promote macrophage and neutrophil chemotaxis and activation and thereby play a role in the pathogenesis of influenza virus-induced acute lung injury. Wild-type (WT) C57BL/6 mice, congenic A1-AdoR knockout (A1-KO) mice, and mice that had undergone reciprocal bone marrow transfer were inoculated intranasally with 10,000 PFU/mouse influenza A/WSN/33 (H1N1) virus. Alternatively, WT mice underwent daily treatment with the A1-AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) from 1 day prior to inoculation. Infection increased bronchoalveolar lining fluid (BALF) adenosine comparably in WT and A1-KO mice. Infection of WT mice resulted in reduced carotid arterial O2 saturation (hypoxemia), lung pathology, pulmonary edema, reduced lung compliance, increased basal airway resistance, and hyperresponsiveness to methacholine. These effects were absent or significantly attenuated in A1-KO mice. Levels of BALF leukocytes, gamma interferon (IFN-γ), and interleukin 10 (IL-10) were significantly reduced in infected A1-KO mice, but levels of KC, IP-10, and MCP-1 were increased. Reciprocal bone marrow transfer resulted in WT-like lung injury severity, but BALF leukocyte levels increased only in WT and A1-KO mice with WT bone barrow. Hypoxemia, pulmonary edema, and levels of BALF alveolar macrophages, neutrophils, IFN-γ, and IL-10 were reduced in DPCPX-treated WT mice. Levels of viral replication did not differ between mouse strains or treatment groups. These findings indicate that adenosine activation of leukocyte A1-AdoRs plays a significant role in their recruitment to the infected lung and contributes to influenza pathogenesis. A1-AdoR inhibitor therapy may therefore be beneficial in patients with influenza virus-induced lung injury. IMPORTANCE Because antiviral drugs are of limited efficacy in patients hospitalized for influenza virus-induced respiratory failure, there is an urgent need for new therapeutics that can limit the progression of lung injury and reduce influenza death rates. We show that influenza A virus infection results in increased production of the nucleoside adenosine in the mouse lung and that activation of A1-subtype adenosine receptors by adenosine contributes significantly to both recruitment of innate immune cells to the lung and development of acute lung injury following influenza virus infection. We also show that treatment with an A1-adenosine receptor antagonist reduces the severity of lung injury in influenza virus-infected mice. Our findings indicate that adenosine plays an important and previously unrecognized role in the innate immune response to influenza virus infection and suggest that drugs which can inhibit either generation of adenosine or activation of A1-adenosine receptors may be beneficial in treating influenza patients hospitalized for respiratory failure.

Heterozygosity for the F508del Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Anion Channel Attenuates Influenza Severity

BACKGROUND Seasonal and pandemic influenza are significant public health concerns. Influenza stimulates respiratory epithelial Cl(-) secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). The purpose of this study was to determine the contribution of this effect to influenza pathogenesis in mice with reduced CFTR activity. METHODS C57BL/6-congenic mice heterozygous for the F508del CFTR mutation (HET) and wild-type (WT) controls were infected intranasally with 10 000 focus-forming units of influenza A/WSN/33 (H1N1) per mouse. Body weight, arterial O2 saturation, and heart rate were monitored daily. Pulmonary edema and lung function parameters were derived from ratios of wet weight to dry weight and the forced-oscillation technique, respectively. Levels of cytokines and chemokines in bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay. RESULTS Relative to WT mice, influenza virus-infected HET mice showed significantly delayed mortality, which was accompanied by attenuated hypoxemia, cardiopulmonary dysfunction, and pulmonary edema. However, viral replication and weight loss did not differ. The protective HET phenotype was correlated with exaggerated alveolar macrophage and interleukin 6 responses to infection and was abrogated by alveolar macrophage depletion, using clodronate liposomes. CONCLUSIONS Reduced CFTR expression modulates the innate immune response to influenza and alters disease pathogenesis. CFTR-mediated Cl(-) secretion is therefore an important host determinant of disease, and CFTR inhibition may be of therapeutic benefit in influenza.

Postinfection A77-1726 Treatment Improves Cardiopulmonary Function in H1N1 Influenza-Infected Mice

Acute respiratory disease is associated with significant morbidity and mortality in influenza. Because antiviral drugs are only effective early in infection, new agents are needed to treat nonvaccinated patients presenting with late-stage disease, particularly those who develop acute respiratory distress syndrome. We found previously that the de novo pyrimidine synthesis inhibitor A77-1726 reversed the influenza-induced impairment of alveolar fluid clearance. Patients with acute respiratory distress syndrome and intact alveolar fluid clearance demonstrate lower mortality than those with compromised fluid clearance. We therefore investigated the effects of treatment with nebulized A77-1726 (67.5 mg/kg) on indices of cardiopulmonary function relevant to the diagnosis of acute respiratory distress syndrome. BALB/cAnNCr mice (8-12 wk old) were inoculated intranasally with 10,000 plaque-forming units/mouse influenza A/WSN/33 (H1N1). Pulse oximetry was performed daily. Alveolar fluid clearance, lung water, and lung mechanics were measured at 2 and 6 days after inoculation in live, ventilated mice by BSA instillation, magnetic resonance imaging, and forced-oscillation techniques, respectively. A77-1726 treatment at 1 day after inoculation delayed mortality. Treatment on Days 1 or 5 reduced viral replication on Day 6, and improved alveolar fluid clearance, peripheral oxygenation, and cardiac function. Nebulized A77-1726 also reversed influenza-induced increases in lung water content and volume, improved pulmonary mechanics, reduced bronchoalveolar lavage fluid ATP and neutrophil content, and increased IL-6 concentrations. The ability of A77-1726 to improve cardiopulmonary function in influenza-infected mice and to reduce the severity of ongoing acute respiratory distress syndrome late in infection suggests that pyrimidine synthesis inhibitors are promising therapeutic candidates for the management of severe influenza.

Respiratory Syncytial Virus Reverses Airway Hyperresponsiveness to Methacholine in Ovalbumin-Sensitized Mice

Each year, approximately 20% of asthmatics in the United States experience acute symptom exacerbations, which commonly result from pulmonary viral infections. The majority of asthma exacerbations in very young children follow infection with respiratory syncytial virus (RSV). However, pathogenic mechanisms underlying induction of asthma exacerbations by RSV are not well understood. We therefore investigated the effect of post-sensitization RSV infection on lung function in ovalbumin (OVA)-sensitized BALB/c mice as a model of RSV asthma exacerbations. OVA sensitization of uninfected female BALB/c mice increased bronchoalveolar lavage fluid (BALF) eosinophil levels and induced airway hyperresponsiveness to the muscarinic agonist methacholine, as measured by the forced-oscillation technique. In contrast, intranasal infection with replication-competent RSV strain A2 for 2–8 days reduced BALF eosinophil counts and reversed airway hyperresponsiveness in a pertussis toxin-sensitive manner. BALF levels of the chemokine keratinocyte cytokine (KC; a murine homolog of interleukin-8) were elevated in OVA-sensitized, RSV-infected mice and reversal of methacholine hyperresponsiveness in these animals was rapidly inhibited by KC neutralization. Hyporesponsiveness could be induced in OVA-sensitized, uninfected mice by recombinant KC or the Gαi agonist melittin. These data suggest that respiratory syncytial virus induces KC-mediated activation of Gαi, resulting in cross-inhibition of Gαq-mediated M3-muscarinic receptor signaling and reversal of airway hyperresponsiveness. As in unsensitized mice, KC therefore appears to play a significant role in induction of airway dysfunction by respiratory syncytial virus. Hence, interleukin-8 may be a promising therapeutic target to normalize lung function in both asthmatics and non-asthmatics with bronchiolitis. However, the OVA-sensitized, RSV-infected mouse may not be an appropriate model for investigating the pathogenesis of viral asthma exacerbations.

The Gold Standard Paradox in Digital Image Analysis: Manual Versus Automated Scoring as Ground Truth

CONTEXT - Novel therapeutics often target complex cellular mechanisms. Increasingly, quantitative methods like digital tissue image analysis (tIA) are required to evaluate correspondingly complex biomarkers to elucidate subtle phenotypes that can inform treatment decisions with these targeted therapies. These tIA systems need a gold standard, or reference method, to establish analytical validity. Conventional, subjective histopathologic scores assigned by an experienced pathologist are the gold standard in anatomic pathology and are an attractive reference method. The pathologists score can establish the ground truth to assess a tIA solutions analytical performance. The paradox of this validation strategy, however, is that tIA is often used to assist pathologists to score complex biomarkers because it is more objective and reproducible than manual evaluation alone by overcoming known biases in a humans visual evaluation of tissue, and because it can generate endpoints that cannot be generated by a human observer. OBJECTIVE - To discuss common visual and cognitive traps known in traditional pathology-based scoring paradigms that may impact characterization of tIA-assisted scoring accuracy, sensitivity, and specificity. DATA SOURCES - This manuscript reviews the current literature from the past decades available for traditional subjective pathology scoring paradigms and known cognitive and visual traps relevant to these scoring paradigms. CONCLUSIONS - Awareness of the gold standard paradox is necessary when using traditional pathologist scores to analytically validate a tIA tool because image analysis is used specifically to overcome known sources of bias in visual assessment of tissue sections.

IKKβ in intestinal epithelial cells regulates allergen-specific IgA and allergic inflammation at distant mucosal sites

Regulation of allergic responses by intestinal epithelial cells (IECs) remains poorly understood. Using a model of oral allergen sensitization in the presence of cholera toxin as adjuvant and mice with cell-specific deletion of inhibitor-κB kinase (IKKβ) in IECs (IKKβΔIEC), we addressed the contribution of IECs to allergic sensitization to ingested antigens and allergic manifestations at distant mucosal site of the airways. Cholera toxin induced higher pro-inflammatory responses and altered the profile of the gut microbiota in IKKβΔIEC mice. Antigen-specific immunoglobulin E (IgE) responses were unaltered in IKKβΔIEC mice, but their IgA antibodies (Abs), T helper type 1 (Th1) and Th17 responses were enhanced. Upon nasal antigen challenge, these mice developed lower levels of allergic lung inflammation, which correlated with higher levels of IgA Abs in the airways. The IKKβΔIEC mice also recruited a higher number of gut-sensitized T cells in the airways after nasal antigen challenge and developed airway hyper-responsiveness, which were suppressed by treatment with anti-interleukin-17A. Fecal microbiota transplant during allergic sensitization reduced Th17 responses in IKKβΔIEC mice, but did not affect IgA Ab responses. In summary, we show that IKKβ in IECs shapes the gut microbiota and immune responses to ingested antigens and influences allergic responses in the airways via regulation of IgA Ab responses.