Lung CD103+dendritic cells and Clec9a signaling are required for neonatal hyperoxia-induced inflammatory responses to rhinovirus infection.

Premature infants, especially those with bronchopulmonary dysplasia (BPD), develop recurrent severe respiratory viral illnesses. We have shown that hyperoxic exposure of immature mice, a model of BPD, increases lung IL-12-producing Clec9a+ CD103+ dendritic cells (DCs), pro-inflammatory responses, and airway hyperreactivity following rhinovirus (RV) infection. However, the requirement for CD103+ DCs and Clec9a, a DAMP receptor that binds necrotic cell cytoskeletal filamentous actin (F-actin), for RV-induced inflammatory responses has not been demonstrated. To test this, 2-day-old C57BL/6J, CD103+ DC-deficient Batf3-/- or Clec9agfp-/- mice were exposed to normoxia or hyperoxia for 14 days. Also, selected mice were treated with neutralizing antibody against CD103. Immediately after hyperoxia, the mice were inoculated with RV intranasally. We found that compared with wild-type mice, hyperoxia-exposed Batf3-/- mice showed reduced levels of IL-12p40, IFN-γ, and TNF-α, fewer IFN-γ-producing CD4+ T cells, and decreased airway responsiveness following RV infection. Similar effects were observed in anti-CD103-treated and Clec9agfp-/- mice. Furthermore, hyperoxia increased airway dead cell number and extracellular F-actin levels. Finally, studies in preterm infants with respiratory distress syndrome showed that tracheal aspirate CLEC9A expression positively correlated with IL12B expression, consistent with the notion that CLEC9A+ cells are responsible for IL-12 production in humans as well as mice. We conclude that CD103+ DCs and Clec9a are required for hyperoxia-induced pro-inflammatory responses to RV infection. In premature infants, Clec9a-mediated activation of CD103+ DCs may promote pro-inflammatory responses to viral infection, thereby driving respiratory morbidity.

Nasal interferon responses to community rhinovirus infections are similar in controls and children with asthma.

BACKGROUND: Rhinovirus (RV) is the main cause of asthma exacerbations in children. Some studies reported that persons with asthma have attenuated interferon (IFN) responses to experimental RV infection compared with healthy individuals. However, responses to community-acquired RV infections in controls and children with asthma have not been compared. OBJECTIVE: To evaluate nasal cytokine responses after natural RV infections in people with asthma and healthy children. METHODS: We compared nasal cytokine expression among controls and children with asthma during healthy, virus-negative surveillance weeks and self-reported RV-positive sick weeks. A total of 14 controls and 21 patients with asthma were studied. Asthma disease severity was based on symptoms and medication use. Viral genome was detected by multiplex polymerase chain reaction. Nasal cytokine protein levels were determined by multiplex assays. RESULTS: Two out of 47 surveillance weeks tested positive for RV, illustrating an asymptomatic infection rate of 5%. A total of 38 of 47 sick weeks (81%) tested positive for the respiratory virus. Of these, 33 (87%) were positive for RV. During well weeks, nasal interleukin 8 (IL-8), IL-12, and IL-1ß levels were higher in children with asthma than controls. Compared with healthy virus-negative surveillance weeks, IL-8, IL-13, and interferon beta increased during colds only in patients with asthma. In both controls and children with asthma, the nasal levels of interferon gamma, interferon lambda-1, IL-1ß, IL-8, and IL-10 increased during RV-positive sick weeks. During RV infection, IL-8, IL-1ß, and tumor necrosis factor-α levels were strongly correlated. CONCLUSION: In both controls and patients with asthma, natural RV infection results in robust type II and III IFN responses.

IL-1ß prevents ILC2 expansion, type 2 cytokine secretion, and mucus metaplasia in response to early-life rhinovirus infection in mice.

BACKGROUND: Early-life wheezing-associated respiratory infection with human rhinovirus (RV) is associated with asthma development. RV infection of 6-day-old immature mice causes mucous metaplasia and airway hyperresponsiveness which is associated with the expansion of IL-13-producing type 2 innate lymphoid cells (ILC2s) and dependent on IL-25 and IL-33. We examined regulation of this asthma-like phenotype by IL-1ß. METHODS: Six-day-old wild-type or NRLP3-/- mice were inoculated with sham or RV-A1B. Selected mice were treated with IL-1 receptor antagonist (IL-1RA), anti-IL-1ß, or recombinant IL-1ß. RESULTS: Rhinovirus infection induced Il25, Il33, Il4, Il5, Il13, muc5ac, and gob5 mRNA expression, ILC2 expansion, mucus metaplasia, and airway hyperresponsiveness. RV also induced lung mRNA and protein expression of pro-IL-1ß and NLRP3 as well as cleavage of caspase-1 and pro-IL-1ß, indicating inflammasome priming and activation. Lung macrophages were a major source of IL-1ß. Inhibition of IL-1ß signaling with IL-1RA, anti-IL-1ß, or NLRP3 KO increased RV-induced type 2 cytokine immune responses, ILC2 number, and mucus metaplasia, while decreasing IL-17 mRNA expression. Treatment with IL-1ß had the opposite effect, decreasing IL-25, IL-33, and mucous metaplasia while increasing IL-17 expression. IL-1ß and IL-17 each suppressed Il25, Il33, and muc5ac mRNA expression in cultured airway epithelial cells. Finally, RV-infected 6-day-old mice showed reduced IL-1ß mRNA and protein expression compared to mature mice. CONCLUSION: Macrophage IL-1ß limits type 2 inflammation and mucous metaplasia following RV infection by suppressing epithelial cell innate cytokine expression. Reduced IL-1ß production in immature animals provides a mechanism permitting asthma development after early-life viral infection.

Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression.

Asthma exacerbations are often caused by rhinovirus (RV). We and others have shown that Toll-like receptor 2 (TLR2), a membrane surface receptor that recognizes bacterial lipopeptides and lipoteichoic acid, is required and sufficient for RV-induced proinflammatory responses in vitro and in vivo. We hypothesized that viral protein-4 (VP4), an internal capsid protein that is myristoylated upon viral replication and externalized upon viral binding, is a ligand for TLR2. Recombinant VP4 and myristoylated VP4 (MyrVP4) were purified by Ni-affinity chromatography. MyrVP4 was also purified from RV-A1B-infected HeLa cells by urea solubilization and anti-VP4 affinity chromatography. Finally, synthetic MyrVP4 was produced by chemical peptide synthesis. MyrVP4-TLR2 interactions were assessed by confocal fluorescence microscopy, fluorescence resonance energy transfer (FRET), and monitoring VP4-induced cytokine mRNA expression in the presence of anti-TLR2 and anti-VP4. MyrVP4 and TLR2 colocalized in TLR2-expressing HEK-293 cells, mouse bone marrow-derived macrophages, human bronchoalveolar macrophages, and human airway epithelial cells. Colocalization was absent in TLR2-null HEK-293 cells and blocked by anti-TLR2 and anti-VP4. Cy3-labeled MyrVP4 and Cy5-labeled anti-TLR2 showed an average fractional FRET efficiency of 0.24 ± 0.05, and Cy5-labeled anti-TLR2 increased and unlabeled MyrVP4 decreased FRET efficiency. MyrVP4-induced chemokine mRNA expression was higher than that elicited by VP4 alone and was attenuated by anti-TLR2 and anti-VP4. Cytokine expression was similarly increased by MyrVP4 purified from RV-infected HeLa cells and synthetic MyrVP4. We conclude that, during RV infection, MyrVP4 and TLR2 interact to generate a proinflammatory response.

Impact of community respiratory viral infections in urban children with asthma.

BACKGROUND: Upper respiratory tract viral infections cause asthma exacerbations in children. However, the impact of natural colds on children with asthma in the community, particularly in the high-risk urban environment, is less well defined. OBJECTIVE: We hypothesized that children with high-symptom upper respiratory viral infections have reduced airway function and greater respiratory tract inflammation than children with virus-positive low-symptom illnesses or virus-negative upper respiratory tract symptoms. METHODS: We studied 53 children with asthma from Detroit, Michigan, during scheduled surveillance periods and self-reported respiratory illnesses for 1 year. Symptom score, spirometry, fraction of exhaled nitric oxide (FeNO), and nasal aspirate biomarkers, and viral nucleic acid and rhinovirus (RV) copy number were assessed. RESULTS: Of 658 aspirates collected, 22.9% of surveillance samples and 33.7% of respiratory illnesses were virus-positive. Compared with the virus-negative asymptomatic condition, children with severe colds (symptom score ≥5) showed reduced forced expiratory flow at 25% to 75% of the pulmonary volume (FEF25%-75%), higher nasal messenger RNA expression of C-X-C motif chemokine ligand (CXCL)-10 and melanoma differentiation-associated protein 5, and higher protein abundance of CXCL8, CXCL10 and C-C motif chemokine ligands (CCL)-2, CCL4, CCL20, and CCL24. Children with mild (symptom score, 1-4) and asymptomatic infections showed normal airway function and fewer biomarker elevations. Virus-negative cold-like illnesses demonstrated increased FeNO, minimal biomarker elevation, and normal airflow. The RV copy number was associated with nasal chemokine levels but not symptom score. CONCLUSION: Urban children with asthma with high-symptom respiratory viral infections have reduced FEF25%-75% and more elevations of nasal biomarkers than children with mild or symptomatic infections, or virus-negative illnesses.

Influence of viral infection on the relationships between airway cytokines and lung function in asthmatic children.

BACKGROUND: Few longitudinal studies examine inflammation and lung function in asthma. We sought to determine the cytokines that reduce airflow, and the influence of respiratory viral infections on these relationships. METHODS: Children underwent home collections of nasal lavage during scheduled surveillance periods and self-reported respiratory illnesses. We studied 53 children for one year, analyzing 392 surveillance samples and 203 samples from 85 respiratory illnesses. Generalized estimated equations were used to evaluate associations between nasal lavage biomarkers (7 mRNAs, 10 proteins), lung function and viral infection. RESULTS: As anticipated, viral infection was associated with increased cytokines and reduced FVC and FEV1. However, we found frequent and strong interactions between biomarkers and virus on lung function. For example, in the absence of viral infection, CXCL10 mRNA, MDA5 mRNA, CXCL10, IL-4, IL-13, CCL4, CCL5, CCL20 and CCL24 were negatively associated with FVC. In contrast, during infection, the opposite relationship was frequently found, with IL-4, IL-13, CCL5, CCL20 and CCL24 levels associated with less severe reductions in both FVC and FEV1. CONCLUSIONS: In asthmatic children, airflow obstruction is driven by specific pro-inflammatory cytokines. In the absence of viral infection, higher cytokine levels are associated with decreasing lung function. However, with infection, there is a reversal in this relationship, with cytokine abundance associated with reduced lung function decline. While nasal samples may not reflect lower airway responses, these data suggest that some aspects of the inflammatory response may be protective against viral infection. This study may have ramifications for the treatment of viral-induced asthma exacerbations.

Hyperoxic Exposure of Immature Mice Increases the Inflammatory Response to Subsequent Rhinovirus Infection: Association with Danger Signals.

Infants with a history of prematurity and bronchopulmonary dysplasia have a high risk of asthma and viral-induced exacerbations later in life. We hypothesized that hyperoxic exposure, a predisposing factor to bronchopulmonary dysplasia, modulates the innate immune response, producing an exaggerated proinflammatory reaction to viral infection. Two- to 3-d-old C57BL/6J mice were exposed to air or 75% oxygen for 14 d. Mice were infected intranasally with rhinovirus (RV) immediately after O2 exposure. Lung mRNA and protein expression, histology, dendritic cells (DCs), and airway responsiveness were assessed 1-12 d postinfection. Tracheal aspirates from premature human infants were collected for mRNA detection. Hyperoxia increased lung IL-12 expression, which persisted up to 12 d postexposure. Hyperoxia-exposed RV-infected mice showed further increases in IL-12 and increased expression of IFN-γ, TNF-α, CCL2, CCL3, and CCL4, as well as increased airway inflammation and responsiveness. In RV-infected, air-exposed mice, the response was not significant. Induced IL-12 expression in hyperoxia-exposed, RV-infected mice was associated with increased IL-12-producing CD103(+) lung DCs. Hyperoxia also increased expression of Clec9a, a CD103(+) DC-specific damaged cell-recognition molecule. Hyperoxia increased levels of ATP metabolites and expression of adenosine receptor A1, further evidence of cell damage and related signaling. In human preterm infants, tracheal aspirate Clec9a expression positively correlated with the level of prematurity. Hyperoxic exposure increases the activation of CD103(+), Clec9a(+) DCs, leading to increased inflammation and airway hyperresponsiveness upon RV infection. In premature infants, danger signal-induced DC activation may promote proinflammatory airway responses, thereby increasing respiratory morbidity.

Suppression of inflammatory cell trafficking and alveolar simplification by the heme oxygenase-1 product carbon monoxide.

Bronchopulmonary dysplasia (BPD), a lung disease of prematurely born infants, is characterized in part by arrested development of pulmonary alveolae. We hypothesized that heme oxygenase (HO-1) and its byproduct carbon monoxide (CO), which are thought to be cytoprotective against redox stress, mitigate lung injury and alveolar simplification in hyperoxia-exposed neonatal mice, a model of BPD. Three-day-old C57BL/6J mice were exposed to air or hyperoxia (FiO2, 75%) in the presence or absence of inhaled CO (250 ppm for 1 h twice daily) for 21 days. Hyperoxic exposure increased mean linear intercept, a measure of alveolar simplification, whereas CO treatment attenuated hypoalveolarization, yielding a normal-appearing lung. Conversely, HO-1-null mice showed exaggerated hyperoxia-induced hypoalveolarization. CO also inhibited hyperoxia-induced pulmonary accumulation of F4/80+, CD11c+, and CD11b+ monocytes and Gr-1+ neutrophils. Furthermore, CO attenuated lung mRNA and protein expression of proinflammatory cytokines, including the monocyte chemoattractant CCL2 in vivo, and decreased hyperoxia-induced type I alveolar epithelial cell CCL2 production in vitro. Hyperoxia-exposed CCL2-null mice, like CO-treated mice, showed attenuated alveolar simplification and lung infiltration of CD11b+ monocytes, consistent with the notion that CO blocks lung epithelial cell cytokine production. We conclude that, in hyperoxia-exposed neonatal mice, inhalation of CO suppresses inflammation and alveolar simplification.

Reduced platelet-derived growth factor receptor expression is a primary feature of human bronchopulmonary dysplasia.

Animal studies have shown that platelet-derived growth factor (PDGF) signaling is required for normal alveolarization. Changes in PDGF receptor (PDGFR) expression in infants with bronchopulmonary dysplasia (BPD), a disease of hypoalveolarization, have not been examined. We hypothesized that PDGFR expression is reduced in neonatal lung mesenchymal stromal cells (MSCs) from infants who develop BPD. MSCs from tracheal aspirates of premature infants requiring mechanical ventilation in the first week of life were studied. MSC migration was assessed in a Boyden chamber. Human lung tissue was obtained from the University of Rochester Neonatal Lung Biorepository. Neonatal mice were exposed to air or 75% oxygen for 14 days. PDGFR expression was quantified by qPCR, immunoblotting, and stereology. MSCs were isolated from 25 neonates (mean gestational age 27.7 wk); 13 developed BPD and 12 did not. MSCs from infants who develop BPD showed lower PDGFR-α and PDGFR-ß mRNA and protein expression and decreased migration to PDGF isoforms. Lungs from infants dying with BPD show thickened alveolar walls and paucity of PDGFR-α-positive cells in the dysmorphic alveolar septa. Similarly, lungs from hyperoxia-exposed neonatal mice showed lower expression of PDGFR-α and PDGFR-ß, with significant reductions in the volume of PDGFR-α-positive alveolar tips. In conclusion, MSCs from infants who develop BPD hold stable alterations in PDGFR gene expression that favor hypoalveolarization. These data demonstrate that defective PDGFR signaling is a primary feature of human BPD.

Macrophage activation state determines the response to rhinovirus infection in a mouse model of allergic asthma.

BACKGROUND: The mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease. METHODS: Eight week-old wild type or IL-4 receptor knockout (IL-4R KO) mice were sensitized and challenged with OVA and inoculated with RV1B or sham HeLa cell lysate. RESULTS: In contrast to OVA-treated wild-type mice with both neutrophilic and eosinophilic airway inflammation, OVA-treated IL-4R KO mice showed increased neutrophilic inflammation with few eosinophils in the airways. Like wild-type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV. There was a shift in lung cytokines from a type 2-predominant response to a type 1 response, including production of IL-12p40 and TNF-α. IL-17A was also increased. RV infection of OVA-treated IL-4R KO mice further increased neutrophilic inflammation. Bronchoalveolar macrophages showed an M1 polarization pattern and ex vivo RV infection increased macrophage production of TNF-α, IFN-γ and IL-12p40. Finally, lung cells from OVA-treated IL-4R KO mice showed reduced CD206+ CD301+ M2 macrophages, decreased IL-13 and increased TNF-α and IL-17A production by F4/80+, CD11b+ macrophages. CONCLUSIONS: OVA-treated IL-4R KO mice show neutrophilic airway inflammation constituting a model of allergic, type 1 cytokine-driven neutrophilic asthma. In the absence of IL-4/IL-13 signaling, RV infection of OVA-treated mice increased type 1 cytokine and IL-17A production from conventionally-activated macrophages, augmenting neutrophilic rather than eosinophilic inflammation. In mice with allergic airways inflammation, IL-4R signaling determines macrophage activation state and the response to subsequent RV infection.

Tuft cells are required for a rhinovirus-induced asthma phenotype in immature mice.

Infection of immature mice with rhinovirus (RV) induces an asthma-like phenotype consisting of type 2 inflammation, mucous metaplasia, eosinophilic inflammation, and airway hyperresponsiveness that is dependent on IL-25 and type 2 innate lymphoid cells (ILC2s). Doublecortin-like kinase 1-positive (DCLK1+) tuft cells are a major source of IL-25. We sought to determine the requirement of tuft cells for the RV-induced asthma phenotype in wild-type mice and mice deficient in Pou2f3, a transcription factor required for tuft cell development. C57BL/6J mice infected with RV-A1B on day 6 of life and RV-A2 on day 13 of life showed increased DCLK1+ tuft cells in the large airways. Compared with wild-type mice, RV-infected Pou2f3-/- mice showed reductions in IL-25 mRNA and protein expression, ILC2 expansion, type 2 cytokine expression, mucous metaplasia, lung eosinophils, and airway methacholine responsiveness. We conclude that airway tuft cells are required for the asthma phenotype observed in immature mice undergoing repeated RV infections. Furthermore, RV-induced tuft cell development provides a mechanism by which early-life viral infections could potentiate type 2 inflammatory responses to future infections.

Macrophage/epithelial cell CCL2 contributes to rhinovirus-induced hyperresponsiveness and inflammation in a mouse model of allergic airways disease.

Human rhinovirus (HRV) infections lead to exacerbations of lower airways disease in asthmatic patients but not in healthy individuals. However, underlying mechanisms remain to be completely elucidated. We hypothesized that the Th2-driven allergic environment enhances HRV-induced CC chemokine production, leading to asthma exacerbations. Ovalbumin (OVA)-sensitized and -challenged mice inoculated with HRV showed significant increases in the expression of lung CC chemokine ligand (CCL)-2/monocyte chemotactic protein (MCP)-1, CCL4/macrophage inflammatory protein (MIP)-1ß, CCL7/MCP-3, CCL19/MIP-3ß, and CCL20/MIP3α compared with mice treated with OVA alone. Inhibition of CCL2 with neutralizing antibody significantly attenuated HRV-induced airways inflammation and hyperresponsiveness in OVA-treated mice. Immunohistochemical stains showed colocalization of CCL2 with HRV in epithelial cells and CD68-positive macrophages, and flow cytometry showed increased CCL2(+), CD11b(+) cells in the lungs of OVA-treated, HRV-infected mice. Compared with lung macrophages from naïve mice, macrophages from OVA-exposed mice expressed significantly more CCL2 in response to HRV infection ex vivo. Pretreatment of mouse lung macrophages and BEAS-2B human bronchial epithelial cells with interleukin (IL)-4 and IL-13 increased HRV-induced CCL2 expression, and mouse lung macrophages from IL-4 receptor knockout mice showed reduced CCL2 expression in response to HRV, suggesting that exposure to these Th2 cytokines plays a role in the altered HRV response. Finally, bronchoalveolar macrophages from children with asthma elaborated more CCL2 upon ex vivo exposure to HRV than cells from nonasthmatic patients. We conclude that CCL2 production by epithelial cells and macrophages contributes to HRV-induced airway hyperresponsiveness and inflammation in a mouse model of allergic airways disease and may play a role in HRV-induced asthma exacerbations.

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness.

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.

Early-life hyperoxia-induced Flt3L drives neonatal lung dendritic cell expansion and proinflammatory responses.

Premature infants with chronic lung disease, bronchopulmonary dysplasia (BPD), develop recurrent cough and wheezing following respiratory viral infections. The mechanisms driving the chronic respiratory symptoms are ill-defined. We have shown that hyperoxic exposure of neonatal mice (a model of BPD) increases the activated lung CD103+ dendritic cells (DCs) and these DCs are required for exaggerated proinflammatory responses to rhinovirus (RV) infection. Since CD103+ DC are essential for specific antiviral responses and their development depends on the growth factor Flt3L, we hypothesized that early-life hyperoxia stimulates Flt3L expression leading to expansion and activation of lung CD103+ DCs and this mediates inflammation. We found that hyperoxia numerically increased and induced proinflammatory transcriptional signatures in neonatal lung CD103+ DCs, as well as CD11bhi DCs. Hyperoxia also increased Flt3L expression. Anti-Flt3L antibody blocked CD103+ DC development in normoxic and hyperoxic conditions, and while it did not affect the baseline number of CD11bhi DCs, it neutralized the effect of hyperoxia on these cells. Anti-Flt3L also inhibited hyperoxia-induced proinflammatory responses to RV. In tracheal aspirates from preterm infants mechanically-ventilated for respiratory distress in the first week of life levels of FLT3L, IL-12p40, IL-12p70 and IFN-γ were higher in infants who went on to develop BPD and FLT3L levels positively correlated with proinflammatory cytokines levels. This work highlights the priming effect of early-life hyperoxia on lung DC development and function and the contribution of Flt3L in driving these effects.

Glycogen synthase kinase-3ß/ß-catenin signaling regulates neonatal lung mesenchymal stromal cell myofibroblastic differentiation.

In bronchopulmonary dysplasia (BPD), alveolar septa are thickened with collagen and α-smooth muscle actin-, transforming growth factor (TGF)-ß-positive myofibroblasts. We examined the biochemical mechanisms underlying myofibroblastic differentiation, focusing on the role of glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin signaling pathway. In the cytoplasm, ß-catenin is phosphorylated on the NH(2) terminus by constitutively active GSK-3ß, favoring its degradation. Upon TGF-ß stimulation, GSK-3ß is phosphorylated and inactivated, allowing ß-catenin to translocate to the nucleus, where it activates transcription of genes involved in myofibroblastic differentiation. We examined the role of ß-catenin in TGF-ß1-induced myofibroblastic differentiation of neonatal lung mesenchymal stromal cells (MSCs) isolated from tracheal aspirates of premature infants with respiratory distress. TGF-ß1 increased ß-catenin expression and nuclear translocation. Transduction of cells with GSK-3ß S9A, a nonphosphorylatable, constitutively active mutant that favors ß-catenin degradation, blocked TGF-ß1-induced myofibroblastic differentiation. Furthermore, transduction of MSCs with ΔN-catenin, a truncation mutant that cannot be phosphorylated on the NH(2) terminus by GSK-3ß and is not degraded, was sufficient for myofibroblastic differentiation. In vivo, hyperoxic exposure of neonatal mice increases expression of ß-catenin in α-smooth muscle actin-positive myofibroblasts. Similar changes were found in lungs of infants with BPD. Finally, low-passage unstimulated MSCs from infants developing BPD showed higher phospho-GSK-3ß, ß-catenin, and α-actin content compared with MSCs from infants not developing this disease, and phospho-GSK-3ß and ß-catenin each correlated with α-actin content. We conclude that phospho-GSK-3ß/ß-catenin signaling regulates α-smooth muscle actin expression, a marker of myofibroblast differentiation, in vitro and in vivo. This pathway appears to be activated in lung mesenchymal cells from patients with BPD.

Increased cytokine response of rhinovirus-infected airway epithelial cells in chronic obstructive pulmonary disease.

RATIONALE: Airway inflammation is a central feature of chronic obstructive pulmonary disease (COPD). COPD exacerbations are often triggered by rhinovirus (RV) infection. OBJECTIVES: We hypothesized that airway epithelial cells from patients with COPD maintain a proinflammatory phenotype compared with control subjects, leading to greater RV responses. METHODS: Cells were isolated from tracheobronchial tissues of 12 patients with COPD and 10 transplant donors. Eight patients with COPD had severe emphysema, three had mild to moderate emphysema, and one had no emphysema. All had moderate to severe airflow obstruction, and six met criteria for chronic bronchitis or had at least one exacerbation the previous year. Cells were grown at air-liquid interface and infected with RV serotype 39. Cytokine and IFN expression was measured by ELISA. Selected genes involved in inflammation, oxidative stress, and proteolysis were assessed by focused gene array and real-time polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Compared with control subjects, cells from patients with COPD demonstrated increased mRNA expression of genes involved in oxidative stress and the response to viral infection, including NOX1, DUOXA2, MMP12, ICAM1, DDX58/RIG-I, STAT1, and STAT2. COPD cells showed elevated baseline and RV-stimulated protein levels of IL-6, IL-8/CXCL8, and growth-related oncogene-alpha/CXCL1. COPD cells demonstrated increased viral titer and copy number after RV infection, despite increased IL-29/IFN-lambda1, IL-28A/IFN-lambda2, and IFN-inducible protein-10/CXCL10 protein levels. Finally, RV-infected COPD cultures showed increased mRNA expression of IL28A/IFNlambda2, IL29/IFNlambda1, IFIH1/MDA5, DDX58/RIG-I, DUOX1, DUOX2, IRF7, STAT1, and STAT2. CONCLUSIONS: Airway epithelial cells from patients with COPD show higher baseline levels of cytokine expression and increased susceptibility to RV infection, despite an increased IFN response.

Rhinovirus C Infection Induces Type 2 Innate Lymphoid Cell Expansion and Eosinophilic Airway Inflammation.

Rhinovirus C (RV-C) infection is associated with severe asthma exacerbations. Since type 2 inflammation is an important disease mechanism in asthma, we hypothesized that RV-C infection, in contrast to RV-A, preferentially stimulates type 2 inflammation, leading to exacerbated eosinophilic inflammation. To test this, we developed a mouse model of RV-C15 airways disease. RV-C15 was generated from the full-length cDNA clone and grown in HeLa-E8 cells expressing human CDHR3. BALB/c mice were inoculated intranasally with 5 x 106 ePFU RV-C15, RV-A1B or sham. Mice inoculated with RV-C15 showed lung viral titers of 1 x 105 TCID50 units 24 h after infection, with levels declining thereafter. IFN-α, ß, γ and λ2 mRNAs peaked 24-72 hrs post-infection. Immunofluorescence verified colocalization of RV-C15, CDHR3 and acetyl-α-tubulin in mouse ciliated airway epithelial cells. Compared to RV-A1B, mice infected with RV-C15 demonstrated higher bronchoalveolar eosinophils, mRNA expression of IL-5, IL-13, IL-25, Muc5ac and Gob5/Clca, protein production of IL-5, IL-13, IL-25, IL-33 and TSLP, and expansion of type 2 innate lymphoid cells. Analogous results were found in mice treated with house dust mite before infection, including increased airway responsiveness. In contrast to Rorafl/fl littermates, RV-C-infected Rorafl/flIl7rcre mice deficient in ILC2s failed to show eosinophilic inflammation or mRNA expression of IL-13, Muc5ac and Muc5b. We conclude that, compared to RV-A1B, RV-C15 infection induces ILC2-dependent type 2 airway inflammation, providing insight into the mechanism of RV-C-induced asthma exacerbations.

Autocrine production of TGF-beta1 promotes myofibroblastic differentiation of neonatal lung mesenchymal stem cells.

We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. We examined the capacity of MSCs to differentiate into myofibroblasts, cells that participate in lung development, injury, and repair. Gene expression was measured by array, qPCR, immunoblot, and immunocytochemistry. Unstimulated MSCs expressed mRNAs encoding contractile (e.g., ACTA2, TAGLN), extracellular matrix (COL1A1 and ELN), and actin-binding (DBN1, PXN) proteins, consistent with a myofibroblast phenotype, although there was little translation into immunoreactive protein. Incubation in serum-free medium increased contractile protein (ACTA2, MYH11) gene expression. MSC-conditioned medium showed substantial levels of TGF-beta1, and treatment of serum-deprived cells with a type I activin receptor-like kinase inhibitor, SB-431542, attenuated the expression of genes encoding contractile and extracellular matrix proteins. Treatment of MSCs with TGF-beta1 further induced the expression of mRNAs encoding contractile (ACTA2, MYH11, TAGLN, DES) and extracellular matrix proteins (FN1, ELN, COL1A1, COL1A2), and increased the protein expression of alpha-smooth muscle actin, myosin heavy chain, and SM22. In contrast, human bone marrow-derived MSCs failed to undergo TGF-beta1-induced myofibroblastic differentiation. Finally, primary cells from tracheal aspirates behaved in an identical manner as later passage cells. We conclude that human neonatal lung MSCs demonstrate an mRNA expression pattern characteristic of myofibroblast progenitor cells. Autocrine production of TGF-beta1 further drives myofibroblastic differentiation, suggesting that, in the absence of other signals, fibrosis represents the "default program" for neonatal lung MSC gene expression. These data are consistent with the notion that MSCs play a key role in neonatal lung injury and repair.

Ovalbumin sensitization and challenge increases the number of lung cells possessing a mesenchymal stromal cell phenotype.

BACKGROUND: Recent studies have indicated the presence of multipotent mesenchymal stromal cells (MSCs) in human lung diseases. Excess airway smooth muscle, myofibroblasts and activated fibroblasts have each been noted in asthma, suggesting that mesenchymal progenitor cells play a role in asthma pathogenesis. We therefore sought to determine whether MSCs are present in the lungs of ovalbumin (OVA)-sensitized and challenged mice, a model of allergic airways disease. METHODS: Balb/c mice were sensitized and challenged with PBS or OVA over a 25 day period. Flow cytometry as well as colony forming and differentiation potential were used to analyze the emergence of MSCs along with gene expression studies using immunochemical analyses, quantitative polymerase chain reaction (qPCR), and gene expression beadchips. RESULTS: A CD45-negative subset of cells expressed Stro-1, Sca-1, CD73 and CD105. Selection for these markers and negative selection against CD45 yielded a population of cells capable of adipogenic, osteogenic and chondrogenic differentiation. Lungs from OVA-treated mice demonstrated a greater average colony forming unit-fibroblast (CFU-F) than control mice. Sorted cells differed from unsorted lung adherent cells, exhibiting a pattern of gene expression nearly identical to bone marrow-derived sorted cells. Finally, cells isolated from the bronchoalveolar lavage of a human asthma patient showed identical patterns of cell surface markers and differentiation potential. CONCLUSIONS: In summary, allergen sensitization and challenge is accompanied by an increase of MSCs resident in the lungs that may regulate inflammatory and fibrotic responses.

CCR2 Mediates Chronic LPS-Induced Pulmonary Inflammation and Hypoalveolarization in a Murine Model of Bronchopulmonary Dysplasia.

The histopathology of bronchopulmonary dysplasia (BPD) includes hypoalveolarization and interstitial thickening due to abnormal myofibroblast accumulation. Chorioamnionitis and sepsis are major risk factors for BPD development. The cellular mechanisms leading to these lung structural abnormalities are poorly understood. We used an animal model with repeated lipopolysaccharide (LPS) administration into the airways of immature mice to simulate prolonged airway exposure to gram-negative bacteria, focusing on the role of C-C chemokine receptor type 2-positive (CCR2+) exudative macrophages (ExMf). Repetitive LPS exposure of immature mice induced persistent hypoalveolarization observed at 4 and 18 days after the last LPS administration. LPS upregulated the expression of lung pro-inflammatory cytokines (TNF-α, IL-17a, IL-6, IL-1ß) and chemokines (CCL2, CCL7, CXCL1, and CXCL2), while the expression of genes involved in lung alveolar and mesenchymal cell development (PDGFR-α, FGF7, FGF10, and SPRY1) was decreased. LPS induced recruitment of ExMf, including CCR2+ ExMf, as well as other myeloid cells like DCs and neutrophils. Lungs of LPS-exposed CCR2-/- mice showed preserved alveolar structure and normal patterns of α-actin and PDGFRα expression at the tips of the secondary alveolar crests. Compared to wild type mice, a significantly lower number of ExMf, including TNF-α+ ExMf were recruited to the lungs of CCR2-/- mice following repetitive LPS exposure. Further, pharmacological inhibition of TLR4 with TAK-242 also blocked the effect of LPS on alveolarization, α-SMA and PDGFRα expression. TNF-α and IL-17a induced α-smooth muscle actin expression in the distal airspaces of E16 fetal mouse lung explants. In human preterm lung mesenchymal stromal cells, TNF-α reduced mRNA and protein expression of PDGFR-α and decreased mRNA expression of WNT2, FOXF2, and SPRY1. Collectively, our findings demonstrate that in immature mice repetitive LPS exposure, through TLR4 signaling increases lung inflammation and impairs lung alveolar growth in a CCR2-dependent manner.