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.

Early-life heterologous rhinovirus infections induce an exaggerated asthma-like phenotype.

BACKGROUND: Early-life wheezing-associated respiratory tract infection by rhinovirus (RV) is a risk factor for asthma development. Infants are infected with many different RV strains per year. OBJECTIVE: We previously showed that RV infection of 6-day-old BALB/c mice induces a mucous metaplasia phenotype that is dependent on type 2 innate lymphoid cells (ILC2s). We hypothesized that early-life RV infection alters the response to subsequent heterologous infection, inducing an exaggerated asthma-like phenotype. METHODS: Wild-type BALB/c mice and Rorafl/flIl7rcre mice lacking ILC2s were treated as follows: (1) sham on day 6 of life plus sham on day 13 of life, (2) RV-A1B on day 6 plus sham on day 13, (3) sham on day 6 plus RV-A2 on day 13, and (4) RV-A1B on day 6 plus RV-A2 on day 13. RESULTS: Mice infected with RV-A1B at day 6 and sham at day 13 showed an increased number of bronchoalveolar lavage eosinophils and increased expression of IL-13 mRNA but not expression of IFN-γ mRNA (which is indicative of a type 2 immune response), whereas mice infected with sham on day 6 and RV-A2 on day 13 of life demonstrated increased IFN-γ expression (which is a mature antiviral response). In contrast, mice infected with RV-A1B on day 6 before RV-A2 infection on day 13 showed increased expression of IL-13, IL-5, Gob5, Muc5b, and Muc5ac mRNA; increased numbers of eosinophils and IL-13-producing ILC2s; and exaggerated mucus metaplasia and airway hyperresponsiveness. Compared with Rorafl/fl mice, Rorafl/flIl7rcre mice showed complete suppression of bronchoalveolar lavage eosinophils and mucous metaplasia. CONCLUSION: Early-life RV infection alters the response to subsequent heterologous infection, inducing an intensified asthma-like phenotype that is dependent on ILC2s.

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.

The Innate Cytokines IL-25, IL-33, and TSLP Cooperate in the Induction of Type 2 Innate Lymphoid Cell Expansion and Mucous Metaplasia in Rhinovirus-Infected Immature Mice.

Early-life respiratory viral infection is a risk factor for asthma development. Rhinovirus (RV) infection of 6-d-old mice, but not mature mice, causes mucous metaplasia and airway hyperresponsiveness that are associated with the expansion of lung type 2 innate lymphoid cells (ILC2s) and are dependent on IL-13 and the innate cytokine IL-25. However, contributions of the other innate cytokines, IL-33 and thymic stromal lymphopoietin (TSLP), to the observed asthma-like phenotype have not been examined. We reasoned that IL-33 and TSLP expression are also induced by RV infection in immature mice and are required for maximum ILC2 expansion and mucous metaplasia. We inoculated 6-d-old BALB/c (wild-type) and TSLP receptor-knockout mice with sham HeLa cell lysate or RV. Selected mice were treated with neutralizing Abs to IL-33 or recombinant IL-33, IL-25, or TSLP. ILC2s were isolated from RV-infected immature mice and treated with innate cytokines ex vivo. RV infection of 6-d-old mice increased IL-33 and TSLP protein abundance. TSLP expression was localized to the airway epithelium, whereas IL-33 was expressed in epithelial and subepithelial cells. RV-induced mucous metaplasia, ILC2 expansion, airway hyperresponsiveness, and epithelial cell IL-25 expression were attenuated by anti-IL-33 treatment and in TSLP receptor-knockout mice. Administration of intranasal IL-33 and TSLP was sufficient for mucous metaplasia. Finally, TSLP was required for maximal ILC2 gene expression in response to IL-25 and IL-33. The generation of mucous metaplasia in immature RV-infected mice involves a complex interplay among the innate cytokines IL-25, IL-33, and TSLP.

Rhinovirus infection induces distinct transcriptome profiles in polarized human macrophages.

Infections with rhinovirus (RV) cause asthma exacerbations. Recent studies suggest that macrophages play a role in asthmatic airway inflammation and the innate immune response to RV infection. Macrophages exhibit phenotypes based on surface markers and gene expression. We hypothesized that macrophage polarization state alters gene expression in response to RV infection. Cells were derived from human peripheral blood derived monocytes. M1 and M2 polarization was carried out by using IFN-γ and IL-4, respectively, and RNA was extracted for Affymetrix Human Gene ST2.1 exon arrays. Selected genes were validated by quantitative (q)PCR. Treatment of nonactivated (M0) macrophages with IFN-γ and IL-4 induced the expression of 252 and 153 distinct genes, respectively, including previously-identified M1 and M2 markers. RV infection of M0 macrophages induced upregulation of 232 genes; pathway analysis showed significant overrepresentation of genes involved in IFN-α/ß signaling and cytokine signaling in the immune system. RV infection induced differential expression of 195 distinct genes in M1-like macrophages but only seven distinct genes in M2-like-polarized cells. In a secondary analysis, comparison between M0-, RV-infected, and M1-like-polarized, RV-infected macrophages revealed differential expression of 227 genes including those associated with asthma and its exacerbation. qPCR demonstrated increased expression of CCL8, CXCL10, TNFSF10, TNFSF18, IL6, NOD2, and GSDMD and reduced expression of VNN1, AGO1, and AGO2. Together, these data show that, in contrast to M2-like-polarized macrophages, gene expression of M1-like macrophages is highly regulated by RV.

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.

IFN-γ Blocks Development of an Asthma Phenotype in Rhinovirus-Infected Baby Mice by Inhibiting Type 2 Innate Lymphoid Cells.

Early-life wheezing-associated infections with rhinovirus (RV) have been associated with asthma development in children. We have shown that RV infection of 6-day-old mice induces mucous metaplasia and airways hyperresponsiveness, which is dependent on IL-13, IL-25, and type 2 innate lymphoid cells (ILC2s). Infection of immature mice fails to induce lung IFN-γ expression, in contrast to mature 8-week-old mice with a robust IFN-γ response, consistent with the notion that deficient IFN-γ production in immature mice permits RV-induced type 2 immune responses. We therefore examined the effects of intranasal IFN-γ administration on RV-induced ILC2 expansion and IL-13 expression in 6-day-old BALB/c and IL-13 reporter mice. Airway responses were assessed by histology, immunofluorescence microscopy, quantitative polymerase chain reaction, ELISA, and flow cytometry. Lung ILC2s were also treated with IFN-γ ex vivo. We found that, compared with untreated RV-infected immature mice, IFN-γ treatment attenuated RV-induced IL-13 and Muc5ac mRNA expression and mucous metaplasia. IFN-γ also reduced ILC2 expansion and the percentage of IL-13-secreting ILC2s. IFN-γ had no effect on the mRNA or protein expression of IL-25, IL-33, or thymic stromal lymphoprotein. Finally, IFN-γ treatment of sorted ILC2s reduced IL-5, IL-13, IL-17RB, ST2, and GATA-3 mRNA expression. We conclude that, in immature mice, IFN-γ inhibits ILC2 expansion and IL-13 expression in vivo and ex vivo, thereby attenuating RV-induced mucous metaplasia. These findings demonstrate the antagonistic function of IFN-γ on ILC2 expansion and gene expression, the absence of which may contribute to the development of an asthma-like phenotype after early-life RV infection.

RORα-dependent type 2 innate lymphoid cells are required and sufficient for mucous metaplasia in immature mice.

Early-life wheezing-associated respiratory tract infection by rhinovirus (RV) is considered a risk factor for asthma development. We have shown that RV infection of 6-day-old BALB/c mice, but not mature mice, induces an asthmalike phenotype that is associated with an increase in the population of type 2 innate lymphoid cells (ILC2s) and dependent on IL-13 and IL-25. We hypothesize that ILC2s are required and sufficient for development of the asthmalike phenotype in immature mice. Mice were infected with RV1B on day 6 of life and treated with vehicle or a chemical inhibitor of retinoic acid receptor-related orphan receptor-α (RORα), SR3335 (15 mg·kg-1·day-1 ip for 7 days). We also infected Rorasg/sg mice without functional ILC2s. ILC2s were identified as negative for lineage markers and positive for cluster of differentiation 25 (CD25)/IL-2Rα and CD127/IL-7Rα. Effects of SR3335 on proliferation and function of cultured ILC2s were determined. Finally, sorted ILC2s were transferred into naïve mice, and lungs were harvested 14 days later for assessment of gene expression and histology. SR3335 decreased the number of RV-induced lung lineage-negative, CD25+, CD127+ ILC2s in immature mice. SR3335 also attenuated lung mRNA expression of IL-13, Muc5ac, and Gob5 as well as mucous metaplasia. We also found reduced expansion of ILC2s in RV-infected Rorasg/sg mice. SR3335 also blocked IL-25 and IL-33-induced ILC2 proliferation and IL-13 production ex vivo. Finally, adoptive transfer of ILC2s led to development of asthmalike phenotype in immature and adult mice. RORα-dependent ILC2s are required and sufficient for type 2 cytokine expression and mucous metaplasia in immature mice.

Toll-like receptor 2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation.

BACKGROUND: We have shown that rhinovirus, a cause of asthma exacerbation, colocalizes with CD68+ and CD11b+ airway macrophages after experimental infection in human subjects. We have also shown that rhinovirus-induced cytokine expression is abolished in Toll-like receptor (TLR2)-/- bone marrow-derived macrophages. OBJECTIVE: We hypothesize that TLR2+ macrophages are required and sufficient for rhinovirus-induced airway inflammation in vivo. METHODS: Naive and ovalbumin (OVA)-sensitized and challenged C57BL/6 wild-type and TLR2-/- mice were infected with RV1B, followed by IgG or anti-TLR2, to determine the requirement and sufficiency of TLR2 for rhinovirus-induced airway responses. Bone marrow chimera experiments using OVA-treated C57BL/6 and TLR2-/- mice were also performed. Finally, naive TLR2-/- mice underwent intranasal transfer of bone marrow-derived wild-type macrophages. RESULTS: RV1B infection of naive wild-type mice induced an influx of airway neutrophils and CD11b+ exudative macrophages, which was reduced in TLR2-/- mice. After allergen exposure, rhinovirus-induced neutrophilic and eosinophilic airway inflammation and hyperresponsiveness were reduced in TLR2-/- and anti-TLR2-treated mice. Transfer of TLR2-/- bone marrow into wild-type, OVA-treated C57BL/6 mice blocked rhinovirus-induced airway responses, whereas transfer of wild-type marrow to TLR2-/- mice restored them. Finally, transfer of wild-type macrophages to naive TLR2-/- mice was sufficient for neutrophilic inflammation after rhinovirus infection, whereas macrophages treated with IL-4 (to induce M2 polarization) were sufficient for eosinophilic inflammation, mucous metaplasia, and airways hyperresponsiveness. CONCLUSIONS: TLR2 is required for early inflammatory responses induced by rhinovirus, and TLR2+ macrophages are sufficient to confer airway inflammation to TLR2-/- mice, with the pattern of inflammation depending on the macrophage activation state.

Rhinovirus Delays Cell Repolarization in a Model of Injured/Regenerating Human Airway Epithelium.

Rhinovirus (RV), which causes exacerbation in patients with chronic airway diseases, readily infects injured airway epithelium and has been reported to delay wound closure. In this study, we examined the effects of RV on cell repolarization and differentiation in a model of injured/regenerating airway epithelium (polarized, undifferentiated cells). RV causes only a transient barrier disruption in a model of normal (mucociliary-differentiated) airway epithelium. However, in the injury/regeneration model, RV prolongs barrier dysfunction and alters the differentiation of cells. The prolonged barrier dysfunction caused by RV was not a result of excessive cell death but was instead associated with epithelial-to-mesenchymal transition (EMT)-like features, such as reduced expression of the apicolateral junction and polarity complex proteins, E-cadherin, occludin, ZO-1, claudins 1 and 4, and Crumbs3 and increased expression of vimentin, a mesenchymal cell marker. The expression of Snail, a transcriptional repressor of tight and adherence junctions, was also up-regulated in RV-infected injured/regenerating airway epithelium, and inhibition of Snail reversed RV-induced EMT-like features. In addition, compared with sham-infected cells, the RV-infected injured/regenerating airway epithelium showed more goblet cells and fewer ciliated cells. Inhibition of epithelial growth factor receptor promoted repolarization of cells by inhibiting Snail and enhancing expression of E-cadherin, occludin, and Crumbs3 proteins, reduced the number of goblet cells, and increased the number of ciliated cells. Together, these results suggest that RV not only disrupts barrier function, but also interferes with normal renewal of injured/regenerating airway epithelium by inducing EMT-like features and subsequent goblet cell hyperplasia.

Toward primary prevention of asthma. Reviewing the evidence for early-life respiratory viral infections as modifiable risk factors to prevent childhood asthma.

A first step in primary disease prevention is identifying common, modifiable risk factors that contribute to a significant proportion of disease development. Infant respiratory viral infection and childhood asthma are the most common acute and chronic diseases of childhood, respectively. Common clinical features and links between these diseases have long been recognized, with early-life respiratory syncytial virus (RSV) and rhinovirus (RV) lower respiratory tract infections (LRTIs) being strongly associated with increased asthma risk. However, there has long been debate over the role of these respiratory viruses in asthma inception. In this article, we systematically review the evidence linking early-life RSV and RV LRTIs with asthma inception and whether they could therefore be targets for primary prevention efforts.

Rhinovirus infection induces interleukin-13 production from CD11b-positive, M2-polarized exudative macrophages.

Rhinovirus (RV) causes asthma exacerbations. Previously, we showed that adherent bronchoalveolar cells from allergen-treated mice produce IL-13 when stimulated with RV ex vivo, implicating cells of the monocyte/macrophage lineage in viral-induced airway inflammation. In this study, we hypothesized that RV infection of allergen-treated mice results in IL-13 production by CD11b+ exudative macrophages in vivo. We sensitized and challenged BALB/c mice with ovalbumin (OVA), after which mice were inoculated with RV or sham HeLa cell lysate. After 1 day, lungs were harvested, and cell suspensions were analyzed by flow cytometry. We repeated this process in IL-13 reporter mice, CD11b-DTR mice in which diphtheria toxin selectively depletes CD11b+ cells, and chemokine receptor 2 (CCR2) null mice. We found that lungs of mice infected with RV alone showed increases in CD45+, CD68+, F4/80+, Ly6C+, and CD11b(high) cells, indicating an influx of inflammatory monocytes and exudative macrophages. The combination of OVA and RV had synergistic effects on the exudative macrophage number. However, CD11b+ cells from OVA-treated, RV-infected mice showed M2 polarization, including expression of CD206 and CD301 and production of IL-13. Similar results were obtained in IL-13 reporter mice. Diphtheria toxin depleted CD11b+, IL-13-producing cells in OVA-treated, RV-infected, CD11b-DTR mice, decreasing airway inflammation and responsiveness. CD11b+, Ly6C+ cells were reduced in CCR2 knockout mice. We conclude that, in contrast to naive mice, RV infection of mice with allergic airways disease induces an influx of IL-13-producing CD11b+ exudative macrophages bearing M2 macrophage markers. This finding further implicates alternatively activated macrophages in RV-induced asthma exacerbations.

Nod-like receptor X-1 is required for rhinovirus-induced barrier dysfunction in airway epithelial cells.

UNLABELLED: Barrier dysfunction of airway epithelium may increase the risk for acquiring secondary infections or allergen sensitization. Both rhinovirus (RV) and polyinosinic-polycytidilic acid [poly(I·C)], a double-stranded RNA (dsRNA) mimetic, cause airway epithelial barrier dysfunction, which is reactive oxygen species (ROS) dependent, implying that dsRNA generated during RV replication is sufficient for disrupting barrier function. We also demonstrated that RV or poly(I·C)-stimulated NADPH oxidase 1 (NOX-1) partially accounts for RV-induced ROS generation. In this study, we identified a dsRNA receptor(s) contributing to RV-induced maximal ROS generation and thus barrier disruption. We demonstrate that genetic silencing of the newly discovered dsRNA receptor Nod-like receptor X-1 (NLRX-1), but not other previously described dsRNA receptors, abrogated RV-induced ROS generation and reduction of transepithelial resistance (R(T)) in polarized airway epithelial cells. In addition, both RV and poly(I·C) stimulated mitochondrial ROS, the generation of which was dependent on NLRX-1. Treatment with Mito-Tempo, an antioxidant targeted to mitochondria, abolished RV-induced mitochondrial ROS generation, reduction in R(T), and bacterial transmigration. Furthermore, RV infection increased NLRX-1 localization to the mitochondria. Additionally, NLRX-1 interacts with RV RNA and poly(I·C) in polarized airway epithelial cells. Finally, we show that NLRX-1 is also required for RV-stimulated NOX-1 expression. These findings suggest a novel mechanism by which RV stimulates generation of ROS, which is required for disruption of airway epithelial barrier function. IMPORTANCE: Rhinovirus (RV), a virus responsible for a majority of common colds, disrupts the barrier function of the airway epithelium by increasing reactive oxygen species (ROS). Poly(I·C), a double-stranded RNA (dsRNA) mimetic, also causes ROS-dependent barrier disruption, implying that the dsRNA intermediate generated during RV replication is sufficient for this process. Here, we demonstrate that both RV RNA and poly(I·C) interact with NLRX-1 (a newly discovered dsRNA receptor) and stimulate mitochondrial ROS. We show for the first time that NLRX-1 is primarily expressed in the cytoplasm and at the apical surface rather than in the mitochondria and that NLRX-1 translocates to mitochondria following RV infection. Together, our results suggest a novel mechanism for RV-induced barrier disruption involving NLRX-1 and mitochondrial ROS. Although ROS is necessary for optimal viral clearance, if not neutralized efficiently, it may increase susceptibility to secondary infections and alter innate immune responses to subsequently inhaled pathogens, allergens, and other environmental factors.

Enhancement of Inducible Nitric Oxide Synthase Activity by Low Molecular Weight Peptides Derived from Protamine: A Potential Therapy for Chronic Rhinosinusitis.

Nitric oxide (NO) is a key immune defense agent that is produced from l-arginine in the airways by leukocytes and airway epithelial cells, primarily via inducible nitric oxide synthase (iNOS). Deficiencies in nasal NO levels have been associated with diseases such as primary ciliary dyskinesia and chronic rhinosinusitis. Herein, we demonstrate a proof-of-concept regarding a potential new therapeutic approach for such disorders. We show that arginine-rich low molecular weight peptides (LMWPs) derived from the FDA-approved protamine (obtained from salmon sperm) are effective at significantly raising NO production in both RAW 264.7 mouse macrophage and LA4 mouse epithelial cell lines. LMWP is produced using a stable, easily produced immobilized thermolysin gel column followed by size-exclusion purification. Monomeric l-arginine induces concentration-dependent increases in NO production in stimulated RAW 264.7 and LA4 cells, as measured by stable nitrite in the cell media. In stimulated RAW 264.7 cells, LMWP significantly increases iNOS expression and total NO production 12-24 h post-treatment compared to cells given equivalent levels of monomeric l-arginine. For stimulated LA4 cells, LMWPs are effective in significantly increasing NO production compared to equivalent l-arginine monomer concentrations over 24 h but do not substantially enhance iNOS expression. The use of the arginase inhibitor S-boronoethyl-l-cysteine in combination with LMWPs results in even higher NO production by stimulated RAW 264.7 cells and LA4 cells. Increases in NO due to LMWPs, compared to l-arginine, occur only after 4 h, which may be due to iNOS elevation rather than increased substrate availability.

Neonatal rhinovirus induces mucous metaplasia and airways hyperresponsiveness through IL-25 and type 2 innate lymphoid cells.

BACKGROUND: Early-life human rhinovirus infection has been linked to asthma development in high-risk infants and children. Nevertheless, the role of rhinovirus infection in the initiation of asthma remains unclear. OBJECTIVE: We hypothesized that, in contrast to infection of mature BALB/c mice, neonatal infection with rhinovirus promotes an IL-25-driven type 2 response, which causes persistent mucous metaplasia and airways hyperresponsiveness. METHODS: Six-day-old and 8-week-old BALB/c mice were inoculated with sham HeLa cell lysate or rhinovirus. Airway responses from 1 to 28 days after infection were assessed by using quantitative PCR, ELISA, histology, immunofluorescence microscopy, flow cytometry, and methacholine responsiveness. Selected mice were treated with a neutralizing antibody to IL-25. RESULTS: Compared with mature mice, rhinovirus infection in neonatal mice increased lung IL-13 and IL-25 production, whereas IFN-γ, IL-12p40, and TNF-α expression was suppressed. In addition, the population of IL-13-secreting type 2 innate lymphoid cells (ILC2s) was expanded with rhinovirus infection in neonatal but not mature mice. ILC2s were the major cell type secreting IL-13 in neonates. Finally, anti-IL-25 neutralizing antibody attenuated ILC2 expansion, mucous hypersecretion, and airways responsiveness. CONCLUSIONS: These findings suggest that early-life viral infection could contribute to asthma development by provoking age-dependent, IL-25-driven type 2 immune responses.

Periostin is required for maximal airways inflammation and hyperresponsiveness in mice.

BACKGROUND: Periostin, a secreted extracellular matrix protein, has been localized to deposits of subepithelial fibrosis in asthmatic patients, and periostin levels have been linked to increases in IL-13. OBJECTIVE: We hypothesized that periostin is required for airway inflammatory responses to a physiologic aeroallergen, house dust mite (HDM). METHODS: We studied F4-F6 B6;129-Postn(tm1Jmol)/J wild-type (Postn(+/+)) and null (Postn(-/-)) mice, as well as C57BL/6 mice treated with either IgM or OC-20 periostin neutralizing antibody. Mice were exposed to 5 doses of HDM intranasally over a 16-day period. RESULTS: HDM increased airways responsiveness in Postn(+/+) but not Postn(-/-) mice. In addition, HDM-treated C57BL/6 mice injected with OC-20 had lower airways responsiveness than HDM-treated mice injected with IgM. Compared with Postn(+/+) mice, Postn(-/-) mice showed decreases in HDM-induced inflammation and mucous metaplasia, as well as reduced IL-4, IL-25, CD68, Gob5, and periostin mRNA expression. OC-20 antibody produced similar results. HDM exposure increased periostin expression in the airway epithelium, subepithelium, smooth muscle and inflammatory cells. OC-20 blocked the HDM-induced IgE response, and T cells incubated with dendritic cells (DCs) from Postn(-/-) mice or treated with OC-20 showed deficient DNA synthesis and IL-13 responses compared with T cells incubated with wild-type DCs. Finally, adoptive transfer of bone marrow-derived DCs from Postn(+/+) mice was sufficient to promote allergic responses in F6 Postn(-/-) littermates. CONCLUSIONS: In mice, periostin is required for maximal airways hyperresponsiveness and inflammation after HDM sensitization and challenge. Periostin is required for maximal HDM-induced T-cell responses.