CDHR3 Asthma-Risk Genotype Affects Susceptibility of Airway Epithelium to Rhinovirus C Infections.

CDHR3 (cadherin-related family member 3) is a transmembrane protein that is highly expressed in airway epithelia and the only known receptor for rhinovirus C (RV-C). A CDHR3 SNP (rs6967330) with G to A base change has been linked to severe exacerbations of asthma and increased susceptibility to RV-C infections in young children. The goals of this study were to determine the subcellular localization of CDHR3 and to test the hypothesis that CDHR3 asthma-risk genotype affects epithelial cell function and susceptibility to RV-C infections of the airway epithelia. We used immunofluorescence imaging, Western blot analysis, and transmission electron microscopy to show CDHR3 subcellular localization in apical cells, including expression in the cilia of airway epithelia. Polymorphisms in CDHR3 rs6967330 locus (G→A) that were previously associated with childhood asthma were related to differences in CDHR3 expression and epithelial cell function. The rs6967330 A allele was associated with higher overall protein expression and RV-C binding and replication compared with the rs6967330 G allele. Furthermore, the rs6967330 A allele was associated with earlier ciliogenesis and higher FOXJ1 expression. Finally, CDHR3 genotype had no significant effects on membrane integrity or ciliary beat function. These findings provide information on the subcellular localization and possible functions of CDHR3 in the airways and link CDHR3 asthma-risk genotype to increased RV-C binding and replication.

Rhinovirus C targets ciliated airway epithelial cells.

BACKGROUND: The Rhinovirus C (RV-C), first identified in 2006, produce high symptom burdens in children and asthmatics, however, their primary target host cell in the airways remains unknown. Our primary hypotheses were that RV-C target ciliated airway epithelial cells (AECs), and that cell specificity is determined by restricted and high expression of the only known RV-C cell-entry factor, cadherin related family member 3 (CDHR3). METHODS: RV-C15 (C15) infection in differentiated human bronchial epithelial cell (HBEC) cultures was assessed using immunofluorescent and time-lapse epifluorescent imaging. Morphology of C15-infected differentiated AECs was assessed by immunohistochemistry. RESULTS: C15 produced a scattered pattern of infection, and infected cells were shed from the epithelium. The percentage of cells infected with C15 varied from 1.4 to 14.7% depending on cell culture conditions. Infected cells had increased staining for markers of ciliated cells (acetylated-alpha-tubulin [aat], p < 0.001) but not markers of goblet cells (wheat germ agglutinin or Muc5AC, p = ns). CDHR3 expression was increased on ciliated epithelial cells, but not other epithelial cells (p < 0.01). C15 infection caused a 27.4% reduction of ciliated cells expressing CDHR3 (p < 0.01). During differentiation of AECs, CDHR3 expression progressively increased and correlated with both RV-C binding and replication. CONCLUSIONS: The RV-C only replicate in ciliated AECs in vitro, leading to infected cell shedding. CDHR3 expression positively correlates with RV-C binding and replication, and is largely confined to ciliated AECs. Our data imply that factors regulating differentiation and CDHR3 production may be important determinants of RV-C illness severity.

In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

Effects of rhinovirus species on viral replication and cytokine production.

BACKGROUND: Epidemiologic studies provide evidence of differential virulence of rhinovirus species (RV). We recently reported that RV-A and RV-C induced more severe illnesses than RV-B, which suggests that the biology of RV-B might be different from RV-A or RV-C. OBJECTIVE: To test the hypothesis that RV-B has lower replication and induces lesser cytokine responses than RV-A or RV-C. METHODS: We cloned full-length cDNA of RV-A16, A36, B52, B72, C2, C15, and C41 from clinical samples and grew clinical isolates of RV-A7 and RV-B6 in cultured cells. Sinus epithelial cells were differentiated at the air-liquid interface. We tested for differences in viral replication in epithelial cells after infection with purified viruses (10(8) RNA copies) and measured virus load by quantitative RT-PCR. We measured lactate dehydrogenase (LDH) concentration as a marker of cellular cytotoxicity, and cytokine and/or chemokine secretion by multiplex ELISA. RESULTS: At 24 hours after infection, the virus load of RV-B (RV-B52, RV-B72, or RV-B6) in adherent cells was lower than that of RV-A or RV-C. The growth kinetics of infection indicated that RV-B types replicate more slowly. Furthermore, RV-B released less LDH than RV-A or RV-C, and induced lower levels of cytokines and chemokines such as CXCL10, even after correction for viral replication. RV-B replicates to lower levels also in primary bronchial epithelial cells. CONCLUSIONS: Our results indicate that RV-B types have lower and slower replication, and lower cellular cytotoxicity and cytokine and/or chemokine production compared with RV-A or RV-C. These characteristics may contribute to reduced severity of illnesses that has been observed with RV-B infections.

Budesonide and formoterol effects on rhinovirus replication and epithelial cell cytokine responses.

BACKGROUND: Combination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells. METHODS: To test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 µM), formoterol (0.1 µM), both, or neither, and then inoculated with RV-16 (5×10(6) plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFa, IFN-ß, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL-28) were analyzed. RESULTS: RV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFa under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma. CONCLUSIONS: Budesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.

Rhinovirus C causes heterogeneous infection and gene expression in airway epithelial cell subsets.

Rhinoviruses infect ciliated airway epithelial cells, and rhinoviruses' nonstructural proteins quickly inhibit and divert cellular processes for viral replication. However, the epithelium can mount a robust innate antiviral immune response. Therefore, we hypothesized that uninfected cells contribute significantly to the antiviral immune response in the airway epithelium. Using single-cell RNA sequencing, we demonstrate that both infected and uninfected cells upregulate antiviral genes (e.g. MX1, IFIT2, IFIH1, and OAS3) with nearly identical kinetics, whereas uninfected non-ciliated cells are the primary source of proinflammatory chemokines. Furthermore, we identified a subset of highly infectable ciliated epithelial cells with minimal interferon responses and determined that interferon responses originate from distinct subsets of ciliated cells with moderate viral replication. These findings suggest that the composition of ciliated airway epithelial cells and coordinated responses of infected and uninfected cells could determine the risk of more severe viral respiratory illnesses in children with asthma, chronic obstructive pulmonary disease, and genetically susceptible individuals.

Rhinovirus increases Moraxella catarrhalis adhesion to the respiratory epithelium.

Rhinovirus causes many types of respiratory illnesses, ranging from minor colds to exacerbations of asthma. Moraxella catarrhalis is an opportunistic pathogen that is increased in abundance during rhinovirus illnesses and asthma exacerbations and is associated with increased severity of illness through mechanisms that are ill-defined. We used a co-infection model of human airway epithelium differentiated at the air-liquid interface to test the hypothesis that rhinovirus infection promotes M. catarrhalis adhesion and survival on the respiratory epithelium. Initial experiments showed that infection with M. catarrhalis alone did not damage the epithelium or induce cytokine production, but increased trans-epithelial electrical resistance, indicative of increased barrier function. In a co-infection model, infection with the more virulent rhinovirus-A and rhinovirus-C, but not the less virulent rhinovirus-B types, increased cell-associated M. catarrhalis. Immunofluorescent staining demonstrated that M. catarrhalis adhered to rhinovirus-infected ciliated epithelial cells and infected cells being extruded from the epithelium. Rhinovirus induced pronounced changes in gene expression and secretion of inflammatory cytokines. In contrast, M. catarrhalis caused minimal effects and did not enhance RV-induced responses. Our results indicate that rhinovirus-A or C infection increases M. catarrhalis survival and cell association while M. catarrhalis infection alone does not cause cytopathology or epithelial inflammation. Our findings suggest that rhinovirus and M. catarrhalis co-infection could promote epithelial damage and more severe illness by amplifying leukocyte inflammatory responses at the epithelial surface.

Club Cell TRPV4 Serves as a Damage Sensor Driving Lung Allergic Inflammation.

Airway epithelium is the first body surface to contact inhaled irritants and report danger. Here, we report how epithelial cells recognize and respond to aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp., because proteases are critical components of many allergens that provoke asthma. In a murine model, Alp1 elicits helper T (Th) cell-dependent lung eosinophilia that is initiated by the rapid response of bronchiolar club cells to Alp1. Alp1 damages bronchiolar cell junctions, which triggers a calcium flux signaled through calcineurin within club cells of the bronchioles, inciting inflammation. In two human cohorts, we link fungal sensitization and/or asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 is also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium, and calcineurin to initiate allergic sensitization.

Basal cells of differentiated bronchial epithelium are more susceptible to rhinovirus infection.

We used an in vitro model of differentiated tracheobronchial epithelium to analyze the susceptibility of different cell types to infection with rhinoviruses (RVs). Primary cells from control subjects were cultured in an air-liquid interface to form differentiated epithelia. Suprabasal and basal fractions were separated after trypsin digestion, and cell suspensions were infected with serotypes RV16 and RV1A. These cell fractions were analyzed for expression of viral capsid protein VP2 (flow cytometry), viral replication (real-time PCR), cytokeratin-14, and intercellular adhesion molecule-1 (ICAM-1). Compared with suprabasal fraction, basal cells had increased percentages of cells staining positive for VP2 (RV1A: 37.8% versus 9.1%, P < 0.01; RV16: 12.0 versus 3.0%, P < 0.05). The average number of viral RNA copies per cell was also higher in basal cells (2.2- and 2.4-fold increase in RV1A- and RV16-infected cells, respectively) compared with suprabasal cells. Furthermore, ICAM-1 was expressed by 33.3% of basal cells, compared with 8.1% of suprabasal cells (P < 0.05). Finally, in culture models of epithelial injury (detached suprabasal cells or scratched surface), there was significantly greater replication of RV1A compared with intact cell layer. These findings demonstrate that basal cells are more susceptible to RV infection than suprabasal cells. For major group RV, this may be in part due to increased expression of ICAM-1; however, minor group RV also replicated more effectively in basal cells. These results suggest the possibility that epithelial cell differentiation is associated with the maturation of antiviral defense mechanisms.

Serial culture of murine primary airway epithelial cells and ex vivo replication of human rhinoviruses.

Human rhinoviruses (HRV) are the primary etiological agents in cold infections, and represent a serious risk to individuals with chronic respiratory disease such as asthma. In order to develop treatment options for HRV infections, murine models are a crucial component in the study of infection mechanisms due to the wide array of reagents and techniques available to study murine immunology. We present here a cell culture system for studying isolated murine epithelial cell responses to HRV. Monolayers of primary mouse airway epithelial cells were maintained in a serial culture system, and the identity and purity of the cell population was confirmed via immunostaining (positive for cytokeratin, negative for vimentin). Infection of these cells with a minor group rhinovirus (HRV-1A) was evidenced by increases in viral RNA, de novo synthesis of viral proteins, and production of infectious virus. This model will be useful in experiments to define mechanisms of viral replication and host/virus interactions within airway epithelial cells.

Identification of amino acids in the HA of H3 influenza viruses that determine infectivity levels in primary swine respiratory epithelial cells.

In the late 1990s, triple reassortant H3N2 influenza A viruses emerged and spread widely within the swine population of the United States. We have shown previously that an isolate representative of this lineage of viruses, A/Swine/Minnesota/593/99 (Sw/MN), has higher infectivity and accelerated replication kinetics in pigs, compared to a human-lineage H3N2 virus isolated from a pig during the same time period, A/Swine/Ontario/00130/97 (Sw/ONT [Landolt, G.A., Karasin, A.I., Phillips, L., Olsen, C.W., 2003. Comparison of the pathogenesis of two genetically different H3N2 influenza A viruses in pigs. J. Clin. Microbiol. 41, 1936-1941]). Additional in vivo experiments using reverse genetics-generated reassortant viruses demonstrated that these phenotypes are dependent upon the HA and/or NA genes (Landolt, G.A., Karasin, A.I., Schutten, M.M., Olsen, C.W., 2006. Restricted infectivity of a human-lineage H3N2 influenza A virus in pigs is hemagglutinin and neuraminidase gene dependent. J. Clin. Microbiol. 44, 297-301). To further study the infectivity of influenza viruses for pigs, we developed a primary swine respiratory epithelial cell (SREC) culture model. In SRECs, Sw/MN infects a significantly higher number of cells compared to Sw/ONT. Using reverse genetics-generated Sw/MN x Sw/ONT reassortant viruses we demonstrate that the infectivity phenotypes of these viruses in SRECs are strongly dependent upon the HA gene. Using chimeras and point directed mutations within the HA genes, we have identified amino acids that, either alone or in combination with other amino acids, impact infectivity. In particular, amino acid 138 is the dominant factor in determining infectivity levels in SRECs.

Microbial volatile communication in human organotypic lung models.

We inhale respiratory pathogens continuously, and the subsequent signaling events between host and microbe are complex, ultimately resulting in clearance of the microbe, stable colonization of the host, or active disease. Traditional in vitro methods are ill-equipped to study these critical events in the context of the lung microenvironment. Here we introduce a microscale organotypic model of the human bronchiole for studying pulmonary infection. By leveraging microscale techniques, the model is designed to approximate the structure of the human bronchiole, containing airway, vascular, and extracellular matrix compartments. To complement direct infection of the organotypic bronchiole, we present a clickable extension that facilitates volatile compound communication between microbial populations and the host model. Using Aspergillus fumigatus, a respiratory pathogen, we characterize the inflammatory response of the organotypic bronchiole to infection. Finally, we demonstrate multikingdom, volatile-mediated communication between the organotypic bronchiole and cultures of Aspergillus fumigatus and Pseudomonas aeruginosa.

Propagation of rhinovirus-C strains in human airway epithelial cells differentiated at air-liquid interface.

Rhinovirus-C (RV-C) were discovered recently using molecular methods. Classical methods failed to detect them since they could not grow in standard cell culture. The complete genome sequences of several RV-C strains are now available but there is little information about their biological characteristics. HRV-C were first grown in organ culture, and more recently, we developed a system for culturing RV-C strains in differentiated epithelial cells of human airway at air-liquid interface (ALI). These cultures supported efficient replication of RV-C strains as determined by quantitative RT-PCR. This system has enabled study of the biological characteristics of RV-C strains, including quantitative research.

Effects of vitamin D on airway epithelial cell morphology and rhinovirus replication.

Vitamin D has been linked to reduced risk of viral respiratory illness. We hypothesized that vitamin D could directly reduce rhinovirus (RV) replication in airway epithelium. Primary human bronchial epithelial cells (hBEC) were treated with vitamin D, and RV replication and gene expression were evaluated by quantitative PCR. Cytokine/chemokine secretion was measured by ELISA, and transepithelial resistance (TER) was determined using a voltohmmeter. Morphology was examined using immunohistochemistry. Vitamin D supplementation had no significant effects on RV replication, but potentiated secretion of CXCL8 and CXCL10 from infected or uninfected cells. Treatment with vitamin D in the form of 1,25(OH)2D caused significant changes in cell morphology, including thickening of the cell layers (median of 46.5 µm [35.0-69.0] vs. 30 µm [24.5-34.2], p<0.01) and proliferation of cytokeratin-5-expressing cells, as demonstrated by immunohistochemical analysis. Similar effects were seen for 25(OH)D. In addition to altering morphology, higher concentrations of vitamin D significantly upregulated small proline-rich protein (SPRR1ß) expression (6.3 fold-induction, p<0.01), suggestive of squamous metaplasia. Vitamin D treatment of hBECs did not alter repair of mechanically induced wounds. Collectively, these findings indicate that vitamin D does not directly affect RV replication in airway epithelial cells, but can influence chemokine synthesis and alters the growth and differentiation of airway epithelial cells.

Biological characteristics and propagation of human rhinovirus-C in differentiated sinus epithelial cells.

Information about the basic biological properties of human rhinovirus-C (HRV-C) viruses is lacking due to difficulties with culturing these viruses. Our objective was to develop a cell culture system to grow HRV-C. Epithelial cells from human sinuses (HSEC) were differentiated at air-liquid interface (ALI). Differentiated cultures supported 1-2 logs growth of HRV-C15 as detected by quantitative RT-PCR. Two distinguishing features of HRVs are acid lability and optimal growth at 33-34 °C. We used this system to show that HRV-C15 is neutralized by low pH (4.5). In contrast to most HRV types, replication of HRV-C15 and HRV-C41 was similar at 34 and 37°C. The HSEC ALI provides a useful tool for quantitative studies of HRV-C replication. The ability of HRV-C to grow equally well at 34°C and 37°C may contribute to the propensity for HRV-C to cause lower airway illnesses in infants and children with asthma.

Allele-specific targeting of microRNAs to HLA-G and risk of asthma.

HLA-G is a nonclassic, class I HLA molecule that has important immunomodulatory properties. Previously, we identified HLA-G as an asthma-susceptibility gene and discovered that the risk of asthma in a child was determined by both the child's HLA-G genotype and the mother's affection status. Here we report a SNP in the 3' untranslated region of HLA-G that influences the targeting of three microRNAs (miRNAs) to this gene, and we suggest that allele-specific targeting of these miRNAs accounts, at least in part, for our earlier observations on HLA-G and the risk of asthma.

Serum and low-density lipoprotein enhance interleukin-8 secretion by airway epithelial cells.

Viral respiratory infections rapidly increase vascular permeability, which leads to the transudation of serum proteins into airway secretions and tissues. To determine whether this process activates airway epithelial cells, bronchial epithelial cells were incubated with serum, and interleukin (IL)-8 secretion and gene expression were examined. As little as 0.1% serum significantly enhanced IL-8 secretion, and maximal secretion (65 +/- 4 ng/ml, 48 h) was observed with 10% serum. Low-density lipoprotein, but not albumin or immunoglobulin G, augmented bronchial epithelial IL-8 secretion, which was partially blocked by a monoclonal antibody specific for the low-density lipoprotein receptor. The IL-8-inducing activity of plasma was also augmented by clotting and platelet activation. Mechanistically, serum activated nuclear factor-kappaB and increased the stability and steady state levels of IL-8 mRNA. In summary, specific components of serum are potent activators of IL-8 mRNA and secretion, and the increased IL-8 production is likely to be a result of both increased transcription and mRNA stability. This effect may represent an innate mechanism for the recruitment of neutrophils to the airway in response to noxious stimuli, such as viral infections, that increase vascular permeability.

CD14-dependent activation of NF-kappaB by filarial parasitic sheath proteins.

Tropical pulmonary eosinophilia is in part caused by the hyperimmune responsiveness of the lung tissue against the antigens of degenerating microfilariae. We have previously shown that the activation of the transcription factor NF-kappaB is essential for the synthesis and release of multiple pro-inflammatory cytokines in HEp-2 human airway epithelial cells following exposure to filarial parasitic sheath proteins (FPS). Neither the antigenic component nor the receptor involved in this activation is known. Herein we provide evidence that FPS activation of NF-kappaB can be augmented by the cell surface expression of CD14. CD14 expression, however, is not sufficient to transduce FPS signals for NF-kappaB activation, since its expression in different cell types does not always furnish the capacity to respond to FPS. We also show that NF-kappaB activation by FPS treatment can be distinguished from that induced by bacterial lipolysaccharide, an agent that can also activate NF-kappaB in a CD14-dependent fashion. These observations suggest that the capacity of certain lung epithelial cells to interact with microfilarial antigens, activate NF-kappaB in a CD14-dependent manner and produce pro-inflammatory cytokines may be a contributory factor to immune responses manifested by tropical pulmonary eosinophilia.

Double-stranded RNA induces the synthesis of specific chemokines by bronchial epithelial cells.

Virus-induced secretion of proinflammatory chemokines (e.g., regulated on activation, normal T cells expressed and secreted [RANTES], interleukin [IL]-8) by airway epithelial cells helps to initiate antiviral responses and airway inflammation by enhancing inflammatory cell recruitment. To define mechanisms for virus-induced chemokine secretion, monolayers of nontransformed bronchial epithelial cells were transfected or incubated with polydeoxyinosinic-deoxycytidylic acid (synthetic double-stranded [ds] RNA), rhinovirus dsRNA, or single-stranded RNA (ssRNA), and the secretion of selected chemokines was determined. Transfection or incubation with dsRNA, but not ssRNA, significantly enhanced secretion of RANTES and IL-8, but not eotaxin or macrophage inflammatory protein-1alpha. Mechanistically, dsRNA induced and activated dsRNA-dependent protein kinase (PKR), and activated nuclear factor-kappaB and p38 mitogen-activated protein kinase. Furthermore, the PKR inhibitor 2-aminopurine significantly blocked dsRNA-induced RANTES and IL-8 secretion, whereas the p38 mitogen-activated protein kinase inhibitor SB203580 suppressed dsRNA-induced IL-8, but not RANTES. These findings indicate that dsRNA selectively induce the secretion of chemokines such as IL-8 and RANTES, and implicate dsRNA-sensitive signaling proteins in this process. Moreover, these data suggest that this may be an important mechanism for the selective secretion of chemokines by viruses (e.g., rhinovirus, respiratory syncytial virus, influenza) that synthesize dsRNA during replication.