Conceived and designed the experiments: LAR RJS SPA YX ACP JEG RLS. Performed the experiments: LAR RJS SPA YX MRH RLS. Analyzed the data: LAR RJS SPA YX ACP JEG RLS. Contributed reagents/materials/analysis tools: MRH ACP. Wrote the paper: LAR RJS ACP JEG RLS.
The authors have declared that no competing interests exist.
Infections with the picornavirus, human rhinovirus (HRV), are a major cause of wheezing illnesses and asthma exacerbations. In developing a murine model of picornaviral airway infection, we noted the absence of murine rhinoviruses and that mice are not natural hosts for HRV. The picornavirus, mengovirus, induces lethal systemic infections in its natural murine hosts, but small genetic differences can profoundly affect picornaviral tropism and virulence. We demonstrate that inhalation of a genetically attenuated mengovirus, vMC0, induces lower respiratory tract infections in mice. After intranasal vMC0 inoculation, lung viral titers increased, peaking at 24 h postinoculation with viral shedding persisting for 5 days, whereas HRV-A01a lung viral titers decreased and were undetectable 24 h after intranasal inoculation. Inhalation of vMC0, but not vehicle or UV-inactivated vMC0, induced an acute respiratory illness, with body weight loss and lower airway inflammation, characterized by increased numbers of airway neutrophils and lymphocytes and elevated pulmonary expression of neutrophil chemoattractant CXCR2 ligands (CXCL1, CXCL2, CXCL5) and interleukin-17A. Mice inoculated with vMC0, compared with those inoculated with vehicle or UV-inactivated vMC0, exhibited increased pulmonary expression of interferon (IFN-α, IFN-β, IFN-λ), viral RNA sensors [toll-like receptor (TLR)3, TLR7, nucleotide-binding oligomerization domain containing 2 (NOD2)], and chemokines associated with HRV infection in humans (CXCL10, CCL2). Inhalation of vMC0, but not vehicle or UV-inactivated vMC0, was accompanied by increased airway fluid myeloperoxidase levels, an indicator of neutrophil activation, increased MUC5B gene expression, and lung edema, a sign of infection-related lung injury. Consistent with experimental HRV inoculations of nonallergic, nonasthmatic human subjects, there were no effects on airway hyperresponsiveness after inhalation of vMC0 by healthy mice. This novel murine model of picornaviral airway infection and inflammation should be useful for defining mechanisms of HRV pathogenesis in humans.
Infections with the picornavirus, human rhinovirus (HRV), are the most frequent cause of the common cold. However, HRV infections, which usually cause self-limiting upper respiratory tract illnesses, are also the leading cause of virus-induced asthma exacerbations
There is considerable evidence that HRV can infect the lower respiratory tract
There are no known murine rhinoviruses, which has significantly hampered the investigation of the mechanisms governing the inflammatory responses to HRV infection and the subsequent development of airway sequelae. Experimental models using either minor receptor group HRV in wild-type mice or major receptor group HRV in mice that are transgenic for human intercellular adhesion molecule-1 (ICAM-1; CD54), the receptor for major group HRV, have been developed recently
Mice are the natural hosts for mengovirus, a picornavirus whose wild-type form causes infections that are more similar to systemic poliovirus infections than to HRV-induced airway infections
After an intranasal inoculation of 106 plaque-forming units (PFU) of attenuated mengovirus, vMC0, a median of 5.4×103 PFU were detected in whole lung homogenates from mice at 0.1 h postinoculation, with viral titers remaining relatively stable at 1 and 3 h postinoculation (
Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0 [A (n = 7 mice per group), B (n = 6 mice per group)], or 5×106 PFU of HRV-A01a (C; n = 4 mice per group). Lungs were harvested at the indicated times, and viral titers in lung homogenates were determined by plaque assays. Data are the total amount of virus present in the lung homogenates (virus concentrations were multiplied by lung homogenate volumes). No virus was detected in lungs from vehicle-inoculated mice. Data are presented as box plots. For one HRV-A01a-inoculated mouse at 3 h postinoculation, a value of 1 PFU was assigned for graphing purposes because virus was undetectable. ND, not detectable.
The induction of type I and III IFN is a key component of the host response to HRV infection
Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). Levels of (A) IFN-α, (B) IFN-β, and (C) IFN-λ protein in BAL fluid on days 1 and 2 postinoculation were determined by ELISA. Data are the total amount of IFN recovered (ELISA values were multiplied by the BAL fluid volume). IFN-λ protein levels below the limit of detection (dotted line) were assigned a value of 5 pg for graphing purposes. Data are presented as box plots. Veh, vehicle; ND, not detectable. *
Pattern recognition receptors, toll-like receptor (TLR)3 and TLR7, have been implicated as viral RNA sensors in the host response to HRV infection
(A–C) Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). (D–F) Mice were treated with anti-neutrophil mAb or control mAb before receiving intranasal inoculations of 106 PFU of vMC0 or vehicle (n = 5–6 mice per group). Levels of (A, D) TLR3, (B, E) TLR7, and (C, F) NOD2 mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. *
Body weight reduction is a sensitive measure of viral respiratory illness in rodents
Percent change in body weight on days 1 (n = 26–30 mice per group), 2 (n = 18–21 mice per group), 3 (n = 14–16 mice per group), and 5 (n = 9 mice per group) after intranasal inoculation with 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle. Data are presented as box plots. *
Intranasal inoculation of vMC0 induced the recruitment of inflammatory cells into the lower airways. Total numbers of BAL cells were significantly elevated on days 1, 3, and 5 postinoculation in mice inoculated with vMC0 compared with those inoculated with UV-inactivated vMC0 or vehicle (
Numbers of (A) total cells, (B) neutrophils, (C) lymphocytes, and (D) macrophages in the BAL fluid harvested at the indicated times from the lungs of mice inoculated with 106 PFU of vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). Data are presented as box plots. †
Giemsa-stained sections of the lungs from mice intransasally inoculated with (A) vehicle, (B) vMC0 (106 PFU) or (C) an equivalent amount of UV-inactivated vMC0. Lungs were harvested on day 2 postinoculation. Magnification, 20X.
The levels of lung-associated neutrophil-specific granule protein, myeloperoxidase (MPO), were significantly elevated on day 1 postinoculation in vMC0-inoculated, compared with vehicle-inoculated, mice, indicating increased neutrophil recruitment to the lung as a whole rather than just enhanced sequestration of neutrophils into the airspace (
Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle. (A) Lung-associated MPO levels on day 1 postinoculation. MPO levels in lung tissue homogenates from mice inoculated with vehicle or vMC0 were determined by ELISA and normalized to total protein levels (n = 4 mice per group). (B) MPO release into airway fluids. BAL fluid was harvested on the indicated days, and MPO levels were determined by ELISA (n = 6 mice per group). Data are the total amount of MPO recovered (ELISA values were multiplied by the BAL fluid volume). MPO levels below the limit of detection were assigned a value of 1 ng for graphing purposes. (C) CXCR2 and (D) IL-17A expression in the lungs on day 1 postinoculation. Levels of CXCR2 and IL-17A mRNA were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels (n = 6 mice per group). Data are presented as box plots. *
Given the significant neutrophilia induced in the lower airways by inhalation of vMC0, the expression of the mouse neutrophil chemoattractant CXCR2 ligands, CXCL1, CXCL2, and CXCL5, was measured. Levels of mRNA in the lung on day 1 postinoculation (
Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). Levels of (A) CXCL1 (KC), (B) CXCL2 (MIP-2), and (C) CXCL5 (LIX) mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. Levels of (D) CXCL1, (E) CXCL2, and (F) CXCL5 protein in BAL fluid on day 2 postinoculation were determined by ELISA. Data are the total amount of chemokine recovered (ELISA values were multiplied by the BAL fluid volume). CXCL2 protein levels below the limit of detection (dotted line) were assigned a value of 4 pg for graphing purposes. Data are presented as box plots. *
Because HRV infection induces high levels of CXCL10 and CCL2 expression
Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 6 mice per group). Levels of (A) CXCL10 (IP-10) and (B) CCL2 (MCP-1) mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. (C) CCL2 protein levels in BAL fluid on day 2 postinoculation were determined by ELISA. Data are the total amount of CCL2 recovered (ELISA values were multiplied by the BAL fluid volume). ND, not detectable. Data are presented as box plots. *
HRV infection is associated with increased mucin expression
Mice received intranasal inoculations of 106 PFU of attenuated mengovirus, vMC0, or vehicle (n = 5 mice per group). Levels of (A) MUC5B and (B) MUC5AC mRNA in lungs on day 1 postinoculation were determined by real-time quantitative RT-PCR and normalized to β-actin mRNA levels. *
To examine whether the lower airway inflammation induced by vMC0 was associated with lung edema, wet:dry lung weight ratios were measured. Wet:dry lung weight ratios were significantly elevated on day 2 postinoculation in the lungs of mice inoculated with vMC0 as compared with vehicle- and UV-vMC0-inoculated mice, indicating the presence of acute lung injury (
Wet:dry lung weight ratios were measured for lungs harvested on day 2 after intranasal inoculation with 106 PFU of attenuated mengovirus, vMC0, an equivalent amount of UV-inactivated vMC0, or vehicle (n = 9 mice per group). Data are presented as box plots; whiskers indicate the 10th and 90th percentiles. *
To examine whether infection of the lower airways with vMC0 induced changes in pulmonary physiology, mice received intranasal inoculations of vMC0, UV-inactivated vMC0, or vehicle, and pulmonary function was measured on day 2 postinoculation. No significant differences were observed among the groups with regard to respiratory system resistance (Rrs) or the input impedance variables, Newtonian resistance (Rn), tissue viscance (G), and elastance (H), either at baseline or in response to methacholine challenge (
Mice received intranasal inoculations of vehicle, 106 PFU of attenuated mengovirus, vMC0 (n = 6–7 mice per group), or an equivalent amount of UV-inactivated vMC0 (n = 4 mice), and on day 2 postinoculation, pulmonary physiology measurements were obtained after exposure to aerosols of normal saline followed by escalating concentrations of methacholine. Values for respiratory system resistance (Rrs) are presented as the group means ± the standard error. There were no significant differences among the groups.
The establishment of useful and varied small animal models to study HRV pathogenesis continues to be an important goal. Diverse animal models can facilitate different types of mechanistic studies and the development of potential new therapeutic strategies
HRV is a picornavirus family member and viral replication is often most efficient in the natural hosts of a virus. Therefore, we have focused on developing small animal models using a picornavirus, an attenuated form of mengovirus, whose natural hosts are rodents. Previously, we described a rat model in which the attenuated mengovirus, vMC0, caused a respiratory infection in rats with several days of viral shedding accompanied by a neutrophilic lower airway inflammatory response
For both of our vMC0 and HRV-A01a inoculations, the amount of infectious virus detected at 0.1 h postinoculation by plaque assay was substantially lower than the inoculum dose. This discrepancy was probably due to deposition of some of the virus in the nasopharynx and gastrointestinal tract as well as the eclipsing of the virus in the lung during the early stage of the infection. Importantly, the starting levels of virus in the lungs were similar for vMC0 and HRV-A01a, but the viral replication and persistence were greater with vMC0.
.Along with the evidence of notable levels of viral replication and persistent viral shedding in the lower respiratory tract, this mouse model has many features in common with HRV infection in humans. Inhalation of live, but not inactivated vMC0, induced signs of illness such as significant body weight loss, which is a hallmark of viral respiratory illnesses in rodents. Infection of the lower respiratory tract with vMC0 resulted in the stimulation of host antiviral pathways much in the same way as HRV. There was a marked increase in Type I and III IFN proteins in the airway fluids as early as 1 day after virus inoculation, which also provides further evidence of viral replication in the lungs after vMC0 infection. Regulation of type I and III IFN production has been implicated as an important checkpoint in the host response to HRV infection
Recruitment of neutrophils and lymphocytes to the airways are common features of vMC0- and HRV-induced airway infections, and the chemokine milieu that was observed in the lung tissue and airway fluids in the vMC0-infected mice has marked similarities to that observed in HRV infections in humans. Increased pulmonary expression of the neutrophil chemoattractant CXCR2 ligands in response to vMC0 infection is consistent with the increased expression of CXCR2 ligands that is observed in response to HRV infection in humans and in mouse models
There was also evidence of acute airway injury, as shown by the development of lung edema, which might be related to the neutrophilic airway inflammation observed in the lower respiratory tract and the evidence for neutrophil activation, i.e., the release of the neutrophil granule protein MPO in the airway fluids. In addition the vMC0 infection stimulated increased pulmonary expression of MUC5B mRNA. It is not clear why MUC5AC mRNA expression was not increased as well. In humans, HRV infection has been shown to stimulate mucin expression
Similar to what we reported in our rat model of vMC0-induced airway infection and inflammation
A potential limitation of using vMC0 in this mouse model of respiratory infection is that, unlike HRV, mengovirus is neurotropic. However, it is important to note that the HRV, as enteroviruses, are closely related to poliovirus, which is also neurotropic. Inhalation of the attenuated mutant of mengovirus, vMC0, induced a self-limited respiratory infection in rodents, demonstrating the plasticity of vMC0 with regard to its tissue tropism. After intranasal inoculation with vMC0, there was no evidence of systemic infection in homogenates of nonrespiratory organs, as tested by plaque assay (not shown), which was consistent with our previous work in rats
The picornaviral airway infection models in rodents involve a single viral inoculation, which is comparable to experimental HRV inoculation studies in human subjects. An HRV infection can trigger a wheezing illness or asthma exacerbation in children and adults
A comparison between our mengovirus infection model and the HRV infection model in mice
In conclusion, we have developed of a robust model of picornavirus-induced airway infection and inflammation in mice. One of this model's key strengths is that it employs a natural pathogen for rodents, mengovirus, which yields viral replication kinetics and magnitudes closer to natural viral respiratory infections. HRV infection models in mice are proving to be useful for the study of HRV-induced airway inflammation but have a weaker component of viral replication. The mengovirus airway infection model should complement these existing models, and should be especially useful for studies where viral replication is an important outcome.
The mice were housed and all experimental procedures were performed in an American Association for Accreditation of Laboratory Animal Care-accredited laboratory animal facility at the University of Wisconsin School of Medicine and Public Health. The study was approved by the University of Wisconsin School of Medicine and Public Health Animal Care and Use Committee (protocol number M00582) and conformed to the Guide for the Care and Use of Laboratory Animals.
Female BALB/cAnNCr mice were purchased from the National Cancer Institute Animal Production Program (Frederick, MD) and used at 6–10 weeks of age for inoculation studies. The mice were housed in microisolator cages within HEPA-filtered isolation cubicles (Britz & Company, Wheatland, WY).
Recombinant vMC0 has been described previously
Mice were inoculated intranasally with vehicle, 106 PFU of vMC0, 106 PFU equivalents of UV-inactivated vMC0, or 5×106 PFU of HRV-A01a under isoflurane anesthesia. For virus titration, lungs were removed from the chest cavity aseptically and weighed. The lungs were homogenized with an automated tissue homogenizer in an appropriate volume of phosphate-buffered saline (PBS) to yield a 10% w/v homogenate. Cell debris were removed by centrifugation, and the supernates were titered for virus by duplicate plating on HeLa cell monolayers
Purified anti-neutrophil mAb 1A8 (mouse Gr-1/Ly-6G-specific) and isotype-matched control mAb 2A3 (rat IgG2a; <0.18 and <0.63 endotoxin units/mg by Limulus amebocyte lysate test, respectively) were purchased from Bio X Cell (West Lebanon, NH). Anti-neutrophil mAb was administered to each mouse by both intraperitoneal and intranasal routes, which has been previously shown to effectively deplete neutrophils in the respiratory tract
Mice were anesthetized and exsanguinated by severing the dorsal aorta. After opening the chest, BAL was performed. The lungs were lavaged with 800 µl of PBS via a tracheal catheter. The lungs were then lavaged again in the same manner, and the BAL fluid samples were combined. The BAL fluid was centrifuged, and the cell pellet was resuspended in 0.2 ml of PBS. The total number of BAL leukocytes was determined with an automated cell counter (model Z1, Beckman Coulter, Hialeah, FL). Differential cell counts were determined by counting 200 leukocytes on cytospin slides stained with Protocol® Wright-Giemsa stain according to the manufacturer's protocol (Fisher Diagnostics, Middletown, VA). BAL fluid was stored at −80°C. For histological assessments of pulmonary inflammation, mouse lungs were filled with 10% formalin, tied off, removed from the chest cavity, and immersed in 10% formalin for 24 h. The tissue was then processed, embedded in paraffin, and cut into 5 µm sections. Sections were stained with a Giemsa stain.
The levels of specific proteins in the BAL fluid or lung homogenates were measured by enzyme-linked immunosorbent assay (ELISA). Mouse CXCL1 (KC, keratinocyte-derived cytokine)-specific, CXCL2 (MIP-2, macrophage inflammatory protein-2)-specific, and IFN-β-specific ELISA kits with sensitivities of 7.8 pg/ml and Mouse IFN-α-specific kits with a sensitivity of 12.5 pg/ml were purchased from Invitrogen (Carlsbad, CA). Mouse CXCL5 (LIX, lipopolysaccharide-induced CXC chemokine)-specific and IFN-λ-specific ELISA kits with sensitivities of 7.8 pg/ml were acquired from R&D Systems (Minneapolis, MN). Mouse CCL2 (MCP-1, monocyte chemoattractant protein-1)-specific ELISA kits with a sensitivity of 7.8 pg/ml were purchased from BD Biosciences (San Diego, CA). Mouse MPO-specific ELISA kits with a sensitivity of 0.4 ng/ml were obtained from Cell Sciences Incorporated (Canton, MA). Total protein levels in lung homogenates were measured by the Coomassie Plus (Bradford) Protein Assay (Pierce, Rockford, IL).
For RNA isolation, mouse lungs were harvested in an RNase-free manner and immediately flash-frozen in liquid nitrogen. Lungs were then powdered under liquid nitrogen with a chilled mortar and pestle. Total RNA was isolated from frozen lung powder with an RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Valencia, CA). The total RNA was reverse transcribed into cDNA with SuperScript® III reverse transcriptase following the manufacturer's instructions (Invitrogen, Carlsbad, CA). The cDNA product was diluted 1∶2.5 fold and used as the template for quantitative real-time PCR, which was performed using an ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, CA). TaqMan® primer and probe sets for TLR3, TLR7, NOD2, CXCL1, CXCL2, CXCL5, CXCL10 (IP-10, IFN-gamma inducible protein 10), CCL2, MUC5B, MUC5AC, and β-actin were purchased from Applied Biosystems. All values were normalized to the endogenous control, β-actin. Standard curves were generated by serial dilutions of cDNA from the lungs of a vMC0-inoculated mouse and used to determine relative expression levels.
To measure lung edema, wet:dry lung weight ratios were determined. Lungs were removed from the chest cavity and immediately weighed to obtain the wet lung weight. All lungs were place in an oven at 65°C for 4 days and then weighed again to determine the dry lung weight.
Mice were anesthetized with pentobarbital (Abbott, North Chicago, IL), intubated via tracheostomy, paralyzed with succinylcholine HCl (Sigma, St. Louis, MO), and ventilated mechanically (flexiVent, SCIREQ, Montreal, QC, Canada). Aerosol challenges were delivered by the ventilator via an inline nebulizer (Aeroneb, SCIREQ) with aerosolized normal saline being followed by methacholine HCl (Sigma) solutions in concentrations of 1, 3, 10, and 30 mg/ml. After each aerosol challenge, measurements of pulmonary physiology were performed by the flexiVent system, alternating measures of Rrs with measures of input impedance variables (Rn, G, and H). For each variable, the highest value occurring after each aerosol challenge was recorded as the response, referenced to the value obtained after saline challenge.
The numbers of total cells, neutrophils, lymphocytes, and macrophages in the BAL fluid were analyzed by an analysis of variance (general linear model), which was followed by planned pairwise comparisons using Fischer's least significant difference test. A residual analysis was employed to test the adequacy of the models. Pulmonary physiology data were analyzed by an analysis of covariance, using the values from the methacholine dose 30 mg/ml as the dependent variable and baseline (after saline) values as the covariate to account for differences in baselines. Nonparametric tests were used to analyze all other data. The Kruskal-Wallis test was used for comparisons among three or more groups and was followed by planned pairwise comparisons using the Mann-Whitney test. For comparisons between two groups, the Mann-Whitney test was used. Box plots depict the median and the interquartile range between the 25th and 75th percentile, and whiskers show the 10th and 90th percentiles. Analyses were performed using the statistical software package SYSTAT 11.0 (Systat Software, Chicago, IL).
We thank Maria Bulat for technical assistance with viral titer measurements.