Orosomucoid like 3 (ORMDL3), a gene localized to chromosome 17q21, has been linked in epidemiologic studies to childhood asthma and rhinovirus (RV) infections. As the single nucleotide polymorphisms linking ORMDL3 to asthma are associated with increased expression of ORMDL3, we have used hORMDL3zp3-Cre mice (which have universal increased expression of human ORMDL3) to determine whether infection of these transgenic mice with RV influences levels of airway inflammation or RV viral load. RV infection of hORMDL3zp3-Cre mice resulted in reduced RV viral load assessed by quantitative real-time PCR (lung and airway epithelium), as well as reduced airway inflammation (total bronchoalveolar lavage cells, neutrophils, macrophages, and lymphocytes) compared with RV-infected wild-type mice. Levels of the antiviral pathways including IFNs (IFN-α, IFN-β, IFN-λ) and RNAse L were significantly increased in the lungs of RV-infected hORMDL3zp3-Cre mice. Levels of the antiviral mouse oligoadenylate synthetase (mOas)1g pathway and RNAse L were upregulated in the lungs of unchallenged hORMDL3zp3-Cre mice. In addition, levels of mOas2, but not mOas1 (mOas1a, mOas1b, mOas1g), or mOas3 pathways were significantly more upregulated by IFNs (IFN-α, IFN-β, IFN-λ) in epithelial cells from hORMDL3zp3-Cre mice compared with RV-infected wild-type mouse epithelial cells. RNAse L–deficient mice infected with RV had increased RV viral load. Overall, these studies suggest that increased levels of ORMDL3 contribute to antiviral defense to RV infection in mice through pathways that may include IFNs (IFN-α, IFN-β, IFN-λ), OAS, and RNAse L.

Orosomucoid like 3 (ORMDL3), a gene localized to chromosome 17q21, has been linked in multiple genome-wide association and non–genome-wide association epidemiologic studies to asthma (1, 2), and has also been linked to the frequency of rhinovirus (RV) wheezing illness in childhood and the subsequent development of childhood asthma (3). Although there are multiple genetic epidemiologic studies linking chromosome 17q21 with asthma (2), there are limited functional studies of ORMDL3 in the lung to better understand how it may contribute to the pathogenesis of asthma precipitated by RV. We have previously reported that in wild-type (WT) mice in vivo, inhalation allergen challenge induces a significant increase in levels of expression of ORMDL3 in airway epithelium and in macrophages (4), cells associated with antiviral pathways that may modulate levels of RV infection. As the single nucleotide polymorphisms (SNP) linking ORMDL3 to asthma and RV infection (3) are associated with increased levels of ORMDL3 expression (5), we used universal ORMDL3 transgenic (TG) mice that express increased levels of human ORMDL3 (6) to determine whether increased levels of expression of ORMDL3 in the lungs in these mice would influence RV viral load, and RV-induced levels of airway inflammation.

From what is currently known of ORMDL3-regulated pathways, RV infection in the context of increased ORMDL3 expression could theoretically either increase airway inflammation [through upregulation of ORMDL3-regulated chemokine pathways (4, 6)], or reduce RV viral load [through ORMDL3 upregulation of antiviral pathways such as oligoadenylate synthetase (OAS) or IFNs (4, 6)] and, as a consequence, reduce airway inflammation. Previous studies have demonstrated that ORMDL3 regulates CXC (CXCL10, CXCL11, IL-8) and CC (CCL20) chemokine pathways in human bronchial epithelium in vitro (4) and in ORMDL3 TG mice in vivo (6). If RV infection of ORMDL3 TG mice triggered chemokine pathways (CC and CXC chemokines) in airway epithelium known to be downstream of ORMDL3 (4, 6), this could result in increased airway inflammation. In contrast, our studies of ORMDL3 transfection into normal human airway epithelial cells demonstrated that increased ORMDL3 induced high levels of antiviral defense pathways including OAS genes (7), which may mediate a reduction in RV viral load and airway inflammation. In humans, the OAS gene family has enzymatically active OAS1, OAS2, and OAS3 (7, 8), as well as OASL, which is devoid of enzymatic activity (7, 8). Our prior studies demonstrated that OAS1, OAS2, and OAS3 were all significantly upregulated in human lung epithelial cells transfected with ORMDL3 (4). The enzymatically active mouse Oas (mOas1a, mOas2, and mOas3) are similar to human OAS (8). OAS catalyze the polymerization of ATP into 2′-5′-linked oligoadenylates, which activate a constitutively expressed latent endonuclease, RNase L, to block viral replication at the level of mRNA degradation (8, 9). OAS1 is upregulated by a wide range of viruses and has been shown to play an antiviral role during viral infections including in studies with the asthma-associated respiratory syncytial virus (RSV) (10). At present it is not known how RV infection of asthmatics with a SNP associated with increased ORMDL3 expression (3) would influence RV viral load or airway inflammation. In this study we have therefore used a mouse model of RV infection to determine whether increased lung expression of ORMDL3 influences RV viral load and levels of airway inflammation.

RV1B was a generous gift of Dr. M. Croft (La Jolla Institute for Allergy and Immunology, La Jolla, CA) (11). The RV1B was propagated in Ohio HeLa cells (European Cell Culture Collection, Porton Down, U.K.) at 33°C, in a humidified, 5% CO2 incubator as described (11). When the full RV1B-induced cytopathic effect developed, HeLa cell lysates were harvested and cellular debris was pelleted by low-speed centrifugation. RV1B in HeLa cell lysates was concentrated and partially purified by centrifugation with a 100,000 m.w. cut-off Centricon filter (2000 rpm at 4°C for 8 h; Millipore) (12, 13). RV1B was titered by plaque assay as described (11). In brief, HeLa cell monolayers inoculated with serial 10-fold dilutions of RV1B were incubated for 3 d at 33°C, and then fixed with 10% formalin. Viral plaques were visualized by staining with crystal violet, counted, and viral titer was expressed as PFU per ml.

hORMDL3zp3-Cre mice on a C57BL/6 background were generated as previously described (6). Female 6–7 wk old hORMDL3zp3-Cre mice, and their WT littermate controls (n = 8 mice per group per experiment; each experiment repeated three times) were lightly anesthetized with isoflurane and inoculated intranasally with 50 μl of RV1B (2.8 × 108 PFU per ml), or an equal volume of sham control. Mice were euthanized 1, 2, or 4 d postinfection. All the mouse experimental protocols described in the 2Materials and Methods were approved by the University of California San Diego Institutional Animal Care and Use Committee.

RV1B viral RNA expression in lung and bronchial epithelial cells was compared with RV1B standard curves and expressed as copies per milligram of RNA using methods previously described (14). In brief, for RNA extractions, lungs were initially snap-frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted from both lungs and bronchial brushing derived airway epithelial cells with RNA STAT-60 (Tel-Test) and reverse transcribed with oligonucleotide and SuperScript II kit (Life Technologies). To study levels of RV1B in airway epithelial cells, epithelial cells were isolated by bronchial brushing as previously described in this laboratory (15). In brief, the bronchial brushing was performed using sterile plastic feeding tubes (Solomon Scientific) modified by removal of the rubber bulb, sanding to create roughness, and autoclaving. The tube was inserted into the right main and left main bronchus. After gentle brushing of each bronchus, tubes containing the brushed epithelial cells were immediately placed in RNA STAT-60 (Tel-Test). We have previously demonstrated that the bronchial brushing epithelial cells are >95% epithelial cells as assessed by ultrastructure, FACS, and quantitative real-time PCR (RT-qPCR) expression of epithelial, but not fibroblast or smooth muscle, genes (15). RT-qPCR for positive-strand viral RNA was conducted using RV1B-specific primers and probes [forward primer, 5′-GTG AAG AGC CSC RTG TGC T-3′; reverse primer, 5′-GCT SCA GGG TTA AGG TTA GCC-3′; probe, 5′-FAM-TGA GTC CTC CGG CCC CTG AAT G-TAMRA-3′ (1113)].

Bronchoalveolar lavage (BAL) fluid was collected by lavaging the lung with 1 ml of PBS via a tracheal catheter (6). Total BAL leukocytes were counted using a hemocytometer. BAL fluid cells were cytospun onto slides, which were then stained with Wright–Giemsa, and differential cell counts performed under a light microscope.

Lungs were processed for immunohistology (paraffin-embedded lung sections) as previously described in this laboratory (4, 6). In brief, lungs were equivalently inflated with an intratracheal injection of the same volume of 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, MO) to preserve the pulmonary architecture. Lung sections were processed for immunohistochemistry to detect neutrophils (anti-mouse neutrophil elastase or species and isotype-matched control Ab; Santa Cruz Biotechnology), CD8+ lymphocytes (anti-mouse CD8 or species and isotype-matched control Ab; GeneTex), or F4/80+ macrophages (anti-mouse F4/80 or species and isotype-matched control Ab; Santa Cruz Biotechnology). The number of peribronchial neutrophils, CD8+ lymphocytes, or macrophages staining positive in the peribronchial space was counted using a light microscope. Results are expressed as the number of peribronchial cells staining positive per bronchiole with a 150–200 mm internal diameter. At least five bronchioles were counted in each slide.

Total RNA was extracted from mouse lungs as described above for quantitation of RV1B mRNA. RT-qPCR to quantitate IFN-α1, IFN-β, and IFN-λ mRNA was performed with TaqMan PCR Master Mix and mouse IFN-α1, IFN-β, and IFN-λ primers (all from Applied Biosystems) as described above for quantitation of RV1B mRNA. Whereas humans express three members of the IFN-λ family, i.e., IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and IFN- λ3 (IL-28B) (16, 17), mice differ from humans in expressing only two IFN-λ isoforms (IFN-λ2, IFN-λ3, but not IFN-λ1) (16, 17). The mouse IFN-λ primers used in this study detected both murine IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B).

Mouse tracheal epithelial cells were obtained by dissection and culture from hORMDL3zp3-Cre mice and WT mice as previously described (18). Cultured tracheal epithelial cells were of >95% purity as assessed by E-cadherin expression on FACS and histologic detection of ciliated epithelial cells. The epithelial cells were incubated at 37°C with 100 ng/ml of IFN-α, IFN-β, or IFN-λ (IFN-λ2 and IFN-λ3 in combination), and the cells were collected for RNA extraction after for 24 h. Total RNA was extracted with RNA-STAT-60 (Tel-Test) and reverse transcribed with oligonucleotide and SuperScript II kit (Life Technologies). RT-qPCR was performed with TaqMan PCR Master Mix and mOas1 (mOas1a, mOas1b, mOas1g), mOas2, and mOas3 primers (all from Life Technology). The relative amounts of transcripts were normalized to those of housekeeping gene (GAPDH) mRNA and compared between the different genes by the ΔΔ cycle threshold method as previously described in this laboratory (4, 6).

RNAse L is constitutively expressed in most cells (9). Total RNA was extracted from mouse lungs of RV-infected WT and RV-infected hORMDL3zp3-Cre mice as described above. RNAse L mRNA in lungs were quantitated by RT-qPCR using TaqMan PCR Master Mix and RNase L primers (all from Applied Biosystems) as described above for quantitation of RV1B mRNA.

RNAse L–deficient mice were generated as previously described (19). RNAse L–deficient mice or WT controls aged 6–7 wk (n = 8 mice per group) were infected with RV1B and lungs processed to determine levels of RV1B mRNA by RT-qPCR as described above. Levels of total BAL cells and BAL differential cell counts were quantitated as described above.

All results are presented as mean ± SEM. A statistical software package (GraphPad Prism, San Diego, CA) was used for the analysis. A t test was used for analysis of two groups. ANOVA analysis was used when more than two groups were compared. A p value <0.05 was considered statistically significant.

RV viral load is maximal in WT mice challenged with RV at day 1 post-RV infection, and RV is not detectable on day 2 or day 4 (Fig. 1). Levels of RV in hORMDL3zp3-Cre mice are significantly reduced compared with WT mice at day 1 in both the lung (p < 0.05) (Fig. 1A), and in bronchial brush epithelial cells (p < 0.005) (Fig. 1B). No RV is detectable at day 2 or day 4 in hORMDL3zp3-Cre mice, results similar to that noted in WT mice (Fig. 1C, 1D). Thus, RV challenge in hORMDL3zp3-Cre mice does not postpone peak viral load to a later timepoint.

FIGURE 1.

RV viral load is reduced in lung and bronchial epithelial cells of RV-infected hORMDL3zp3-Cre mice. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 1, 2, or 4 d postinfection. Levels of RV1B viral load were quantitated in the lungs (A), and bronchial brushing epithelial cells (B) by RT-qPCR. Levels of RV viral load were significantly lower in the lungs (p < 0.05), and the bronchial epithelium (p < 0.005) of hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV. RV was not detected in the lungs of WT or hORMDL3zp3-Cre mice at 2 (C), or 4 d (D), post-RV infection.

FIGURE 1.

RV viral load is reduced in lung and bronchial epithelial cells of RV-infected hORMDL3zp3-Cre mice. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 1, 2, or 4 d postinfection. Levels of RV1B viral load were quantitated in the lungs (A), and bronchial brushing epithelial cells (B) by RT-qPCR. Levels of RV viral load were significantly lower in the lungs (p < 0.05), and the bronchial epithelium (p < 0.005) of hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV. RV was not detected in the lungs of WT or hORMDL3zp3-Cre mice at 2 (C), or 4 d (D), post-RV infection.

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WT mice had a significant increase in the total number of BAL cells (p < 0.0001) (Fig. 2A), BAL neutrophils (p < 0.0001) (Fig. 2B), BAL macrophages (p < 0.005) (Fig. 2C), and BAL lymphocytes (p < 0.05) (Fig. 2D) on day 1 post-RV infection. The total number of BAL cells was significantly lower in hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV (p < 0.0001) (Fig. 2A). In addition, the total number of BAL neutrophils (p < 0.0001) (Fig. 2B), and BAL macrophages (p < 0.001) (Fig. 2C) was significantly lower in hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV.

FIGURE 2.

RV-induced BAL inflammation is reduced in hORMDL3zp3-Cre mice. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 1, 2, or 4 d postinfection. BAL fluid was collected by lavaging the lung with 1 ml of PBS via a tracheal catheter. Total BAL leukocytes were counted using a hemocytometer. BAL fluid cells were cytospun onto slides that were then stained with Wright–Giemsa and differential cell counts performed under a light microscope. One day post-RV infection in WT mice there was a significant increase in the total number of BAL cells (p < 0.0001) (A), BAL neutrophils (p < 0.0001) (B), BAL macrophages (p < 0.005) (C), and BAL lymphocytes (p < 0.05) (D). The total number of BAL cells were significantly lower in hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV (p < 0.0001) (A). In addition, the total number of BAL neutrophils (p < 0.0001) (B), and BAL macrophages (p < 0.001) (C) were significantly lower in hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV. Levels of BAL lymphocytes were significantly lower in hORMDL3zp3-Cre mice compared with WT mice 4 d postinfection with RV (p < 0.001) (D).

FIGURE 2.

RV-induced BAL inflammation is reduced in hORMDL3zp3-Cre mice. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 1, 2, or 4 d postinfection. BAL fluid was collected by lavaging the lung with 1 ml of PBS via a tracheal catheter. Total BAL leukocytes were counted using a hemocytometer. BAL fluid cells were cytospun onto slides that were then stained with Wright–Giemsa and differential cell counts performed under a light microscope. One day post-RV infection in WT mice there was a significant increase in the total number of BAL cells (p < 0.0001) (A), BAL neutrophils (p < 0.0001) (B), BAL macrophages (p < 0.005) (C), and BAL lymphocytes (p < 0.05) (D). The total number of BAL cells were significantly lower in hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV (p < 0.0001) (A). In addition, the total number of BAL neutrophils (p < 0.0001) (B), and BAL macrophages (p < 0.001) (C) were significantly lower in hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV. Levels of BAL lymphocytes were significantly lower in hORMDL3zp3-Cre mice compared with WT mice 4 d postinfection with RV (p < 0.001) (D).

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WT mice infected with RV had a significant increase in BAL neutrophils (peaks on day 1) (Fig. 2A), and a significant increase in BAL lymphocytes (peaks on day 4) (Fig. 2D). At day 2 and day 4 post-RV infection, total BAL cell inflammation, as well as BAL neutrophil and BAL macrophage inflammation were reduced in both hORMDL3zp3-Cre mice and WT mice (Fig. 2A, 2B, 2D). There was no increased BAL inflammation (total cell, neutrophils, macrophages, or lymphocytes) in hORMDL3zp3-Cre mice compared with WT mice at day 2 and day 4 to suggest that ORMDL3 overexpression induced increased BAL inflammation at later timepoints following RV inoculation (Fig. 2). Indeed the number of BAL lymphocytes at 4 d was reduced in hORMDL3zp3-Cre mice compared with WT mice (p < 0.001) (Fig. 2C).

WT mice had a significant increase in the total number of peribronchial neutrophils (p < 0.0001) (Fig. 3A–E), peribronchial F4/80+ macrophages (p < 0.001) (Fig. 3F–J), and peribronchial CD8+ lymphocytes (p < 0.05) (Fig. 3K–O), on day 1 post-RV infection as assessed by immunohistochemistry. The total number of peribronchial neutrophils (p < 0.0001) (Fig. 3A–E), peribronchial F4/80+ macrophages (p < 0.05) (Fig. 3F–J), and peribronchial CD8+ lymphocytes (p < 0.05) (Fig. 3K–O) was significantly lower in hORMDL3zp3-Cre mice compared with WT mice 1 d postinfection with RV.

FIGURE 3.

RV-induced peribronchial inflammation is reduced in lungs of hORMDL3zp3-Cre mice. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 1, 2, or 4 d postinfection. Lung sections were processed for immunohistochemistry to detect neutrophils (anti-mouse neutrophil elastase Ab), CD8+ lymphocytes (anti-mouse CD8 Ab), or macrophages (anti-mouse F4/80 Ab). The number of neutrophils, CD8+ lymphocytes, or macrophages staining positive in the peribronchial space was counted using a light microscope. Results are expressed as the number of peribronchial neutrophils, CD8+ lymphocytes, or macrophages per bronchiole with 150–200 mm internal diameter. At least five bronchioles were counted in each slide. RV infection in WT mice induced a significant increase in the total number of peribronchial neutrophils (p < 0.0001) (AE), peribronchial macrophages (p < 0.05) (FJ), and peribronchial CD8+ lymphocytes (p < 0.05) (KO) 1 d postinfection with RV. The total number of peribronchial neutrophils (p < 0.0001) (A–E) and peribronchial macrophages (p < 0.05) (F–J) were significantly lower in hORMDL3zp3-Cre mice infected with RV compared with WT mice 1 d postinfection with RV. Levels of CD8+ lymphocytes were significantly lower in hORMDL3zp3-Cre mice infected with RV compared with WT mice 1, 2, and 4 d postinfection with RV (p < 0.05) (K–O).

FIGURE 3.

RV-induced peribronchial inflammation is reduced in lungs of hORMDL3zp3-Cre mice. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 1, 2, or 4 d postinfection. Lung sections were processed for immunohistochemistry to detect neutrophils (anti-mouse neutrophil elastase Ab), CD8+ lymphocytes (anti-mouse CD8 Ab), or macrophages (anti-mouse F4/80 Ab). The number of neutrophils, CD8+ lymphocytes, or macrophages staining positive in the peribronchial space was counted using a light microscope. Results are expressed as the number of peribronchial neutrophils, CD8+ lymphocytes, or macrophages per bronchiole with 150–200 mm internal diameter. At least five bronchioles were counted in each slide. RV infection in WT mice induced a significant increase in the total number of peribronchial neutrophils (p < 0.0001) (AE), peribronchial macrophages (p < 0.05) (FJ), and peribronchial CD8+ lymphocytes (p < 0.05) (KO) 1 d postinfection with RV. The total number of peribronchial neutrophils (p < 0.0001) (A–E) and peribronchial macrophages (p < 0.05) (F–J) were significantly lower in hORMDL3zp3-Cre mice infected with RV compared with WT mice 1 d postinfection with RV. Levels of CD8+ lymphocytes were significantly lower in hORMDL3zp3-Cre mice infected with RV compared with WT mice 1, 2, and 4 d postinfection with RV (p < 0.05) (K–O).

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We also quantitated levels of airway inflammation at later timepoints following RV inoculation (2 and 4 d) (Fig. 3) to compare with day 1. WT mice infected with RV had a significant increase in peribronchial neutrophils (peaks on day 1) (Fig. 3E), and a significant increase in BAL lymphocytes (peaks on day 4) (Fig. 3O). At day 2 and day 4 post-RV infection, peribronchial neutrophil (Fig. 3A–E) and peribronchial macrophage (Fig. 3F–J) inflammation were reduced in both hORMDL3zp3-Cre mice and WT mice. There was no increased peribronchial inflammation (neutrophils, macrophages, or CD8+ lymphocytes) in hORMDL3zp3-Cre mice compared with WT mice at day 2 and day 4 postinfection with RV to suggest that ORMDL3 overexpression induced increased peribronchial inflammation at later timepoints following RV inoculation. Indeed, the number of peribronchial CD8+ lymphocytes at 2 and 4 d post-RV infection was reduced in hORMDL3zp3-Cre mice compared with WT mice (p < 0.05) (Fig. 3K–O).

RV1B infection in WT mice induced an increase in lung levels of IFN-α1 (p < 0.05), IFN-β (p < 0.05), and IFN-λ (p < 0.05) as assessed by RT-qPCR (Fig. 4). However, RV1B infection in hORMDL3zp3-Cre mice induced a significantly enhanced increase compared with RV1B-infected WT mice in lung levels of IFN-α1 (232-fold increase versus 7-fold increase) (hORMDL3zp3-Cre RV1B versus WT RV1B) (p < 0.05), IFN-β (143-fold increase versus 16-fold increase) (hORMDL3zp3-Cre RV1B versus WT RV1B) (p < 0.05), and IFN-λ (232-fold increase versus 48-fold increase) (hORMDL3zp3-Cre RV1B versus WT RV1B) (p < 0.05) as assessed by RT-qPCR (Fig. 4).

FIGURE 4.

Increased lung levels of IFN-α1, IFN-β, and IFN-λ in RV1B-infected hORMDL3zp3-Cre mice. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 24 h postinfection. Lungs were processed to detect levels of IFN-α1 (A), IFN-β (B), and IFN-λ (C) mRNA by RT-qPCR. RV1B infection in WT mice induced an increase in lung levels of IFN-α1 (p < 0.05), IFN-β (p < 0.05), and IFN-λ (p < 0.05). RV1B infection in hORMDL3zp3-Cre mice induced a significantly enhanced increase in lung levels of IFN-α1 (p < 0.05), IFN-β (p < 0.05), and IFN-λ (p < 0.05) (RV1B hORMDL3zp3-Cre mice versus RV1B WT mice).

FIGURE 4.

Increased lung levels of IFN-α1, IFN-β, and IFN-λ in RV1B-infected hORMDL3zp3-Cre mice. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 24 h postinfection. Lungs were processed to detect levels of IFN-α1 (A), IFN-β (B), and IFN-λ (C) mRNA by RT-qPCR. RV1B infection in WT mice induced an increase in lung levels of IFN-α1 (p < 0.05), IFN-β (p < 0.05), and IFN-λ (p < 0.05). RV1B infection in hORMDL3zp3-Cre mice induced a significantly enhanced increase in lung levels of IFN-α1 (p < 0.05), IFN-β (p < 0.05), and IFN-λ (p < 0.05) (RV1B hORMDL3zp3-Cre mice versus RV1B WT mice).

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As lung levels of IFN-α, IFN-β, and IFN-λ were significantly increased in RV1B-infected hORMDL3zp3-Cre mice compared with RV1B-infected WT mice, we examined whether hORMDL3zp3-Cre mouse tracheal epithelial cells would have an increased ability to express the antiviral mOas pathway when stimulated in vitro with IFN-α, IFN-β, or IFN-λ. These studies demonstrated that levels of mOas2 increased in WT mouse tracheal epithelial cells stimulated in vitro with IFN-α (p < 0.05) (Fig. 5A), IFN-β (p < 0.05) (Fig .5C), or IFN-λ (p < 0.05) (Fig. 5E) as assessed by RT-qPCR. In WT epithelial cells the level of induction of mOas was greatest with IFN-β (749-fold), compared with IFN-α (130-fold) and IFN-λ (60-fold) (Fig. 5A, 5C, 5E). However, IFN stimulation in vitro of epithelial cells derived from the hORMDL3zp3-Cre mice induced a significantly enhanced increase compared with IFN-stimulated WT mouse epithelial cells in levels of mOas2 when stimulated with IFN-α (399-fold increase versus 130-fold increase) (hORMDL3zp3-Cre versus WT) (p < 0.05), IFN-β (1351-fold increase versus 749-fold increase) (hORMDL3zp3-Cre versus WT) (p < 0.05), or IFN-λ (666-fold increase versus 60-fold increase) (hORMDL3zp3-Cre versus WT) (p < 0.02) as assessed by RT-qPCR (Fig. 5A, 5C, 5E). In contrast to the enhanced mOas2 response to IFNs in hORMDL3zp3-Cre compared with WT mice, there was a similar increase in mOas1 (mOas1a, mOas1b, mOas1g), and mOas3 in response to IFN-α (Fig. 5B), and IFN-β (Fig. 5D) in epithelial cells derived from hORMDL3zp3-Cre compared with WT mice. IFN-λ (Fig. 5F) also induced a similar increase in mOas1b, mOas1g, and mOas3 in epithelial cells derived from hORMDL3zp3-Cre compared with WT mice. However, in contrast to IFN-α and IFN-β, IFN-λ (Fig. 5F) did not increase levels of mOas1a in epithelial cells derived from hORMDL3zp3-Cre compared with WT mice (p < 0.05). Baseline expression of mOas1g (but not mOas1a, mOas1b, mOas2, or mOas3) in tracheal epithelial cells (p < 0.05) (Fig. 5B) and lungs (p < 0.02) (Fig. 5G) of hORMDL3zp3-Cre mice was statistically significantly higher than baseline expression of mOas1g in WT mice (p < 0.05).

FIGURE 5.

Levels of mOas2 in hORMDL3zp3-Cre lung airway epithelial cells are significantly increased upon stimulation with IFN-α, IFN-β, and IFN-λ. Mouse lung epithelial cells derived from WT or hORMDL3zp3-Cre mice were incubated for 24 h in vitro with 100 ng/ml of IFN-α (A and B), IFN-β (C and D), or IFN-λ (E and F). Levels of mOas1 (Oas1a, Oas1b, Oas1g), mOas2, and mOas3 mRNA were quantitated by RT-qPCR. Levels of mOas2 increased in WT mouse lung epithelial cells stimulated with IFN-α (p < 0.05) (A), IFN-β (p < 0.05) (C), or IFN-λ (p < 0.05) (E). IFN stimulation of epithelial cells derived from the lungs of hORMDL3zp3-Cre mice induced a significantly enhanced increase in levels of mOas2 compared with WT mice when stimulated with IFN-α (p < 0.05), IFN-β (p < 0.05), or IFN-λ) (p < 0.05) (hORMDL3zp3-Cre versus WT) (A, C, and E). There was a similar increase in mOas1 (mOas1a, mOas1b, mOas1g), and mOas3 in response to IFN-α (B), IFN-β (D), and IFN-λ (F) in epithelial cells derived from hORMDL3zp3-Cre mice compared with WT mice. Baseline expression of mOas1g (but not mOas1a, mOas1b, mOas2, or mOas3) in epithelial cells (B), and lungs (G) of hORMDL3zp3-Cre mice is higher than baseline expression in WT mice (p < 0.05).

FIGURE 5.

Levels of mOas2 in hORMDL3zp3-Cre lung airway epithelial cells are significantly increased upon stimulation with IFN-α, IFN-β, and IFN-λ. Mouse lung epithelial cells derived from WT or hORMDL3zp3-Cre mice were incubated for 24 h in vitro with 100 ng/ml of IFN-α (A and B), IFN-β (C and D), or IFN-λ (E and F). Levels of mOas1 (Oas1a, Oas1b, Oas1g), mOas2, and mOas3 mRNA were quantitated by RT-qPCR. Levels of mOas2 increased in WT mouse lung epithelial cells stimulated with IFN-α (p < 0.05) (A), IFN-β (p < 0.05) (C), or IFN-λ (p < 0.05) (E). IFN stimulation of epithelial cells derived from the lungs of hORMDL3zp3-Cre mice induced a significantly enhanced increase in levels of mOas2 compared with WT mice when stimulated with IFN-α (p < 0.05), IFN-β (p < 0.05), or IFN-λ) (p < 0.05) (hORMDL3zp3-Cre versus WT) (A, C, and E). There was a similar increase in mOas1 (mOas1a, mOas1b, mOas1g), and mOas3 in response to IFN-α (B), IFN-β (D), and IFN-λ (F) in epithelial cells derived from hORMDL3zp3-Cre mice compared with WT mice. Baseline expression of mOas1g (but not mOas1a, mOas1b, mOas2, or mOas3) in epithelial cells (B), and lungs (G) of hORMDL3zp3-Cre mice is higher than baseline expression in WT mice (p < 0.05).

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There was a statistically significant baseline upregulation of RNAse L mRNA in the lungs of hORMDL3zp3-Cre compared with WT mice prior to RV infection (p < 0.05) (Fig. 6A). Levels of RNAse L quantitated by RT-qPCR were higher in the lungs of hORMDL3zp3-Cre mice infected with RV compared with WT mice infected with RV (p < 0.02) (Fig. 6A).

FIGURE 6.

RV-infected RNase L–deficient mice have increased RV viral load and neutrophil inflammation. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 24 h postinfection. There is a statistically significant baseline upregulation of RNase L mRNA in the lungs of hORMDL3zp3-Cre compared with WT mice prior to RV infection (p < 0.05) (A). Levels of RNase L quantitated by RT-qPCR were significantly higher in the lungs of hORMDL3zp3-Cre mice infected with RV compared with WT mice infected with RV (p < 0.02) (A). WT and RNase L–deficient mice were infected with RV1B and sacrificed 24 h postinfection. Levels of RV viral load as assessed by RT-qPCR were significantly higher in the lungs of RNase L–deficient mice infected with RV compared with WT mice infected with RV (p < 0.05) (B). The total number of BAL cells (p < 0.02) (C) and BAL neutrophils (p < 0.02) (D) was significantly higher in RNase L–deficient mice infected with RV compared with WT mice infected with RV. The levels of BAL macrophages (E) and BAL lymphocytes (F) were similar in RV-infected RNase L–deficient mice and RV-infected WT mice.

FIGURE 6.

RV-infected RNase L–deficient mice have increased RV viral load and neutrophil inflammation. WT and hORMDL3zp3-Cre mice were infected with RV1B and sacrificed 24 h postinfection. There is a statistically significant baseline upregulation of RNase L mRNA in the lungs of hORMDL3zp3-Cre compared with WT mice prior to RV infection (p < 0.05) (A). Levels of RNase L quantitated by RT-qPCR were significantly higher in the lungs of hORMDL3zp3-Cre mice infected with RV compared with WT mice infected with RV (p < 0.02) (A). WT and RNase L–deficient mice were infected with RV1B and sacrificed 24 h postinfection. Levels of RV viral load as assessed by RT-qPCR were significantly higher in the lungs of RNase L–deficient mice infected with RV compared with WT mice infected with RV (p < 0.05) (B). The total number of BAL cells (p < 0.02) (C) and BAL neutrophils (p < 0.02) (D) was significantly higher in RNase L–deficient mice infected with RV compared with WT mice infected with RV. The levels of BAL macrophages (E) and BAL lymphocytes (F) were similar in RV-infected RNase L–deficient mice and RV-infected WT mice.

Close modal

Levels of RV viral load as assessed by RT-qPCR were significantly higher in the lungs of RNAse L–deficient mice infected with RV compared with WT mice infected with RV (p < 0.05) (Fig. 6B).

The total number of BAL cells was significantly higher in RNAse L–deficient mice compared with WT mice infected with RV (p < 0.02) (Fig. 6C). In addition, the total number of BAL neutrophils were significantly higher in RNAse L–deficient mice compared with WT mice infected with RV (p < 0.02) (Fig. 6D). The levels of BAL macrophages (Fig. 6E) and BAL lymphocytes (Fig. 6F) were similar in RV-infected RNAse L–deficient mice and RV-infected WT mice.

This study demonstrates that RV infection of hORMDL3zp3-Cre mice (which have increased levels of expression of human ORMDL3) results in reduced RV viral load in the lung and airway epithelium, as well as reduced airway inflammation (total BAL cells, neutrophils, macrophages, and CD8+ lymphocytes) compared with RV-infected WT mice. The potential pathway(s) by which increased levels of ORMDL3 reduce RV viral load may either be mediated by inhibition of RV viral replication and/or increase in RV viral clearance. Although the mouse model of RV infection we have used induces increases in IFN-α, IFN-β, and IFN-λ suggestive of RV replication, similar mouse models of RV infection have low levels of RV replication (14). As we have measured RV viral load and not RV replication, further studies are needed to determine the relative contribution of inhibition of RV viral replication and/or increase in RV viral clearance to the reduced RV viral load we have detected in hORMDL3zp3-Cre mice. ORMDL3 upregulates levels of the antiviral pathways including IFNs (IFN-α, IFN-β, IFN-λ), RNase L, mOas2, and mOas1g. In particular, RV-challenged hORMDL3zp3-Cre mice had a significantly enhanced lung IFN-α, IFN-β, and IFN-λ response compared with WT mice challenged with RV. IFNs are a family of proteins with antiviral properties that are produced and released in response to viral infections including RV, and function in establishing both an antiviral state in host cells such as epithelial cells infected with RV, and activate immune cells to clear the RV infection. IFNs bind to cell-surface IFN receptors on viral-infected cells to activate antiviral pathways. For example, IFN-α and IFN-β bind to the IFN-α/β receptor complex IFNAR1/2 (20), and IFN-λ to the IL-10Rβ/IL28Rα, which can induce OAS. This catalyzes the polymerization of ATP into 2′-5′-linked oligoadenylates, which activate a constitutively expressed latent endonuclease, RNase L, to cleave viral and cellular ssRNAs, thereby blocking viral replication (Fig. 7). The 2′-5′A-OAS-RNase L pathway is typically latent but is tightly regulated by type I IFNs (including IFN-α and IFN-β) and type III IFNs (including IFN-λ) (21, 22). Active OASs then synthesize 2′-5′ linked oligoadenylates from ATP, which bind to inactive RNase L and, in the presence of ADP or ATP, activates its enzymatic activity (21, 22). Active RNase L then recognizes and cleaves ssRNA, which can result in inhibition of cellular and viral protein synthesis, autophagy, and apoptosis to prevent virus replication (21, 22). Although ORMDL3 induces RNase L, and RNase –deficient mice have increased RV viral load, further study is needed to determine whether hORMDL3zp3-Cre mice reduction in RV viral load is mediated by Oas and RNase L. Future experiments in which hORMDL3zp3-Cre mice are crossed with either RNase L–deficient or OAS–deficient mice and are studied using a mouse protocol with more robust RV viral replication (23) will help to define whether there is role for the ORMDL3-Oas-RNase L pathway in reducing RV viral replication, and/or whether ORMDL3, Oas, and RNase L contribute to RV viral clearance.

FIGURE 7.

ORMDL3, IFN, OAS, and RNase L antiviral pathway. A potential antiviral pathway induced by ORMDL3. ORMDL3 induces expression of both OAS and IFN. ORMDL3 induce OAS (blue arrows 1–2), which activates RNase L (arrow 3) to reduce RV load arrow (arrow 4). ORMDL3 also induces IFN (green arrow; a), which through autocrine pathway binds to its receptor (arrow b) and also activates OAS antiviral pathway (arrow c).

FIGURE 7.

ORMDL3, IFN, OAS, and RNase L antiviral pathway. A potential antiviral pathway induced by ORMDL3. ORMDL3 induces expression of both OAS and IFN. ORMDL3 induce OAS (blue arrows 1–2), which activates RNase L (arrow 3) to reduce RV load arrow (arrow 4). ORMDL3 also induces IFN (green arrow; a), which through autocrine pathway binds to its receptor (arrow b) and also activates OAS antiviral pathway (arrow c).

Close modal

In addition to IFNs activating mOas, our studies have shown that in vitro epithelial cells with increased expression of ORMDL3 exhibit enhanced activation of mOas2 in response to IFNs and may therefore contribute to the RNAse L antiviral pathway. Thus, ORMDL3 may exert antiviral effects by both activating IFNs as well as by IFNs, inducing mOas2 in cells expressing increased levels of ORMDL3 (Fig. 7). Thus, the SNP on chromosome 17q21 associated with increased ORMDL3 expression may have been evolutionarily conserved to function as an innate antiviral defense pathway. At present our studies have only demonstrated that ORMDL3 plays a role in reducing RV viral load and further studies are needed to determine whether ORMDL3 also plays a role in reducing viral load of other important respiratory viruses such as RSV and influenza. Our prior studies demonstrated that human bronchial epithelial cells transfected with ORMDL3 upregulated OAS1, OAS2, and OAS3 (4, 6), whereas current studies demonstrate that incubation of mouse tracheal epithelial cells derived from hORMDL3zp3-Cre mice with IFNs (IFN-α, IFN-β, or IFN-λ) resulted in a significantly enhanced increase in mOas2 compared with WT mice. In contrast, IFNs (IFN-α, IFN-β, or IFN-λ) did not enhance the mOas1 (mOas1a, mOas1b, mOas1g) or mOas3 response of murine tracheal epithelial cells from hORMDL3zp3-Cre mice compared with WT mice. There was also a constitutive increase in mOas1g (but not mOas1a, mOas1b, mOas2, or mOas3) in mouse tracheal epithelial cells, and mouse lungs, derived from hORMDL3zp3-Cre mice compared with WT mice. In addition, there was a statistically significant upregulation of lung RNase L mRNA in hORMDL3zp3-Cre compared with WT mice at baseline, without RV infection. Further studies will need to investigate whether the baseline increase in expression of mOas1g and RNase L in the lungs of hORMDL3zp3-Cre mice enables a very early activation of RNase L and degradation of RV RNA. Of the eight mouse oas1 genes (oas1a-oas1h), only oas1a and oas1g are believed to be catalytically active (7). Mouse oas1b encodes a catalytically inactive OAS but it has an alternative antiviral activity (7, 8, 24). Our studies of ORMDL3 thus demonstrate differences in the ORMDL3 regulation of OAS family members in human (OAS1, OAS2, OAS3) and mouse (mOas2, mOas1g) cells, which may relate to differences in mouse and human OAS amino acid sequence identity and function (mouse Oas1c and human OAS1 share 52% identity; mouse Oas2 and human OAS2 share 61% identity; mouse Oas3 and human OAS3 share 66% identity) (7, 8, 24), in methods used to increase expression of ORMDL3 in human cells (transfection) and mouse cells (hORMDL3zp3-Cre mice), or in the cell types studied (bronchial epithelial cells in humans versus tracheal epithelial cells in mice).

Although our studies in mice demonstrate that RV induces innate IFNs, there is controversy in human studies as to whether asthmatics have a defective innate IFN response. Some (25, 26) but not all studies (2729) suggest that airway epithelial cells derived from asthmatics have a reduced capacity for innate IFN synthesis in response to RV infection compared with normal airway epithelial cells. For example, a deficiency in expression of each of IFN-α, IFN-β, and IFN-λ has been described in asthma (25, 26). However, these deficiencies in expression of IFN-α, IFN-β, and IFN-λ have not been confirmed in all studies of asthmatics (27, 28). Studies have also shown increased levels of IFN-λ in the upper airway of wheezing asthmatic children compared with nonwheezing asthmatic children with an upper respiratory infection (29). In addition, during a viral infection in asthmatics, increased levels of IFN-α, IFN-β, and IFN-λ have been detected, which correlate with more severe asthma symptoms and the presence of an asthma exacerbation (30). In contrast, after the resolution of a viral infection in asthmatics, lower levels of IFN-λ are related to more severe asthma symptoms (30). At present it is not known what accounts for some but not all studies demonstrating a deficient IFN response in asthma. Whether genetics, asthma medications, the timing of measuring IFN responses (i.e., during the infection versus after resolution of the infection), or other factors influence the results in the different studies requires further investigation.

Epidemiologic studies in birth cohorts of children at high risk for the development of asthma have shown that there is a significant gene (chromosome 17q21) and environment (RV infection wheezing illness) interaction in early childhood associated with the development of asthma (3). The combined effect of RV infection in the first 3 y of life with a SNP on chromosome 17q21 was significantly associated with the development of asthma evaluated from age 6 (3). The SNP on chromosome 17q21 was associated with the number of human RV wheezing illnesses in the first 3 y of life, but not with the number of wheezing illnesses from another respiratory virus RSV (3). At present it is unknown whether the RV infection in early childhood is causal in the development of asthma, or only unmasks an underlying propensity to asthma with no direct effect on asthma risk (31). Our study in hORMDL3zp3-Cre mice infected with RV in vivo does not suggest that these mice have a deficient ability to clear RV infection. In addition, hORMDL3zp3-Cre mice infected with RV in vivo did not have more severe airway inflammation that may predispose humans to the development of asthma in childhood. Previous studies of RV infection in childhood have clearly linked the SNP on chromosome 17q21 with the frequency of RV wheezing illness in the first 3 y of life and the development of asthma (3). There are several potential explanations as to why hORMDL3zp3-Cre mice infected with RV in vivo may have less severe RV infection, rather than increased infection as might be predicted based on the human RV and chromosome 17q21 studies. For example, the SNP linking chromosome 17q21 to RV and the development of asthma includes four genes (ORMDL3, GSDMB, IKZF3, ZPB2) and not only ORMDL3 (2). Thus, it might be possible that GSDMB, IKZF3, or ZPB2 rather than ORMDL3 could be the gene contributing to the development of RV-induced wheezing illness and asthma in early childhood. It should also be noted that the mouse model we have used has several differences from the human study linking ORMDL3 to RV-induced wheezing illness and development of asthma. The mouse model evaluated the RV viral load and airway inflammatory response to an acute RV infection. The human RV and chromosome 17q21 genetic epidemiology study examined whether RV infection influenced the development of asthma several years later in children at high risk for the development of asthma who had an RV-induced wheezing illness (not investigated in the mouse study). RV infection also differs in humans and mice in that in mice we have used a model of RV1b infection that gains entry into the cell using the low-density lipoprotein receptor (32), which is not the predominant receptor used by the majority of the >160 RV serotypes (32). In contrast, in humans, RV utilizes three major types of cellular membrane glycoproteins to gain entry into the host cell including ICAM 1 (ICAM-1) (the majority of RV-A and RV-B), low-density lipoprotein receptor family members (12 RV-A types), and CDHR3 (RV-C) (32).

Thus, in summary, in this study we demonstrated that RV infection of hORMDL3zp3-Cre mice resulted in reduced RV viral load (lung and airway epithelium), as well as reduced airway inflammation (total BAL cells, neutrophils, macrophages, and CD8+ lymphocytes) compared with RV-infected WT mice. Levels of the antiviral pathways including IFNs (IFN-α, IFN-β, IFN-λ) and RNase L were significantly increased in the lungs of RV-infected hORMDL3zp3-Cre mice. In addition, levels of the antiviral mOas2 pathway, but not the mOas1 (mOas1a, mOas1b, mOas1g), or mOas3 pathways were significantly more upregulated by IFNs (IFN-α, IFN-β, IFN-λ) in epithelial cells from hORMDL3zp3-Cre mice compared with RV-infected WT mouse epithelial cells. RNase L–deficient mice infected with RV had increased RV viral load, implying an important role for RNase L in reducing RV viral load. Overall, these studies suggest that increased levels of ORMDL3 contribute to antiviral defense to RV infection in mice through pathways that may include IFNs (IFN-α, IFN-β, IFN-λ), OAS, and RNase L.

We acknowledge the generous provision of RNase L–deficient mice by Robert Silverman (Cleveland Clinic Lerner Research Institute, Cleveland).

This work was supported by National Institutes of Health Grants AI 107779, AI 38425, AI 07535, and AI 07469 (to D.H.B.). M.K. was supported by National Institutes of Health Grant T32 AI07469.

Abbreviations used in this article:

     
  • BAL

    bronchoalveolar lavage

  •  
  • mOas

    mouse Oas

  •  
  • OAS

    oligoadenylate synthetase

  •  
  • ORMDL3

    orosomucoid like 3

  •  
  • RSV

    respiratory syncytial virus

  •  
  • RT-qPCR

    quantitative real-time PCR

  •  
  • RV

    rhinovirus

  •  
  • SNP

    single nucleotide polymorphism

  •  
  • TG

    transgenic

  •  
  • WT

    wild-type.

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The authors have no financial conflicts of interest.