Respiratory enterovirus (like parainfluenza virus) can cause chronic lung disease if protection by airway epithelial STAT1 is lost

Abstract

Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell capacity for IFN signal transduction is presumed to protect against respiratory viral infection. However, it has been difficult to fully test these concepts given the absence of tools to analyze IFN signaling specific to airway epithelial cells in vivo. To address these issues, we generated a new line of transgenic mice with Cre-driver genes (Foxj1 and Scgb1a1) for a floxed-Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1^f/f mice) to target the master IFN-signal regulator STAT1 in airway epithelial cells and tested these mice for control of infection due to mouse parainfluenza (Sendai) virus (SeV) and human enterovirus (EV)-D68. Indeed, both types of infections showed increases in viral titers and severity of acute illness in Foxj1-Scgb1a1-Cre-Stat1^f/f mice and conventional Stat1^−/− mice compared to wild-type mice. In concert, the chronic lung disease that develops after SeV infection was also increased in Foxj1-Scgb1a1-Cre-Stat1^f/f and Stat1^−/− mice, marked by airway and adjacent parenchymal immune cell infiltration and mucus production for at least 7 wk after infection. Unexpectedly, relatively mild EV-D68 infection also progressed to chronic lung disease in Foxj1-Scgb1a1-Cre-Stat1^f/f and Stat1^−/− mice but was limited (like viral replication) to airways. The results thereby provide proof-of-concept for a critical role of barrier epithelial cells in protection from acute illness and chronic disease after viral infection and suggest a specific role for airway epithelial cells given the limitation of EV-D68 replication and acute and chronic manifestations of disease primarily to airway tissue.

Introduction

Normal function of interferon signal transduction is generally accepted as a requirement for defense against viral infections in general, and respiratory viral infections in particular (1, 2). This proposal is often based on the function of the STAT1 transcription factor that is essential for each type of IFN signaling pathway. For example, STAT1-similar to IFNR-deficiencies in transgenic mouse models results in a marked increase in susceptibility to viral infection (3–6), including respiratory viral infection (7, 8). The central role of STAT1 in anti-viral defense is further highlighted by observations that viruses often selectively target STAT1 function to subvert the immune response (9). Moreover, molecular enhancement of STAT1 function can provide increased protection against viral infection compared to the normal physiological settings of the IFN signaling system in cell and mouse models (10). In addition, susceptibility to viral infection is also markedly increased in humans with mutations that inactivate STAT1 gene function (11–13). Together, the critical nature of STAT1 function for anti-viral host defense is well established. What is less certain is the cellular site of action for STAT1 in the setting of infection and the implications for STAT1 function beyond the time of the acute infectious illness.

In that regard, another conventional concept for the immune system is the role of barrier epithelial cells in host defense against viral infection and the special role of airway epithelial cells in defense against respiratory viral infection. Here again, some of the best evidence for this concept was gained from loss-of-function mouse models. In particular, conventional Stat1^−/− mice show increased susceptibility to severe infection with mouse parainfluenza virus also known as Sendai virus (SeV), and further, Stat1^−/− mice that are reconstituted with wild-type bone marrow cells indicate that STAT1-expressing stromal cells rather than immune cells protect mice against acute illness, marked by weight loss and viral replication after this type of infection (7). Given that SeV infection can be readily detected in airway epithelial cells, these experiments provided initial evidence for these cells to account for the stromal cell function of STAT1 as a control point for respiratory viral infection. However, the experimental tools for this study were not sufficient to localize protection precisely to epithelial cells versus other stromal cells or even further to the airway epithelial cell subset since bone marrow chimeras do not provide epithelial cell-specific targeting and SeV infection is not likely to be restricted to airway epithelial cells. In addition, this initial study did not assess the development of chronic lung disease that might develop after clearance of infectious virus in relation to epithelial cells or any other cell type. This issue of long-term post-viral disease is vital to understanding how acute viral infections might initiate or exacerbate chronic airway disease, including the link between viral infection and asthma, chronic obstructive pulmonary disease (COPD), and asthma-COPD overlap syndrome in humans and whether or not the connection includes host defense compromise such as deficiencies of IFN production and/or signaling function (14, 15).

To address these issues, the present experiments were initiated to define the phenotype for STAT1 deficiency specifically in barrier epithelial cells that might serve as the primary host cells during viral infection. Within this cell population in the airway epithelium, specific candidates for viral host cells include ciliated cells that can be marked with Foxj1 gene expression and club cells with Scgb1a1 gene expression (16, 17). Accordingly, we generated a new line of transgenic mice with airway epithelial cell Cre-driver genes (Foxj1 and Scgb1a1) for a floxed-Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1^f/f mice) to determine whether STAT1 function in airway epithelial cells was required for controlling respiratory infection with SeV. We also extended this mouse model to studies of a human viral pathogen in the form of enterovirus D68 (EV-D68), a member of the Picornaviridae family that (unlike other enteroviruses) shares characteristics with human rhinovirus (RV) members of this family. In concert with shared viral features, EV-D68 can also cause respiratory infection, and this infection can result in severe illness in susceptible populations, particularly asthmatics (18–23) as well as acute flaccid myelitis in other patients, based possibly on neuronal receptor binding (24). The severity of EV-D68-driven illness might be based on viral capacity to subvert the IFN-based immune response (25, 26), raising the possibility that any further IFN-deficiency might no longer influence the infection. In addition, to our knowledge, EV-D68 infection is limited to an acute illness. Thus, EV-D68 unlike SeV (27–30) and influenza A virus (31) has not yet been linked to the development of chronic disease that persists after clearance of infectious virus, particularly long-term respiratory disease.

Here we also aimed to define the relationship of STAT1 function and any change in acute illness to the development of chronic lung disease as a model of viral induction, exacerbation, and/or progression of the chronic airway disease. As noted above, this issue is particularly relevant to the virus-linked airway disease found in humans with asthma, COPD, or asthma-COPD overlap syndrome (32, 33). Accordingly, we focused on the cardinal features of this type of disease, i.e., airway inflammation (marked by immune cell infiltration), mucus production, (marked by mucin Muc5ac expression), hyperreactivity (marked by baseline and methacholine-induced lung resistance), and fibrosis (marked by trichrome staining) as relevant endpoints that might develop as a consequence of viral infection. We were particularly interested in airway mucus production since it appears to be linked to the earliest decline in lung function (34) and later exacerbation, progression, and death in asthma (35–39) and COPD (40–42) and to viral reprogramming of airway progenitor epithelial cells (APECs) towards mucous cells (32). Our results indicate that barrier epithelial cell capacity for STAT1 function is critical to protect against acute illness and the subsequent chronic lung disease that develops after both SeV and EV-D68 infections. The findings thereby provide proof-of-concept for the key role of barrier epithelial cells in the short-term and long-term host responses to viral infection, linking severity of acute infection to the development of chronic disease. In addition, limitation of EV-D68 infection to airway epithelial cells suggests that STAT1 protects at the level of airway versus alveolar epithelial barrier cells in the case of disease confined to airways.

Materials and Methods

Mice

Wild-type C57BL/6J mice (000664) and Stat1^−/− mice (012606) mice were obtained from The Jackson Laboratory. To generate mice with Stat1-flox gene expression (Stat1^f/f), C57BL/6J genomic tail DNA was used to generate a 1.6-kb genomic DNA fragment containing Stat1 exons 10–12, a 1.6-kb fragment upstream of exon 10 containing a loxP sequence as the left arm, and a 2.5 kb fragment downstream of exon 12 as the right arm using PCR-based amplification with primers derived from MGI:103063 and defined in Supplemental Table I. The Stat1 targeting vector was assembled in a pSVloxfrtneofrt plasmid containing one loxP site and a Frt-flanked PGK promoter-driven neo gene. The homologous regions of the final vector consisted of a 1.6 kb genomic fragment, one loxP, exon 10, 11 and 12, loxP-frt-neo-frt and 2.5 kb genomic DNA fragment containing exon 13. The vector was transfected into C57BL/6-LacZ embryonic stem cells (B6/BLU, ATCC SCRC-1019), and positive cell clones were microinjected into foster C57BL/6J mice to produce chimeric mice that were bred with C57BL/6J mice for germline transmission of the Stat1-flox allele based on LacZ gene expression detected with X-gal staining of RBCs as described previously (43, 44). The frt-flanked neo gene cassette was removed by breeding to Flp-deleter mice (005703), and offspring were mated with wild-type C57BL/6J mice and screened for absence of the Flp transgene and presence of two loxP sites. Heterozygotes were bred to obtain homozygous Stat1^f/f mice that were used to prepare genomic DNA that was digested with restriction enzymes, size-separated on a 0.8% agarose Tris-Acetate-EDTA (pH 8.0) gel, and transferred onto a Biodyne nylon membrane (Pall Life Sciences) for Southern blot with 820- and 500-bp P³²-radiolabeled fragments immediately upstream of the left arm and downstream of the right arm, respectively, to validate homologous recombinants. The Stat1^f/f mice were bred to CAG-Cre mice (019099) to generate CAG-Cre-Stat1^f/f mice that were used to determine STAT1 expression across mouse tissues. The Foxj1-Cre mice (44) (to target ciliated cells) and Scgb1a1-Cre mice (45) (to target primarily club cells) were crossed to ROSA26^mTmG reporter mice (007576) to define Cre-mediated recombination efficiency. Finally, Foxj1-Scgb1a1-Cre mice were crossed to Stat1^f/f mice to generate Foxj1-Scgb1a1-Cre-Stat1^f/f mice for studies of viral infection. All mice were maintained on a C57BL/6J background and were co-housed in a barrier facility using cages fitted with micro-isolator lids. Animal husbandry and experimental procedures were approved by the Animal Studies Committees of Washington University School of Medicine in accordance with the guidelines from the National Institutes of Health.

Tissue staining and microscopy

For Cre-recombination experiments, lungs from Foxj1-Cre-ROSA26^mTmG and Scgb1a1-Cre-ROSA26^mTmG were fixed with 10% formalin, embedded in paraffin, cut into 5-μm sections, and adhered to charged slides. Sections were deparaffinized in Fisherbrand CitroSolv (Fisher), hydrated, and treated with heat-activated antigen unmasking solution (Vector Laboratories). Immunostaining was performed using primary chicken anti-GFP antibody (Abcam), mouse anti-Foxj1 mAb from S. Brody (Washington University) (46), or goat anti-Scgb1a1 Ab (sc-9772, Santa Cruz). Primary Ab was detected with secondary Ab labeled with Alexa Fluor 594 for Foxj1 and Scgb1a1 and with Alexa Fluor 488 for GFP. Co-staining for GFP and Sftpc was performed with lung tissue that was cryopreserved in OCT compound (Tissue-Tek, Sakura Fine Tek, Torrance, CA) and then cut into 6-μm-thick sections as described previously (44), and primary rabbit anti-Sftpc Ab (ab40879, Abcam) and Alexa Fluor 647-labeled Ab (Thermo Fisher Scientific). All sections were stained with DAPI and were imaged using an Olympus BX51 microscope with a charge-coupled device camera interfaced to MagniFire software from Olympus (Melville, NY) for conventional imaging or a Zeiss LSM 880 laser scanning microscope for confocal imaging. Staining was quantified in whole lung sections using a NanoZoomer S60 slide scanner (Hamamatsu) and Image J software (47).

For viral infection experiments, immunostaining was performed using primary mouse anti-Stat1 Ab (610116, BD Biosciences), chicken anti-SeV Ab (10100648, Charles River Laboratories), mouse anti-β-Tubulin IV mAb (T7941, Sigma), goat anti-Scgb1a1 Ab (sc-9772, Santa Cruz), rabbit anti-Sftpc Ab (ab40879, Abcam), rabbit anti-EV-D68 VP1 Ab (GTX132313, GeneTex), mouse anti-MUC5AC mAb (clone 45M1, MA 5–12175, Thermo Fisher Scientific), and rabbit anti-human CLCA1 Ab (aa 33–63). Primary Abs were detected with secondary Abs labeled with Alexa Fluor 488 (Thermo Fisher Scientific) or Alexa Fluor 594-labeled Ab (Thermo Fisher Scientific) followed by DAPI staining and immunofluorescence microscopy. Sections were also stained with hematoxylin and eosin or PAS and hematoxylin. PAS⁺ staining was quantified with settings specific for the purple-magenta-tone of mucins, Muc5ac⁺ immunostaining was quantified with a setting specific for brown tone of mucins, and Clca1⁺ and Muc5ac⁺ immunostaining were quantified based on fluorescence signal, each using whole lung sections and the image analysis system described above.

Virus preparation and infection

SeV was obtained from ATCC (Sendai/52 Fushimi strain, ATCC VR-105) and prepared and titered by plaque-forming assay as described previously (28). EV-D68 was identified in a clinical sample of bronchoalveolar lavage fluid with PCR and gene sequencing and was then used as a template to generate four overlapping fragments that covered the entire viral genome with RT-PCR. The fragments were cloned into a shuttle vector, and plasmid clones corresponding to the established genome sequence were placed downstream of the T7 RNA polymerase promoter and hammerhead ribozyme to produce transcripts with accurate 5’-ends (48) and poly(A) tail at the 3’end of the complete EV-D68 genome. The full-length EV-D68 genome contained identical nucleotide sequence to EV-D68 detected in the clinical sample. To generate infectious virus, the full-length genome plasmid was linearized by NsiI restriction enzyme digestion, and the RNA was transcribed in vitro using the MEGAscript kit (Invitrogen, CA). The DNA template was eliminated by treatment of the transcription mixture with TRIzol (Invitrogen, CA) and two rounds of DNase treatment and RNeasy (QIAgen) columns. HeLa cells were transfected with the transcribed RNA (3μg) by electroporation using GenePulser Xcell (Bio-Rad, CA). Electroporated cells were placed in a P100 dish and harvested at 24 h post-electroporation. Virus was purified by centrifugation through a sucrose cushion as described previously (49) and viral stocks were quantified using PCR assay for viral RNA and plaque-forming assay for adapted virus that is cytopathic in RD cells (ATCC CCL-136) to define equivalent PFU (ePFU) values as described previously (50).

Mice were infected with SeV (5 × 10⁵ viruses based on qPCR assay for viral RNA equivalent to 1 × 10⁵ PFU based on plaque-forming assay for infectious virus) or EV-D68 (1.5 × 10⁸ viruses equivalent to 0.5 × 10⁶ ePFU). Dosing was performing intranasally using SeV or EV-D68 in 30 μl of PBS or an equivalent amount of UV-inactivated virus (as a control for viral replication) or PBS alone under ketamine/xylazine anesthesia at 6–9 wk of age for SeV or 4 wk of age for EV-D68. Mouse age for SeV was based on previous work (27–30) and for EV-D68 was aimed at using younger but still adult mice to potentially increase susceptibility to infection just as done in other recent reports (51–53). Our approach thereby enables direct comparison across publications. Moreover, lung titers of EV-D68 RNA were no different for infection at 4 wk versus 6–9 wk of age for wild-type or Scgb1a1-Foxj1-Cre-Stat1^f/f mice (data not shown). Results from male and female mice were pooled since no significant differences were found between sexes as reported initially for SeV (54) and confirmed for SeV and EV-D68 in preliminary studies (data not shown). Mouse lungs were frozen at −70 °C for homogenization in PBS with a cell disrupter (Mini-Beadbeater-96, Biospec Products) followed by viral plaque-forming assay to track PFU level normalized to gm of lung tissue (recognizing that one mouse lung weighs approximately 100 mg) (55). Viral titers for stock solutions and lung infections were monitored by PCR assay for EV-D68 and SeV using primers defined in Supplemental Table I.

RNA analysis

Total RNA was isolated from lung tissue using the RNeasy Plus Mini kit (Qiagen) and converted to cDNA using the High-Capacity cDNA Archive kit (Life Technologies) and random hexamer primers (Applied Biosystems). Levels of viral RNA were monitored using a real-time qPCR assay for EV-D68 capsid protein (VP1) gene or SeV nucleoprotein (NP) gene with forward and reverse primers and MGB probes described in Supplemental Table I. To quantify the level of viral RNA, segments of the EV-D68 genome (nt 1–2706) or SeV genome (nt 10–1715) were cloned into plasmid pCR2.1 using the TA cloning kit (ThermoFisher Scientific) and used as standards. Lung levels of mucin Muc5ac mRNA were determined using real-time quantitative PCR (qPCR) assay with probes described in Supplemental Table I. All target mRNA and viral RNA levels were normalized to Gapdh mRNA level using the TaqMan Rodent GAPDH Control Kit. All mRNA values were expressed as fold-change normalized to Gapdh mRNA.

Western blot analysis

Mouse tissues were homogenized in 1% Nonidet P-40, 0.05M Tris, pH 8.0, 250 mM NaCl, and 1 mM EDTA containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Extracted protein was subjected to western blotting with mouse anti-Stat1 Ab (BD Biosciences) and anti-Gapdh mAb (MAB374, MilliporeSigma) detected with horseradish peroxidase-conjugated horse anti-mouse IgG Ab (7076, Cell Signaling) and enhanced chemiluminescence as described previously (10).

Airway reactivity

Airway reactivity to nebulized methacholine (Sigma, St. Louis, MO) was determined by measurements of respiratory system resistance (R_RS) using the FinePointe Resistance and Compliance system (DSI Buxco Research Systems, Wilmington, NC) as described previously (28, 29). Mice were anesthetized, ventilated via tracheostomy, and sequentially challenged with aerosolized PBS (baseline) followed by doubling doses of methacholine ranging from 0.6125 to 20 mg/ml. Methacholine was delivered at 3-min intervals using an in-line nebulizer (Aerogen Laboratory; 2.4–4 μm particle size). Resistance values were recorded during a 3-min period following each challenge. Data were manually verified, and spurious epochs removed from analysis.

Statistical analysis

Unless stated otherwise, all data are presented as mean ± SEM and two-tailed unpaired Student’s t-test was used to assess statistical significance between means. In all cases, the threshold for statistical significance was set at a P value less than 0.05. Airway reactivity for viral infection versus UV-inactivated virus condition was assessed using two-way analysis of variance and Bonferroni post-hoc correction.

Results

Generation of epithelial cell-specific Stat1 gene knockout mice

To determine if epithelial STAT1 function controls viral infection, we generated transgenic mice that expressed a floxed Stat1 allele (Stat1^f/f) using the gene targeting scheme described in Materials and Methods and depicted in Fig. 1A. Mutant allele expression in heterozygous Stat1^wt/f mice was confirmed using Southern blotting for tail DNA (Fig. 1B) In addition, crossing homozygous Stat1^f/f mice to Flp-deleter mice and then to CAG-Cre mice resulted in Cre-mediated recombination with loss of STAT1 protein across lung, liver, and splenic tissues similar to conventional Stat1^−/− mice based on western blotting (Fig. 1C). Together, these results validated the establishment of transgenic Stat1^f/f mice with Flp gene deletion (hereafter referred to as Stat1^f/f mice) for further study of STAT1 function in vivo. These mice exhibited normal birth frequency, development, and longevity without any histological changes in a full-organ survey that included lung tissue.

FIGURE 1.

Generation of epithelial-cell STAT1-deficient Foxj1-Scgb1a1-Cre-Stat1^f/f mice. (A) Schematic of mouse wild-type (WT) Stat1 gene, Stat1 gene targeting construct, Stat1-floxed allele, and final Cre-modified Stat1^flox/flox (Cre-Stat1^f/f) gene locus. (B) Southern blot of tail DNA from heterozygous Stat1^wt/f and control WT mice after treatment with restriction enzymes KpnI (Stat1^f/f allele size of 9 kb, WT allele size of 7 kb) or BamHI (Stat1^f/f allele size of 3 kb, WT allele size of 11 kb). (C) Western blot of tissue homogenates from indicated mouse strains using anti-STAT1 or anti-Gapdh Ab. (D) Breeding scheme for Foxj1-Cre or Scgb1a1-Cre mice cross to CAG-ROSA26^mTmG mice to generate epithelial cell-specific CAG-GFP reporter mouse strains (Foxj1-Cre-ROSA26^mTmg and Scgb1a1-Cre-ROSA26^mTmG). (E) Immunostaining for Foxj1, Scgb1a1, Sfptc, and GFP and counterstain with DAPI of lung sections from Foxj1-Cre-ROSA26^mTmG or Scgb1a1-ROSA26^mTmG. Scale bar, 100 μm. (F) Quantitation of immunostaining for conditions in (E) along with control ROSA26^mTmG mice. For (B,C,E,F), values are representative of 3 separate experiments (n≥8 mice per condition in each experiment). For (F), * indicates p<0.05.

To define conditions for epithelial cell deletion of the Stat1 gene, we crossed Foxj1-Cre mice (that target ciliated cells) and Scgb1a1-Cre mice (that target club cells) to ROSA26^mTmG reporter mice (Fig. 1D). Lung sections from Foxj1-Cre-ROSA26^mTmG mice immunostained with anti-Foxj1 and anti-GFP Abs showed co-localization (Fig. 1E) and a high level of co-staining (94.8% of Fojx1 cells were GFP⁺) based on image analysis (Fig. 1F). Lung sections from Scgb1a1-Cre-ROSA26^mTmG mice immunostained for Scgb1a1, Sftpc, and GFP showed significant albeit lower levels of co-localization (Fig. 1E) and a correspondingly significant but lower level of co-staining (63.4% of Scgb1a1⁺ cells and 38.6% of Sftpc⁺ cells were GFP⁺) based on image analysis (Fig. 1F). Each of these results was consistent with our previous analysis of Foxj1-Cre mice and reports of Scgb1a1-Cre activity in club and alveolar type II (AT2) cells (56). Further, the results provided a basis for establishing combined Foxj1-Scgb1a1-Cre-Stat1^f/f mice to define the role of barrier epithelial cells in STAT1 function for host defense against viral infection.

Epithelial STAT1 function defends against acute SeV infection

Our initial analysis of STAT1 function showed that body weight loss was significantly enhanced in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice after SeV infection (Fig. 2A). In addition, body weight was decreased slightly but significantly in Stat1^−/− mice compared to Foxj1-Scgb1a1-Cre-Stat1^f/f mice such that Stat1^−/− mice were euthanized at 8 d after infection when body weight became <70% of the initial baseline value. Similarly, survival rates were significantly decreased in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice after SeV infection and were decreased even further in Stat1^−/− mice after SeV infection (Fig. 2B). These changes were accompanied by increases in viral titers based on lung levels of SeV-NP RNA in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice, and in turn in Stat1^−/− mice compared to Foxj1-Scgb1a1-Cre-Stat1^f/f mice (Fig. 2C). Similar increases in viral titers were found based on infectious virus using plaque-forming assay (Fig. 2C). In concert with body weight, survival, and viral titer data, we also observed increases in the levels of viral immunostaining (Fig. 2D) and immune cell infiltration (Fig. 2E) in Foxj1-Scgb1a1-Cre-Stat1^f/f mice and Stat1^−/− mice compared to wild-type mice after SeV infection. Viral and immune cell infiltration was localized primarily to airway epithelial tissue at 3 d after infection but extended to alveolar sites by 5 d after infection, consistent with replication in both airway and alveolar epithelial cells.

FIGURE 2.

STAT1 deficiency in epithelial cells increases severity of SeV infection. (A) Body weights for WT, Stat1^−/−, and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at the indicated times after infection with SeV (1 × 10⁵ PFU given intranasally) or equivalent control SeV-UV. (B) Survival rates for conditions in (A). (C) Lung levels of SeV-NP RNA using PCR assay and corresponding levels of infectious SeV titers using plaque-forming assay for WT, Stat1^−/−, and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at the indicated times after infection with SeV. (D) Immunostaining for SeV and counterstaining with DAPI in lung sections for conditions in (C). Scale bar, 400 μm. (E) Hematoxylin and eosin staining in lung sections for conditions in (C). Scale bar, 200 μm. For (A-E), values are representative of 3 separate experiments (n≥8 mice per condition in each experiment). For (A-B), * indicates p<0.05 by ANOVA except for survival rates by Kaplan-Meier analysis, and for (C-E), X signifies data that could not be obtained due to non-survival conditions.

To address this issue, we also used SeV co-staining with epithelial cell markers to more precisely define the site of SeV replication. In that regard, immunostaining for SeV and STAT1 was markedly increased at 3 d after SeV infection and SeV immunostaining was co-localized with STAT1 as well as ciliated cell marker β-Tubulin IV, club cell marker of Scgb1a1, and AT2 cell marker Sftpc at 3 d after infection (Fig. 3A). These results were consistent with SeV replication in ciliated cell, club cell, and AT2 cells leading to induction of STAT1 expression at these cell sites. We also found that STAT1 immunostaining was lost in ciliated cells in Foxj1-Cre-Stat1^f/f mice and in both club cells and AT2 cells in Scgb1a1-Cre-Stat1^f/f mice at 3 d after SeV infection (Fig. 3B), consistent with the patterns of Cre recombination observed in Foxj1-Cre-ROSA26^mTmG and Scgb1a1-Cre-ROSA26^mTmG reporter mice (Fig. 1D). Moreover, the efficiencies for Cre-mediated Stat1 gene deletion in airway and alveolar epithelial cells (63% in club cells and 39% in AT2 cells) fit with the partial increase in severity of infection in Foxj1-Scgb1a1-Cre-Stat1^f/f compared to the full increase in Stat1^−/− mice. Nonetheless, the results provide a system for epithelial cell-specific deletion of Stat1 gene expression and consequent evidence that loss of STAT1 function in barrier epithelial cells leads to increased viral replication and severity of infection.

FIGURE 3.

SeV replication and STAT1 induction co-localizes to epithelial cells. (A) Immunostaining for SeV and STAT1, β-Tubulin IV, Scgb1a1, or Sftpc and counterstaining with DAPI in lung sections from WT mice at 3 d after infection with SeV. Scale bar, 100 μm. (B) Immunostaining for STAT1 and β-Tubulin IV, Scgb1a1, or Sftpc and counterstaining with DAPI in lung sections from Foxj1-Cre-Stat1^f/f, Scgb1a1-Cre-Stat1^f/f, and WT mice at 3 d after infection with SeV. Arrows indicate epithelial cells (green staining) without STAT1 (red staining) in the context of Stat1 gene deletion. Scale bar, 100 μm. For (A,B), values are representative of 3 separate experiments (n≥8 mice per condition in each experiment).

Epithelial STAT1 function protects against SeV-induced chronic lung disease

Given the capacity of SeV infection to trigger long-term lung disease (27–30), we also continued study of Foxj1-Scgb1a1-Cre-Stat1^f/f and wild-type mice that survived the acute illness due to SeV infection. Here we found that the degree of chronic lung disease was significantly increased in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice marked by airway and adjacent parenchymal immune cell infiltration based on hematoxylin⁺ staining in hematoxylin and eosin stained lung sections (Fig. 4A) and mucus production marked by PAS⁺ staining and mucin Muc5ac⁺ immunostaining (Fig. 4B) in lung sections at 49 d after SeV infection. Quantitative image analysis of whole lung sections demonstrated that PAS⁺ and Muc5ac⁺ areas were significantly increased in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice (Fig. 4C, 4D, 4E). In concert with these observations, we found that increases in Muc5ac expression were closely coordinated with increases in Clca1 expression in airway mucous cells based on immunofluorescence microscopy (Fig. 5A). Quantitative image analysis of whole lung sections confirmed that Clca1⁺ and Muc5ac⁺ areas were significantly increased in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice (Fig. 5B, 5C). Together, these results indicated that epithelial cell STAT1 defense against acute SeV infection also translated to protection against chronic lung disease that develops after clearance of SeV infection.

FIGURE 4.

STAT1 deficiency in epithelial cells increases susceptibility to chronic lung disease after SeV infection. (A) Hematoxylin and eosin staining of lung sections from WT and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 49 d after infection with SeV or SeV-UV. Scale bar, 500 μm. (B) PAS staining and Muc5ac immunostaining for conditions in (A). Scale bar=400 μm. (C) PAS staining and Muc5ac immunostaining for conditions in (A). Scale bar, 500 μm. (D) PAS staining and Muc5ac immunostaining for conditions in (A). Scale bar, 500 μm. (E) Quantitation of PAS staining and Muc5ac immunostaining from conditions in (C, D). For (A-E), data are representative of 3 separate experiments (n≥8 mice per condition in each experiment). For (E), * indicates p<0.05.

FIGURE 5.

STAT1 deficiency in epithelial cells increases susceptibility to chronic lung disease after SeV infection. (A)Immunostaining for Clca1 and Muc5ac and counterstaining with DAPI in lung sections from WT and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 49 d after infection with SeV or SeV-UV. Scale bar, 100 μm. (B) Corresponding whole lung sections for Clca1 immunostaining for conditions in (A). Scale bar, 500 μm. (C) Quantitation of Clca1 and Muc5ac immunostaining in whole lung sections for conditions in (A). For (A-C), data are representative of 3 separate experiments (n≥8 mice per condition in each experiment). For (C), * indicates p<0.05.

STAT1 function defends against acute EV-D68 infection

We next aimed to extend our studies of epithelial cell STAT1 to defense against respiratory viruses that are pathogenic in humans, especially in relation to severe airway disease. Given the high levels of EV-D68 replication (1×10⁸-fold increase in viral titer) in primary-culture human airway epithelial cells (data not shown), we selected this virus for further study in mice in vivo. Initial experiments showed no significant body weight loss in Stat1^−/−, Foxj1-Scgb1a1-Cre-Stat1^f/f, or wild-type mice after EV-D68 infection (data not shown). Nonetheless, we detected significant increases in viral titer monitored by the level of EV-D68-VP1 RNA with the largest increase in lung tissue and smaller increases in tracheal, nasal, and pharyngeal tissue in wild-type mice (Fig. 6A). In addition, we detected a time-dependent increase in EV-D68-VP1 RNA in lung tissue with significant increases in viral RNA in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice, and in turn in Stat1^−/− mice compared to Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 0.5–12 d after infection (Fig. 6B). Both time- and strain-dependence of EV-D68 titers were similar to the pattern that we found for SeV titers in lung tissue (Fig. 2C). However, the level of replication for EV-D68 was less than SeV, e.g., 1×10¹-fold versus 1×10⁸-fold increase in viral RNA levels in the lung at 1 h versus 1–3 d after infection in Stat1^−/− mice (Fig. 2C and Fig. 6B). Viral titer for EV-D68 was 5.7×10⁷ whereas SeV was 1.2×10⁴ viral RNA copies at 1 h after infection. However, this initial difference is expected since the inoculum for EV-D68 was 1.5×10⁸ whereas SeV was 5.0×10⁵ viral RNA copies, and the 1-h values reflect the inoculum amount since there is no detectable viral RNA replication until 4–14 h after infection (57, 58). In contrast, the viral titer at later time points is lower for EV-D68 than for SeV, but this result is also expected given the lower level of replication for a non-adapted human pathogen compared to a native rodent pathogen in a mouse host.

FIGURE 6.

STAT1-deficiency in epithelial cells increases severity of EV-D68 infection. (A) Tissue levels of EV-D68-VP1 RNA in WT mice at 1 d after infection with EV-D68 (0.5 × 10⁶ ePFU given intranasally) or an equivalent amount of control EV-D68-UV. (B) Lung levels of EV-D68-VP1 RNA for WT, Stat1^−/−, and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at the indicated times after infection with EV-D68. (C) Immunostaining for EV-D68 with DAPI counterstaining in lung sections from Stat1^−/− mice at 3 d after infection with EV-D68 or EV-D68-UV. Scale bar, 100 μm. (D) Immunostaining for EV-D68 and Scgb1a1 or β-Tubulin IV and counterstaining with DAPI in lung sections for conditions in (C) imaged with conventional and confocal microscopy. Arrows indicate cells co-staining for EV-D68 and Scgb1a1 or β-Tubulin IV. Scale bars, 50 μm. (E) Hematoxylin and eosin staining in lung sections for conditions in (A). Scale bar, 500 μm. (F) Hematoxylin and eosin staining in airway sections for conditions in (A). Scale bar, 200 μm. For (A-E), data are representative of 3 separate experiments (n≥8 mice per condition in each experiment). For (A,C), * indicates p<0.05.

In concert with the increase in viral levels in lung tissue, we also observed immunostaining for EV-D68 in airway epithelium at 3 d after infection in wild-type, Stat1^−/−, and Foxj1-Scgb1a1-Cre-Stat1^f/f mice (Fig. 6C). Moreover, EV-D68 was localized selectively to airway club cells based on co-staining with Scgb1a1 and to airway ciliated cells based on co-staining with β-Tubulin IV using conventional and confocal microscopy (Fig. 6D). Similarly, hematoxylin-eosin staining of lung sections showed immune cell infiltration mainly into the airway epithelium and adjacent subepithelial tissue at 3–8 d after infection in whole lung sections (Fig. 6E) and at higher magnification of airway epithelial sections (Fig. 6E) in all three strains of mice. Similar to viral levels, immune cell infiltration of airways was increased in Stat1^−/− and Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice at 3–8 d after EV-D68 infection (Fig. 6F). Together, these results provided evidence of productive infection with EV-D68 in mice that was confined at least primarily to airway epithelial cells and was sensitive to loss of STAT1 function in lung epithelial cells, most likely the same set of host airway epithelial cells.

Epithelial STAT1 function protects against EV-D68-induced chronic lung disease

To define whether EV-D68 infection might unexpectedly trigger long-term lung disease, we studied the possibility that excess mucus production might develop as a marker of chronic lung disease after this type of viral infection. Indeed, PAS staining of lung sections showed increases in PAS⁺ cells (consistent with the morphology of mucous cells) in the airway epithelium generally with adjacent immune cell infiltration in Stat1^−/− and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 8, 21, and 49 d after EV-D68 infection (Fig. 7A). Similarly, Muc5ac immunostaining demonstrated specific increases in Muc5ac⁺ mucous cells in the airway epithelium under these same experimental conditions (Fig. 7B). Corresponding staining of whole lung sections demonstrated that histopathologic changes marked by mucus production areas were confined to airways after EV-D68 infection (Fig. 7B, 7C). Quantitative image analysis of whole lung sections further confirmed that PAS⁺ and Muc5ac⁺ areas were significantly increased in Stat1^−/− and to a lesser degree in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice at 8–49 d after EV-D68 infection (Fig. 7C, 7D, 7E). In concert with the evidence of chronic lung disease from tissue staining, we also found significant increases in lung levels of Muc5ac mRNA in Stat1^−/− and to a lesser degree in Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice at 8–49 d after EV-D68 infection (Fig. 7F). In concert with these observations, we again found that increases in Muc5ac expression were linked to increases in Clca1 expression in airway mucous cells based on immunofluorescence microscopy (Fig. 8A). Quantitative image analysis of whole lung sections confirmed that Clca1⁺ and Muc5ac⁺ areas were significantly increased in Stat1^−/− and to a lesser degree in Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 49 d after EV-D68 infection (Fig. 8B, 8C).

FIGURE 7.

STAT1 deficiency in epithelial cells increases susceptibility to chronic lung disease after EV-D68 infection. (A) PAS-hematoxylin staining of lung sections from WT, Stat1^−/−, and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 8, 21 and 49 d after infection with EV-D68. Scale bar, 100 μm. (B) Muc5ac immunostaining with hematoxylin counterstaining from the conditions in (A). Scale bar, 100 μm. (C) PAS and hematoxylin stained lung sections from WT, Stat1^−/−, Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 49 d after infection with EV-D68 or EV-D68-UV. Scale bar, 500 μm. (D) Muc5ac and hematoxylin stained lung sections for conditions in (C). Scale bar, 500 μm. (E) Quantitation of PAS⁺ and Muc5ac⁺ staining areas for conditions in (C, D). (F) Lung levels of Muc5ac mRNA for conditions in (E). For (A-F), data are representative of 3 separate experiments (n≥8 mice per condition in each experiment). For (E,F), * indicates p<0.05.

FIGURE 8.

STAT1 deficiency in epithelial cells increases susceptibility to chronic lung disease after EV-D68 infection. (A) Immunostaining for Clca1 and Muc5ac and counterstaining with DAPI in lung sections for WT, Stat1^−/−, Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 49 d after infection with EV-D68 or EV-D68-UV. Scale bar, 100 μm. (B) Corresponding whole lung sections for Clca1 immunostaining from Stat1^−/− mice for conditions in (A). Scale bar, 500 μm. (C) Quantitation of Clca1 and Muc5ac immunostaining in whole lung sections for conditions in (A). For (A-C), data are representative of 3 separate experiments (n≥8 mice per condition in each experiment). For (C), * indicates p<0.05.

In addition to immune cell infiltration and excess mucus production, we also found significant increases in airway reactivity to inhaled methacholine in Stat1^−/− mice at 8 and 49 d after infection with EV-D68 compared to EV-D68-UV (Fig. 9A). Similar to the pattern observed for regulation of Muc5ac expression, the development of airway hyper-reactivity was not detectable in wild-type mice and was also found at only the highest concentration of methacholine in Foxj1-Scgb1a1-Cre-Stat1^f/f mice (Fig. 9A). We detected no significant increase in baseline respiratory system resistance (R_RS) at 8 or 49 d after viral infection in wild-type, Stat1^−/−, or Foxj1-Scgb1a1-Cre-Stat1^f/f mice (data not shown), consistent with our studies of SeV and IAV infections (28, 29, 31). To further address the comparison between the post-EV-D68 mouse model with chronic lung disease in humans, we also assessed the development of fibrosis as a feature of airway disease. Here we detected a significant increase in Gomori trichrome staining for the blue-tone of collagen expression in Stat1^−/− mice at 49 d after EV-D68 infection (Fig. 9B). Corresponding staining of whole lung sections demonstrated that trichrome⁺ staining was confined to airways (Fig. 9C), and quantitative image analysis of these sections confirmed that trichrome⁺ areas were significantly increased in Stat1^−/− but not Foxj1-Scgb1a1-Cre-Stat1^f/f or wild-type mice at 49 d after EV-D68 infection (Fig. 9D). Together, the findings show that key features of airway disease, i.e., immune cell infiltration, mucus production, hyper-reactivity, and fibrosis can be triggered in response to EV-D68 infection if protection from STAT1 function in barrier epithelial cells is lost. The localized nature of the acute infection, chronic disease, and STAT1 deficiency in the EV-D68 model further suggests that the critical function of STAT1 to protect against these outcomes is linked in particular to airway epithelial cells.

FIGURE 9.

STAT1 deficiency in epithelial cells increases susceptibility to chronic lung disease marked by airway hyperreactivity and fibrosis after EV-D68 infection. (A) Levels of airway reactivity using response of respiratory system resistance (R_RS) to inhaled methacholine (MCh) in WT, Stat1^−/−, and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 8 d and 49 d after infection with EV-D68 or EV-D68-UV. (B) Gomori trichrome staining of lung sections from Stat^−/− mice at 49 d after infection with EV-D68 or EV-D68-UV. Scale bar, 200 μm. (C) Gomori trichome staining of whole lung sections for conditions in (B) with yellow colorization indicating computer-assigned collagen-containing light-blue⁺ staining areas. Scale bar, 500 μm. (D) Quantitation of trichome light-blue⁺ staining areas in lung sections for WT, Stat1^−/−, and Foxj1-Scgb1a1-Cre-Stat1^f/f mice at 49 d after infection with EV-D68 or EV-D68-UV. For (A,D), values are representative of 3 separate experiments (n=5–8 mice per condition in each experiment). For (A), * indicates p<0.05 by ANOVA for entire MCh concentration-response and ** indicates p<0.05 by post-hoc Bonferroni for single MCh concentration versus EV-D68-UV condition. For (D), * indicates p<0.05.

Discussion

The present study develops key evidence that STAT1 function in barrier epithelial cells, and in particular airway epithelial cells, provides a fundamental mechanism for host defense against respiratory viral infection and in turn a critical safeguard against chronic lung disease that develops after clearance of infectious virus. These concepts derive from a series of specific findings: (1) loss of STAT1 function in barrier epithelial cells is achieved with newly developed Foxj1-Scgb1a1-Cre-Stat1^f/f transgenic mice and results in increased viral titers of both mouse and human viral pathogens (SeV and EV-D68, respectively); (2) increases in viral titers caused by epithelial STAT1 deficiency is associated with more severe acute infectious illness marked by exaggerated weight loss after SeV and immune cell infiltration after SeV and EV-D68 infections; (3) loss of STAT1 control over acute infection also leads to more severe manifestations of the chronic lung disease that can develop after clearance of SeV and EV-D68 infection and is marked by immune cell infiltration and mucus production for at least as long as 7 wk after infection; (4) STAT1-deficiency limited to epithelial cells coupled with the limitation of acute infection to airway epithelial cells (ciliated and club cells) and chronic disease to airways in the case of EV-D68 infection further suggests that STAT1 protection is specific at least in part to airway epithelial cells within the lung epithelial barrier. Here we discuss the novelty and significance of these findings for the acute infection, chronic lung disease, and implications for comparable conditions in humans.

In regard to the acute infection, we expected that global loss of STAT1 function and compromise in interferon signal transduction would lead to an increase in viral replication and titer as we described previously for SeV (7). Similarly, there was a transient delay in viral clearance in Stat^−/− mice after SARS-CoV infection that persisted in bone marrow chimeras with STAT1 deficiency in stromal cells (59). Thus, as expected, we observed that both SeV and EV-D68 titers were significantly increased in conventional Stat1^−/− mice compared to wild-type mice. For both types of viruses, we also found an increase in viral titer in Foxj1-Scgb1a1-Cre-Stat1^f/f transgenic mice. Of note, the level of compromise was less than for Foxj1-Scgb1a1-Cre-Stat1^f/f transgenic mice than Stat1^−/− mice, but this result can be explained by the fact that the viruses infect both Foxj1+ ciliated cells and Scgb1a1⁺ club cells and the efficiency Cre recombination for the Scgb1a1-driver is only 63% in this cell population, consistent with previous reports by others (56). Moreover, in the case of SeV infection, the virus also infects distal Sftpc⁺ AT2 cells, and the efficiency is only 39%, thereby providing for further viral replication. In contrast, EV-D68 infection is limited to airway epithelial cells based on immunostaining for viral protein that selectively co-localizes with club cell and ciliated cell proteins in the airway epithelium. Thus, a protective effect under these conditions indicates that airway epithelial cells (rather than lung epithelial barrier cells in general) must provide a key protective site. This finding establishes long-sought evidence for this concept.

In regard to chronic lung disease, we found that the increased severity of acute infection translated to more likely and severe development of chronic lung disease that persisted after clearance of infectious virus for both SeV and EV-D68. In the case of SeV infection, the signs of chronic lung disease such as mucus production are not evident until 21 d after infection and become maximal at 49 d after infection (27–30). In contrast, for EV-D68 infection, mucus production develops during the acute infection illness at 8 d after infection and persists for at least 49 d after infection. However, this apparent difference might reflect the acute destruction of epithelium after more severe infection with SeV and hence the inability to manifest epithelial cell differentiation towards mucous cells until there is epithelial repair by 8–12 d after infection (60). Indeed, in the case of EV-D68 infection, there were no significant signs of chronic disease unless the severity of infection was enhanced through the loss of STAT1 function using Stat1^−/− or Foxj1-Scgb1a1-Cre-Stat1^f/f mice. Nonetheless, despite the relatively mild nature of the acute infection with EV-D68, it is striking that signs of chronic airway disease marked by excess immune cell infiltration and mucous cell metaplasia persisted for at least 7 wk after initial infection. The limitation of EV-D68 replication to airway club and ciliated cells further provides for a specific role of airway epithelial cells in host defense and protection from lung disease. Moreover, the focal nature of the disease likely reflects localized sites of viral replication in the absence of IFN-dependent control and in turn localized reprogramming of host cells that is needed for long-term virus-induced disease.

Our findings also significantly advance previous studies of SeV, EV-D68, and related respiratory viruses in mouse models. For example, our own studies of SeV provided initial evidence that STAT1 function was required to control viral replication and acute illness based on STAT1 expression in the stromal cell compartment, but did not study epithelial cell-specific approaches or the development of chronic disease (7). Similarly, our studies of SARS-coronavirus detected increases in viral titer in Stat1^−/− mice but did not define any phenotype beyond 9 d after infection (59). Similarly, a previous study detected airway neutrophil infiltration and hyperreactivity at 2 d after EV-D68 infection in female, wild-type Balbc/J mice but did not study host defense or chronic disease (61). This transient effect was slightly suppressed with anti-IL-17A blocking antibody, consistent with neutrophil-linked hyperreactivity found after short-term airway injury even in response to non-infectious agents (62), but the immune basis for subsequent longer-term disease after EV-D68 infection still needs to be defined. In contrast to the case for EV-D68, there is considerable evidence for SeV and IAV infections that the immune mechanism for post-viral disease is based on a prolonged type 2 immune response (and not an IL-17 response) that proceeds from epithelial stem cell expansion to innate immune cell activation and feed-forward to airway progenitor epithelial cell (APEC) differentiation towards mucous cells (28–31, 63). Further studies will be needed to determine whether the same mechanism drives chronic disease after EV-D68 infection. Nonetheless, the present finding for induction of Clca1 gene expression, which is highly dependent on IL-13 stimulation (29, 31, 64, 65), suggests that the type 2 immune response might also drive chronic mucus production and airway hyper-reactivity after EV-D68 similar to SeV and IAV infections (28–31). The same mechanism is also proposed to mediate short-term airway disease after human rhinovirus (RV) infections in mice (66).

In contrast, the present model for EV-D68 infection exhibits significant viral replication, e.g., at least a 1×10¹-fold increase from initial viral dose and 2–3×10³-10⁴-fold higher viral titers for 3–8 d in Stat1^−/− and Foxj1-Scgb1a1-Cre-Stat1^f/f mice compared to wild-type mice. The model thereby represents an advance over mouse models for infections with closely related RVs (RV-A, RV-B, and RV-C strains) that replicate poorly in mice with peak viral titer that is generally 1×10³-fold lower than the initial inoculum and is characterized by viral clearance at 4 d after infection (67–70). These results highlight the frontline role of anti-viral host defense at the airway epithelial cell barrier in controlling infection, but this system is complemented by the actions of immune cells and in particular the adaptive immune response that allows for eventual clearance of the virus via cytotoxic T cell and antibody-producing B cell and T cell function. Presumably, at least some aspects of this adaptive immune response do not depend on IFN signal transduction and are therefore sufficient to clear infection even when initial defenses are compromised. Nonetheless, the persistently increased viral loads found with epithelial cell compromise appear to be enough to cause chronic lung disease based on the present mouse model.

The clearance of infectious virus also raises the issue of persistent viral-RNA remnants that are likely non-infectious but might still have a role in post-viral disease. In that context, we reported (and confirm here) that viral-RNA remnants are found long after clearance of infectious virus in the case of SeV (28), and we found the same persistence more recently for influenza A virus (31). In addition, our report for IAV includes data for active viral RNA replication with detection of both positive and negative-strand viral RNA and for localization of these viral-RNA remnants to sites of chronic lung disease. In each case, viral-RNA remnants are not detectable after infection with UV-inactivated virus, thereby further excluding any artifact of the PCR-based detection assay Moreover, many other labs have reported similar persistence for other RNA viruses, including RSV (73), measles virus (74), Ebola virus (75), and chikungunya virus (76). The role of active viral-RNA remnants in chronic disease is a subject of intense investigation in our lab and others. Notably, we do not detect persistence of EV-D68 RNA despite the development of chronic lung disease, suggesting that viral-RNA remnants are a marker versus a cause of long-term disease.

The present findings have significant implications for both acute illness and chronic disease that develops after viral infection and respiratory viral infection in particular in humans. In particular, EV-D68 can cause severe acute illness in humans, particularly asthmatics. The present model might therefore provide evidence that IFN-signaling deficiency is a factor in susceptibility to this type of infection. The basis for any possible IFN deficiency in asthma is less certain, but differences based on the type of infection might be highly relevant given the varying capacity of different viruses to subvert IFN production and signaling, including EV-D68 and SeV (25, 26, 71). In that context, the results provide the intriguing possibility that EV-D68 similar to SeV and IAV might be particularly prone to cause the severity of acute infection that leads to chronic disease. This experimental relationship fits with studies of viral infection in children where the type of virus does not correlate with the development of subsequent asthma (72). Additional work must particularly address, the likely scheme for viral reprogramming of APECs towards mucous cells and the possible upstream and downstream influence of immune cells. The present findings should serve as a new experimental model for these directions based on viral infections that provide significant replication and severity of illness that are key to chronic disease in the mouse model and likely in comparable airway disease due to asthma and chronic obstructive pulmonary disease.

Together, the present study establishes a new experimental model that provides proof-of-concept for the role of barrier epithelial cells in defense against respiratory viral infection based on the capacity for STAT1-dependent IFN signal transduction. The model provides for protection against acute infectious illness as well as the fundamental features of chronic respiratory disease, i.e., airway inflammation, mucus production, hyperreactivity, and fibrosis. Thus, the model should allow further development of precise therapeutic strategies for acute and chronic lung disease in humans, ranging from gene-based to small-molecule IFN-signal enhancers to target upstream events (10, 77, 78) to cytokine- and kinase-inhibitor strategies to target downstream events such as mucous cell metaplasia (65, 79, 80). These approaches will require definition of expression and function of epithelial cell-derived cytokines in the post-EV-D68 model as was done for SeV infection and was translated to humans with COPD (29). In fact, comparison of mechanism for post-viral lung disease after EV-D68 versus SeV and IAV and related RVs will likely prove useful for stratification of patients with viral exacerbations of chronic lung disease.

Abbreviations used in this article:

Clca1

chloride channel accessory 1

COPD

chronic obstructive pulmonary disease

EV

enterovirus

IAV

influenza A virus

PAS

periodic acid-Schiff

qPCR

quantitative PCR

RV

rhinovirus

MUC5AC

mucin 5AC

SeV

Sendai virus