Early-life pulmonary viral infection leads to long-term functional and lower airway structural changes in the lungs

Abstract

Early-life respiratory virus infections have been correlated with enhanced development of childhood asthma. In particular, significant numbers of respiratory syncytial virus (RSV)-hospitalized infants go on to develop lung disease. It has been suggested that early-life viral infections may lead to altered lung development or repair that negatively impacts lung function later in life. Our data demonstrate that early-life RSV infection modifies lung structure, leading to decreased lung function. At 5 wk postneonatal RSV infection, significant defects are observed in baseline pulmonary function test (PFT) parameters consistent with decreased lung function as well as enlarged alveolar spaces. Lung function changes in the early-life RSV-infected group continue at 3 mo of age. The altered PFT and structural changes induced by early-life RSV were mitigated in TSLPR^−/− mice that have previously been shown to have reduced immune cell accumulation associated with a persistent Th2 environment. Importantly, long-term effects were demonstrated using a secondary RSV infection 3 mo following the initial early-life RSV infection and led to significant additional defects in lung function, with severe mucus deposition within the airways, and consolidation of the alveolar spaces. These studies suggest that early-life respiratory viral infection leads to alterations in lung structure/repair that predispose to diminished lung function later in life.

NEW & NOTEWORTHY These studies outline a novel finding that early-life respiratory virus infection can alter lung structure and function long-term. Importantly, the data also indicate that there are critical links between inflammatory responses and subsequent events that produce a more severe pathogenic response later in life. The findings provide additional data to support that early-life infections during lung development can alter the trajectory of airway function.

INTRODUCTION

Respiratory syncytial virus (RSV), a ubiquitous human pathogen, infects nearly all children by the age of two (1, 2). It produces severe lower respiratory tract disease, including bronchiolitis, and is characterized by excessive mucus production and immune-mediated lung damage (1, 3–5). In addition to severe disease following initial RSV infection, the immune system is persistently altered following a severe early-life infection, which impacts future disease later in life (6–11). Consequently, severe RSV disease has been linked with the development of childhood wheezing and asthma (12–14). Studies with neonatal RSV infection demonstrate persistent changes in the lung that include mucus production and increased immune cells, including dendritic cells (DC), remodeling, and innate lymphoid type-2 cells (ILC2), that contribute to Th2 responses and future exacerbations within the lung and mitigated in TSLPR^−/− animals (15–21). These latter studies primarily explored the upper airway function in response to a methacholine challenge, whereas the present studies have utilized extensive baseline pulmonary function testing without provocation.

Many lung pathologies are associated with pulmonary function defects. For instance, BPD, a severe lung complication due to premature birth, leads to loss of lung function both immediately following birth as well as throughout life (22). The loss of lung function during the first 2 years of life in humans may never recover to full capacity, although lung development continues through puberty and into early adulthood (23, 24). Lung capacity defects early in life would be exacerbated in an environment that continues to respond inappropriately to environmental and pathogenic (viral) stimuli, thus maintaining and increasing lung structural defects.

The data described in these studies support and extend these concepts by examining parameters of lung function that reflect upper and lower airway alterations. We observe significant baseline lung functional defects across multiple parameters in the lungs of early-life RSV-infected mice that persist for at least 3 mo postinfection. These results support the clinical data suggesting that infants who have suffered a severe early-life RSV infection have dampened lung function throughout life that is exacerbated upon secondary exposures. Thus, the novel findings in these studies are that a severe viral infection during a critical time in lung development alters the lung structure and function that likely has long-term consequences.

MATERIALS AND METHODS

Animals

All experiments involving the use of animals were approved by the University of Michigan animal care and use committee (Protocol PRO0010620, exp. 02/10/2025). Male and female BALB/c mice, 6–8 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME) and used as breeders for experimental animals and adult studies. TSLPR^−/− mice were bred in-house and originally provided by S. F. Ziegler (Benaroya Institute, Seattle, WA). Male neonatal mice born in-house were used for all early-life experiments. All mice were maintained under standard pathogen-free conditions.

RSV Infection

A chimeric RSV A2 strain with recombinant Line19 fusion protein was used for all experiments as previously described (25). Male Balb/c mice were infected intranasally (5 µL/animal) with 3 × 10⁵ pfu of RSV A2/L19-F at 7 days of age. Viral stocks were grown in Hep-2 cells and concentrations were determined by plaque assay. Virus was ultracentrifuged (100,000 g for 30 min at 4°C) and resuspended in sterile saline before use.

Secondary RSV Infection

Mice were infected at 3 mo post-early-life RSV infection with a chimeric RSV A2 strain with recombinant Line19 fusion protein used for all experiments as previously described (25). Naïve age-matched 3-mo-old mice were used as adult single RSV infection controls.

PFT Analysis

Long-term analysis of lung function was performed at 5 wk postinfection, 3 mo postinfection, and following secondary RSV-infection. Mice were subjected to a series of pulmonary function tests (PFT) on a specialized PFT Controller (DSI, Harvard Bioscience, Inc.). Anesthetized and trached mice were then placed in the supine position on the bed within the plethysmograph. The tracheotomy tube was connected to the face plate of the instrument where fresh air was provided through mechanical ventilation, and a series of maneuvers were performed to measure parameters associated with RC (resistance and compliance), FV (flow-volume), and PV (pressure-volume).

Lung Histology

Whole lungs were perfused with 10 mL of saline to remove blood and gravity inflated from a height of ∼25 cm with 10% formalin. Lungs and heart were carefully removed and placed in 10% formalin overnight. Five-micrometer lung sections were stained with hematoxylin and eosin (H&E) for alveolarization measurement or left unstained for immunohistochemistry.

Following secondary RSV infection, formalin-fixed lung sections were stained with H&E, periodic acid-Schiff (PAS), or trichrome. Photomicrographs were captured using a Zeiss Axio Imager Z1 and AxioVision 4.8 software (Zeiss, Munich, Germany).

Alveolarization Measurement

Alveolarization size was measured using Fiji analysis software. The measurements were automated but monitored to assure that only alveolar spaces were measured and did not include vessels. H&E-stained lung sections were blind coded, and four images were captured in the upper left, lower left, upper right, and lower right section of the lungs at ×200 magnification using a Zeiss Axio Imager Z1 and AxioVision 4.8 software (Zeiss, Munich, Germany).

Immunohistochemistry

Slides were deparaffinized with Histoclear (VWR) and rehydrated in fresh ddH₂O. Antigen retrieval was performed using DAKO target retrieval solution (Agilent Technologies, Santa Clara, CA) using a pressure cooker for 25 min. Slides were placed on ice for 10 min. Slides were blocked using 5% donkey serum in TTBS and incubated at room temperature (RT) in a humidified chamber for at least 1 h. Blocking buffer was removed and primary antibody, diluted in block solution, was added and incubated overnight at 4°C. Slides were washed three times with TTBS (5 min each wash) and secondary antibody was added and incubated at RT for 2 h. DAPI solution was then added and incubated at RT for 10 min. Slides were rinsed three times with PBS, dried, and mounted using ProLong Gold (Invitrogen) and coverslipped. ProLong Gold was allowed to cure at least overnight in the dark at RT followed by storage at 4°C until image capture. Images were collected using fluorescent microscopy, and overlays were performed using LCmicro software. The AEC1 cells fluorescent staining used a hamster anti-podoplanin (Jackson Immuno, Cat. No. 8.1.1; 1:100 dilution), followed by a secondary goat-anti-hamster-FITC (Jackson Immuno, Cat. No. 107-095-142; 1:500 dilution). The AEC2 cells were stained using a primary rabbit-anti-ProSPC (Millipore, Cat. No. AB3786: 1:500 dilution), followed by a secondary donkey-anti-rabbit-Cy3 (Jackson Immuno, Cat. No. 711-166-152; 1:500 dilution).

Mucus Scoring Analysis

Slides from PAS-stained lungs were blind-coded and scored by an individual observer to quantify mucus on a scale of 1–4. Scoring is as follows: 1 = Minimal/No Mucus; 2 = Slight: Multiple airways with goblet cell hyperplasia and mucus; 3 = Moderate: Multiple airways with significant mucus and some plugging; 4 = Severe: significant Mucus plugging.

Flow Cytometry

The left lung was removed, and single cells were isolated by enzymatic digestion with 1 mg/mL of collagenase (Roche) and 20 U/mL of DNaseI (Sigma, St. Louis, MO) in RPMI 1640 + 10% FCS for 60 min at 37°C. Tissues were further dispersed through an 18-gauge needle (5-mL syringe), RBCs were lysed, and samples were filtered twice through 100-μm nylon mesh. Fc receptors were blocked with purified anti-CD16/32 (clone 93; BioLegend, San Diego, CA). Surface markers were identified using Abs (clones) against the following antigens, all from BioLegend: CD45 (30-F11), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD69 (H1.2F3), CD90 (53-2.1), ST2 (D1H9), CD103 (2E7), CD11c (N418), CD11b (M1/70), Ly6C (HK1.4). For innate lymphoid cell staining, lineage markers were anti-CD3, CD11b, B220, Gr-1, and TER119. ILC2: CD45+/Lin−/CD90+/ST2+. Data were collected using a NovoCyte flow cytometer (ACEA Bioscience, Inc. San Diego, CA). Data analysis was performed using FlowJo software (Tree Star, OR). Gating was performed as previously described (15, 26).

Statistical Analysis

Data are presented as mean values ± SE. Comparison of two groups was performed with an unpaired, two-tailed Student’s t test. Comparison of three or more groups was analyzed by one-way ANOVA, followed by a two-tailed Student’s post hoc t test for individual comparisons. A P value <0.05 was considered significant.

RESULTS

Neonatal RSV Infected Mice Assessed for Functional Alteration

Decreases in lung function are indicative of a damaged or remodeled lung and is a symptom of childhood asthma. To determine whether neonatal RSV infection could lead to persistent alterations in pulmonary function, we infected neonatal mice at 7 days of age (EL-RSV) and performed pulmonary function tests (PFT) using spontaneous and ventilated maneuvers of anesthetized and tracheotomized mice at 5 wk postinfection. There is no evidence of RSV infection at 5 wk postinfection, either by plaque assay or PCR of mRNA for RSV gene expression (data not shown). Our previous studies identified that male mice have a more severe EL-RSV response than female mice (15), having more severe allergy exacerbations and long-term complications that allowed us to focus on male mice. Significant defects in the compliance and resistance of the lung following an early-life RSV infection, including reduction in dynamic lung compliance (Cdyn), were observed (Fig. 1A), indicative of a stiff lung phenotype. Correlating with a loss of lung compliance, increases in lung resistance (Rl), compliant pressure (dPpl), and maximum pressure (dPmax) were also observed in the lungs at 5 wk postneonatal RSV infection (Fig. 1, B and C). To further evaluate persistent pulmonary defects following early-life RSV infection, the flow-volume relationships were also determined at 5 wk postneonatal RSV. We observed decreases in the amount of airflow upon exhalation (forced vital capacity, FVC) in neonatally infected mice (Fig. 1D). Furthermore, the forced expiratory volume (FEV) was also significantly reduced 35–44% over all time parameters tested (Fig. 1E). Clinically, FEV decreases of 15% or more are indicative of asthma. Importantly, the ratio of FEV to FVC is not altered following EL-RSV (Fig. 1F). No change in FEV/FVC, along with the reduction of both FEV and FVC, is suggestive of a restrictive lung phenotype. A significant decrease in the static inspiratory and expiratory capacities of the lung in the mice given an early-life RSV infection compared with controls was also observed (Fig. 1G). Compliance of the lung was also significantly decreased upon measurement of Cchord (Chord compliance) and compliance at 50% vital capacity (Cfvc50) (Fig. 1H). These data indicate that even in a relaxed state, lungs from an early-life RSV infection have a decreased ability to function.

Figure 1.

Neonatal respiratory syncytial virus (RSV) infection leads to severe pulmonary function deficiencies. Neonatal male mice were infected at 7 days of age (EL-RSV) and pulmonary function tests (PFT) were performed using plethysmography of tracheotomized mice at 5 wk postinfection compared with sex- and age-matched controls. A–C: measurements of lung compliance and resistance. D–F: flow-volume relationships measuring dynamic lung properties. G and H: static inhalation and exhalation capacities of the lung. n = 6 mice/group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. dPpl, compliant pressure; dPmax, maximum pressure; FVC, forced vital capacity; Cchord, Chord compliance; Cfvc50, compliance at 50% vital capacity.

Alveolarization Assessment in Neonatal RSV Infected Mice

Alterations of lung function have been correlated with defects in the structural development of the lung, including alteration of alveolar development and remodeling (27, 28). To determine whether the decreased lung function was accompanied by changes in alveolar development, alveoli at 5 wk post-early-life RSV infection were assessed in formalin-fixed gravity inflated lungs. Histopathology indicates an increase in the size of the alveolar spaces in EL-RSV mice compared with uninfected controls (Fig. 2, A and B). Quantitation of the alveolar spaces in EL-RSV mice showed increased total size of alveoli (Fig. 2C) with fewer total number of alveoli (Fig. 2D) compared with uninfected controls. Immunofluorescent staining of the epithelium supports these findings (Fig. 2, E and F) along with a correlating decrease in the number of airway epithelial type 2 cells enumerated by flow cytometry in enzyme dispersed lungs (Fig. 2G). Thus, reduction in the development/repair of the alveoli may be contributing to altered lung function following early-life RSV.

Figure 2.

Alveolarization and extracellular matrix (ECM) components are altered following neonatal respiratory syncytial virus (RSV) infection. Neonatal male mice were infected at 7 days of age (EL-RSV), and lungs were examined at 5 wk postinfection compared with sex- and age-matched controls. A and B: whole lungs were gravity inflated from a height of ∼25 cm using 10% formalin for 5 min and then removed intact from the sternum. Hematoxylin & eosin (H&E) staining was performed and images captured at ×200 magnification. Representative images shown. C and D: H&E stained lung sections were blind-coded and four images captured from the upper left, lower left, upper right, and lower right sections of the lung and alveolar size, and number were quantified using Fiji software. E and F: immunohistochemistry staining was performed on five-micrometer lung sections to detect alveolar type-1 cells (green) and alveolar type-2 cells (red). G: whole lungs were collected and processed into single cell suspension and alveolar type-2 cells were quantified using flow cytometry. *P < 0.05, **P < 0.01; n = 5 mice/group.

Neonatal RSV Infection Leads to Long-Term Severe Pulmonary Function Deficiencies

To further evaluate persistent pulmonary defects following early-life RSV infection, lung function was evaluated at 3 mo postneonatal RSV infection. Significant defects involved in the compliance and resistance of the lung similar to those observed at 5 wk postinfection including a significant reduction in dynamic lung compliance (Cdyn) with correlative increases in lung resistance (Rl), compliant pressure (dPpl), and maximum pressure (dPmax) (Fig. 3, A–C) continued to be prevalent. In addition, we observed decreases in dynamic breathing parameters, including FVC in the mice that were neonatally infected with RSV (Fig. 3D). Furthermore, the FEV₁₀₀ continues to be significantly reduced by 24% with an increased FEV₁₀₀/FVC ratio (Fig. 3, E and F), indicating a restrictive lung phenotype. Similar to the 5-wk postinfection data, we also observe a significant decrease in the static inspiratory and expiratory capacities (VC) of the lung in the mice given an early-life RSV infection compared with age/sex-matched controls (Fig. 3G). Compliance of the lung shown through analysis of Cchord and Cfvc50 were also significantly altered (Fig. 3H). These data indicate that the lungs of mice following an early-life RSV infection have a decreased lung function even at 3 mo postinfection.

Figure 3.

Neonatal respiratory syncytial virus (RSV) infection leads to long-term severe pulmonary function deficiencies. Neonatal male mice were infected at 7 days of age (EL-RSV) and pulmonary function tests (PFT) were performed using plethysmography of tracheotomized mice at 3 mo postinfection compared with sex- and age-matched controls. A–C: measurements of lung compliance and resistance. D–F: flow-volume relationships measuring dynamic lung properties. G and H: static inhalation and exhalation capacities of the lung. ***P < 0.001, ****P < 0.0001; n = 4 mice/group. dPpl, compliant pressure; dPmax, maximum pressure; FEV, forced expiratory volume; FVC, forced vital capacity; Cchord, Chord compliance; Cfvc50, compliance at 50% vital capacity.

Early-Life RSV Infection Induced Lung Changes in TSLPR^−/− Mice

Previously published studies have identified that neonatal RSV infection can induce susceptibility to allergic responses later in life and was associated with TSLP-induced responses and alteration of matrix deposition (15, 18, 29, 30). These responses were characterized by altered transcriptional profiles and chromatin remodeling associated with decreased type I IFN production in bone marrow-derived DC, strongly suggesting an altered immune phenotype. In the present studies, we have extended this to include an association of TSLP-induced responses with the baseline airway functional and structural changes induced by early-life RSV infection. The data presented in Fig. 4 clearly identify that the lung function, while again changed in wild-type (WT) mice given an early-life RSV infection, is not altered in TSLPR-deficient mice. In particular, parameters observed in the previous data at 4 wk and 3 mo post-RSV infection, including Cdyn (Fig. 4A), resistance (Fig. 4B), and pressure measurements (Fig. 4C), as well as FEV and FVC (Fig. 4D), and lung capacities (Fig. 4F) and compliance (Fig. 4G) measures are all unchanged and similar to uninfected mice in the TSLPR^−/− animals. Furthermore, the histopathologic examination (Fig. 4, H–K) and blinded measurement of alveolar size (Fig. 4L) demonstrate no changes in the airway structure in the TSLPR^−/− animals. Together, these data, with our previous data on TSLPR and its impact on immune responses (15), further suggest a relationship between inflammatory immune responses and lung structure/function that may be most critical in early life when the airways and lungs are developing.

Figure 4.

Early-life respiratory syncytial virus (RSV) infection induced lung changes are mitigated in TSLPR^−/− mice. Neonatal Balb/c wild-type (WT) and TSLPR^−/− male mice were infected at 7 days of age (EL-RSV), and pulmonary function tests (PFT) were performed using plethysmography of tracheotomized mice at 5 wk postinfection compared with sex- and age-matched controls. A–C: measurements of lung compliance and resistance. D and E: flow-volume relationships measuring dynamic lung properties. F and G: static capacities of the lung. H–K: whole lungs were gravity inflated from a height of ∼25 cm using 10% formalin for 5 min and then removed intact from the sternum. Hematoxylin & eosin (H&E) staining was performed and images captured at ×200 magnification. Representative images shown. L: H&E stained lung sections were blind-coded and four images captured from the upper left, lower left, upper right, and lower right sections of the lung, and alveolar size was quantified using Fiji software. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n = 5 mice/group. dPpl, compliant pressure; dPmax, maximum pressure; FEV, forced expiratory volume; FVC, forced vital capacity; Cchord, Chord compliance; Cfvc50, compliance at 50% vital capacity.

Neonatal RSV Infection Lung Function Defects and Structure Alteration upon Secondary RSV Infection

It has been well established that children infected during early life with RSV can develop exacerbated reactions, including wheezing and difficulty breathing, upon future respiratory virus infection (31, 32). Therefore, to examine long-term consequences of early-life RSV, neonatally infected mice were reinfected at 3 mo postinfection, and analysis was performed 7 days postinfection (Fig. 5A). The PFT analyses demonstrate extensive exacerbation of lung function defects, including resistance/compliance (Fig. 5, B–D), flow-volume dynamic breathing (Fig. 5, E–G), and pressure-volume static functions (Fig. 5, H and I). These defects include significant loss of both dynamic and static lung compliance (Fig. 5, B and I) accompanied by increased lung resistance and inflation pressure (Fig. 5, C and D) with decreased dynamic and static inspiratory and expiratory capacities (Fig. 5, E and H). Importantly, FEV was significantly reduced by >20% in the reinfected animals (Fig. 5F) with an increased FEV/FVC ratio (Fig. 5G), again indicating a restrictive lung phenotype. To examine the structural architecture of the lungs, trichrome staining was performed, and visual observation of the alveolar spaces indicates a loss of integrity within the lungs and severe consolidation of alveolar spaces throughout the lungs of the reinfected mice (Fig. 5, J–L). These data support clinical data that indicate that early-life RSV infection can lead to reoccurring pulmonary function complications upon subsequent infection cycles, even at later life periods.

Figure 5.

Neonatal respiratory syncytial virus (RSV) infection primes for severe lung functional defects and structural destruction upon secondary RSV infection. Neonatal male mice were infected at 7 days of age and a second RSV infection performed at 3 mo post-early-life infection (EL-RSV + Adult RSV) and compared with age-matched adult mice given a single RSV infection at 3 mo of age (Adult only RSV) and naïve mice. Analysis was then performed at 7 days post adult RSV infection. A: experimental design. B–I: pulmonary function tests (PFT) were performed using plethysmography of tracheotomized mice. J–L: the two middle lobes of the right lung were removed and fixed in formalin and embedded in paraffin. Five-micrometer sections were stained with trichrome to visualize collagen deposition and alveolar consolidation. Representative images shown at ×200 magnification. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n= 5 mice/group. dPpl, compliant pressure; dPmax, maximum pressure; FEV, forced expiratory volume; FVC, forced vital capacity; Cchord, Chord compliance; Cfvc50, compliance at 50% vital capacity. [Image A created with a licensed version of BioRender.com.]

In addition to lung functional complications, children who previously had severe disease following RSV infection during early life have been shown to develop enhanced immunopathology upon future respiratory viral exposures later in life (33, 34). Histological observations within the lung using periodic-acid Schiff (PAS) staining indicate significant airway occlusion by mucus deposition (Fig. 6, A–C) correlated by significantly enhanced mucus scoring (Fig. 6D). H&E staining further shows severe infiltration of inflammatory cells into the lungs of the reinfected mice (Fig. 6, E–G). Flow cytometry analysis indicates that the increased inflammatory cells are mostly CD103+ dendritic cells (DC), CD4+/CD8+ T cells (total and CD69+ activated/memory), and innate lymphoid type-2 cells (ILC2) (Fig. 6, H–K) compared with adult only RSV. These data support neonatal RSV infection leading to enhanced immunopathology following secondary infection at a much later time due to infiltration of inflammatory cells that leads to mucus deposition/airway occlusion that accompanies severe lung function defects.

Figure 6.

Previous neonatal respiratory syncytial virus (RSV) infection leads to enhanced recruitment of inflammatory cells and mucus deposition upon reinfection with RSV 3 mo later. Neonatal male mice were infected at 7 days of age and a second RSV infection performed at 3 mo post-early-life infection (EL-RSV + Adult RSV) and compared with age-matched adult mice given a single RSV infection at 3 mo of age (Adult only RSV) and naïve mice. Analysis was then performed at 7 days post adult RSV infection. A–C: the two middle lobes of the right lung were removed and fixed in formalin and embedded in paraffin. Five-micrometer sections were stained with periodic-acid Schiff (PAS) to visualize mucus deposition. Representative images shown at ×200 magnification. D: slides containing PAS-stained lung sections were blind coded and scored by an individual observer to quantify mucus on a scale of 1–4. 1 = Minimal/No Mucus; 2 = Slight: Multiple airways with goblet cell hyperplasia and mucus; 3 = Moderate: Multiple airways with significant mucus and some plugging; 4 = Severe: significant Mucus plugging. E–G: the two middle lobes of the right lung were removed and fixed in formalin and embedded in paraffin. Five-micrometer sections were stained with hematoxylin & Eosin (H&E) to visualize inflammatory immune cell infiltration. Representative images shown at ×200 magnification. H–K: the left lung was removed and dissociated into a single cell suspension and stained for flow cytometry analysis to quantify immune cell populations within the lung. *P < 0.05, **P < 0.01; n = 5 mice/group. DC, dendritic cells; ILC2, innate lymphoid type-2 cells.

DISCUSSION

The long-term effects of early-life pathogenic exposures within the lung may be associated with pulmonary complications throughout life. One of the first clinically significant pathogens encountered in life is RSV, with nearly all children infected by 2 years of age. Immunologic memory following RSV infection is often suboptimal, skewed toward pathogenic responses, and insufficient for blocking reinfection throughout life (35–37). In addition, severe early-life RSV infection is associated with childhood wheezing related to respiratory viral infection exacerbation, allergies, and asthma, that is accompanied by compromised lung function (13, 38–40). Interestingly, males are predisposed compared with females for severe clinical disease in infants as well in preclinical modeling of severe RSV (15, 41–43) and allowed our studies to focus on male mice. Several studies have demonstrated altered lung changes in mice upon early-life RSV infection that have been correlated to changes in immune cell numbers and responses upon allergen or RSV rechallenge (20, 44–49). These latter studies have primarily examined upper airway responsiveness upon a methacholine challenge promoting a hyper-responsive effect. Studies to uncover the mechanisms of lung functional complications have suggested numerous causes, including immune cell function and altered repair. The findings reported in the present studies expand upon these ideas and indicate that following an early-life RSV infection, 1) baseline lung function is impaired long-term, 2) lower airway alveolar development is altered, and 3) enhanced immunopthology and lung remodeling accompany pulmonary function defects upon reinfection. The link between immune cell activation and lung structural alterations were further strengthened by the absence of lung structural changes in early-life infected TSLPR^−/− mice that have been previously shown to lack inflammatory DC and ILC2 cell accumulation in the lung post-early-life RSV infection (15).

In the clinic, diagnosis of obstructive versus restrictive lung disease is accomplished by comparing the FEV/FVC ratio with a decreased indication of an obstructive phenotype and increased or unchanged FEV/FVC indicative of a restrictive lung phenotype (50, 51). Our studies identified that long-term functional defects in the lung following early-life RSV are associated with a more restrictive lung phenotype, indicated by increased lung resistance with decreased compliance of the lung, decreased FEV and FVC absolute values, and an unchanged FEV/FVC ratio (51–53). However, the fact that the studies also indicate persistent upper airway mucus suggests a potential mixed restrictive and obstructive phenotype. The inappropriate pathogenic response to viral infection later in life would further promote lung function defects that could continue to reinforce and worsen the altered lung structure and obstructive phenotype. In a study examining 81 children hospitalized for RSV bronchiolitis in infancy, an association with a restrictive pattern of lung function later in life (median age of ∼12 yr) was observed, further demonstrating a correlation of lung alteration by early-life RSV infection (54). The consequence of this functional change into adulthood is presently unclear, but a recent large clinical study on early-life lower respiratory infection has indicated that those patients have twice the risk of dying prematurely from respiratory disease (55).

In humans and mice, lung development begins in utero and continues throughout childhood (56). Alveolarization continues into adulthood with maximum alveolarization occurring between 2 and 3 yr in humans (57–59). In the mouse lung, alveolarization peaks between birth and 14 days of age. It is of interest to note that this peak in alveolarization corresponds with an influx in type 2 immune cells (ILC2) and IL-13 production, which enhance lung development (23, 60) and are greatly enhanced during RSV infection (16, 18, 19). Other studies have suggested that remodeling of airways can occur after RSV and other respiratory virus infection (21, 45, 61–64). Extracellular matrix (ECM) dysregulation could explain the restrictive phenotype of the lungs and an area to explore in the future. Perhaps the persistent presence of ILC2 and other inflammatory immune cells in the lung post-RSV infection, as shown in previous studies (15), promotes the altered development/repair responses and alters the lung long-term. This latter inflammatory process is altered if specific innate immune mediator pathways, IL-1, IL-33, and TSLP, are blocked (15, 18, 29, 30, 65–71) and in these studies, TSLP pathway blockade ameliorates the altered lung structure/function defects.

In patients of all ages, exposure to pathogens within the lung leads to altered lung function, including asthmatics that suffer from severe exacerbations during viral infections (72–74). These exacerbations can be caused by a persistent type 2 immune environment and/or structural alterations due to persistent injury. Previous studies have shown that a persistent increase in inflammatory cells is observed following early-life RSV infection that includes DC and ILC2 (15, 18, 19, 47, 75). The detrimental role of increased inflammatory DC and ILC2 following RSV infection may be twofold: 1) as inducers of pathogenic inflammation and 2) in altering the structural/developmental processes of the lungs in neonates. ILC2 appears to have a role in normal lung development but studies have also identified that DC can enhance ILC2 function leading to pathogenic effects. The DC- and ILC2-mediated inflammatory events post-RSV infection were shown to be associated with persistent expression of TSLP as well as IL-33 in the lung that can lead to a Th2 immune environment (15, 18, 66, 76–82). The TSLPR^−/− mice did not develop altered lung structure/function changes with early-life RSV infection corresponding to the previously observed lack of immune cell (DC, ILC2) accumulation (15). Our current reinfection studies expand upon these concepts showing that a secondary RSV infection at 3 mo post-early-life infection leads to exacerbated lung dysfunction, increased mucus, and increased inflammatory cell infiltration (e.g., T cells/DC/ILC2).

Although multiple early-life events likely also trigger a similar altered lung phenotype including BPD (83–86), these studies identify a causative role for early-life respiratory virus infection leading to lung dysfunction that may initiate a long-term and possibly a lifetime of structural alteration within the lung. Importantly, the structural defects of the early-life infected mice largely mimic the clinical setting where severe exacerbations are observed in children for several years following a severe early-life respiratory viral infection, especially RSV. The mechanisms for these changes are likely complex and associated with an inappropriate immune environment, specific cellular responses, and control of lung mesenchymal/epithelial cell maturation.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This research was funded by the National Institutes of Health, Grant Nos. HL158001-01 (to C.-A.M.), R35HL150682 (to N.W.L.), and RO1AI138348 (to N.W.L.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.-A.M., W.F., D.M.W., S.F.Z., R.L.Z., and N.W.L. conceived and designed research; C.-A.M., W.F., S.M.H., S.B.M., A.J.R., K.Y., and D.M.W. performed experiments; C.-A.M., W.F., A.J.R., K.Y., R.L.Z., and N.W.L., analyzed data; C.-A.M., W.F., D.M.W., S.F.Z., and N.W.L. interpreted results of experiments; C.-A.M., W.F., and N.W.L. prepared figures; C.-A.M., W.F., and N.W.L. drafted manuscript; C.-A.M., W.F., S.M.H., R.L.Z., and N.W.L., edited and revised manuscript; C.-A.M., W.F., S.M.H., S.B.M., A.J.R., K.Y., D.M.W., S.F.Z., R.L.Z., and N.W.L. approved final version of manuscript.