Exposure to combustion derived particulate matter exacerbates influenza infection in neonatal mice by inhibiting IL22 production.

BACKGROUND: Particulate matter (PM) containing environmentally persistent free radicals (EPFRs) are formed during various combustion processes, including the thermal remediation of hazardous wastes. Exposure to PM adversely affects respiratory health in infants and is associated with increased morbidity and mortality due to acute lower respiratory tract infections. We previously reported that early-life exposure to PM damages the lung epithelium and suppresses immune responses to influenza virus (Flu) infection, thereby enhancing Flu severity. Interleukin 22 (IL22) is important in resolving lung injury following Flu infection. In the current study, we determined the effects of PM exposure on pulmonary IL22 responses using our neonatal mouse model of Flu infection. RESULTS: Exposure to PM resulted in an immediate (0.5-1-day post-exposure; dpe) increase in IL22 expression in the lungs of C57BL/6 neonatal mice; however, this IL22 expression was not maintained and failed to increase with either continued exposure to PM or subsequent Flu infection of PM-exposed mice. This contrasts with increased IL22 expression in age-matched mice exposed to vehicle and Flu infected. Activation of the aryl hydrocarbon receptor (AhR), which mediates the induction and release of IL22 from immune cells, was also transiently increased with PM exposure. The microbiome plays a major role in maintaining epithelial integrity and immune responses by producing various metabolites that act as ligands for AhR. Exposure to PM induced lung microbiota dysbiosis and altered the levels of indole, a microbial metabolite. Treatment with recombinant IL22 or indole-3-carboxaldehyde (I3A) prevented PM associated lung injury. In addition, I3A treatment also protected against increased mortality in Flu-infected mice exposed to PMs. CONCLUSIONS: Together, these data suggest that exposure to PMs results in failure to sustain IL22 levels and an inability to induce IL22 upon Flu infection. Insufficient levels of IL22 may be responsible for aberrant epithelial repair and immune responses, leading to increased Flu severity in areas of high PM.

Respiratory Syncytial Virus Disease Severity Is Associated with Distinct CD8+ T-Cell Profiles.

Rationale: Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants worldwide. Although T-helper type 2 (Th2) cell pathology is implicated in severe disease, the mechanisms underlying the development of immunopathology are incompletely understood.Objectives: We aimed to identify local immune responses associated with severe RSV in infants. Our hypothesis was that disease severity would correlate with enhanced Th2 cellular responses.Methods: Nasal aspirates were collected from infants hospitalized with severe (admitted to the pediatric ICU) or moderate (maintained in the general ward) RSV disease at 5 to 9 days after enrollment. The immune response was investigated by evaluating T-lymphocyte cellularity, cytokine concentration, and viral load.Measurements and Main Results: Patients with severe disease had increased proportions of CD8 (cluster of differentiation 8)-positive T cells expressing IL-4 (Tc2) and reduced proportions of CD8+ T cells expressing IFNγ (Tc1). Nasal aspirates from patients with severe disease had reduced concentrations of IL-17. Patients with greater frequencies of Tc1, CD8+ T cells expressing IL-17 (Tc17), and CD4+ T cells expressing IL-17 (Th17) had shorter durations of hospitalization.Conclusions: Severe RSV disease was associated with distinct T-cell profiles. Tc1, Tc17, and Th17 were associated with shorter hospital stay and may play a protective role, whereas Tc2 cells may play a previously underappreciated role in pathology.

Altered gut microbiota in infants is associated with respiratory syncytial virus disease severity.

BACKGROUND: Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infections in infants. There are still no vaccines or specific antiviral therapies against RSV, mainly due to the inadequate understanding of RSV pathogenesis. Recent data suggest a role for gut microbiota community structure in determining RSV disease severity. Our objective was to determine the gut microbial profile associated with severe RSV patients, which could be used to help identify at-risk patients and develop therapeutically protective microbial assemblages that may stimulate immuno-protection. RESULTS: We enrolled 95 infants from Le Bonheur during the 2014 to 2016 RSV season. Of these, 37 were well-babies and 58 were hospitalized with RSV. Of the RSV infected babies, 53 remained in the pediatric ward (moderate) and 5 were moved to the pediatric intensive care unit at a later date (severe). Stool samples were collected within 72 h of admission; and the composition of gut microbiota was evaluated via 16S sequencing of fecal DNA. There was a significant enrichment in S24_7, Clostridiales, Odoribacteraceae, Lactobacillaceae, and Actinomyces in RSV (moderate and severe) vs. controls. Patients with severe RSV disease had slightly lower alpha diversity (richness and evenness of the bacterial community) of the gut microbiota compared to patients with moderate RSV and healthy controls. Beta diversity (overall microbial composition) was significantly different between all RSV patients (moderate and severe) compared to controls and had significant microbial composition separating all three groups (control, moderate RSV, and severe RSV). CONCLUSIONS: Collectively, these data demonstrate that a unique gut microbial profile is associated with RSV disease and with severe RSV disease with admission to the pediatric intensive care unit. More mechanistic experiments are needed to determine whether the differences observed in gut microbiota are the cause or consequences of severe RSV disease.

Elevated Levels of Type 2 Respiratory Innate Lymphoid Cells in Human Infants with Severe Respiratory Syncytial Virus Bronchiolitis.

Rationale: Studies of the immune responses at the site of respiratory syncytial virus (RSV) infection are sparse despite nearly five decades of research into understanding RSV disease.Objectives: To investigate the role of mucosal innate immune responses to RSV and respiratory viral load in infants hospitalized with the natural disease.Methods: Cytokines, viral load, and type 2 innate lymphoid cell (ILC2) levels in nasal aspirates, collected within 24 hours of enrollment, from infants hospitalized with RSV infection were quantified.Measurements and Main Results: RSV severity in infants was categorized based on admission to the general ward (moderate) or the pediatric ICU (severe). Evaluable subjects included 30 patients with severe and 63 patients with moderate disease (median age, 74 d; range, 9-297 d). ILC2s were found in the nasal aspirates of patients with severe disease (0.051% of total respiratory CD45+ cells) to a significantly greater extent than in patients with moderate disease (0.018%, P = 0.004). Levels of IL-4, IL-13, IL-33, and IL-1ß were significantly higher in nasal aspirates of patients with severe disease compared with those of patients with moderate disease. Factors associated with disease severity were gestational age (odds ratio, 0.49; 95% confidence interval, 0.29-0.82; P = 0.007) and IL-4 (odds ratio, 9.67; 95% confidence interval, 2.45-38.15; P = 0.001).Conclusions: This study shows, for the first time, that elevated levels of ILC2s is associated with infant RSV severity. The findings highlight the dominance of type-2 responses to RSV infection in infants and suggest an important role of ILC2 in shaping the immune response early during RSV infection.

Androgen receptor agonists increase lean mass, improve cardiopulmonary functions and extend survival in preclinical models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease that predominantly affects boys as a result of mutation(s) in the dystrophin gene. DMD is characterized by musculoskeletal and cardiopulmonary complications, resulting in shorter life-span. Boys afflicted by DMD typically exhibit symptoms within 3-5 years of age and declining physical functions before attaining puberty. We hypothesized that rapidly deteriorating health of pre-pubertal boys with DMD could be due to diminished anabolic actions of androgens in muscle, and that intervention with an androgen receptor (AR) agonist will reverse musculoskeletal complications and extend survival. While castration of dystrophin and utrophin double mutant (mdx-dm) mice to mimic pre-pubertal nadir androgen condition resulted in premature death, maintenance of androgen levels extended the survival. Non-steroidal selective-AR modulator, GTx-026, which selectively builds muscle and bone was tested in X-linked muscular dystrophy mice (mdx). GTx-026 significantly increased body weight, lean mass and grip strength by 60-80% over vehicle-treated mdx mice. While vehicle-treated castrated mdx mice exhibited cardiopulmonary impairment and fibrosis of heart and lungs, GTx-026 returned cardiopulmonary function and intensity of fibrosis to healthy control levels. GTx-026 elicits its musculoskeletal effects through pathways that are distinct from dystrophin-regulated pathways, making AR agonists ideal candidates for combination approaches. While castration of mdx-dm mice resulted in weaker muscle and shorter survival, GTx-026 treatment increased the muscle mass, function and survival, indicating that androgens are important for extended survival. These preclinical results support the importance of androgens and the need for intervention with AR agonists to treat DMD-affected boys.

New mouse model of pulmonary hypertension induced by respiratory syncytial virus bronchiolitis.

Pulmonary hypertension (PH) has been observed in up to 75% of infants with moderate to severe respiratory syncytial virus (RSV) bronchiolitis and is associated with significant morbidity and mortality in infants with congenital heart disease. The purpose of the present study was to establish a mouse model of PH secondary to RSV bronchiolitis that mimics the disease etiology as it occurs in infants. Neonatal mice were infected with RSV at 5 days of age and then reinfected 4 wk later. Serum-free medium was administered to age-matched mice as a control. Echocardiography and right ventricular systolic pressure (RVSP) measurements via right jugular vein catheterization were conducted 5 and 6 days after the second infection, respectively. Peripheral capillary oxygen saturation monitoring did not indicate hypoxia at 2-4 days post-RSV infection, before reinfection, and at 2-7 days after reinfection. RSV-infected mice had significantly higher RVSP than control mice. Pulsed-wave Doppler recording of the pulmonary blood flow by echocardiogram demonstrated a significantly shortened pulmonary artery acceleration time and decreased pulmonary artery acceleration time-to-ejection time ratio in RSV-infected mice. Morphometry showed that RSV-infected mice exhibited a significantly higher pulmonary artery medial wall thickness and had an increased number of muscularized pulmonary arteries compared with control mice. These findings, confirmed by RVSP measurements, demonstrate the development of PH in the lungs of mice infected with RSV as neonates. This animal model can be used to study the pathogenesis of PH secondary to RSV bronchiolitis and to assess the effect of treatment interventions. NEW & NOTEWORTHY This is the first mouse model of respiratory syncytial virus-induced pulmonary hypertension, to our knowledge. This model will allow us to decipher molecular mechanisms responsible for the pathogenesis of pulmonary hypertension secondary to respiratory syncytial virus bronchiolitis with the use of knockout and/or transgenic animals and to monitor therapeutic effects with echocardiography.

Crawling with Virus: Translational Insights from a Neonatal Mouse Model on the Pathogenesis of Respiratory Syncytial Virus in Infants.

The infant immune response to respiratory syncytial virus (RSV) remains incompletely understood. Here we review the use of a neonatal mouse model of RSV infection to mimic severe infection in human infants. We describe numerous age-specific responses, organized by cell type, observed in RSV-infected neonatal mice and draw comparisons (when possible) to human infants.

Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33.

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.

Regulatory T cells and IL10 suppress pulmonary host defense during early-life exposure to radical containing combustion derived ultrafine particulate matter.

BACKGROUND: Exposure to elevated levels of particulate matter (PM) is associated with increased risk of morbidity and mortality due to respiratory tract viral infections in infants. Recent identification of environmentally persistent free radicals (EPFRs) in the PM from a variety of combustion sources suggests its role in the enhancement of disease severity of lower respiratory tract infections (LRTI). Our previous studies demonstrated that acute exposure to EPFRs induces pulmonary immunosuppression allowing for enhanced influenza disease severity. Here, we determine the mechanism of EPFR-induced immunosuppression and its impact on the immune response towards influenza infection. METHODS: Neonatal mice (3 days old) were acutely exposed to DCB (combustion derived PM with chemisorbed EPFR) for seven consecutive days. Four days post-exposure (dpe), mice were infected with influenza virus. Pulmonary T cell phenotypes including regulatory T cells (Tregs) were analyzed by flow cytometry. The role of IL10 in EPFR-induced exacerbation of influenza disease severity was determined by administering recombinant IL10 (rIL10) to wild type mice or by using IL10 deficient (IL10-/-) neonatal mice. Mice were assessed for morbidity by measuring percent weight change and pulmonary viral load. RESULTS: Neonatal mice exposed to EPFRs had a significant increase in pulmonary Tregs and the immunosuppressive cytokine IL10 following influenza infection, which coincided with decreased protective T cell responses to influenza infection at 6 dpi. Depletion of Tregs in EPFR-exposed neonatal mice resulted in increased protective, adaptive T cell responses, whereas adoptive transfer of Tregs from EPFR-exposed neonates to air-exposed neonatal mice suppressed adaptive T cell responses towards influenza infection. Further, treatment with rIL10 could recapitulate EPFR-induced exacerbation of morbidity and pulmonary viral load compared to air exposed and influenza infected mice, whereas, EPFR-exposed IL10-/- neonates exhibited significant reductions in morbidity, pulmonary viral load and adaptive T cell responses following influenza infection. CONCLUSIONS: Neonatal exposure to EPFRs induced Tregs and IL10 resulting in suppressed adaptive T cell responses and enhanced influenza disease severity in neonatal mice. Depletion of Tregs increased adaptive T cell responses and deficiency of IL10 reduced morbidity and conferred enhanced protection against influenza virus.

Impaired gamma delta T cell-derived IL-17A and inflammasome activation during early respiratory syncytial virus infection in infants.

Respiratory syncytial virus (RSV) infection remains a significant global health burden disproportionately affecting infants and leading to long-term lung disease. Interleukin (IL)-17A has been shown to be involved in regulating viral and allergic lung inflammatory responses, which has led to a more recent interest in its role in RSV infection. Using a neonatal mouse model of RSV, we demonstrate that neonates fail to develop IL-17A responses compared with adult mice; the main immediate IL-17A contributor in adults were γδ T cells. Antibody neutralization of IL-17A in adult mice caused increased lung inflammation and airway mucus from RSV, whereas exogenous IL-17A administration to RSV-infected neonates caused decreased inflammation but no change in airway mucus. We also observed a lack of pro-inflammatory cytokine production (IL-1ß, IL-6) from infected neonates. Using human cord blood mononuclear cells (CBMCs) and adult peripheral blood mononuclear cells (PBMCs), we compared inflammasome activation by direct retinoic acid-inducible gene I agonism; CBMCs failed to induce pro-inflammatory cytokines or IL-17A(+) γδ T cells compared with PBMCs. Our results indicate that RSV disease severity is in part mediated by a lack of inflammasome activation and IL-17A production in neonates.

Limited type I interferons and plasmacytoid dendritic cells during neonatal respiratory syncytial virus infection permit immunopathogenesis upon reinfection.

UNLABELLED: Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN-α-treated neonatal mice, suggesting dual mechanisms of action. IMPORTANCE: Respiratory syncytial virus (RSV) is the most significant cause of lower respiratory tract infection in infancy worldwide. Despite the dire need, we have failed to produce efficacious RSV vaccines or therapeutics. Part of the reason for this failure is our lack of understanding of how RSV interacts with the infant immune system to suppress the development of protective immunity. In the study described in the present paper, we used a neonatal mouse model, which more closely mimics human infants, to study the role of the innate immune system, particularly type I interferons (IFNs) and plasmacytoid dendritic cells (pDCs), in the pathogenesis of RSV infection. RSV infection in neonates induced limited type I IFN and pDC responses. IFN-α treatment or adoptive transfer of adult pDCs capable of producing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. These data suggest that IFN-α is a promising target for future RSV vaccine design.

Building a better neonatal mouse model to understand infant respiratory syncytial virus disease.

BACKGROUND: Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infection in infants; and severe RSV infection in infants is associated with asthma development. Today, there are still no vaccines or specific antiviral therapies against RSV. The mechanisms of RSV pathogenesis in infants remain elusive. This is partly due to the fact that the largely-used mouse model is semi-permissive for RSV. The present study sought to determine if a better neonatal mouse model of RSV infection could be obtained using a chimeric virus in which the F protein of A2 strain was replaced with the F protein from the line 19 clinical isolate (rA2-19F). METHODS: Five-day-old pups were infected with the standard laboratory strain A2 or rA2-19F and various immunological and pathophysiological parameters were measured at different time points post infection, including lung histology, bronchoalveolar lavage fluid (BALF) cellularity and cytokines, pulmonary T cell profile, and lung viral load. A cohort of infected neonates were allowed to mature to adulthood and reinfected. Pulmonary function, BALF cellularity and cytokines, and T cell profiles were measured at 6 days post reinfection. RESULTS: The rA2-19F strain in neonatal mice caused substantial lung pathology including interstitial inflammation and airway mucus production, while A2 caused minimal inflammation and mucus production. Pulmonary inflammation was characterized by enhanced Th2 and reduced Th1 and effector CD8(+) T cells compared to A2. As with primary infection, reinfection with rA2-19F induced similar but exaggerated Th2 and reduced Th1 and effector CD8(+) T cell responses. These immune responses were associated with increased airway hyperreactivity, mucus hyperproduction and eosinophilia that was greater than that observed with A2 reinfection. Pulmonary viral load during primary infection was higher with rA2-19F than A2. CONCLUSIONS: Therefore, rA2-19F caused enhanced lung pathology and Th2 and reduced effector CD8(+) T cell responses compared to A2 during initial infection in neonatal mice and these responses were exacerbated upon reinfection. The exact mechanism is unknown but appears to be associated with increased pulmonary viral load in rA2-19F vs. A2 infected neonatal lungs. The rA2-19F strain represents a better neonatal mouse model of RSV infection.

Hepatitis E antibodies in laboratory rabbits from 2 US vendors.

We tested laboratory rabbits from 2 US vendors for antibodies against hepatitis E virus (HEV); Seroprevalences were 40% and 50%. Retrospective analysis of an ocular herpes simplex 1 experiment demonstrated that HEV seropositivity had no effect on experiment outcome. HEV probably is widespread in research rabbits, but effects on research remain unknown.

Exposure to combustion generated environmentally persistent free radicals enhances severity of influenza virus infection.

BACKGROUND: Exposures to elevated levels of particulate matter (PM) enhance severity of influenza virus infection in infants. The biological mechanism responsible for this phenomenon is unknown. The recent identification of environmentally persistent free radicals (EPFRs) associated with PM from a variety of combustion sources suggests its role in the enhancement of influenza disease severity. METHODS: Neonatal mice (< seven days of age) were exposed to DCB230 (combustion derived PM with a chemisorbed EPFR), DCB50 (non-EPFR PM sample), or air for 30 minutes/day for seven consecutive days. Four days post-exposure, neonates were infected with influenza intranasally at 1.25 TCID50/neonate. Neonates were assessed for morbidity (% weight gain, peak pulmonary viral load, and viral clearance) and percent survival. Lungs were isolated and assessed for oxidative stress (8-isoprostanes and glutathione levels), adaptive immune response to influenza, and regulatory T cells (Tregs). The role of the EPFR was also assessed by use of transgenic mice expressing human superoxide dismutase 2. RESULTS: Neonates exposed to EPFRs had significantly enhanced morbidity and decreased survival following influenza infection. Increased oxidative stress was also observed in EPFR exposed neonates. This correlated with increased pulmonary Tregs and dampened protective T cell responses to influenza infection. Reduction of EPFR-induced oxidative stress attenuated these effects. CONCLUSIONS: Neonatal exposure to EPFR containing PM resulted in pulmonary oxidative stress and enhanced influenza disease severity. EPFR-induced oxidative stress resulted in increased presence of Tregs in the lungs and subsequent suppression of adaptive immune response to influenza.

Synergistic interactions of TLR2/6 and TLR9 induce a high level of resistance to lung infection in mice.

Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have focused historically on antibiotic development and vaccine-enhanced adaptive immunity. However, we have reported recently that the lungs' innate defenses can be induced therapeutically by inhalation of a bacterial lysate that protects mice against otherwise lethal pneumonia. In this study, we tested in mice the hypothesis that TLRs are required for this antimicrobial phenomenon and found that resistance could not be induced in the absence of the TLR signaling adaptor protein MyD88. We then attempted to recapitulate the protection afforded by the bacterial lysate by stimulating the lung epithelium with aerosolized synthetic TLR ligands. Although most single or combination treatments yielded no protection, simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent gram-positive and gram-negative pathogens. Protection was associated with rapid pathogen killing in the lungs, and pathogen killing could be induced from lung epithelial cells in isolation. Taken together, these data demonstrate the requirement for TLRs in inducible resistance against pneumonia, reveal a remarkable, unanticipated synergistic interaction of TLR2/6 and TLR9, reinforce the emerging evidence supporting the antimicrobial capacity of the lung epithelium, and may provide the basis for a novel clinical therapeutic that can protect patients against pneumonia during periods of peak vulnerability.

Maternal exposure to combustion generated PM inhibits pulmonary Th1 maturation and concomitantly enhances postnatal asthma development in offspring.

BACKGROUND: Epidemiological studies suggest that maternal exposure to environmental hazards, such as particulate matter, is associated with increased incidence of asthma in childhood. We hypothesized that maternal exposure to combustion derived ultrafine particles containing persistent free radicals (MCP230) disrupts the development of the infant immune system and results in aberrant immune responses to allergens and enhances asthma severity. METHODS: Pregnant C57/BL6 mice received MCP230 or saline by oropharyngeal aspiration on gestational days 10 and 17. Three days after the second administration, blood was collected from MCP230 or saline treated dams and 8-isoprostanes in the serum were measured to assess maternal oxidative stress. Pulmonary T cell populations were assayed in the infant mice at six days, three and six weeks of postnatal age. When the infant mice matured to adults (i.e. six weeks of age), an asthma model was established with ovalbumin (OVA). Airway inflammation, mucus production and airway hyperresponsiveness were then examined. RESULTS: Maternal exposure to MCP230 induced systemic oxidative stress. The development of pulmonary T helper (Th1/Th2/Th17) and T regulatory (Treg) cells were inhibited in the infant offspring from MCP230-exposed dams. As the offspring matured, the development of Th2 and Treg cells recovered and eventually became equivalent to that of offspring from non-exposed dams. However, Th1 and Th17 cells remained attenuated through 6 weeks of age. Following OVA sensitization and challenge, mice from MCP230-exposed dams exhibited greater airway hyperresponsiveness, eosinophilia and pulmonary Th2 responses compared to offspring from non-exposed dams. CONCLUSIONS: Our data suggest that maternal exposure to MCP230 enhances postnatal asthma development in mice, which might be related to the inhibition of pulmonary Th1 maturation and systemic oxidative stress in the dams.

Immunomodulation with IL-4R alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease.

Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants worldwide. Severe RSV infections in infants cause bronchiolitis, wheeze, and/or cough and significantly increase the risk for developing asthma. RSV pathogenesis is thought to be due to a Th2-type immune response initiated in response to RSV infection, specifically in the infant. Using a neonatal mouse system as an appropriate model for human infants, we sought to determine whether local inhibition of IL-4Rα expression during primary RSV infection in the neonate would prevent Th2-skewed responses to secondary RSV infection and improve long-term pulmonary function. To reduce IL-4Rα expression, antisense oligonucleotides (ASOs) specific for IL-4Rα were administered intranasally to neonatal mice at the time of primary infection. Mice were initially infected with RSV at 1 wk of age and were reinfected at 6 wk of age. Administration of IL-4Rα ASOs during primary RSV infection in neonatal mice abolished the pulmonary dysfunction normally observed following reinfection in the adult. This ablation of pulmonary dysfunction correlated with a persistent rebalancing of the Th cell compartment with decreased Th2 responses (i.e., reduced goblet cell hyperplasia, Th2 cells, and cytokine secretion) and increased Th1 responses (i.e., elevated Th1 cell numbers and type I Abs and cytokines). Our data support our hypothesis that a reduction in the Th2 immune response during primary infection in neonates prevents Th2-mediated pulmonary pathology initially and upon reinfection and further suggest that vaccine strategies incorporating IL-4Rα ASOs may be of significant benefit to infants.

Inchoate CD8+ T cell responses in neonatal mice permit influenza-induced persistent pulmonary dysfunction.

Influenza infection remains a significant cause of pulmonary morbidity and mortality worldwide, with the highest hospitalization and mortality rates occurring in infants and elder adults. The mechanisms inducing this considerable morbidity and mortality are largely unknown. To address this question, we established a neonatal mouse model of influenza infection to test the hypothesis that the immaturity of the neonatal immune system is responsible for the severe pulmonary disease observed in infants. Seven-day-old mice were infected with influenza A virus (H1N1) and allowed to mature. As adults, these mice showed enhanced airway hyperreactivity, chronic pulmonary inflammation, and diffuse emphysematous-type lesions in the lungs. The adaptive immune responses of the neonates were much weaker than those of adults. This insufficiency appeared to be in both magnitude and functionality and was most apparent in the CD8(+) T cell population. To determine the role of neonatal CD8(+) T cells in disease outcome, adult, naive CD8(+) T cells were adoptively transferred into neonates before infection. Neonatal mice receiving the adult CD8(+) T cells had significantly lower pulmonary viral titers and greatly improved pulmonary function as adults (airway resistance similar to SHAM). Additional adoptive transfer studies using adult CD8(+) T cells from IFN-gamma-deficient mice demonstrated the importance of IFN-gamma from CD8(+) T cells in controlling the infection and in determining disease outcome. Our data indicate that neonates are more vulnerable to severe infections due to immaturity of their immune system and emphasize the importance of vaccination in infants.

Type I Interferon Potentiates IgA Immunity to Respiratory Syncytial Virus Infection During Infancy.

Respiratory syncytial virus (RSV) infection is the most frequent cause of hospitalization in infants and young children worldwide. Although mucosal RSV vaccines can reduce RSV disease burden, little is known about mucosal immune response capabilities in children. Neonatal or adult mice were infected with RSV; a subset of neonatal mice received interferon alpha (IFN-α) (intranasal) prior to RSV infection. B cells, B cell activating factor (BAFF) and IgA were measured by flow cytometry. RSV specific IgA was measured in nasal washes. Nasal associated lymphoid tissue (NALT) and lungs were stained for BAFF and IgA. Herein, we show in a mouse model of RSV infection that IFN-α plays a dual role as an antiviral and immune modulator and age-related differences in IgA production upon RSV infection can be overcome by IFN-α administration. IFN-α administration before RSV infection in neonatal mice increased RSV-specific IgA production in the nasal mucosa and induced expression of the B-cell activating factor BAFF in NALT. These findings are important, as mucosal antibodies at the infection site, and not serum antibodies, have been shown to protect human adults from experimental RSV infection.

IL-4Rα on dendritic cells in neonates and Th2 immunopathology in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is one of the leading causes of bronchiolitis in children, and severe RSV infection early in life has been associated with asthma development. Using a neonatal mouse model, we have shown that down-regulation of IL-4 receptor α (IL-4Rα) with antisense oligonucleotides in the lung during neonatal infection protected from RSV immunopathophysiology. Significant down-regulation of IL-4Rα was observed on pulmonary CD11b+ myeloid dendritic cells (mDCs) suggesting a role for IL-4Rα on mDCs in the immunopathogenesis of neonatal RSV infection. Here, we demonstrated that neonatal CD11b+ mDCs expressed higher levels of IL-4Rα than their adult counterparts. Because CD11b+ mDCs mainly present antigens to CD4+ T cells, we hypothesized that increased expression of IL-4Rα on neonatal CD11b+ mDCs was responsible for Th2 - biased RSV immunopathophysiology. Indeed, when IL-4Rα was selectively deleted from CD11b+ mDCs, the immunopathophysiology typically observed following RSV reinfection was ablated, including Th2 inflammation, airway-mucus hyperproduction, and pulmonary dysfunction. Further, overexpression of IL-4Rα on adult CD11b+ DCs and their adoptive transfer into adult mice was able to recapitulate the Th2-biased RSV immunopathology typically observed only in neonates infected with RSV. IL-4Rα levels on CD11c+ cells were inversely correlated with maturation status of CD11b+ mDCs upon RSV infection. Our data demonstrate that developmentally regulated IL-4Rα expression is critical for the maturity of pulmonary CD11b+ mDCs and the Th2-biased immunopathogenesis of neonatal RSV infection.