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1.  IFN-γ Production during Initial Infection Determines the Outcome of Reinfection with Respiratory Syncytial Virus 
Rationale: Severe respiratory syncytial virus (RSV) bronchiolitis has been associated with deficient IFN-γ production in humans, but the role of this cytokine in determining the outcome of reinfection is unknown.
Objectives: To define the role of IFN-γ in the development of RSV-mediated airway hyperresponsiveness (AHR) and lung histopathology in mice.
Methods: Wild-type (WT) and IFN-γ knockout mice were infected with RSV in the newborn or weaning stages and reinfected 5 weeks later. Airway responses were assessed on Day 6 after the primary or secondary infection.
Measurements and Main Results: Both WT and IFN-γ knockout mice developed similar levels of AHR and airway inflammation after primary infection. After reinfection, IFN-γ knockout mice, but not WT mice, developed AHR, airway eosinophilia, and mucus hyperproduction. Intranasal administration of IFN-γ during primary infection but not during reinfection prevented the development of these altered airway responses on reinfection in IFN-γ knockout mice. Adoptive transfer of WT T cells into IFN-γ knockout mice before primary infection restored IFN-γ production in the lungs and prevented the development of altered airway responses on reinfection. Treatment of mice with IFN-γ during primary neonatal infection prevented the enhancement of AHR and the development of airway eosinophilia and mucus hyperproduction on reinfection.
Conclusions: IFN-γ production during primary RSV infection is critical to the development of protection against AHR and lung histopathology on reinfection. Provision of IFN-γ during primary infection in infancy may be a potential therapeutic approach to alter the course of RSV-mediated long-term sequelae.
doi:10.1164/rccm.200612-1890OC
PMCID: PMC2204078  PMID: 17962634
respiratory syncytial virus; interferon-γ; asthma; airway hyperresponsiveness; mice
2.  Delayed Sequelae of Neonatal Respiratory Syncytial Virus Infection Are Dependent on Cells of the Innate Immune System 
Journal of Virology  2014;88(1):604-611.
Infection with respiratory syncytial virus (RSV) in neonatal mice leads to exacerbated disease if mice are reinfected with the same virus as adults. Both T cells and the host major histocompatibility complex genotype contribute to this phenomenon, but the part played by innate immunity has not been defined. Since macrophages and natural killer (NK) cells play key roles in regulating inflammation during RSV infection of adult mice, we studied the role of these cells in exacerbated inflammation following neonatal RSV sensitization/adult reinfection. Compared to mice undergoing primary infection as adults, neonatally sensitized mice showed enhanced airway fluid levels of interleukin-6 (IL-6), alpha interferon (IFN-α), CXCL1 (keratinocyte chemoattractant/KC), and tumor necrosis factor alpha (TNF-α) at 12 to 24 h after reinfection and IL-4, IL-5, IFN-γ, and CCL11 (eotaxin) at day 4 after reinfection. Weight loss during reinfection was accompanied by an initial influx of NK cells and granulocytes into the airways and lungs, followed by T cells. NK cell depletion during reinfection attenuated weight loss but did not alter T cell responses. Depletion of alveolar macrophages with inhaled clodronate liposomes reduced both NK and T cell numbers and attenuated weight loss. These findings indicate a hitherto unappreciated role for the innate immune response in governing the pathogenic recall responses to RSV infection.
doi:10.1128/JVI.02620-13
PMCID: PMC3911760  PMID: 24173217
3.  The Role of T Cells in the Enhancement of Respiratory Syncytial Virus Infection Severity during Adult Reinfection of Neonatally Sensitized Mice▿  
Journal of Virology  2008;82(8):4115-4124.
Respiratory syncytial virus (RSV) is the major cause of infantile bronchiolitis and hospitalization. Severe RSV disease is associated with the development of wheezing in later life. In a mouse model of the delayed effects of RSV, the age at primary infection determines responses to reinfection in adulthood. During primary RSV infection, neonatal BALB/c mice developed only mild disease and recruited CD8 cells that were defective in gamma interferon production. Secondary reinfection of neonatally primed mice caused enhanced inflammation and profuse lung T-cell recruitment. CD4 cell depletion during secondary RSV challenge attenuated disease (measured by weight loss); depletion of CD8 cells also markedly attenuated disease severity but enhanced lung eosinophilia, and depletion of both CD4 and CD8 cells together completely abrogated weight loss. Depletion of CD8 (but not CD4) cells during primary neonatal infection was protective against weight loss during adult challenge. Therefore, T cells, in particular CD8 T cells, play a central role in the outcome of neonatal infection by enhancing disease during secondary challenge. These findings demonstrate a crucial role for T cells in the regulation of immune responses after neonatal infection.
doi:10.1128/JVI.02313-07
PMCID: PMC2293007  PMID: 18272579
4.  Age at First Viral Infection Determines the Pattern of T Cell–mediated Disease during Reinfection in Adulthood 
The Journal of Experimental Medicine  2002;196(10):1381-1386.
Infants experiencing severe respiratory syncytial virus (RSV) bronchiolitis have an increased frequency of wheeze and asthma in later childhood. Since most severe RSV infections occur between the 8th and 24th postnatal week, we examined whether age at first infection determines the balance of cytokine production and lung pathology during subsequent rechallenge. Primary RSV infection in newborn mice followed the same viral kinetics as in adults but was associated with reduced and delayed IFN-γ responses. To study rechallenge, mice were infected at 1 day or 1, 4, or 8 weeks of age and reinfected at 12 weeks. Neonatal priming produced more severe weight loss and increased inflammatory cell recruitment (including T helper 2 cells and eosinophils) during reinfection, whereas delayed priming led to enhanced interferon γ production and less severe disease during reinfection. These results show the crucial importance of age at first infection in determining the outcome of reinfection and suggest that the environment of the neonatal lung is a major determinant of cytokine production and disease patterns in later life. Thus, simply delaying RSV infection beyond infancy might reduce subsequent respiratory morbidity in later childhood.
doi:10.1084/jem.20020943
PMCID: PMC2193991  PMID: 12438429
bronchiolitis; asthma; immunity; pneumovirinae; virus
5.  The Role of RSV Infection in Asthma Initiation and Progression: Findings in a Mouse Model 
Pulmonary Medicine  2011;2011:748038.
Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract diseases (bronchiolitis and pneumonia) during infancy and early childhood. There is increasing evidence which indicates that severe pulmonary disease caused by RSV infection in infancy is associated with recurrent wheezing and development of asthma later in childhood. However, the underlying mechanisms linking RSV infection to persistent airway hyperresponsiveness and dysfunction are not fully defined. To study these processes in ways which are not available in humans, animal models have been established and have provided valuable insight into the pathophysiology of RSV-induced disease. In this paper, we discuss experimental models of RSV infection in mice and highlight a new investigative approach in which mice are initially infected as neonates and then reinfected later in life. The findings shed light on the mechanisms underlying the association between early severe RSV infection and development of asthma later in childhood.
doi:10.1155/2011/748038
PMCID: PMC3135221  PMID: 21766019
6.  An immunocompromised BALB/c mouse model for respiratory syncytial virus infection 
Virology Journal  2005;2:3.
Background
Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which can be fatal, especially in immunocompromised patients. The BALB/c mouse, currently used as a model for studying RSV immunopathology, is semi-permissive to the virus. A mouse model that more closely mimics human RSV infection is needed. Since immunocompromised conditions increase risk of RSV infection, the possibility of enhancing RSV infection in the BALB/c mouse by pretreatment with cyclophosphamide was examined in this study. BALB/c mice were treated with cyclophosphamide (CYP) and five days later, they were infected with RSV intranasally. Pulmonary RSV titers, inflammation and airway hyperresponsiveness were measured five days after infection.
Results
CYP-treated mice show higher RSV titers in their lungs of than the untreated mice. Also, a decreased percentage of macrophages and an increased number of lymphocytes and neutrophils were present in the BAL of CYP-treated mice compared to controls. The CYP-treated group also exhibited augmented bronchoalveolar and interstitial pulmonary inflammation. The increased RSV infection in CYP-treated mice was accompanied by elevated expression of IL-10, IL-12 and IFN-γ mRNAs and proteins compared to controls. Examination of CYP-treated mice before RSV infection showed that CYP treatment significantly decreased both IFN-γ and IL-12 expression.
Conclusions
These results demonstrate that CYP-treated BALB/c mice provide a better model for studying RSV immunopathology and that decreased production of IL-12 and IFN-γ are important determinants of susceptibility to RSV infection.
doi:10.1186/1743-422X-2-3
PMCID: PMC549044  PMID: 15701174
7.  Responsiveness to respiratory syncytial virus in neonates is mediated through thymic stromal lymphopoietin and OX40 ligand 
Background
Recent studies revealed a critical role for thymic stromal lymphopoietin (TSLP) released from epithelial cells and OX40 ligand (OX40L) expressed on dendritic cells (DCs) in TH2 priming and polarization.
Objectives
We sought to determine the importance of the TSLP-OX40L axis in neonatal respiratory syncytial virus (RSV) infection.
Methods
Mice were initially infected with RSV as neonates or adults and reinfected 5 weeks later. Anti-OX40L or anti-TSLP were administered during primary or secondary infection. Outcomes included assessment of airway function and inflammation and expression of OX40L, TSLP, and IL-12.
Results
OX40L was expressed mainly on CD11c+MHC class II (MHCII)+CD11b+ DCs but not CD103+ DCs. Treatment of neonates with OX40L antibody during primary RSV infection prevented the subsequent enhancement of airway hyperresponsiveness and the development of airway eosinophilia and mucus hyperproduction on reinfection. Administration of anti-TSLP before neonatal RSV infection reduced the accumulation of lung DCs, decreased OX40L expression on lung DCs, and attenuated the enhancement of airway responses after reinfection.
Conclusions
In mice initially infected as neonates, TSLP expression induced by RSV infection is an important upstream event that controls OX40L expression, lung DC migration, and TH2 polarization, accounting for the enhanced response on reinfection.
doi:10.1016/j.jaci.2012.08.033
PMCID: PMC3593657  PMID: 23036746
Respiratory syncytial virus; OX40 ligand; thymic stromal lymphopoietin
8.  Exposure of neonates to Respiratory Syncytial Virus is critical in determining subsequent airway response in adults 
Respiratory Research  2006;7(1):107.
Background
Respiratory syncytial virus (RSV) is the most common cause of acute bronchiolitis in infants and the elderly. Furthermore, epidemiological data suggest that RSV infection during infancy is a potent trigger of subsequent wheeze and asthma development. However, the mechanism by which RSV contributes to asthma is complex and remains largely unknown. A recent study indicates that the age of initial RSV infection is a key factor in determining airway response to RSV rechallenge. We hypothesized that severe RSV infection during neonatal development significantly alters lung structure and the pulmonary immune micro-environment; and thus, neonatal RSV infection is crucial in the development of or predisposition to allergic inflammatory diseases such as asthma.
Methods
To investigate this hypothesis the present study was conducted in a neonatal mouse model of RSV-induced pulmonary inflammation and airway dysfunction. Seven-day-old mice were infected with RSV (2 × 105 TCID50/g body weight) and allowed to mature to adulthood. To determine if neonatal RSV infection predisposed adult animals to enhanced pathophysiological responses to allergens, these mice were then sensitized and challenged with ovalbumin. Various endpoints including lung function, histopathology, cytokine production, and cellularity in bronchoalveolar lavage were examined.
Results
RSV infection in neonates alone led to inflammatory airway disease characterized by airway hyperreactivity, peribronchial and perivascular inflammation, and subepithelial fibrosis in adults. If early RSV infection was followed by allergen exposure, this pulmonary phenotype was exacerbated. The initial response to neonatal RSV infection resulted in increased TNF-α levels in bronchoalveolar lavage. Interestingly, increased levels of IL-13 and mucus hyperproduction were observed almost three months after the initial infection with RSV.
Conclusion
Neonatal RSV exposure results in long term pulmonary inflammation and exacerbates allergic airways disease. The early increase in TNF-α in the bronchoalveolar lavage implicates this inflammatory cytokine in orchestrating these events. Finally, the data presented emphasize IL-13 and TNF-α as potential therapeutic targets for treating RSV induced-asthma.
doi:10.1186/1465-9921-7-107
PMCID: PMC1563465  PMID: 16893457
9.  A Novel 5-Lipoxygenase-Activating Protein Inhibitor, AM679, Reduces Inflammation in the Respiratory Syncytial Virus-Infected Mouse Eye▿  
Clinical and Vaccine Immunology : CVI  2009;16(11):1654-1659.
Respiratory syncytial virus (RSV) is an important cause of viral respiratory disease in children, and RSV bronchiolitis has been associated with the development of asthma in childhood. RSV spreads from the eye and nose to the human respiratory tract. Correlative studies of humans and direct infection studies of BALB/c mice have established the eye as a significant pathway of entry of RSV to the lung. At the same time, RSV infection of the eye produces symptoms resembling allergic conjunctivitis. Cysteinyl leukotrienes (CysLTs) are known promoters of allergy and inflammation, and the first step in their biogenesis from arachidonic acid is catalyzed by 5-lipoxygenase (5-LO) in concert with the 5-LO-activating protein (FLAP). We have recently developed a novel compound, AM679, which is a topically applied and potent inhibitor of FLAP. Here we show with the BALB/c mouse eye RSV infection model that AM679 markedly reduced the RSV-driven ocular pathology as well as the synthesis of CysLTs in the eye. In addition, AM679 decreased the production of the Th2 cell cytokine interleukin-4 but did not increase the viral load in the eye or the lung. These results suggest that FLAP inhibitors may be therapeutic for RSV-driven eye disease and possibly other inflammatory eye indications.
doi:10.1128/CVI.00220-09
PMCID: PMC2772391  PMID: 19759251
10.  Comparative Effects of Two Neutralizing Anti-Respiratory Syncytial Virus (RSV) Monoclonal Antibodies in the RSV Murine Model: Time versus Potency 
Antimicrobial Agents and Chemotherapy  2005;49(11):4700-4707.
Respiratory syncytial virus (RSV) is the leading viral pathogen responsible for bronchiolitis and pneumonia in infants and young children worldwide. We have previously shown in the mouse model that treatment with an anti-RSV neutralizing monoclonal antibody (MAb) against the F glycoprotein of RSV, palivizumab, decreased lung inflammation, airway obstruction, and postmethacholine airway hyperresponsiveness. MEDI-524, or Numax, is a new MAb derived from palivizumab with enhanced neutralizing activity against RSV. We compared the effects of these two MAbs on different markers of disease severity using the murine model of RSV infection. BALB/c mice were intranasally inoculated with RSV A2. Palivizumab or MEDI-524 was administered once at either 24 h before or 48 h after RSV inoculation. Regardless of the time of administration, all treated mice showed significantly decreased RSV loads in bronchoalveolar lavage samples measured by plaque assay. Only MEDI-524 given at −24 h significantly decreased lung RSV RNA loads on days 5 and 28 after RSV inoculation. Pulmonary histopathologic scores, airway obstruction, and postmethacholine airway hyperresponsiveness were significantly reduced in mice treated with MEDI-524 at 24 h before inoculation, compared with untreated controls and the other regimens evaluated. MEDI-524 was superior to palivizumab on several outcome variables of RSV disease assessed in the mouse model: viral replication, inflammatory and clinical markers of acute disease severity, and long-term pulmonary abnormalities.
doi:10.1128/AAC.49.11.4700-4707.2005
PMCID: PMC1280119  PMID: 16251314
11.  Resveratrol-Mediated Gamma Interferon Reduction Prevents Airway Inflammation and Airway Hyperresponsiveness in Respiratory Syncytial Virus-Infected Immunocompromised Mice ▿  
Journal of Virology  2011;85(24):13061-13068.
Respiratory syncytial virus (RSV) is the most important cause of severe, lower respiratory tract infections in infants, and RSV infections have been associated with chronic wheezing and asthma during childhood. However, the mechanism of RSV-induced airway inflammation and airway hyperresponsiveness (AHR) is poorly understood. Furthermore, there are presently neither effective vaccines nor drugs available for the prevention or treatment of RSV infections. In this study, we investigated the effect of the plant extract resveratrol as a means of preventing airway inflammation and attenuating RSV-induced AHR. Our data showed that resveratrol reduced RSV lung titers and the number of infiltrating lymphocytes present in bronchoalveolar lavage fluid (BALF) and reduced inflammation. Furthermore, resveratrol attenuated airway responses to methacholine following RSV infection and significantly decreased gamma interferon (IFN-γ) levels in BALF of RSV-infected mice. Data presented in this report demonstrated that resveratrol controlled Toll-like receptor 3 (TLR3) expression, inhibited the TRIF signaling pathway, and induced M2 receptor expression following RSV infection. These data support a role for the use of resveratrol as a means of reducing IFN-γ levels associated with RSV-mediated airway inflammation and AHR, which may be mediated via TLR3 signaling.
doi:10.1128/JVI.05869-11
PMCID: PMC3233156  PMID: 21937650
12.  Th17 cytokines are critical for RSV associated airway hyperreponsiveness through regulation by complement C3a and tachykinins1 
Respiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. In mice, acute RSV infection causes airway hyperresponsiveness (AHR), inflammation, and mucus hypersecretion. Infected cells induce complement activation, producing the anaphylatoxin C3a. Here we show RSV infected wild type mice produce Th17 cytokines, a response not previously associated with viral infections. Mice deficient in the C3aR (C3aR1−/−) fail to develop AHR following acute RSV infection, and production of Th17 cytokines was significantly attenuated. Tachykinin production has also been implicated in RSV pathophysiology, and tachykinin receptor null mice (TACR1−/−) were similarly protected from developing AHR. These animals were also deficient in production of Th17 cytokines. Tachykinin release was absent in C3aR1−/− mice, while C3a levels were unchanged in TACR1−/− animals. Thus, our data reveal a crucial sequence following acute RSV infection where initial C3a production causes tachykinin release, followed by activation of the IL-17A pathway. Deficiency of either receptor affords protection from AHR, identifying two potential therapeutic targets.
doi:10.4049/jimmunol.1101789
PMCID: PMC3186836  PMID: 21918196
respiratory syncytial virus; C3a anaphylatoxin; complement; inflammation; IL17A; airway hyperresponsiveness; tachykinins; substance P; hemokinin-1
13.  Early-life viral infection and allergen exposure interact to induce an asthmatic phenotype in mice 
Respiratory Research  2010;11(1):14.
Background
Early-life respiratory viral infections, notably with respiratory syncytial virus (RSV), increase the risk of subsequent development of childhood asthma. The purpose of this study was to assess whether early-life infection with a species-specific model of RSV and subsequent allergen exposure predisposed to the development of features of asthma.
Methods
We employed a unique combination of animal models in which BALB/c mice were neonatally infected with pneumonia virus of mice (PVM, which replicates severe RSV disease in human infants) and following recovery, were intranasally sensitised with ovalbumin. Animals received low-level challenge with aerosolised antigen for 4 weeks to elicit changes of chronic asthma, followed by a single moderate-level challenge to induce an exacerbation of inflammation. We then assessed airway inflammation, epithelial changes characteristic of remodelling, airway hyperresponsiveness (AHR) and host immunological responses.
Results
Allergic airway inflammation, including recruitment of eosinophils, was prominent only in animals that had recovered from neonatal infection with PVM and then been sensitised and chronically challenged with antigen. Furthermore, only these mice exhibited an augmented Th2-biased immune response, including elevated serum levels of anti-ovalbumin IgE and IgG1 as well as increased relative expression of Th2-associated cytokines IL-4, IL-5 and IL-13. By comparison, development of AHR and mucous cell change were associated with recovery from PVM infection, regardless of subsequent allergen challenge. Increased expression of IL-25, which could contribute to induction of a Th2 response, was demonstrable in the lung following PVM infection. Signalling via the IL-4 receptor α chain was crucial to the development of allergic inflammation, mucous cell change and AHR, because all of these were absent in receptor-deficient mice. In contrast, changes of remodelling were evident in mice that received chronic allergen challenge, regardless of neonatal PVM infection, and were not dependent on signalling via the IL-4 receptor.
Conclusion
In this mouse model, interaction between early-life viral infection and allergen sensitisation/challenge is essential for development of the characteristic features of childhood asthma, including allergic inflammation and a Th2-biased immune response.
doi:10.1186/1465-9921-11-14
PMCID: PMC2842242  PMID: 20122285
14.  Respiratory Syncytial Virus Infection and G and/or SH Protein Expression Contribute to Substance P, Which Mediates Inflammation and Enhanced Pulmonary Disease in BALB/c Mice 
Journal of Virology  2000;74(4):1614-1622.
A distinct clinical presentation of respiratory syncytial virus (RSV) infection of humans is bronchiolitis, which has clinical features similar to those of asthma. Substance P (SP), a tachykinin neuropeptide, has been associated with neurogenic inflammation and asthma; therefore, we chose to examine SP-induced inflammation with RSV infection. In this study, we examined the production of pulmonary SP associated with RSV infection of BALB/c mice and the effect of anti-SP F(ab)2 antibodies on the pulmonary inflammatory response. The peak production of pulmonary SP occurred between days 3 and 5 following primary RSV infection and day 1 after secondary infection. Treatment of RSV-infected mice with anti-SP F(ab)2 antibodies suggested that SP may alter the natural killer cell response to primary and secondary infection. In mice challenged after formalin-inactivated RSV vaccination, SP appears to markedly enhance pulmonary eosinophilia as well as increase polymorphonuclear cell trafficking to the lung. Based on studies with a strain of RSV that lacks the G and SH genes, the SP response to RSV infection appears to be associated with G and/or SH protein expression. These data suggest that SP may be an important contributor to the inflammatory response to RSV infection and that anti-SP F(ab)2 antibodies might be used to ameliorate RSV-associated disease.
PMCID: PMC111635  PMID: 10644330
15.  Gene Expression Differences in Lungs of Mice during Secondary Immune Responses to Respiratory Syncytial Virus Infection▿ †  
Journal of Virology  2010;84(18):9584-9594.
Vaccine-induced immunity has been shown to alter the course of a respiratory syncytial virus (RSV) infection both in murine models and in humans. To elucidate which mechanisms underlie the effect of vaccine-induced immunity on the course of RSV infection, transcription profiles in the lungs of RSV-infected mice were examined by microarray analysis. Three models were used: RSV reinfection as a model for natural immunity, RSV challenge after formalin-inactivated RSV vaccination as a model for vaccine-enhanced disease, and RSV challenge following vaccination with recombinant RSV virus lacking the G gene (ΔG-RSV) as a model for vaccine-induced immunity. Gene transcription profiles, histopathology, and viral loads were analyzed at 1, 2, and 5 days after RSV challenge. On the first 2 days after challenge, all mice displayed an expression pattern in the lung similar of that found in primary infection, showing a strong innate immune response. On day 5 after RSV reinfection or after challenge following ΔG-RSV vaccination, the innate immune response was waning. In contrast, in mice with vaccine-enhanced disease, the innate immune response 5 days after RSV challenge was still present even though viral replication was diminished. In addition, only in this group was Th2 gene expression induced. These findings support a hypothesis that vaccine-enhanced disease is mediated by prolonged innate immune responses and Th2 polarization in the absence of viral replication.
doi:10.1128/JVI.00302-10
PMCID: PMC2937637  PMID: 20592085
16.  Respiratory syncytial virus-induced acute and chronic airway disease is independent of genetic background: An experimental murine model 
Virology Journal  2005;2:46.
Background
Respiratory syncytial virus (RSV) is the leading respiratory viral pathogen in young children worldwide. RSV disease is associated with acute airway obstruction (AO), long-term airway hyperresponsiveness (AHR), and chronic lung inflammation. Using two different mouse strains, this study was designed to determine whether RSV disease patterns are host-dependent. C57BL/6 and BALB/c mice were inoculated with RSV and followed for 77 days. RSV loads were measured by plaque assay and polymerase chain reaction (PCR) in bronchoalveolar lavage (BAL) and whole lung samples; cytokines were measured in BAL samples. Lung inflammation was evaluated with a histopathologic score (HPS), and AO and AHR were determined by plethysmography.
Results
Viral load dynamics, histopathologic score (HPS), cytokine concentrations, AO and long-term AHR were similar in both strains of RSV-infected mice, although RSV-infected C57BL/6 mice developed significantly greater AO compared with RSV-infected BALB/c mice on day 5. PCR detected RSV RNA in BAL samples of RSV infected mice until day 42, and in whole lung samples through day 77. BAL concentrations of cytokines TNF-α, IFN-γ, and chemokines MIG, RANTES and MIP-1α were significantly elevated in both strains of RSV-infected mice compared with their respective controls. Viral load measured by PCR significantly correlated with disease severity on days 14 and 21.
Conclusion
RSV-induced acute and chronic airway disease is independent of genetic background.
doi:10.1186/1743-422X-2-46
PMCID: PMC1183251  PMID: 15916706
Viral pneumonia; mouse model; airway hyperresponsiveness; PCR; cytokines
17.  Combined fluticasone propionate and salmeterol reduces RSV infection more effectively than either of them alone in allergen-sensitized mice 
Virology Journal  2006;3:32.
Background
Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in infants and is a risk factor for the development of asthma. Allergic asthmatics are more susceptible to RSV infection and viral exacerbation.
Methods
Since the effectiveness of corticosteroids in treating RSV infection has been controversial, we tested fluticasone propionate (FP) and salmeterol (Sal) alone versus FP plus Sal (FPS) on RSV-induced airway inflammation. Mice were sensitized and challenged with ovalbumin (OVA) and infected with RSV. Following infection they were treated with FP, Sal, or FPS intranasally and airway hyperreactivity (AHR), inflammation and RSV titers were examined.
Results
The group treated with FPS showed significantly lower AHR compared to the group treated with FP or Sal alone. The group treated with FP alone showed slightly decreased (non-significant) AHR compared to controls. Treatment with FPS resulted in significant decreases in the percentage of eosinophils and neutrophils in bronchoalveolar lavage fluid and in lung pathology compared to FP or Sal. FP alone decreased eosinophils but not neutrophils or lymphocytes, while Sal alone decreased eosinophils and neutrophils but not lymphocytes. FPS treatment of mice infected with RSV in the absence of allergen sensitization resulted in a 50% decrease of RSV titer in the lung and a reduction in neutrophils compared to FP or Sal.
Conclusion
Together, these results indicate that fluticasone in combination with salmeterol is a more effective treatment for decreasing airway hyperreactivity and inflammation than either of them alone in allergen-sensitized, RSV-infected mice.
doi:10.1186/1743-422X-3-32
PMCID: PMC1488829  PMID: 16719922
18.  A role for airway remodeling during respiratory syncytial virus infection 
Respiratory Research  2005;6(1):122.
Background
Severe respiratory syncytial virus infection (RSV) during infancy has been shown to be a major risk factor for the development of subsequent wheeze. However, the reasons for this link remain unclear. The objective of this research was to determine the consequences of early exposure to RSV and allergen in the development of subsequent airway hyperreactivity (AHR) using a developmental time point in the mouse that parallels that of the human neonate.
Methods
Weanling mice were sensitized and challenged with ovalbumin (Ova) and/or infected with RSV. Eight days after the last allergen challenge, various pathophysiological endpoints were examined.
Results
AHR in response to methacholine was enhanced only in weanling mice exposed to Ova and subsequently infected with RSV. The increase in AHR appeared to be unrelated to pulmonary RSV titer. Total bronchoalveolar lavage cellularity in these mice increased approximately two-fold relative to Ova alone and was attributable to increases in eosinophil and lymphocyte numbers. Enhanced pulmonary pathologies including persistent mucus production and subepithelial fibrosis were observed. Interestingly, these data correlated with transient increases in TNF-α, IFN-γ, IL-5, and IL-2.
Conclusion
The observed changes in pulmonary structure may provide an explanation for epidemiological data suggesting that early exposure to allergens and RSV have long-term physiological consequences. Furthermore, the data presented here highlight the importance of preventative strategies against RSV infection of atopic individuals during neonatal development.
doi:10.1186/1465-9921-6-122
PMCID: PMC1283984  PMID: 16242038
respiratory syncytial virus; pulmonary; inflammation; age factors; asthma; mice
19.  Immunomodulation with IL-4 Receptor-α Antisense Oligonucleotide Prevents RSV-Mediated Pulmonary Disease1 
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 of 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 if 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 (ASO) specific for IL-4Rα were administered intranasally to neonatal mice at the time of primary infection. Mice were initially infected with RSV at one week of age and reinfected at six weeks of age. Administration of IL-4Rα ASO 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 and Th2 cells and cytokine secretion) and increased Th1 responses (i.e. elevated Th1 cell numbers and type I antibodies 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 ASO may be of significant benefit to infants.
doi:10.4049/jimmunol.1000484
PMCID: PMC3063095  PMID: 20861354
20.  Respiratory Syncytial Virus Persistence in the Lungs Correlates with Airway Hyperreactivity in the Mouse Model 
The Journal of infectious diseases  2008;198(10):1435-1443.
Background
Previous studies in mice showed that respiratory syncytial virus (RSV) infection was associated with RSV RNA persistence. This study was designed to characterize the significance of RSV RNA persistence and its relation to RSV-induced chronic airway disease.
Methods
Mice were inoculated with live RSV, UV light–treated RSV, heat-inactivated RSV, or medium. Bronchoal-veolar lavage fluid samples were obtained and lung specimens were harvested on days 1, 5, and 42 after inoculation to assess lung inflammation, lung mRNA expression of interleukin (IL)–4, IL-5, IL-15, and interferon (IFN)–γ; RSV loads were assessed by culture and real-time polymerase chain reaction (PCR) and correlated with pulmonary function.
Results
During the acute phase of infection, RSV loads as indicated by culture and PCR were significantly higher in mice inoculated with live RSV. On day 42, RSV RNA remained detectable only in mice inoculated with live or UV light–treated RSV. Lung inflammation, IFN-γ:IL-4 mRNA expression ratios, airway obstruction (AO), and airway hyperreactivity (AHR) were significantly increased in mice inoculated with live RSV. AO on day 5 and AHR on day 42 were significantly correlated with RSV RNA copy number in lung samples.
Conclusions
Infection with live RSV induced acute and chronic airway disease that was associated with a predominantly Th-1 immune response and RSV RNA persistence that significantly correlated with pulmonary function abnormalities.
doi:10.1086/592714
PMCID: PMC3689551  PMID: 18828742
21.  Interleukin 18 Coexpression during Respiratory Syncytial Virus Infection Results in Enhanced Disease Mediated by Natural Killer Cells▿  
Journal of Virology  2010;84(8):4073-4082.
Respiratory syncytial virus (RSV) causes bronchiolitis, the main cause of infantile hospitalization. Immunity against reinfection is poor, and there is great interest in boosting vaccine responses using live vectors expressing host cytokines. We therefore constructed a recombinant RSV expressing murine interleukin 18 (RSV/IL-18), a cytokine capable of inducing strong antiviral immune responses. In vitro RSV/IL-18 replicated at wild-type levels and produced soluble IL-18. In naïve BALB/c mice, RSV/IL-18 infection significantly increased both IL-18 mRNA and protein and attenuated the peak viral load 3-fold. Despite a reduced viral load, RSV/IL-18 infection caused a biphasic weight loss at days 2 and 6 postinfection that was not seen in wild-type infection. Day 2 disease was associated with enhanced pulmonary natural killer (NK) cell numbers and activity and was prevented by NK cell depletion during infection; day 6 disease was correlated with CD8 T-cell recruitment and was enhanced by NK cell depletion. IL-18 expression during priming also enhanced RSV-specific antibody responses and T-cell responses on secondary RSV infection. Therefore, while IL-18 boosted antiviral immunity and reduced the viral load, its coexpression worsened disease. This is the first recombinant RSV with this property, and these are the first studies to demonstrate that NK cells can induce pathology during pulmonary viral infections.
doi:10.1128/JVI.02014-09
PMCID: PMC2849516  PMID: 20130064
22.  Pulmonary Vγ4+ γδ T Cells Have Proinflammatory and Antiviral Effects in Viral Lung Disease1 
Host defenses, while effecting viral clearance, contribute substantially to inflammation and disease. This double action is a substantial obstacle to the development of safe and effective vaccines against many agents, particularly respiratory syncytial virus (RSV). RSV is a common cold virus and the major cause of infantile bronchiolitis worldwide. The role of αβ T cells in RSV-driven immunopathology is well studied, but little is known about the role of “unconventional” T cells. During primary RSV challenge of BALB/c mice, some Vγ7+ γδ T cells were present; however, immunization with a live vaccinia vector expressing RSV F protein substantially enhanced Vγ4+ γδ T cell influx after RSV infection. Harvested early, these cells produced IFN-γ, TNF, and RANTES after ex vivo stimulation. By contrast, those recruited 5 days after challenge made IL-4, IL-5, and IL-10. Depletion of γδ T cells in vivo reduced lung inflammation and disease severity and slightly increased peak viral replication but did not prevent viral clearance. These studies demonstrate a novel role for γδ T cells in the development of immunopathology and cellular influx into the lungs after immunization and RSV challenge. Though a minor population, γδ T cells have a critical influence on disease and are an attractive interventional target in the alleviation of viral lung disease.
PMCID: PMC2646375  PMID: 19124761
23.  Stimulation of Immature Lung Macrophages with Intranasal Interferon Gamma in a Novel Neonatal Mouse Model of Respiratory Syncytial Virus Infection 
PLoS ONE  2012;7(7):e40499.
Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral death in infants. Reduced CD8 T-cells and negligible interferon gamma (IFNγ) in the airway are associated with severe infant RSV disease, yet there is an abundance of alveolar macrophages (AM) and neutrophils. However, it is unclear, based on our current understanding of macrophage functional heterogeneity, if immature AM improve viral clearance or contribute to inflammation and airway obstruction in the IFNγ-deficient neonatal lung environment. The aim of the current study was to define the age-dependent AM phenotype during neonatal RSV infection and investigate their differentiation to classically activated macrophages (CAM) using i.n. IFNγ in the context of improving viral clearance. Neonatal and adult BALB/cJ mice were infected with 1×106 plaque forming units (PFU)/gram (g) RSV line 19 and their AM responses compared. Adult mice showed a rapid and robust CAM response, indicated by increases in major histocompatibility complex class II (MHC II), CD86, CCR7, and a reduction in mannose receptor (MR). Neonatal mice showed a delayed and reduced CAM response, likely due to undetectable IFNγ production. Intranasal (i.n.) treatment with recombinant mouse IFNγ (rIFNγ) increased the expression of CAM markers on neonatal AM, reduced viral lung titers, and improved weight gain compared to untreated controls with no detectable increase in CD4 or CD8 T-cell infiltration. In vitro infection of J774A.1 macrophages with RSV induced an alternatively activated macrophage (AAM) phenotype however, when macrophages were first primed with IFNγ, a CAM phenotype was induced and RSV spread to adjacent Hep-2 cells was reduced. These studies demonstrate that the neonatal AM response to RSV infection is abundant and immature, but can be exogenously stimulated to express the antimicrobial phenotype, CAM, with i.n. rIFNγ.
doi:10.1371/journal.pone.0040499
PMCID: PMC3391240  PMID: 22792355
24.  Chinchilla and Murine Models of Upper Respiratory Tract Infections with Respiratory Syncytial Virus 
Journal of Virology  2005;79(10):6035-6042.
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and the elderly. While the primary infection is the most serious, reinfection of the upper airway throughout life is the rule. Although relatively little is known about either RSV infection of the upper respiratory tract or host mucosal immunity to RSV, recent literature suggests that RSV is the predominant viral pathogen predisposing to bacterial otitis media (OM). Herein, we describe mouse and chinchilla models of RSV infection of the nasopharynx and Eustachian tube. Both rodent hosts were susceptible to RSV infection of the upper airway following intranasal challenge; however, the chinchilla proved to be more permissive than the mouse. The chinchilla model will likely be extremely useful to test the role of RSV in bacterial OM and the efficacy of RSV vaccine candidates designed to provide mucosal and cytotoxic T-lymphocyte immunity. Ultimately, we hope to investigate the relative ability of these candidates to potentially protect against viral predisposal to bacterial OM.
doi:10.1128/JVI.79.10.6035-6042.2005
PMCID: PMC1091680  PMID: 15857989
25.  Enhanced virulence, airway inflammation and impaired lung function induced by respiratory syncytial virus deficient in secreted G protein 
Thorax  2004;59(6):517-521.
Background: Respiratory syncytial virus (RSV) infection can cause bronchial hyperresponsiveness and asthma exacerbations. In mice it results in airway inflammation and airway hyperresponsiveness. Since viral factors influencing these responses are not well defined, a study was undertaken to investigate the role of secreted G protein of human RSV in determining virulence, inflammatory responses, and changes in lung function.
Methods: BALB/c mice were infected with a spontaneous mutant of RSV deficient in secreted G protein (RSV-ΔsG) or with wild type RSV (RSV-WT). Viral titres, numbers of pulmonary inflammatory cells, and concentrations of interferon (IFN)-γ, interleukin (IL)-4, IL-5 and IL-10 in bronchoalveolar lavage (BAL) fluid were determined. Airway function was assessed at baseline and following methacholine provocation using barometric whole body plethysmography.
Result: Following infection with RSV-ΔsG, viral titres were increased 50-fold compared with RSV-WT. Influx of eosinophils and macrophages to the lung and concentrations of IFN-γ and IL-10 in BAL fluid were also significantly higher following infection with RSV-ΔsG. Airway function, both at baseline and after methacholine provocation, was significantly decreased following infection with RSV-ΔsG compared with RSV-WT.
Conclusion: Secreted G protein is likely to be a regulatory factor in RSV infection limiting infectivity of the virus, inflammatory responses in the lungs, and reduction in lung function.
doi:10.1136/thx.2003.017343
PMCID: PMC1747027  PMID: 15170038

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