Ana M. Gomez

Respiratory Syncytial Virus Induces Pneumonia, Cytokine Response, Airway Obstruction, and Chronic Inflammatory Infiltrates Associated with Long-Term Airway Hyperresponsiveness in Mice

BACKGROUND Respiratory syncytial virus (RSV) infection is associated with acute morbidity (e.g., pneumonia and airway obstruction [AO]) and long-term complications (e.g., airway hyperresponsiveness [AHR]). We present a comprehensive evaluation of the acute and chronic phases of RSV respiratory tract infection, using a mouse model. METHODS BALB/c mice were inoculated with RSV and monitored for 154 days. RSV loads and cytokines were measured in bronchoalveolar lavage (BAL) samples. Pneumonia severity was assessed using a standard histopathologic score, and pulmonary function was determined by plethysmography. RESULTS RSV-infected mice exhibited viral replication that peaked on day 4-5 and became undetectable by day 7. These mice developed acute pneumonia (peak days, 4-5) and chronic pulmonary inflammatory infiltrates that lasted up to 154 days after inoculation. BAL concentrations of tumor necrosis factor- alpha, interleukin (IL)-6, interferon- gamma, IL-4, IL-10, KC (an IL-8 homologue), MIG (CXCL9), RANTES, macrophage inflammatory protein-1 alpha, and eotaxin were significantly higher in RSV-infected mice than in control mice. RSV-infected mice developed acute AO during the first week of infection that persisted for 42 days. RSV-infected mice also showed significant AHR in response to methacholine up to 154 days. CONCLUSION This model provides a means to investigate the immunopathogenesis of RSV infection and its association with reactive airway disease.

MAVS and MyD88 are essential for innate immunity but not cytotoxic T lymphocyte response against respiratory syncytial virus

Infection by RNA viruses is detected by the host through Toll-like receptors or RIG-I-like receptors. Toll-like receptors and RIG-I-like receptors signal through the adaptors MyD88 and MAVS, respectively, to induce type I IFNs (IFN-I) and other antiviral molecules, which are thought to be essential for activating the adaptive immune system. We investigated the role of these adaptors in innate and adaptive immune responses against respiratory syncytial virus (RSV), a common human pathogen. Deletion of Mavs abolished the induction of IFN-I and other proinflammatory cytokines by RSV. Genome-wide expression profiling in the lung showed that the vast majority of RSV-induced genes depended on MAVS. Although Myd88 deficiency did not affect most RSV-induced genes, mice lacking both adaptors harbored a higher and more prolonged viral load and exhibited more severe pulmonary disease than those lacking either adaptor alone. Surprisingly, Myd88−/−Mavs−/− mice were able to activate a subset of pulmonary dendritic cells that traffic to the draining lymph node in response to RSV. These mice subsequently mounted a normal cytotoxic T-lymphocyte response and demonstrated delayed but effective viral clearance. These results provide an example of a normal and effective adaptive immune response in the absence of innate immunity mediated by MAVS and MyD88.

Comparative Effects of Two Neutralizing Anti-Respiratory Syncytial Virus (RSV) Monoclonal Antibodies in the RSV Murine Model: Time versus Potency

ABSTRACT 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.

Anti-Respiratory Syncytial Virus (RSV) Neutralizing Antibody Decreases Lung Inflammation, Airway Obstruction, and Airway Hyperresponsiveness in a Murine RSV Model

ABSTRACT Numerous studies have described a strong association between respiratory syncytial virus (RSV) infection in infancy and the development of recurrent wheezing and airway hyperresponsiveness. We evaluated the effect of an anti-RSV neutralizing monoclonal antibody (palivizumab) on different aspects of RSV disease by using a murine model. BALB/c mice were intranasally inoculated with RSV A2. Palivizumab or an isotype-matched control antibody was administered once at 24 h before inoculation, 1 h after inoculation, or 48 h after inoculation. Regardless of the timing of administration, all mice treated with the neutralizing antibody showed significantly decreased RSV loads in bronchoalveolar lavage (BAL) and lung specimens compared with those of infected controls. Pulmonary histopathologic scores, airway obstruction measured by plethysmography, and airway hyperresponsiveness after methacholine challenge were significantly reduced in mice treated with the anti-RSV antibody 24 h before inoculation compared with those for untreated controls. Concentrations of interferon-gamma, interleukin-10, macrophage inflammatory protein 1α, regulated on activation normal T-cell expressed and secreted (RANTES), and eotaxin in BAL fluids were also significantly reduced in mice treated with palivizumab 24 h before inoculation. This study demonstrates that reduced RSV replication was associated with significant modulation of inflammatory and clinical markers of acute disease severity and significant improvement of the long-term pulmonary abnormalities. Studies to determine whether strategies aimed at preventing or reducing RSV replication could decrease the long-term morbidity associated with RSV infection in children should be considered.

Human Metapneumovirus Infection Induces Long-Term Pulmonary Inflammation Associated with Airway Obstruction and Hyperresponsiveness in Mice

BACKGROUND Human metapneumovirus (hMPV) is a newly described paramyxovirus that is associated with bronchiolitis, pneumonia, and asthma exacerbation. The objective of the present work was to study the duration of pulmonary inflammation and the functional consequences of infection with hMPV by use of a BALB/c mouse model. METHODS BALB/c mice were inoculated with 1 x 10(8) TCID(50) of hMPV type A (C-85473), and viral persistence in lungs was assessed by reverse-transcription polymerase chain reaction for 154 days after infection. Pulmonary inflammation was characterized in histopathological experiments by use of a validated scoring system, and periodic acid-Schiff (PAS) staining of lung sections was used to document increased mucus production, also until day 154. Finally, respiratory functions were analyzed by taking plethysmographic measurements until day 70. RESULTS Persistence of viral RNA and significant pulmonary inflammation were noted until day 154, whereas the findings for PAS staining suggested that mucus production was increased only until day 12. Maximal breathing difficulties occurred on day 5, and airway obstruction and hyperresponsiveness were still significant until at least day 70. CONCLUSION Acute hMPV infection in BALB/c mice is associated with long-term pulmonary inflammation that leads to significant obstructive disease of the airways. This animal model will be of a great benefit in the evaluation of novel therapeutic and prophylactic modalities.

The Impact of Steroids Given with Macrolide Therapy on Experimental Mycoplasma pneumoniae Respiratory Infection

BACKGROUND Systemic steroids have been advocated in addition to antimicrobial therapy for severe Mycoplasma pneumoniae pneumonia. We evaluated the efficacy of clarithromycin, dexamethasone, and combination therapy for M. pneumoniae respiratory infection. METHODS Mice infected with M. pneumoniae were treated with clarithromycin, dexamethasone, combined clarithromycin/dexamethasone, or placebo daily; mice were evaluated at baseline and after 1, 3, and 6 days of therapy. Outcome variables included M. pneumoniae culture, lung histopathologic score (HPS), and bronchoalveolar lavage cytokine, chemokine, and growth factor concentrations. RESULTS Clarithromycin monotherapy resulted in the greatest reductions in M. pneumoniae concentrations. After 3 days of treatment, combination therapy significantly reduced lung HPS compared with placebo, clarithromycin, and dexamethasone alone, whereas, after 6 days of therapy, clarithromycin alone and combination therapy significantly reduced lung HPS compared with placebo. Concentrations of interleukin (IL)-12 p40, RANTES, macrophage chemotactic protein-1, and cytokine-induced neutrophil chemoattractant were significantly lower in mice treated with clarithromycin alone and/or combination therapy compared with dexamethasone alone and/or placebo; combination therapy resulted in a significantly greater reduction than clarithromycin alone for IL-12 p40 and RANTES. CONCLUSIONS Although monotherapy with clarithromycin had the greatest effect on reducing concentrations of M. pneumoniae, combination therapy had the greatest effect on decreasing levels of cytokines and chemokines as well as pulmonary histologic inflammation.

Decreased innate immune cytokine responses correlate with disease severity in children with respiratory syncytial virus and human rhinovirus bronchiolitis.

The immunopathogenesis of respiratory syncytial virus (RSV) and human rhinovirus lower respiratory tract infections in children remains to be defined. We measured nasal wash concentrations of 29 cytokines in infants with RSV or human rhinovirus lower respiratory tract infections. Concentrations of interferon-&ggr; in RSV and innate immunity cytokines in both infections inversely correlated with disease severity.

Characterization of Inflammation in Syphilitic Villitis and in Villitis of Unknown Etiology

Chronic villitis is a histologic diagnosis that may be either associated with infection, or termed villitis of undetermined etiology (VUE). The lymphocytic infiltrate in VUE has been reported to consist of maternal lymphocytes, but the origin of the lymphocytic infiltrate in infectious villitis has not been identified. The purpose of our study was to compare the maternal vs. fetal origin of the infiltrating lymphocytes in VUE and syphilitic villitis, and to expand the immunophenotypic data provided by previous studies. Paraffin-embedded placentas from four males with VUE and two males with syphilitic villitis were subjected to fluorescence in situ hybridization (FISH) for the X and Y chromosomes. Serial sections were stained with antibodies to CD3, CD4, CD8, CD68, HLA-DR, and CD20. Quantitation of the relative number of cells marking with each antibody was done for four villi in each slide. CD3 lymphocytes predominated in both VUE and syphilitic villitis, with slightly more CD8 cells compared to CD4 cells. CD68 and HLA-DR positive cells were as frequent as CD3 cells, and B-lymphocytes were rare. Maternal cells were the predominant intravillous population in both VUE and syphilitic villitis, and neutrophils in syphilitic villitis were also maternal. These data indicate that the immune response in both syphilitic villitis and VUE is similar, raising the possibility of a similar immunopathogenetic pathway.

Respiratory syncytial virus-induced acute and chronic airway disease is independent of genetic background: An experimental murine model

BackgroundRespiratory 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.ResultsViral 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.ConclusionRSV-induced acute and chronic airway disease is independent of genetic background.

Effect of dexamethasone on respiratory syncytial virus-induced lung inflammation in children: results of a randomized, placebo controlled clinical trial

Inflammatory mediators play a major role in the pathogenesis of respiratory syncytial virus (RSV) infection. The objective of this study was to evaluate the effect of i.v. dexamethasone on cytokine concentrations in tracheal aspirates (TA) of children with severe RSV disease and to correlate them with disease severity. Twenty‐five cytokines were measured in TA obtained from children <2 yr old intubated for severe RSV disease, and enrolled in a double‐blind study of i.v. dexamethasone (0.5 mg/kg; n = 22) vs. placebo (n = 19). Cytokine concentrations, measured at baseline and days 1 and 5 post‐randomization using a multiplex assay, were compared within both treatment groups and correlated with: (i) tracheal white blood cell counts, (ii) tracheal RSV loads by culture and (iii) parameters of disease severity, including number of days of requirement for mechanical ventilation, intensive care unit (ICU), and hospitalization. At baseline interleukin (IL)‐13 and IL‐15 concentrations were significantly higher in the dexamethasone treatment group. On day 1 post‐treatment, only MCP‐1, eotaxin and IL‐6 concentrations were significantly different but higher in the placebo group. On day 5: IL‐13, IL‐7, IL‐8 and MIP‐1α concentrations were higher in dexamethasone‐treated patients. In both groups MIP‐1β inversely correlated with the days of ventilator support; MIP‐1α, MIP‐1β and eotaxin inversely correlated with ICU days; and IL‐6 inversely correlated with hospitalization regardless of the treatment assigned. Systemic administration of dexamethasone did not have a consistent effect on TA concentrations of pro‐inflammatory cytokines. This may help explain, at least in part, the lack of clinical benefit of steroid treatment in children with severe RSV bronchiolitis.