Sivanarayana Mandalapu

Influenza A Virus Exacerbates Staphylococcus aureus Pneumonia in Mice by Attenuating Antimicrobial Peptide Production

Influenza A represents a significant cause of morbidity and mortality worldwide. Bacterial complications of influenza A confer the greatest risk to patients. TH17 pathway inhibition has been implicated as a mechanism by which influenza A alters bacterial host defense. Here we show that preceding influenza causes persistent Staphylococcus aureus infection and suppression of TH17 pathway activation in mice. Influenza does not inhibit S. aureus binding and uptake by phagocytic cells but instead attenuates S. aureus induced TH17 related antimicrobial peptides necessary for bacterial clearance in the lung. Importantly, exogenous lipocalin 2 rescued viral exacerbation of S. aureus infection and decreased free iron levels in the bronchoalveolar lavage from mice coinfected with S. aureus and influenza. These findings indicate a novel mechanism by which influenza A inhibits TH17 immunity and increases susceptibility to secondary bacterial pneumonia. Identification of new mechanisms in the pathogenesis of bacterial pneumonia could lead to future therapeutic targets.

The complex relationship between inflammation and lung function in severe asthma

Asthma is a common respiratory disease affecting ∼300 million people worldwide. Airway inflammation is thought to contribute to asthma pathogenesis, but the direct relationship between inflammation and airway hyperresponsiveness (AHR) remains unclear. This study investigates the role of inflammation in a steroid-insensitive, severe allergic airway disease model and in severe asthmatics stratified by inflammatory profile. First, we used the T-helper (TH)-17 cells adoptive transfer mouse model of asthma to induce pulmonary inflammation, which was lessened by tumor necrosis factor (TNF)-α neutralization or neutrophil depletion. Although decreased airspace inflammation following TNFα neutralization and neutrophil depletion rescued lung compliance, neither intervention improved AHR to methacholine, and tissue inflammation remained elevated when compared with control. Further, sputum samples were collected and analyzed from 41 severe asthmatics. In severe asthmatics with elevated levels of sputum neutrophils, but low levels of eosinophils, increased inflammatory markers did not correlate with worsened lung function. This subset of asthmatics also had significantly higher levels of TH17-related cytokines in their sputum compared with severe asthmatics with other inflammatory phenotypes. Overall, this work suggests that lung compliance may be linked with cellular inflammation in the airspace, whereas T-cell-driven AHR may be associated with tissue inflammation and other pulmonary factors.

Influenza-induced type I interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice

Suppression of type 17 immunity by type I interferon (IFN) during influenza A infection has been shown to enhance susceptibility to secondary bacterial pneumonia. Although this mechanism has been described in coinfection with gram-positive bacteria, it is unclear whether similar mechanisms may impair lung defense against gram-negative infections. Furthermore, precise delineation of the duration of type I IFN-associated susceptibility to bacterial infection remains underexplored. Therefore, we investigated the effects of preceding influenza A virus infection on subsequent challenge with the gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa and the temporal association between IFN expression with susceptibility to Staphylococcus aureus challenge in a mouse model of influenza and bacterial coinfection. Here we demonstrate that preceding influenza A virus led to increased lung E. coli and P. aeruginosa bacterial burden, which was associated with suppression of type 17 immunity and attenuation of antimicrobial peptide expression. Enhanced susceptibility to S. aureus coinfection ceased at day 14 of influenza infection, when influenza-associated type I IFN levels had returned to baseline levels, further suggesting a key role for type I IFN in coinfection pathogenesis. These findings further implicate type I IFN-associated suppression of type 17 immunity and antimicrobial peptide production as a conserved mechanism for enhanced susceptibility to both gram-positive and gram-negative bacterial coinfection during influenza infection.

The role of IL-27 in susceptibility to post-influenza Staphylococcus aureus pneumonia

BackgroundInfluenza is a common respiratory virus and Staphylococcus aureus frequently causes secondary pneumonia during influenza infection, leading to increased morbidity and mortality. Influenza has been found to attenuate subsequent Type 17 immunity, enhancing susceptibility to secondary bacterial infections. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection.MethodsA murine model of influenza and Staphylococcus aureus infection was used to mimic human viral, bacterial co-infection. C57BL/6 wild-type, IL-27 receptor α knock-out, and IL-10 knock-out mice were infected with Influenza H1N1 (A/PR/8/34) or vehicle for 6xa0days followed by challenge with Staphylococcus aureus or vehicle for 24xa0hours. Lung inflammation, bacterial burden, gene expression, and cytokine production were determined.ResultsIL-27 receptor α knock-out mice challenged with influenza A had increased morbidity compared to controls, but no change in viral burden. IL-27 receptor α knock-out mice infected with influenza displayed significantly decreased IL-10 production compared to wild-type. IL-27 receptor α knock-out mice co-infected with influenza and S. aureus had improved bacterial clearance compared to wild-type controls. Importantly, there were significantly increased Type 17 responses and decreased IL-10 production in IL-27 receptor α knock-out mice. Dual infected IL-10−/− mice had significantly less bacterial burden compared to dual infected WT mice.ConclusionsThese data reveal that IL-27 regulates enhanced susceptibility to S. aureus pneumonia following influenza infection, potentially through the induction of IL-10 and suppression of IL-17.

A Novel Outbred Mouse Model of 2009 Pandemic Influenza and Bacterial Co-Infection Severity

Influenza viruses pose a significant health risk and annually impose a great cost to patients and the health care system. The molecular determinants of influenza severity, often exacerbated by secondary bacterial infection, are largely unclear. We generated a novel outbred mouse model of influenza virus, Staphylococcus aureus, and co-infection utilizing influenza A/CA/07/2009 virus and S. aureus (USA300). Outbred mice displayed a wide range of pathologic phenotypes following influenza virus or co-infection ranging broadly in severity. Influenza viral burden positively correlated with weight loss although lung histopathology did not. Inflammatory cytokines including IL-6, TNF-α, G-CSF, and CXCL10 positively correlated with both weight loss and viral burden. In S. aureus infection, IL-1β, G-CSF, TNF-α, and IL-6 positively correlated with weight loss and bacterial burden. In co-infection, IL-1β production correlated with decreased weight loss suggesting a protective role. The data demonstrate an approach to identify biomarkers of severe disease and to understand pathogenic mechanisms in pneumonia.

Vitamin D supplementation decreases Aspergillus fumigatus specific Th2 responses in CF patients with aspergillus sensitization: a phase one open-label study

BackgroundPatients with cystic fibrosis (CF) complicated by allergic bronchopulmonary aspergillosis (ABPA) are vitamin D deficient and in vitro treatment with 1,25 (OH)2 vitamin D3 of CD4+ cells from CF patients with ABPA decreases Aspergillus fumigatus(Af)-induced Th2 responses. This Phase I clinical trial investigated the safety and effectiveness of daily vitamin D3 supplementation in CF patients with ABPA to reduce allergic responses and ABPA symptoms, and increase serum vitamin D levels.MethodsSeven patients ages 12xa0years and older with a clinical diagnosis of CF and ABPA with current evidence of Af sensitization received 4000xa0IU vitamin D3 (cholecalciferol) daily for 24xa0weeks. The primary outcome of the study was safety followed by the Aspergillus induced IL-13 response in CD4+ T cells to test the hypothesis that vitamin D supplementation is safe and reduces Aspergillus induced IL-13 responses in CD4+ T cells. Secondary outcomes included total IgE, Aspergillus- specific IgE, vitamin D levels, FEV1, urinary calcium/creatinine ratio, and cytokine production by Aspergillus-stimulated peripheral blood T cells.ResultsSix months of vitamin D3 supplementation resulted in significant increases in serum 25-(OH) vitamin D level, and the treatment was well tolerated without evidence of vitamin D toxicity or hypercalcemia. There were no serious adverse events. Daily vitamin D supplementation led to significantly decreased Aspergillus induced IL-13 responses between the baseline visit and that at 24xa0weeks (pu2009=u20090.04). Aspergillus-specific IgE level was also significantly decreased after 8 (pu2009=u20090.035) and 24xa0weeks of daily vitamin D supplementation (pu2009=u20090.04).Conclusions4000xa0IU vitamin D3 daily over a 24-week period is well tolerated in CF patients with a history ABPA and current evidence of Th2 immunity to Af. . Daily vitamin D supplementation was associated with reduced Aspergillus induced IL-13 responses from peripheral. . CD4+ T cells and Aspergillus-specific IgE levels, as well as increased serum vitamin D levels. This treatment was well tolerated and the study supports further investigation of the use of vitamin D supplementation in Th2 mediated diseases.Trial registrationThis trial was registered at www.clinicaltrials.gov as NCT01222273.

Bromodomain and Extra-Terminal Protein Inhibition Attenuates Neutrophil-dominant Allergic Airway Disease

Atopic asthma is a prevalent respiratory disease that is characterized by inflammation, mucus hypersecretion, and airway hyperresponsiveness. The complexity of this heterogeneous disorder has commanded the need to better define asthma phenotypes based on underlying molecular mechanisms of disease. Although classically viewed as a type 2-regulated disease, type 17 helper T (Th17) cells are known to be influential in asthma pathogenesis, predominantly in asthmatics with neutrophilia and severe refractory disease. Bromodomain and extra-terminal domain (BET) chromatin adaptors serve as immunomodulators by directly regulating Th17 responses and Th17-mediated pathology in murine models of autoimmunity and infection. Based on this, we hypothesized that BET proteins may also play an essential role in neutrophil-dominant allergic airway disease. Using a murine model of neutrophil-dominant allergic airway disease, we demonstrate that BET inhibition limits pulmonary inflammation and alters the Th17-related inflammatory milieu in the lungs. In addition, inhibition of BET proteins improved lung function (specifically quasi-static lung compliance and tissue elastance) and reduced mucus production in airways. Overall, these studies show that BET proteins may have a critical role in asthma pathogenesis by altering type 17 inflammation, and thus interfering with BET-dependent chromatin signaling may provide clinical benefits to patients suffering from asthma.

STAT2 Signaling Regulates Macrophage Phenotype During Influenza and Bacterial Super-Infection

Influenza is a common respiratory virus that infects between 5 and 20% of the US population and results in 30,000 deaths annually. A primary cause of influenza-associated death is secondary bacterial pneumonia. We have previously shown that influenza induces type I interferon (IFN)-mediated inhibition of Type 17 immune responses, resulting in exacerbation of bacterial burden during influenza and Staphylococcus aureus super-infection. In this study, we investigated the role of STAT2 signaling during influenza and influenza-bacterial super-infection in mice. Influenza-infected STAT2−/− mice had increased morbidity, viral burden, and inflammation when compared to wild-type mice. Despite an exaggerated inflammatory response to influenza infection, we found increased bacterial control and survival in STAT2 deficient mice during influenza-MRSA super-infection compared to controls. Further, we found that increased bacterial clearance during influenza-MRSA super-infection is not due to rescue of Type 17 immunity. Absence of STAT2 was associated with increased accumulation of M1, M2 and M1/M2 co-expressing macrophages during influenza-bacterial super-infection. Neutralization of IFNγ (M1) and/or Arginase 1 (M2) impaired bacterial clearance in Stat2−/− mice during super-infection, demonstrating that pulmonary macrophages expressing a mixed M1/M2 phenotype promote bacterial control during influenza-bacterial super-infection. Together, these results suggest that the STAT2 signaling is involved in suppressing macrophage activation and bacterial control during influenza-bacterial super-infection. Further, these studies reveal novel mechanistic insight into the roles of macrophage subpopulations in pulmonary host defense.

123Influenza-induced Susceptibility to Secondary Bacterial Pneumonia Is Rescued in STAT2-/- Mice Via an IL-23 Independent Mechanism

Background. Secondary bacterial pneumonia is a well-recognized complication of influenza infection. In mice, Th17 activation is critical for host bacterial lung defense but is attenuated by preceding influenza, resulting in exacerbation of pneumonia. Mice lacking the type I interferon (IFN) receptor are protected from impaired bacterial clearance, suggesting a role for type I IFN in co-infection pathogenesis. Type I IFN is upregulated by influenza, inhibits dendritic cell production of Th17-activating IL23 and IL-1β, and utilizes STAT1 and STAT2 for signaling. We explored the requirement for STAT2 in influenza-associated impairment of lung bacterial clearance in a mouse model of influenza A and methicillin-resistant Staphylococcus aureus(MRSA) co-infection using STAT2-/mice. Methods. Adult male wild-type (WT) and STAT2-/mice were infected with influenza A/PR/8/34 H1N1 or vehicle. On day 6, mice were challenged with vehicle or MRSA (USA300). Mice were sacrificed on day 7; bronchoalveolar lavage (BAL) was performed for cell count and lungs were harvested for flow cytometry, bacterial burden, cytokine level, and gene expression. Results. STAT2-/mice had more weight loss compared to WT on day 6 of influenza infection (89.5% vs 84.2% of starting weight); on day 7, STAT2-/mice had significantly increased influenza viral burden and a higher percentage of neutrophils in BAL fluid (72% vs 40%), although total cell counts did not differ. Compared to mice infected with MRSA alone, WT mice with co-infection had increased bacterial burden that was not seen in STAT2-/mice. Furthermore, influenza-infected STAT2-/mice had significantly prolonged survival following challenge with MRSA at a dose lethal to WT mice (median survival, 30 vs 18 hours). In WT mice, IL-17 and IL-22 producing CD4T cells were reduced in co-infected mice compared to MRSA alone, but these cells were preserved in STAT2-/mice. However, no differences in IFN-β or IL-23 gene expression or IL-23 concentration were detected in whole lung, although IL-22 gene expression was increased. Conclusion. STAT2-/mice are protected from impaired clearance of MRSA from the lung and from mortality during influenza co-infection despite increased influenza severity. This effect may be mediated by IL-22 rescue that is IL-23 independent, but the mechanism requires further elucidation. Disclosures. All authors: No reported disclosures.