Date of Award

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Jonathan A. McCullers, M.D.

Committee

B. Keith English, M.D. Paul G. Thomas, Ph.D. Christopher M. Waters, Ph.D. Richard J. Webby, Ph.D.

Keywords

Influenza, Alveolar Macrophages, Streptococcus pneumoniae, Secondary Bacterial Pneumonia, Immunomodulation, Corticosteroids.

Abstract

Viruses such as influenza suppress host immune function by a variety of methods. This may result in a significant morbidity through several pathways, including facilitation of secondary bacterial pneumonia from pathogens such as Streptococcus pneumoniae. Lungresident alveolar macrophages (AMs) act as the first line of innate cellular immunity against respiratory bacterial pathogens, including pneumococcus. Therefore, they represent an attractive target for study Before investigating the impact of influenza infection on resident AMs, we first characterized different subsets of lung-resident macrophages in naïve mice using a novel in vivo labeling approach in conjunction with multicolor flow cytometric analysis and confocal microscopic examination. A stable fluorescent dye, PKH26-PCL, was administered intranasally to selectively label the lung-resident macrophages in a well-established murine model prior to influenza infection. We determined the turnover kinetics of the lung-resident macrophage subsets during the course of influenza infection. More than 90% of resident AMs were lost in the first week after influenza, while the remaining cells had a necrotic phenotype. To establish the impact of this innate immune defect, influenza-infected mice were challenged with a small dose of Streptococcus pneumoniae. Early AM-mediated bacterial clearance was significantly impaired during the AM depletion phase in influenza-infected mice – about 50% of the initial bacterial inoculum could be harvested from the alveolar airspaces 3 hours later. In mock-infected mice, by contrast, more than 95% of inocula up-to-50-fold higher was efficiently cleared. Co-infection during the AM depletion phase caused significant body weight loss and mortality. Two weeks after influenza, the AM population was fully replenished with successful re-establishment of the early innate host protection. Local GM-CSF treatment induced partial expansion of resident AMs during influenza infection. Thus, it led to partial restoring of the impaired early bacterial clearance with efficient protection against secondary pneumococcal pneumonia. We conclude that a novel immunosuppression mechanism occurs during influenza infection through the resident AM depletion. Among other potential effects, this establishes a niche for secondary pneumococcal infection by altering early cellular innate immunity in the lungs resulting in pneumococcal outgrowth and lethal pneumonia. This novel mechanism will inform development of novel therapeutic approaches to restore lung innate immunity against bacterial super-infections. Secondary bacterial pneumonia (SBP) is a leading cause of the increased hospitalizations and mortality during influenza epidemics and pandemics despite routine use of standard antibiotics. Antibiotic-induced immunopathology associated with bacterial cell wall lysis has been suggested to contribute to these poor outcomes. Using Streptococcus pneumoniae in a wellestablished murine model of SBP following influenza, we stratified disease severity based on the pneumococcal load in the lungs via in vivo bioluminescence imaging. Ampicillin treatment cured mice with mild pneumonia but was ineffective against severely pneumonic mice, despite effective bacterial killing. This treatment failure makes it crucial to explore immunmodulation approaches that can prevent the aggravated lung immunopathology during antibiotic treatment of severe SBP. Therefore, we tested the efficacy of the standard anti-inflammatory drug dexamethasone as an adjunctive corticosteroid therapy. Adjunctive dexamethasone treatment significantly improved ampicillin-induced immunopathology and survival outcomes in mice with severe SBP. However, early dexamethasone therapy during primary influenza infection impaired the adaptive immunity in the lungs as manifest by increased viral titers, with an associated loss of its protective functions in SBP. The clinical use of corticosteroids as an adjunctive therapy for treating pneumonia is still under debate. However, our findings support adjunctive clinical use of corticosteroids in severe cases of community-acquired pneumonia. Nonetheless, dexamethasone treatment has drawbacks implied by delayed body weight recovery in dexamethasone-treated mice, which may explain the published controversy on the corticosteroid efficacy in terms of disease morbidity. This relative success of our animal model of SBP to simulate the clinical therapeutic settings in humans will help explore novel immunomodulation approaches to improve the poor outcomes of antibiotic treatment of severe community-acquired pneumonia.

DOI

10.21007/etd.cghs.2013.0108

Comments

Six month embargo expired June 2014

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