Cirrhosis-Induced Defects in Innate Puemonary Defenses Against Streptococcus Pneumoniae
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Authors
Propst-Graham, Katie
Issue Date
2007-02
Volume
Issue
Type
Thesis
Language
en_US
Keywords
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Abstract
Streptococcus pneumoniae is the most common cause of community-acquired pneumonia, and this pathogen is especially detrimental for certain population groups, such as cirrhotic patients. It is well known that mortality from pneumococcal pneumonia is very high in those with cirrhosis, even when they receive intensive care support and appropriate treatment. However, the defects in host defense responsible for the increased morbidity and mortality from pneumococci in these patients have not been clearly elucidated. In addition, the impact of pneumococcal virulence factors, such as the toxin pneumolysin, on pulmonary defenses within cirrhotics is largely unknown. The purpose of this research was to identify defect(s) within innate host defenses in the cirrhotic host, and to examine the impact of pneumolysin on these defenses. The innate defenses analyzed within this research included mucociliary clearance, early pre-neutrophil- and later neutrophil-mediated defenses, and in vivo complement deposition inside the lungs.
Cirrhosis was induced in male Sprague-Dawley rats by weekly gavage with carbon tetrachloride, whereas control rats were gavaged weekly with PBS. All cirrhotic rats used for experimentation had a visible accumulation of ascites fluid for at least two consecutive weeks. To examine mucociliary clearance, an intranasal mortality trial was conducted in cirrhotic and control rats with type 3 S. pneumoniae ATCC 6303. The cirrhotic rats demonstrated increased mortality from S. pneumoniae following intranasal challenge. However, a comparison with mortality studies previously conducted by our laboratory following transtracheal challenge did not indicate additional defects in mucociliary clearance in the cirrhotic animals. To further analyze mucociliary clearance and to determine the impact of pneumolysin on this host defense, bacterial movement from the nasopharynx to the lungs and ciliary beat frequency on tracheal epithelial cells were measured following intranasal infection with isogenic pneumolysin-producing (Ply+) and pneumolysin-deficient (Ply-) WU2-derived type 3 S. pneumoniae strains. Ciliary beat frequency and movement of pneumococci into the lungs were not different in cirrhotic vs. control rats, nor were they impacted by pneumolysin production.
Early pre-neutrophil pulmonary defenses were analyzed in vivo by quantification of non-neutrophil-mediated killing of the Ply+ and Ply- WU2 S. pneumoniae strains. Rats were infected transtracheally, and pre-neutrophil killing was quantified 1 hour later by a standard plate counting technique. Killing of the Ply+ strain was significantly decreased in the cirrhotic rats, indicating a defect in pre-neutrophil pulmonary defense in the cirrhotic host. By contrast, the cirrhotic rats killed the Ply- strain as well as control animals, indicating that pneumolysin contributes to pneumococcal resistance against killing by these defenses in the cirrhotic host. Pneumolysin did not impair killing in the control rats, indicating this virulence factor may not be as effective against pre-neutrophil defenses in the normal host. To examine the mechanisms responsible for this defect in the cirrhotic host, anti-pneumococcal proteins in alveolar lining fluid were quantified by enzyme linked immunosorbent assay. Cirrhotic rats were found to have significantly decreased lysozyme and complement component C3 levels compared to the control animals.
Pulmonary neutrophil function was analyzed using a similar assay to that used to quantify pre-neutrophil killing, except that neutrophils were pre-recruited into the lungs by transtracheal instillation of Escherichia coli lipopolysaccharide that shuts down preneutrophil-mediated killing. After allowing 5 hours for neutrophil recruitment, cirrhotic and control rats were transtracheally infected with either the Ply+ or Ply- WU2 S. pneumoniae and killing was quantified as described above. Neutrophil-mediated killing of the WU2-derived strains was not impaired in the cirrhotic rats, nor was it affected by pneumolysin production. Since these results were in disagreement with previous research by our laboratory, the assay was repeated using S. pneumoniae ATCC 6303. In that case, neutrophil killing was impaired in the cirrhotic animals, verifying our previous data and indicating that strain differences influence neutrophil-mediated killing, even among strains of the same capsular type.
To help elucidate the mechanism for decreased neutrophil-mediated killing of S. pneumoniae ATCC 6303 in the cirrhotic host, complement deposition on pneumococci within the lungs was measured using a novel in vivo assay recently developed in our laboratory. Cirrhotic and control rats were infected transtracheally with S. pneumoniae ATCC 6303, and the pneumococci were then recovered from the lungs 15 minutes later by bronchoalveolar lavage. Flow cytometry was used to determine the percentage of the bacteria recovered that had C3 bound to their surface. Our results indicated there may be reduced binding of C3 to pneumococci within the lungs of the cirrhotic animals, although additional animals will be required to determine if the difference is statistically significant.
The primary conclusions from these studies are that increased mortality from pneumococcal pneumonia in the cirrhotic rat model is likely related to defects in both early pre-neutrophil- and neutrophil-mediated killing of pneumococci within the lungs. Pneumococcal production of pneumolysin impairs pre-neutrophil defenses, but only in the cirrhotic host. Reduced pre-neutrophil defense in the cirrhotic rats is believed to be the result of decreased anti-pneumococcal proteins in alveolar lining fluid. We also believe pneumolysin may impair neutrophil-mediated killing of S. pneumoniae, but proof of this will require the creation of additional pneumolysin-deficient pneumococcal strains. Mean complement deposition on pneumococci was lower within the lungs of the cirrhotic rats, which we believe to play a role in the reduced neutrophil-mediated killing of S. pneumoniae ATCC 6303 measured in the cirrhotic animals. These findings contribute to a better understanding of the defects in innate host defenses that lead to increased mortality from pneumococcal infection in the cirrhotic host. They also help elucidate the impact of pneumolysin on innate pulmonary defenses in all hosts.
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Publisher
Creighton University
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Copyright is retained by the Author.
A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.