Cirrhosis-induced defects in innate pulmonary defenses against Streptococcus pneumoniae

Katie L Propst-Graham 2 Laurel C Preheim 0 1 2 Elizabeth A Vander Top 2 Mary U Snitily 1 Martha J Gentry-Nielsen 1 2 0 Department of Medicine, University of Nebraska College of Medicine , Omaha, Nebraska , USA 1 Research Service, Omaha Veterans Affairs Medical Center , Omaha, Nebraska , USA 2 Department of Medical Microbiology and Immunology, Creighton University School of Medicine , Omaha, Nebraska , USA Background: The risk of mortality from pneumonia caused by Streptococcus pneumoniae is increased in patients with cirrhosis. However, the specific pneumococcal virulence factors and host immune defects responsible for this finding have not been clearly established. This study used a cirrhotic rat model of pneumococcal pneumonia to identify defect(s) in innate pulmonary defenses in the cirrhotic host and to determine the impact of the pneumococcal toxin pneumolysin on these defenses in the setting of severe cirrhosis. Results: No cirrhosis-associated defects in mucociliary clearance of pneumococci were found in these studies, but early intrapulmonary killing of the organisms before the arrival of neutrophils was significantly impaired. This defect was exacerbated by pneumolysin production in cirrhotic but not in control rats. Neutrophil-mediated killing of a particularly virulent type 3 pneumococcal strain also was significantly diminished within the lungs of cirrhotic rats with ascites. Levels of lysozyme and complement component C3 were both significantly reduced in bronchoalveolar lavage fluid from cirrhotic rats. Finally, complement deposition was reduced on the surface of pneumococci recovered from the lungs of cirrhotic rats in comparison to organisms recovered from the lungs of control animals. Conclusion: Increased mortality from pneumococcal pneumonia in this cirrhotic host is related to defects in both early pre-neutrophil- and later neutrophil-mediated pulmonary killing of the organisms. The fact that pneumolysin production impaired pre-neutrophil-mediated pneumococcal killing in cirrhotic but not control rats suggests that pneumolysin may be particularly detrimental to this defense mechanism in the severely cirrhotic host. The decrease in neutrophil-mediated killing of pneumococci within the lungs of the cirrhotic host is related to insufficient deposition of host proteins such as complement C3 on their surfaces. Pneumolysin likely plays a role in complement consumption within the lungs. Our studies, however, were unable to determine whether pneumolysin more negatively impacted this defense mechanism in cirrhotic than in control rats. These findings contribute to our understanding of the defects in innate pulmonary defenses that lead to increased mortality from pneumococcal pneumonia in the severely cirrhotic host. They also suggest that pneumolysin may be a particularly potent pneumococcal virulence factor in the setting of cirrhosis. - Background Pneumonia is among the top ten causes of death in the United States and the most common cause of death due to infection [1,2]. The Gram positive bacterium Streptococcus pneumoniae is the leading cause of communityacquired pneumonia [3], and is an increasing concern due to the emergence of multi-antibiotic resistant strains [4,5]. Certain patient populations are at a particularly increased risk for pneumococcal infection, including young children, the elderly, and patients with immunodeficiencies or other medical conditions such as cirrhosis [6-9]. Cirrhosis is one of the most common causes of acquired immunodeficiency, and it is well known that mortality from pneumococcal pneumonia is very high in cirrhotics, even with appropriate intensive care support and antibiotic treatment [10-12]. In one particular multi-center study, cirrhosis of the liver increased the risk of death from invasive pneumococcal pneumonia more than any other condition analyzed [11]. However, the specific defect(s) in host defense responsible for this increased morbidity and mortality have not been clearly elucidated. In order to study the interaction between S. pneumoniae and innate host defenses in the cirrhotic host, our laboratory was the first to develop a cirrhotic rat model of pneumococcal pneumonia [13]. The progression of pneumococcal pneumonia requires that the organism evade a wide array of innate pulmonary host defenses. Once S. pneumoniae colonizes the airways, the mucociliary clearance apparatus, composed of mucus entrapment and the beating of ciliated epithelial cells lining the bronchi, prevents pneumococci from moving down into the lower respiratory tract. If pneumococci evade this host defense and enter the lungs, they become deposited on the alveolar lining layer. This thin aqueous layer covering the surface of the pulmonary epithelium contains numerous proteins and peptides possessing antibacterial activity. Lysozyme, lactoferrin, -defensins, and surfactant within alveolar lining fluid constitute an important immediate defense against pneumococci prior to the recruitment of neutrophils [14-16]. Resident alveolar macrophages also phagocytose and kill many types of organisms that escape mucociliary clearance. However, previous research has shown that alveolar macrophages do not efficiently phagocytose and kill highly encapsulated type 3 S. pneumoniae [17-19]. For example, we showed that only 11% of alveolar macrophages from the lungs of either cirrhotic or control rats infected with type 3 S. pneumoniae had pneumococci associated with them [20]. The inability of macrophages to kill this organism was further confirmed in a murine model of pneumococcal pneumonia in which alveolar macrophage depletion failed to reduce pneumococcal clearance from the lungs [21]. Recruited pulmonary neutrophils are an important host defense against pneumococci. However, for phagocytosis and efficient neutrophil-mediated defense against S. pneumoniae, the organisms must be opsonized, notably with fragments of complement component C3 [22,23]. Pneumococci possess multiple virulence factors that facilitate evasion of host defenses [24,25]. One such virulence factor, pneumolysin, is a 53-kDa protein made by all clinical S. pneumoniae isolates. Pneumolysin is a pore-forming toxin that can be released from lysed or intact pneumococci during late log phase [26]. Pneumolysin has both cytotoxic and complement-activating activities [27,28]. The cytotoxic activity affects a wide array of host cells and contributes to lung damage during the early stages of pneumococcal pneumonia [29-31]. The complementactivating activity activates and consumes complement proteins [32], thus diverting complement away from the bacterial surface. This activity prevents efficient opsonophagocytosis and killing by host phagocytes [33]. Our laboratory has previously shown that, like their human counterparts, cirrhotic rats with ascites are hypocomplementemic [34] and are more susceptible to pneumococcal infection. They have increased mortality compared to control animals (...truncated)