C5a in defense against murine Gram-negative pneumonia

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DESCRIPTION (provided by applicant): This project is studying how the anaphylatoxin C5a, a protein generated by the immune system upon first interacting with invading microorganisms, helps support host defenses in the lung during acute Gram negative pneumonia. This disease is a serious threat to hospitalized, post-operative, and immunocompromised patients and the bacteria that cause it are increasingly resistant to broad-spectrum antibiotics. Using a murine model of lung infection, our goal is to better define the role of C5a so that improved therapies against Gram-negative pneumonia might be developed. The first aim of the project examines how C5a enhances the in vitro responses of alveolar macrophages to the clinically important pathogen Pseudomonas aeruginosa and will measure C5a's effect on phagocytosis, respiratory burst, bacterial killing, and release of proinflammatory mediators. Parallel studies will examine C5a's effect against this pathogen in whole blood. The second aim will study how structures on the surface of Gram-negative bacteria may alter the generation and ultimately the effectiveness of C5a. These studies take advantage of a mutant of a virulent strain of Klebsiella pneumoniae in which the gene responsible for initiating synthesis of the surface carbohydrate O-antigen has been deleted. Wild-type and mutant strains will be compared in terms of their ability to promote the generation of C5a and to alter the responses of alveolar macrophages. Parallel studies will be performed using a strain of E. coli which has been transformed to synthesize the Klebsiella O-antigen to determine if expression of this complement countermeasure can convey virulence to an otherwise nonpathogenic bacteria. The final aim of the proposal will examine 3 strategies to boost the level of C5a produced in the lung during acute infection. These experiments will examine intratracheal therapy with a protein derived from cobra venom which can generate C5a in the absence of an invading pathogen, an inhibitor of the enzyme carboxypeptidase-N (an important deactivator of C5a), and lastly recombinant murine C5a. It is hoped that the results will better define the role of C5a in host defense during Gram-negative lung infection, will increase understanding of how Gram-negative organisms evade complement-mediated lung defenses, and will determine whether novel C5a-enhancing therapies might be used to assist host defense during acute bacterial pneumonia.