In vitro modeling of Coxiella burnetii-alveolar macrophage interactions and the role of pulmonary surfactant protein D : a dissertation /

Bibliographic Details
Main Author:	Soltysiak, Kelly Ann
Format:	Thesis Book
Language:	English
Published:	[College Station, Tex.] : [Texas A&M University System Health Science Center], [2013]
Subjects:	Coxiella burnetii. Pulmonary Surfactant-Associated Protein D. Q Fever > etiology. Major Biomedical Sciences.

Description
Abstract:	ABSTRACT: Coxiella burnetii is the etiologic bacterial agent of Q Fever, a febrile illness of zoonotic origin. Natural infection occurs when aerosolized bacteria are inhaled and the bacteria have a tropism for alveolar macrophages. The alveolar space of the lungs is comprised of structural alveolar epithelial cells, type II alveolar epithelial cells which secrete surfactants, dendritic cells and macrophages. The murine alveolar macrophage cell line MH-S was created as an improved model for macrophages in this environment through immortalization due to infection of purified murine alveolar macrophages with SV40. Macrophages in the alveolar space are bathed in pulmonary surfactant. Surfactant protein D (SP-D) is a C-type lectin involved in innate immunity in the lungs. It can bind to the carbohydrate moieties of invading pathogens and aggregate bacteria or viruses for enhanced clearance by macrophages. Macrophages can be stimulated to classical and alternative activation states based upon external or exogenous stimuli. M1, or classical activation results in production and secretion of inflammatory cytokines, including IL-1[beta], IL-12, and TNF[alpha]. M2, or alternative activation results in production and secretion of anti-inflammatory cytokines including IL-4 and IL-10. We investigated the role SP-D plays in C. burnetii infection of alveolar macrophages by characterization of C. burnetii infection in alveolar macrophages. Using the MH-S alveolar macrophage cell line, transcriptional kinetics were measured to determine the macrophage activation state. Infection with Phase I RSA493 and Phase II RSA439 resulted in increased transcription of il-1 [beta], il-6, il-12p40, tnf[alpha], and gm-csf over uninfected controls in the first 8 hours of infection. Inducible nitric oxide (nos2) transcription was transient for cells infected with C. burnetii, but sustained for cells treated with LPS. A nitric oxide secretion assay demonstrated C. burnetii infection resulted in barely detectible levels of nitric oxide, but significant secretion after LPS treatment. Transcription was not stimulated in M2 gene il-4 and was only transient with il-10. ELISA analysis confirmed production IL-6, IL-2, and TNF[alpha]. Mature IL-1[beta] was produced by MH-S cells infected with C. burnetii, however it was not secreted, indicating the alveolar macrophages were activated towards a classical, or M1, activation state upon infection with C. burnetii. SP-D interactions with C. burnetii were characterized during infection of MH-S cells. SP-D binds to C. burnetii in a calcium-dependent manner. SP-D treatment does not result in changes in M1 or M2 cytokine transcription in MH-S, antimicrobial effects, or aggregation of C. burnetii. SP-D pretreatment results in a significant reduction of phagocytosis and an increase in attachment of the bacteria. This indicated that C. burnetii does not experience negative consequences from interactions with this particular component of the pulmonary innate immune system. Combining the results from the studies presented here the MH-S cell line is a physiologically relevant in vitro model for C. burnetii-alveolar macrophage interactions and for acute C. burnetii infection. Future studies might include determining the mechanism for C. burnetii infection suppressing secretion of mature IL-1[beta] and nitric oxide from alveolar macrophages. Additional studies should also include biochemical characterization of interactions between C. burnetii, SP-D and alveolar macrophage receptors.
Item Description:	Vita. "Major Subject: Biomedical Science". "Submitted to the Office of Research and Graduate Studies of The Texas A&M University System Health Science Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2013." Approved as to style and content by: James E. Samuel, Jeffrey D. Cirillo, Paul de Figueiredo, Vernon Tesh.
Physical Description:	xi, 104 leaves : illustrations (mostly color) ; 28 cm.
Bibliography:	Includes bibliographical references (leaves 89-98).