title: Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells.
abstract: The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates.
authors.affiliation: Laboratory of Mucosal Pathogens and Cellular Immunology, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: Savannah
authors.initials: S
authors.lastname: Maggio
authors.affiliation: Microscopy and Imaging Core Facility, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Takeda
authors.affiliation: National Research Council Canada, Human Health Therapeutics Portfolio, Ontario, Canada.
authors.forename: Felicity
authors.initials: F
authors.lastname: Stark
authors.affiliation: Laboratory of Mucosal Pathogens and Cellular Immunology, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: Anda I
authors.initials: AI
authors.lastname: Meierovics
authors.affiliation: National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Idalia
authors.initials: I
authors.lastname: Yabe
authors.affiliation: Laboratory of Mucosal Pathogens and Cellular Immunology, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: Siobhan C
authors.initials: SC
authors.lastname: Cowley
mesh_terms: D000818:Animals; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism* / Q000382:microbiology*; D005604:Francisella tularensis / Q000254:growth & development*; D007371:Interferon-gamma / Q000378:metabolism; D020381:Interleukin-17 / Q000378:metabolism; D008168:Lung / Q000378:metabolism* / Q000382:microbiology*; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000378:metabolism; D048069:Tumor Necrosis Factors / Q000378:metabolism; D014774:Virulence / Q000502:physiology
chemical_list: D020381:Interleukin-17; D026361:Reactive Nitrogen Species; D048069:Tumor Necrosis Factors; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse