title: Robust growth of avirulent phase II Coxiella burnetii in bone marrow-derived murine macrophages.
abstract: Published data show that murine bone marrow-derived macrophages (BMDM) restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. Growth restriction of phase II bacteria is thought to result from potentiated recognition of pathogen-associated molecular patterns, which leads to production of inhibitory effector molecules. Past studies have used conditioned medium from L-929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of conditioned medium, such as variable amounts of type I interferons, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the C. burnetii Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6J mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria were readily internalized by BMDM, and replicated within degradative, LAMP1-positive vacuoles to achieve roughly 3 logs of growth over 6 days. Uninfected BMDM did not appreciably express CD38 or Egr2, markers of classically (M1) and alternatively (M2) activated macrophages, respectively, nor did infection change the lack of polarization. In accordance with an M0 phenotype, infected BMDM produced moderate amounts of TNF and nitric oxide. Similar NMII growth results were obtained using C57BL/6J myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein 1 (Nramp1) C57BL/6J knock-in mice were transduced with ER-Hoxb8, and macrophages were derived from immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the Nramp1 transporter does not negatively impact NMII growth. Results with NMII were recapitulated in primary macrophages where C57BL/6J Nramp1+ BMDM efficiently killed Salmonella enterica serovar Typhimurium. M-CSF differentiated murine macrophages from bone marrow and conditional ER-Hoxb8 myeloid progenitors will be useful ex vivo models for studying Coxiella-macrophage interactions.
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Diane C
authors.initials: DC
authors.lastname: Cockrell
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Carrie M
authors.initials: CM
authors.lastname: Long
authors.affiliation: Innate Immunity and Pathogenesis Unit, Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Shelly J
authors.initials: SJ
authors.lastname: Robertson
authors.affiliation: Plague Section, Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Shannon
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Heather E
authors.initials: HE
authors.lastname: Miller
authors.affiliation: Retroviral Immunology Section, Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Lara
authors.initials: L
authors.lastname: Myers
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Larson
authors.affiliation: Salmonella-Host Cell Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Tregei
authors.initials: T
authors.lastname: Starr
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Beare
authors.affiliation: Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.
authors.forename: Robert A
authors.identifier: http://orcid.org/0000-0001-6870-4619
authors.initials: RA
authors.lastname: Heinzen
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000378:metabolism / Q000382:microbiology*; D002478:Cells, Cultured; D003115:Colony-Stimulating Factors / Q000378:metabolism; D016997:Coxiella burnetii / Q000254:growth & development* / Q000472:pathogenicity; D005260:Female; D008264:Macrophages / Q000166:cytology / Q000378:metabolism / Q000382:microbiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D011778:Q Fever / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D003115:Colony-Stimulating Factors; D014409:Tumor Necrosis Factor-alpha