title: Inhibition of Francisella tularensis LVS infection of macrophages results in a reduced inflammatory response: evaluation of a therapeutic strategy for intracellular bacteria.
abstract: Francisella tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. A flow cytometric assay was developed to measure bacterial uptake, using a fluorescein isothiocyanate-labelled anti-F. tularensis lipopolysaccharide antibody in conjunction with antibodies to cell surface markers, in order to determine the specific cell phenotypes that were positive for the bacteria. Several phagocytic inhibitors were evaluated in macrophage cell lines and a lung homogenate assay to determine whether the uptake of F. tularensis strain LVS could be altered. Our data show that cytochalasin B, LY294002, wortmannin, nocodazole, MG132 and XVA143 inhibitors reduced LVS uptake by >50% in these assays without having significant cytotoxic effects. Furthermore, a reduction in the inflammatory cytokines monocyte chemoattractant protein-1, interleukin-6 and tumour necrosis factor-α was found in the supernatant of lung tissue infected with LVS when the inhibitory compounds were present. Similarly, there was an alteration in bacterial uptake and a reduction in the inflammatory cytokine response following the administration of wortmannin to LVS-infected mice. Although wortmannin treatment alone did not correlate with the enhanced survival of LVS-infected mice, these inhibitors may have utility in combination therapeutic approaches or against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.
authors.affiliation: Defence Science and Technology Laboratory, Biomedical Sciences Department, Salisbury, Wiltshire, UK. rvdelia@dstl.gov.uk
authors.forename: Riccardo
authors.initials: R
authors.lastname: D'Elia
authors.forename: Dominic C
authors.initials: DC
authors.lastname: Jenner
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Laws
authors.forename: Margaret G M
authors.initials: MG
authors.lastname: Stokes
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Jackson
authors.forename: Angela E
authors.initials: AE
authors.lastname: Essex-Lopresti
authors.forename: Helen S
authors.initials: HS
authors.lastname: Atkins
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005604:Francisella tularensis / Q000187:drug effects / Q000276:immunology* / Q000472:pathogenicity; D008168:Lung / Q000187:drug effects / Q000276:immunology / Q000382:microbiology; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010587:Phagocytosis / Q000187:drug effects / Q000276:immunology; D018410:Pneumonia, Bacterial / Q000276:immunology* / Q000382:microbiology*; D018709:Statistics, Nonparametric; D014406:Tularemia / Q000188:drug therapy / Q000276:immunology* / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D016207:Cytokines