Measurement of Macrophage‐Mediated Killing of Intracellular Bacteria, Including Francisella and Mycobacteria

Karen L. Elkins1, Siobhán C. Cowley1, J. Wayne Conlan2

1 Laboratory of Mycobacterial Diseases and Cellular Immunology, Division of Bacterial, Parasitic, and Allergenic Products, CBER/U.S. FDA, Rockville, Maryland, 2 National Research Council of Canada, Institute for Biological Sciences, Ottawa, Ontario, Canada
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.25
DOI:  10.1002/0471142735.im1425s93
Online Posting Date:  April, 2011
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Abstract

Macrophages activated by T cell cytokines are a critical defense mechanism against intracellular bacterial pathogens. This unit presents two general methods for assessing the capacity of mouse macrophages, activated with either soluble cytokines or whole immune T lymphocytes, to control or reduce numbers of intracellular bacteria residing within them. “Measurement of killing” is inferred from a reduction in the number of colony‐forming units (cfu) of bacteria at the end of a culture period, compared to the input numbers of cfu at initiation of culture, to the peak numbers of cfu measured during culture, or to a control group in which killing is expected to be poor. Curr. Protoc. Immunol. 93:14.25.1‐14.25.13. © 2011 by John Wiley & Sons, Inc.

Keywords: Francisella; Mycobacteria; intracellular bacteria; macrophage; cytokine; intracellular killing; T lymphocytes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: IFN‐γ/TNF‐α‐Mediated Activation of Mouse Macrophages for Intracellular Killing of F. tularensis LVS
  • Basic Protocol 2: T Cell–Mediated Control of Intramacrophage Growth of F. tularensis LVS
  • Alternate Protocol 1: T Cell–Mediated Control of Intramacrophage Growth of Mycobacterium tuberculosis
  • Support Protocol 1: Preparation of F. tularensis LVS Bacterial Stocks
  • Support Protocol 2: Generation of LVS‐Immune T Lymphocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: IFN‐γ/TNF‐α‐Mediated Activation of Mouse Macrophages for Intracellular Killing of F. tularensis LVS

  Materials
  • Confluent monolayer of mouse BMM (unit 14.1), cultured without any antibiotics in the medium for at least three days before beginning the assay.
  • Complete tissue culture medium (for BMM and cocultures) with 10% FBS (see recipe), with and without 50 µg/ml gentamicin sulfate (e.g., Sigma)
  • Wash buffer: phosphate‐buffered saline (PBS; appendix 2A) with 2% fetal bovine serum (FBS; appendix 2A)
  • Frozen stock of F. tularensis LVS bacteria ( protocol 4)
  • Recombinant IFN‐γ (various sources, e.g., BD Pharmingen)
  • Recombinant TNF‐α (various sources, e.g., BD Pharmingen)
  • Mueller‐Hinton agar plates (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 24‐well tissue culture plates
  • 37°C, 5% CO 2 humidified incubator
NOTE: Use distilled water, preferably water for injection (WFI), where water is called for.

Basic Protocol 2: T Cell–Mediated Control of Intramacrophage Growth of F. tularensis LVS

  Materials
  • Source of naive, control, and LVS‐immune lymphocytes, e.g., splenic lymphoctyes from LVS‐immune mice ( protocol 5; also see unit 19.14)
  • Additional reagents and equipment for measuring intracellular killing of F. tularensis LVS by mouse macrophages (see protocol 1)

Alternate Protocol 1: T Cell–Mediated Control of Intramacrophage Growth of Mycobacterium tuberculosis

  • Frozen stocks of M. tuberculosis. bacteria (unit 19.5)
  • Distilled water (preferably WFI, water for injection) with 0.01% (w/v) saponin
  • 7H11 agar plates (unit 19.5)
  • PBS with 0.05% (v/v) Tween 80
  • Source of naive, control, and M. tuberculosis—immune lymphocytes ( protocol 5), e.g., splenic lymphoctyes from M. bovis BCG–vaccinated mice (unit 19.5)
  • Phosphate‐buffered saline (PBS; appendix 2A)
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Figures

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Literature Cited

   Baker, C.N., Hollis, D.G., and Thornsberry, C. 1985. Anti‐microbial susceptibility testing of Francisella tularensis with a modified Mueller‐Hinton broth. J. Clin. Micro. 22:212‐215.
   Barel, M., Hovanessian, A.G., Meibom, K., Briand, J.P., Dupuis, M., and Charbit, A. 2008. A novel receptor‐ligand pathway for entry of Francisella tularensis in monocyte‐like THP‐1 cells: Interaction between surface nucleolin and bacterial elongation factor Tu. BMC Microbiol. 8:145.
   Bosio, C.M., Bielefeldt‐Ohmann, H., and Belisle, J.T. 2007. Active suppression of the pulmonary immune response by Francisella tularensis Schu4. J. Immunol. 178:4538‐4547.
   Bosio, C.M. and Elkins, K.L. 2001. Susceptibility to secondary Francisella tularensis LVS infection in B cell deficient mice is associated with neutrophilia but not with defects in specific T cell mediated immunity. Infect. Immun. 69:194‐203.
   Carlson, P.E. Jr., Carroll, J.A., O'Dee, D.M., and Nau, G.J. 2007. Modulation of virulence factors in Francisella tularensis determines human macrophage responses. Microb. Pathog. 42:204‐214.
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   Clemens, D.L. and Horwitz, M.A. 2007. Uptake and intracellular fate of Francisella tularensis in human macrophages. Ann. N.Y. Acad. Sci. 1105:160‐186.
   Collazo, C.M., Meierovics, A.I., De Pascalis, R., Wu, T.H., Lyons, C.R., and Elkins, K.L. 2009. T cells from lungs and livers of Francisella tularensis–immune mice control the growth of intracellular bacteria. Infect. Immun. 77:2010‐2021.
   Cowley, S.C. and Elkins, K.L. 2003a. CD4+ T cells mediate IFN‐gamma‐independent control of Mycobacterium tuberculosis infection both in vitro and in vivo. J. Immunol. 171:4689‐4699.
   Cowley, S.C. and Elkins, K.L. 2003b. Multiple T cell subsets control Francisella tularensis LVS intracellular growth without stimulation through macrophage interferon gamma receptors. J Exp Med 198:379‐389.
   Cowley, S.C., Myltseva, S.V., and Nano, F.E. 1996. Phase variation in Francisella tularensis affecting intracellular growth, lipopolysaccharide antigenicity and nitric oxide production. Mol. Microbiol. 20:867‐874.
   Cowley, S.C., Hamilton, E., Frelinger, J.A., Su, J., Forman, J., and Elkins, K.L. 2005. CD4–CD8– T cells control intracellular bacterial infections both in vitro and in vivo. J. Exp. Med. 202:309‐319.
   Cowley, S., Goldberg, M.F., Ho, J.A., Sedgwick, J.D., and Elkins, K.L. 2008. Membrane TNF‐α is a suboptimal regulator of nitric oxide production but effectively controls apoptosis during Francisella tularensis LVS infection. J. Infect. Dis. 198:284‐292.
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   Eigelsbach, H.T. and Downs, C.M. 1961. Prophylactic effectiveness of live and killed tularemia vaccines. J. Immunol. 87:415‐425.
   Eigelsbach, H.T., Hunter, D.H., Janssen, W.A., Dangerfield, H.G., and Rabinowitz, S.G. 1975. Murine model for study of cell‐mediated immunity: Protection against death from fully virulent Francisella tularensis infection. Infect. Immun. 12:999‐1005.
   Elkins, K.L., Cooper, A., Colombini, S.M., Cowley, S.C., and Kieffer, T.L. 2002. In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 sub‐ of Interleukin‐12 (IL‐12) but not on IL‐12 p70. Infect. Immun. 70:1936‐1948.
   Elkins, K.L., Cowley, S.C., and Bosio, C.M. 2003. Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain. Microbes Infect. 5:132‐142.
   Hazlett, K.R., Caldon, S.D., McArthur, D.G., Cirillo, K.A., Kirimanjeswara, G.S., Magguilli, M.L., Malik, M., Shah, A., Broderick, S., Golovliov, I., Metzger, D.W., Rajan, K., Sellati, T.J., and Loegering, D.J. 2008. Adaptation of Francisella tularensis to the mammalian environment is governed by cues which can be mimicked in vitro. Infect. Immun. 76:4479‐4488.
   Kolibab, K., Parra, M., Yang, A.L., Perera, L.P., Derrick, S.C., and Morris, S.L. 2009. A practical in vitro growth inhibition assay for the evaluation of TB vaccines. Vaccine 28:317‐322.
   Mackaness, G.B. 1962. Cellular resistance to infection. J. Exp. Med. 116:381‐406.
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   Mackaness, G.B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129:973‐992.
   Pasetti, M.F., Cuberos, L., Horn, T.L., Shearer, J.D., Matthews, S.J., House, R.V., and Sztein, M.B. 2008. An improved Francisella tularensis live vaccine strain (LVS) is well tolerated and highly immunogenic when administered to rabbits in escalating doses using various immunization routes. Vaccine 26:1773‐1785.
   Pierini, L.M. 2006. Uptake of serum‐opsonized Francisella tularensis by macrophages can be mediated by class A scavenger receptors. Cell Microbiol. 8:1361‐1370.
   Sandström, G. 1994. The tularaemia vaccine. J. Chem. Technol. Biotechnol. 59:315‐320.
   Schulert, G.S. and Allen, L.A. 2006. Differential infection of mononuclear phagocytes by Francisella tularensis: Role of the macrophage mannose receptor. J. Leukoc. Biol. 80:563‐571.
   Sjöstedt, A. 2007. Tularemia: History, epidemiology, pathogen physiology, and clinical manifestations. Ann. N.Y. Acad. Sci. 1105:1‐29.
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Key References
   Bosio and Elkins, 2001. See above.
  This publication describes the original development of the coculture assay using LVS, and includes data regarding choices in many of the key parameters.
   Cowley and Elkins, 2003a. See above.
  This publication describes the original development of the coculture assay using M. tuberculosis.
   Elkins et al., 2002. See above.
  This publication compares the activities of T cells from IL‐12 p35 and p40 knockout mice in the coculture assay, illustrating minimal differences in the ability of each type to control intramacrophage LVS growth despite the dramatic difference in vivo in the outcome of intradermal LVS infection.
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