ABSTRACT
Murine bone marrow derived macrophages (BMM) are valuable tools to investigate macrophage functions such as cytokine production and bactericidal activities from different strains of mice. In most studies BMM are generated and characterised using cell culture systems with fetal calf serum (FCS) as an essential supplement. Since serum contains varying amounts of undefined components influencing the maturation and polarisation process of BMM there is a need for a more standardised methodology. The aim of the present study was to establish a cell culture system for the generation of murine BMM under standardised serum free conditions. The use of a newly developed compositionally defined serum supplement enabled us to gain mature BMM from BALB/c and C57BL/6 mice expressing the myeloid marker F4/80, CD11b and MOMA-2. Under these serum-free conditions LPS and IFN-gamma stimulated C57BL/6 BMM released more IL-12 and nitric oxide (NO) compared to BALB/c BMM whereas the latter cells produced higher levels of IL-10 and MCP-1 after LPS stimulation. Serum-free generated C57BL/6 BMM showed enhanced bactericidal activity against the Gram-negative rod Burkholderia pseudomallei compared to BALB/c BMM. In conclusion the serum-free generation of BMM described in this study will assure more standardised and reproducible conditions for the future characterisation of murine BMM.
Subject(s)
Bone Marrow Cells/cytology , Cell Culture Techniques/methods , Culture Media, Serum-Free , Macrophages/cytology , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Burkholderia pseudomallei/immunology , Chemokine CCL2/metabolism , Interleukin-10/metabolism , Interleukin-12/metabolism , Lipopolysaccharides , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nitric Oxide/metabolismABSTRACT
The gram-negative rod Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease which is endemic in tropical and subtropical areas. The bacterium multiplies intracellularly within the cytosol, induces the formation of actin tails, and can spread directly from cell to cell. Recently, it has been shown that B. pseudomallei can induce caspase-1-dependent cell death in macrophages. The aim of the present study was to further elucidate the role of caspase-1 during B. pseudomallei infection. In vivo experiments with caspase-1(-/-) mice revealed a high susceptibility to B. pseudomallei challenge. This phenotype was associated with a significantly higher bacterial burden 2 days after infection and decreased gamma interferon (IFN-gamma) and interleukin-18 cytokine levels 24 h after infection compared to control animals. caspase-1(-/-) bone marrow-derived macrophages (BMM) exhibited strong caspase-3 expression and reduced cell damage compared to wild-type (WT) cells during early B. pseudomallei infection, indicating "classical" apoptosis, whereas WT BMM showed signs of rapid caspase-1-dependent cell death. Moreover, we found that caspase-1(-/-) BMM had a strongly increased bacterial burden compared to WT cells 3 h after infection under conditions where no difference in cell death could be observed between both cell populations at this time point. We therefore suggest that caspase-1-dependent rapid cell death might contribute to resistance by reducing the intracellular niche for B. pseudomallei, but, in addition, caspase-1 might also have a role in controlling intracellular replication of B. pseudomallei in macrophages. Moreover, caspase-1-dependent IFN-gamma production is likely to contribute to resistance in murine melioidosis.
Subject(s)
Burkholderia pseudomallei/pathogenicity , Caspase 1/metabolism , Melioidosis/immunology , Animals , Apoptosis , Bone Marrow Cells/cytology , Burkholderia pseudomallei/growth & development , Burkholderia pseudomallei/physiology , Down-Regulation , Female , Humans , Interferon-gamma/metabolism , Interleukin-18/metabolism , Macrophages/cytology , Macrophages/microbiology , Male , Melioidosis/microbiology , Melioidosis/mortality , Mice , Mice, Inbred C57BLABSTRACT
Staphylococcus aureus is one of the most common causes of hospital-acquired infections. At the same time, 25% of healthy persons are symptom-free S. aureus carriers, and they have an increased risk of developing nosocomial S. aureus septicemia. Paradoxically, their prognosis is much better than that of noncarriers. We compared the antibody profiles for carriers and noncarriers toward S. aureus superantigens. In carriers, we found high titers of neutralizing antibodies specific for those superantigens that are expressed by their colonizing strain. The results show that carriage status confers strain-specific humoral immunity, which may contribute to protection during S. aureus septicemia.
