NO killing of B. mallei Figure 1. Antimicrobial activity of murine and human macrophage-like cell lines against B. mallei. Intracellular survival of B. mallei was studied over a 20 h period in murine (i.e., J774 and AMJ) and human (i.e., THP1 and U937) macrophage-like cell lines. Data from 8–14 independent observations gathered in 3 separate experiments are represented in box-and-whiskers plots as median, intraquartile and total ranges. doi:10.1371/journal.pone.0001976.g001 Student’s t-test was used for statistical analysis and the data considered statistically significant when p,0.05. Results Survival of B. mallei in human and murine cell lines The number of log phase B. mallei internalized by J774 cells increased as a function of time, reaching approximately 106 CFU/ 105 macrophages 2 h after infection. Because of this delay phagocytosis, all subsequent Burkholderia infections, unless indicat- ed, were carried out for 2 h before gentamicin was added to the medium. Mice offer a wide variety of genetic reagents that facilitate investigations of O2-dependent host defenses in host– pathogen interactions. Therefore, we deemed it important to compare the intracellular growth of B. mallei in a number of murine (J774 and alveolar AMJ-C11 macrophages) and human (i.e., THP1 and U937) cell lines (fig. 1). Remarkably, all murine and human macrophage-like cell lines tested killed B. mallei with very similar kinetics. These findings indicate that murine macrophages are a relevant model in which to study B. mallei- phagocyte interactions. IFNc enhances killing of B. mallei by macrophages It has been shown that IFNc enhances the antimicrobial activity of macrophages against the opportunistic pathogen B. pseudomallei. However, to our knowledge, no data are available on the role that IFNc plays in intracellular resistance to B. mallei. Because IFNc has recently been shown to be critical for resistance of mice to B. mallei [24], we tested the effect of IFNc treatment on the anti- Burkholderia activity of J774 cells (fig. 2). IFNc-primed macrophages were significantly (p,0.01) more efficient at killing B. mallei than unstimulated controls. The number of B. mallei recovered from IFNc-treated macrophages was at least 10-fold lower than controls (fig. 2). In some cases, IFNc-primed J774 cells completely eliminated the bacteria from the cell cultures. These data demonstrate that IFNc enhances the innate resistance of murine macrophages to B. mallei. Reactive oxygen and nitrogen species differentially contribute to the anti-B. mallei activity of IFNc-primed macrophages ROS and RNS are key components of the antimicrobial arsenal of the innate and the activated response of macrophages against a variety of intracellular pathogens [25]. To test the importance of ROS and RNS in the killing of B. mallei, macrophages were obtained from C57BL/6 mice and their congenic gp91phox2/2, iNOS2/2 and gp91phox2/2/iNOS2/2 immunodeficient strains. Unstimulated macrophages isolated from C57BL/6 mice killed over 99% of B. mallei within 5 h of infection (fig. 3A). Strikingly, singly or doubly immunodeficient macrophages lacking gp91phox and/or iNOS hemoproteins exhibited similar bactericidal activity as that of unstimulated wild-type controls. These findings suggest that neither ROS nor RNS appear to play a significant role in the early killing of B. mallei by unstimulated macrophages. Similar to the improved killing seen in the J774 cell line, IFNc increased (p,0.001) the anti-B. mallei activity of primary macrophages isolated from C57BL/6 mice. It should be noted that some of the IFNc-primed macrophages completely eliminated B. mallei from the cultures, a phenomenon that was seen in all of the IFNc- primed, gp91phox-deficient macrophages (fig. 3B). These findings suggest that ROS are dispensable for the anti-B. mallei activity of IFNc-primed macrophages. Conversely, RNS contributed to the anti-B. mallei arsenal of IFNc-activated macrophages (fig. 3B). In fact, this intracellular pathogen was recovered in significantly higher numbers from both IFNc-primed, iNOS2/2 and iNOS2/2 /gp91phox2/2 macrophages than from wild-type or gp91phox2/2 controls. The killing activity of IFNc-treated, iNOS- or iNOS/ gp91phox- deficient macrophages was similar to that of unstimu- lated wild-type controls. Collectively, these data indicate that RNS but not ROS mediate the anti-Burkholderia activity of IFNc-primed Figure 2. Anti-Burkholderia activity of IFNc-primed macrophages. The intracellular survival of B. mallei was assessed in control and IFNc- primed J774 cells. Selected groups of macrophages were treated with 200 U/ml IFNc 16 h before infection. Data from 6 independent observations gathered on 2 separate experiments are represented in box-and-whiskers plots as median, intraquartile and total ranges. *P,0.01 compared to unstimulated control macrophages. doi:10.1371/journal.pone.0001976.g002 PLoS ONE | www.plosone.org 4 April 2008 | Volume 3 | Issue 4 | e1976