Increased Virulence in M. massiliense Infection Figure 3. Lung histology from M. massiliense infected IFNc-KO mice. Panel A, A-H shows representative lung histopathology from the M. massiliense CIP 108297- and epidemic CRM-0019-infected animals on day 30 and 60 of the infection. Tissue sections are stained with hematoxylin and eosin. The lower photograph depicts the magnified region denoted by the square (upper photograph). CRM-0019-infected mice (panel A: D and H) showed an increased rate of granuloma (arrows) progression and involvement of larger areas of the lung compared to the control mice (panel A: B and E). Panel B shows representative lung acid fast staining bacilli at day 60 after M. massiliense CIP 108297 and CRM-0019 infection. Total magnification, panel A [A, C, E, G] = 10x; panel A [B, D, F, H] = 20x; panel B = 100x. doi:10.1371/journal.pone.0024726.g003 analysis of alveolar macrophages (CD11b+) and dendritic cell (CD11c+) populations from lungs and spleens of M. massiliense CRM-0019-infected mice compared to CIP 108297-infected mice. Figure 4 A shows representative dot plots of lung cells from the isotype control, reference CIP 108297 and the outbreak M. massiliense CRM-0019 strain after 30 days of infection in mice primarily gated on CD11b+ macrophages cells expression of IL- 12. As shown in Figure 4 B and C, macrophages and dendritic cells from the CRM-0019-infected mice showed reduced numbers of lung and spleen IL-12 and TNFa producing macrophage and dendritic cells compared to the reference strain CIP 108297. CD4+ and CD8+ T cell cytokine expression during Mycobacterium massiliense infection We next sought to investigate the differences in the CD4+ and CD8+ T cells in the lungs and spleens of mice infected with the epidemic strain M. massiliense CRM-0019 compared to reference strain CIP 108297 after 30 and 60 days of infection. We evaluated CD4+ and CD8+ T cells ability to produce the protective cytokine TNFa required for efficient granuloma formation [34]. We also evaluated CD4+ and CD8+ T cells ability to induce immunosup- pressive cytokines such as IL-4 which is considered to increase host susceptibility to intracellular pathogens [20]. Figure 5 A shows representative dot plots of lung cells from the isotype control, reference CIP 108297 and the outbreak M. massiliense CRM-0019 strain after 30 days of infection in mice primarily gated on CD4+ T cells expression of TNFa. Mice infected with CRM-0019 showed significant reduced numbers of lung and spleen TNFa–expressing CD4+ T cells by day 30 of infection compared to the reference strain (Figure 5 B-C). However, animals infected with the epidemic strain during chronic infection showed similar level of TNFa–expressing CD4+ and CD8+ T cells in the spleen as the reference strain (Figure 5 C). The two types of infected mice did not differ in lung and spleen IL-4–expressing CD4+ and CD8+ T cells (data not shown). CD4+Foxp3 regulatory T cell expression during Mycobacterium massiliense infection To try to understand these differences seen between the epidemic strain CRM-0019 and the reference M. massiliense strain CIP 108297, we performed studies in which we re-examined the T cell response. Figure 6 A shows representative dot plots of lung cells from the isotype control, reference CIP 108297 and the outbreak M. massiliense CRM-0019 strain after 30 days of infection in mice primarily gated on CD4+ T cells expression of Foxp3+. The results in Figure 6 B clearly show an increased number of CD4+Foxp3+IL-10+ T cells in the lungs and spleens of mice infected with the epidemic strain compared to the reference strain at both time points. In fact, compared to CIP 108297-infected PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24726