Increased Virulence in M. massiliense Infection Figure 4. Decreased macrophage and dendritic cell expression of IL-12 and TNFa in the lungs and spleens of IFNc-KO mice infected with the epidemic strain M. massiliense CRM-0019. Mice infected with M. massiliense CIP 108297 (closed square) and the epidemic strain M. massiliense CRM-0019 (open circle) were assayed by flow cytometry on days 30 and 60. Panel 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. Panels B and C show the total number of macrophage (CD11b+) and dendritic (CD11c+) cells expressing IL-12 or TNFa in the lungs (A) and spleens (B) of both types of infected mice. Results are expressed as the mean number of CD11b+IL-12+, CD11b+ TNFa +,CD11c+IL-12+, CD11c+ TNFa + (6 SEM, n = 5) T cells in the lungs and spleens. Student t-test, *p,0.050. doi:10.1371/journal.pone.0024726.g004 mice, those infected with CRM-0019 demonstrated increased numbers of both CD4+Foxp3+ T cells and CD4+Foxp3+ IL10+ T cells in the lungs and spleens during the course of infection. Discussion Owing to its relatively recent recognition as a novel Mycobac- terium species [2], not much is known about the virulence and pathogenicity of M. massiliense. The limited information presently available suggests that the clinical manifestations of the diseases caused by M. massiliense in humans are similar to those caused by M. abscessus [14]. The results of this study clearly show that M. massiliense clinical isolates with different genetic backgrounds can significantly differ in their ability to replicate and persist inside macrophages and induce distinct immune responses contributing to the different virulence phenotypes in mice. The Brazilian epidemic BRA100 strain M. massiliense CRM-0019 clearly replicates more efficiently than the reference strain CIP 108297 in mouse bone marrow macrophages resulting in rapid cell lysis. It is also more virulent for GKO mice than is strain CIP 108297, persisting in the lungs and spleens and inducing more severe lung and spleen pathology. Mice infected with strain CIP 108297 eventually clear the infection showing an absence of residual bacilli and organ pathology 60 days post-infection. Several reasons may account for the enhanced persistence of CRM-0019 in vivo including the increased ability of this isolate to replicate and survive inside macrophages and, potentially, our novel observation here that this particular M. massiliense isolate expands a regulatory T cell response that attempts to dampen the progression of organ pathology associated with bacterial persistence but also suppresses the protective TNFa and IL-12 [TH1] response required for mycobacterial containment and eventual elimination. In this regard, we found evidence that immunodeficient GKO mice administered the epidemic BRA100 strain M. massiliense CRM-0019 through the aerosol route expand a weak TH1 response, compared to the reference strain CIP 108297. Recent studies have shown that immunocompetent C57BL/6 and BALB/ c mice infected with a large intravenous dose of a M. massiliense BRA100 isolate closely related to CRM-0019 developed a robust TH1 response consisting of macrophage, dendritic cell and natural killer cell activation, induced by IL-12 and IL-17 [35]. However, there are important differences between their study and ours which raise new interesting questions. First, they intravenously infected their immunocompetent mice with a very large inoculum PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24726