Increased Virulence in M. massiliense Infection Figure 5. Decreased early CD4+ and CD8+ T cell expression of TNFa in the lungs and spleens of IFNc-KO mice infected with the epidemic strain M. massiliense CRM-0019. Mice infected with an aerosol dose of M. massiliense CIP 108297 (closed square) and 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 CD4+ T cells expression of TNFa. Panels B and C show decreased CD4+ and CD8+ T cell expression of TNFa in the lungs (A) and spleens (B) of mice infected with the epidemic strain CRM- 0019 compared to CIP 108297 (6 SEM, n = 5). Results are expressed as the mean number of CD4+TNFa+, CD8+TNFa+, (6 SEM, n = 5) T cells in the lungs and spleens. Student t-test, *p,0.050. doi:10.1371/journal.pone.0024726.g005 of M. massiliense CRM-0019 (16106 organisms per mice, with concentration level reaching 107 CFU in the liver, spleen and lungs [35]. Thus, although their murine model of intravenous infection revealed important immunological findings, it may not reflect the typical bacterial numbers nor immune responses elicited by immunodeficient humans during a nosocomial, wound or aerosol infection [7,21]. Prior studies have shown that increased doses of NTM can induce increasingly robust immune responses [20]. Second, an intravenous infection which kinetically spreads to all organs rapidly causing rapid bacterial dissemination does not reflect bacilli deposited in soft tissue during a surgical procedure nor a pulmonary infection [36]. Our model was developed to use a dose of bacteria and an immunodeficient animal model which best reflects those individuals which generally become infected with such opportunistic pathogens as those from the M. abscessus-M. massiliense group. The apparent lack of TH1 immunity during aerosol infection with the outbreak strain M. massiliense CRM-0019 was due to reduced numbers of CD11b+ cells producing TNF-a and IL-12 cells at the site of infection. In addition, CD11c+ dendritic cells were also expressing less TNF-a and IL-12. Importantly, in our comparative evaluation of T cell responses in M. massiliense CRM- 0019 and CIP 108297-infected mice, we observed reduced numbers of CD4+ T cells secreting TNFa in the lungs and spleens of CRM-0019-infected mice at 30 days, while at the same time increased numbers of CD4+Foxp3+IL-10+ CD4 cells was observed. This is the first observation that non-tuberculous rapidly-growing mycobacteria are capable of inducing the PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24726