presence of brefeldin A (10 mg/ml) for the last 2 h, then divided into two pools for double staining with IL-4 and IFN-c or TNF and IFN-c for analysis by ICS as described elsewhere [34,51]. Generation of BM-DC Bone marrow (BM) cells were isolated by flushing murine femurs and tibia with culture medium and differentiated into dendritic cells (DC) in the presence of GM-CSF (50 ng/ml; a gift from DNAX Research Institute). Real-time quantitative PCR cDNA was synthesized and analyzed by real-time PCR using specific oligonucleotides as described previously [51,52]. Target gene mRNA expression was quantified using SYBR green (Applied Biosystems) and normalized to the ubiquitin mRNA levels. Cytokine ELISA Blood serum from infected mice and supernatants from in vitro cultures of NFATp2/2 and WT primary cells were harvested at indicated time points and TNF and IFN-c secretion was assessed by ELISA using mouse TNF and mouse IFN-c Kits (R&D Systems). Statistics GraphPad Prism (GraphPad Software, La Jolla California USA, www.graphpad.com) was used to prepare Kaplan Meier survival curves, calculate Log-rank test, and median time to morbidity. A students t-test was used to determine the difference in ELISA values between treatment and genotyped groups. Results NFATp-deficient mice are more susceptible to MTb infection We infected 20 WT and 16 NFATp2/2 mice with the aerosolized clinical MTb strain HN878 and compared the survival of the animals. We note that five random mice were sacrificed after initial infection to demonstrate that the appropriate range (149 out of a range of 20–200) of MTb colony-forming units (CFU) had been delivered. Infected mice were then placed in a time-to- death experiment and were regularly weighed and bled. Any animals reaching the endpoint established for TB infection according to the protocol guidelines were sacrificed and at that time their lung, spleen, and serum were collected for CFU determination, histological examination, and cytokine analysis. Since the NFATp2/2 mice were bred on the relatively TB- resistant BALB/c background [43,53], BALB/c mice were employed as the control strain, and the MTb strain HN878, which has previously been shown to cause accelerated death in murine TB models [49,54], was used to infect the mice. As shown in Figure 1A, NFATp2/2 mice had a dramatically decreased median TB survival rate (119 days) as compared to WT mice (205 days, p = 0.0042). Consistent with this finding, the CFU count in the lungs of NFATp2/2 animals was approximately three orders of magnitude higher (8.66108 CFU/lung) at the symp- tomatic endpoint as compared to the CFU count in lungs of WT mice (86105 CFU/lung) sacrificed at the same time. Furthermore, at this morbid stage, the lungs of NFATp2/2 mice revealed rampant infection with bacilli outside of granulomas and massive cellular infiltration of the airways (Figure 1B). By contrast, lungs from WT animals at the same time post-infection showed lymphocytic infiltration without exuberant bacterial growth or NFATp and TB Infection Figure 1. Increased susceptibility of NFATp2/2 mice to aerosolized M. tuberculosis. A. Survival of BALB/c (WT) vs. NFATp2/2 mice after infection with MTb. 20 WT and 16 NFATp2/2 mice were infected with the aerosolized clinical MTb strain HN878 (,149 CFU) and monitored for survival. B and C. Lung pathology. (B) NFATp2/2 and (C) WT mice were infected with MTb, and lungs taken from premortal NFATp2/2 mice and from the WT mice sacrificed at the same time were fixed in 10% neutral buffered Formalin. Fixed tissues were embedded in paraffin, and 6–10 mm sections were H&E or Ziehl-Neelsen acid-fast stained as indicated. doi:10.1371/journal.pone.0041427.g001 evidence of vital tissue damage (Figure 1C), indicating the inability of the NFATp2/2 mice to maintain control of the infection. Dysregulation of IFN-c and TNF mRNA levels in the lungs of MTb-infected NFATp2/2 mice. Animals deficient in IFN-c and TNF or their receptors are highly susceptible to TB [55,56]; however, the cellular source of the TNF that is important in TB containment has not been identified. Given that transcription of the Th1 cytokine IFN-c and T cell-derived TNF have previously been shown to be NFATp-dependent [34,45,48], we next examined lungs from MTb-infected WT and NFATp-deficient PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41427