mice and determined the levels of IFN-c and TNF mRNA. For comparison, we also evaluated mRNA levels of the canonical Th2 cytokine gene IL-4 in NFATp2/2 and wild-type mice, since mice deficient in IL-4, including mice of the BALB/c background, do not display decreased survival following MTb infection [56,57,58]. We harvested lungs from two to three WT and two to three NFATp-deficient mice that were sacrificed between two and ten weeks after TB infection, then purified total RNA, and performed real-time PCR using IFN-c-, TNF-, or IL-4-specific primers. Levels of IFN-c mRNA in the lungs of WT and NFATp2/2 animals showed the most marked difference at 6 weeks post-TB infection, at which point the IFN-c mRNA levels were significantly lower in NFATp2/2 mice, following relatively reduced levels of IFN-c mRNA in NFATp2/2 mice observed at 4 weeks post-TB infection (Figure 2A). Given the necessity of an intact IFN-c signaling pathway to control the early stages of murine TB infection [59], these data are consistent with the enhanced mortality of the NFATp2/2 mice. By contrast, TNF mRNA levels in the lungs of NFATp2/2 mice markedly peaked 4 weeks post-TB infection, becoming significantly higher than the corre- sponding levels in WT mice. By 8 weeks post-infection, however, WT mice TNF mRNA levels were significantly higher than those observed in NFATp2/2 mice (Figure 2B). This is consistent with enhanced TNF levels secondary to a dramatic inflammatory response derived from a non-T cell source. In addition, this dysregulated immune response evident in the lungs of NFATp2/2 mice is also reflected in the expression of IL-4. Levels of IL-4 mRNA in the lungs were very low up to 8 weeks post-TB infection regardless of the presence of NFATp (Figure 2C), consistent with previous studies in WT mice [56,58]. However, NFATp2/2 mice showed a marked increase in IL-4 mRNA in the lungs at 10 weeks post-MTb infection (Figure 2C). This is reminiscent of earlier studies of splenocytes from this NFATp2/2 mouse strain in which, despite an initial defect in early IL-4 transcription, increased production of IL-4 was observed over the course of the immune response, coinciding with enhanced Th2 development [43]. In order to rule out that the aberrant cytokine expression was secondary to perturbed leukocyte recruitment to the lungs in the NFATp2/2 mice, we next analysed the cell types present in the lungs of age-matched uninfected and MTb-infected NFATp2/2 and WT mice over the course of the first 6 weeks post-infection. As shown in Figure 3, the timing and number of recruitment of total leukocytes in the lungs of the NFATp2/2 and WT mice were similar (Figure 3A), with only a modest increase the number of CD11b+ cells (Figure 3B), indicative of the monocyte lineage, recruited to the lungs of NFATp2/2 mice relative to WT mice (1.65610661.56105 vs. 1.25610661.16105 cells, respectively, at 6 weeks post-MTb infection). The timing and number of recruitment of total lymphocytes (Figure 3C), CD3+ cells (Figure 3D), and CD4+ cells (Figure 3E) were also similar in NFATp2/2 and WT mice, with a slight increase in the number of CD8+ cells (Figure 3F) recruited to the lungs of WT mice relative to NFATp2/2 mice (8.06610568.26104 cells vs. 6.3610565.16104, respectively, at 6 weeks post-MTb infection). Thus, the observed dysregulation of IFN-c or TNF mRNA in the lungs of NFATp2/2 mice was not correlated with differential recruitment of different immune cell populations to the lung. Taken together, these results demonstrate that production of IFN- c and TNF are dysregulated in the lungs of NFATp-deficient mice following MTb infection, particularly within the first six weeks. Serum TNF levels are increased in NFATp-deficient and wild-type mice during MTb infection and correlated with disease severity. As shown in Figure 4, significantly higher levels of circulating TNF were detected in the serum of NFATp2/2 mice NFATp and TB Infection Figure 2. Distinct dynamics of cytokine mRNA expression in the lungs of MTb-infected NFATp2/2 and WT mice. NFATp2/2 and WT mice were infected with MTb and total RNA was purified from the lungs at 2, 4, 6, 8 (8.4), and 10 weeks post-infection. (A) IFN-c, (B) TNF, and (C) IL-4 mRNA was measured by real-time PCR and values were normalized to ubiquitin mRNA. Error bars indicate mean of 3 samples 6SD. doi:10.1371/journal.pone.0041427.g002 at 4 weeks after infection, concordant with the increased TNF mRNA levels in NFATp2/2 lungs at this time point, and they were again elevated at 16 weeks post-infection when the animals began to show clinical signs of severe disease. By contrast, at 16 weeks after infection, WT animals still appeared healthy and maintained lower TNF levels (Figure 4). Notably, near their respective median times of death (in NFATp2/2 (13 to 20 weeks) or WT (30–36 weeks)), when CFU in the lungs were equivalent in the two groups of mice, there were no significant differences in TNF levels, which were elevated in both groups (Figure 4). Thus, increased serum levels of TNF were correlated with the severity of disease post-infection in both NFATp2/2 mice and WT mice, and the higher TNF levels in NFATp2/2 mice were consistent with their more rapid disease progression and mortality. PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41427