NFATp and TB Infection harvested at different time points post-induction as indicated. Purified total RNA was analyzed by real-time PCR with TNF and IFN-c-specific primers, and cellular supernatants were used to measure TNF and IFN-c protein levels by ELISA. doi:10.1371/journal.pone.0041427.g005 TNF neutralizing agent, etanercept (Enbrel), which contains the p75 TNF receptor binding domain attached to the human IgG1 hinge and Fc domains and reversibly binds both human and murine trimeric TNF [64]. At 6 and 12 weeks after infection, 16 Figure 6. TNF, IFN-c, and IL-4 production by CD4+ Th1 and Th2 cells from NFATp-deficient and WT mice. Primary CD4+ T cells from NFATp2/2 or WT mice were polarized into Th1 and Th2 populations for six days and subsequently stimulated with anti-CD3 plus anti-CD28 for 4 h before intracellular cytokine staining (ICS) for IL-4 and IFN-c (A) or TNF and IFN-c (B). NFATp-dependent production of IFN-c protein by Th1 cells and IL-4 protein by Th2 cells is evident, while both Th1 and Th2 cells produce TNF protein with a more marked decrease in TNF expression in Th2 cells from NFATp2/2 mice. doi:10.1371/journal.pone.0041427.g006 WT and 16 NFATp2/2 mice were given twice-weekly intraper- itoneal injections of Enbrel (0.4 mg/kg) and their survival was monitored. 16 WT and 16 NFATp2/2 mice served as controls and were injected at the same times with PBS. In separate experiments, both a human IgG1 antibody group and a PBS treated group were used as negative controls for the groups receiving Enbrel injections; irrelevant IgG treatment yielded results similar to PBS treatment (data not shown). In addition, uninfected NFATp2/2 mice were given intraperitoneal injections of Enbrel, PBS, or the IgG and no reduction in survival was noted. At 6 weeks post-infection, prior to treatment with Enbrel, CFU in the lung and spleen of NFATp2/2 and WT mice were approximately equivalent (Figure 7A). As shown in Figure 7B, anti-TNF treatment administered early (beginning at 6 weeks post-infection) further significantly decreased NFATp2/2 survival by 20 to 28 days (median survival for NFATp2/2+PBS mice was 145 vs. 117 for NFATp2/2+Enbrel mice; p = 0.003). This result is consistent with blockage of TNF production leading to the increased mortality originating from monocytic cells such as DC that produce TNF in an NFATp- independent manner. Blocking TNF with Enbrel administration decreased the median survival of WT mice (200 vs. 220 days; p = 0.08), but not to the same extent as it did in the NFATp2/2 deficient mice. The limited effect of Enbrel treatment may be due to clearance of both Enbrel and the irrelevant antibody by the mouse immune system. Even when TNF was blocked in the WT mice, they still had significantly greater survival than the NFATp2/2 mice. These results are consistent with other factors such as IFN-c playing a significant part in the enhanced mortality observed in NFATp2/2 mice (Figure 7B). Strikingly, when anti-TNF treatment was administered late (beginning at 12 weeks post-infection), it had no significant effect on disease progression in either WT (median survival 225 days without vs. 232 days with Enbrel) or NFATp2/2 (median survival 130.5 days without vs. 132 days with Enbrel) mice (Figure 7C) indicating that TNF’s role in host defense is most important early in infection. This result is consistent with the high TNF levels observed in the mice at the time of disease onset, which is reflective of the severity of disease. Thus, NFATp is essential for clearance of MTb even in the presence of high levels of TNF. Furthermore, the effect of NFATp is broader than its impact upon T cell-derived TNF production, consistent with our observation of the significant reduction of IFN-c levels in the lungs and T cells of the NFATp2/2 mice. Discussion Here, we have established a role for one of the NFAT family of transcription factors, NFATp, in protection against susceptibility to TB via its key role in regulating IFN-c and T cell-derived TNF. The NFAT proteins regulate a wide variety of genes involved in the immune response, organ and tissue development, and T cell fate [46,65,66,67,68]. Studies employing gene disruption in mice have demonstrated that NFATp is specifically required for the expression of T cell-derived TNF [34,43,48] and IFN-c [45]. IFN- c is a central cytokine in the host Th1 response to MTb infection in humans as well as mice [6,10,28,55], and patients with genetic disruptions in IFN-c or the a chain of its receptor, IFN-cR, display increased susceptibility to mycobacterial infection [5]. Several studies in mice have shown that TNF is essential to control infection [37,41,56,69,70,71] and in humans the treatment of PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e41427