Figure 7. Time-dependent protective role of TNF during MTb infection. A. Comparable CFU counts in lungs and spleens from WT and NFATp2/2 mice infected with aerosolized MTb. B and C. NFATp2/2 mice and their BALB/c control mice (WT) were infected with aerosolized HN878 MTb strain at ,100 CFU/mouse as described [50]. At 6 (B) and 12 (C) weeks post-infection, 16 WT and 16 NFATp2/2 were given twice- weekly intraperitoneal 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. doi:10.1371/journal.pone.0041427.g007 autoimmune disease with a TNF blocking antibody results in the activation of latent TB infection [7,38]. The cellular source of the TNF, however, was not previously known. Extensive work performed in in vivo mouse models have demonstrated that anti-MTb immunity in mice is mediated predominantly by CD4+ Th1 cells with the aid of CD8+ T cells and involves IFN-c signaling [1,3,56,72,73]. Most of these studies are based on the fact that mice that do not produce IFN-c fail to inhibit MTb growth and rapidly succumb to the infection. Mechanistically, several studies have demonstrated that the impairment of the Th1 cytokine response and IFN-c production leads to faulty anti-bacterial function in macrophages at sites of MTb infection [74,75]. An elegant study using mice deficient in NFATp and TB Infection transcription factor T-bet, the master regulatory factor directing commitment to the Th1 lineage [24,25], demonstrated that their increased susceptibility to TB was associated with decreased transcription of the T-bet-dependent IFN-c gene [26]. The dramatically increased susceptibility to MTb infection that we observe in NFATp2/2 mice is consistent with their greatly impaired expression of IFN-c in the lungs and CD4+ T cells, from Th1 cells in particular. Thus, our data from NFATp2/2 mice further link the defect of IFN-c gene expression with increased susceptibility to MTb. The regulation of IFN-c gene expression by NFATp is part of a larger network of transcriptional regulation that directs differen- tiation of na¨ıve T helper cells into Th1 and Th2 cells [46]. At early stages of T cell activation, NFATp binds to the promoters of both the IFN-c and IL-4 genes, but this binding is later restricted to the IFN-c promoter in Th1 cells and the IL-4 promoter in Th2 cells. Changes in NFAT binding are accompanied by alterations in chromatin organization as assayed by DNase hypersensitivity and histone acetylation at the IFN-c and IL-4 loci [76,77,78]. The TNF locus also exhibits DNase hypersensitive sites and activation- dependent histone acetylation in T cells [27,34,79,80,81] as well as activation-dependent intrachromosomal interactions that involve NFATp-dependent promoter and enhancer regions [34,82]. In the experiments presented here, we have shown that na¨ıve or total CD4+ T cells from NFATp2/2 mice are impaired in their ability to produce IFN-c and TNF mRNA and protein in response to MTb infection. We have further delineated the expression of these cytokines in CD4+ T helper cell subsets from NFATp2/2 mice: Th1-specific expression of IFN-c is impaired and the expression of TNF in Th1 and Th2 cells is decreased, to a greater extent in Th2 cells. Unlike IFN-c, which is secreted by Th1 cells, NK cells, and NKT cells, TNF is expressed in a wide range of cell types, including T cells, B cells, DCs, macrophages, mast cells, and fibroblasts, and its cell type- and stimulus-specific regulation utilizes distinct signal transduction pathways, transcription factors, and coactivators [27]. We have previously shown that NFATp is a key activator in TNF gene regulation in T cells stimulated by calcium flux or via their antigen receptor, but not in monocytes and macrophages stimulated by LPS or MTb [29,31,32,33,34,83]. In an in vivo toxic shock model, NFATp deficiency renders mice resistant to superantigen-induced T cell-mediated toxic shock without affecting their susceptibility to LPS-induced macrophage- mediated shock [48]. Here we have shown that in NFATp2/2 mice, while TNF mRNA and protein production is decreased in activated CD4+ T cells, BM-DC stimulated by MTb are not significantly affected in their ability to produce TNF mRNA and protein. This is consistent with earlier reports that transcriptional activation of the TNF gene promoter is NFAT-independent in a murine DC line [84], as is the case in a murine macrophage cell line [33], in contrast to NFAT-dependent TNF expression in T cells [29,31,34,83,85,86], B cells [29,87,88,89], and mast cells [84,90]. The results presented here therefore provide further in vivo demonstration of the cell type-specific regulation of the TNF gene. In the context of previous data, these results reinforce the idea that NFAT-independent TNF expression in cells such as DCs and cells of the monocyte/macrophage lineage underlie the early peak of TNF expression we observe in the lungs and serum of NFATp2/2 mice. Although T cell-specific expression of both TNF and IFN-c was significantly impaired in the NFATp2/2 mice used in our study, the pattern of dysregulation of TNF expression in the lungs of NFATp2/2 mice was distinct from that of IFN-c. While IFN-c mRNA levels in the lungs of MTb-infected NFATp2/2 mice were PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e41427