decreased relative to WT mice up to 6 weeks post-TB infection, expression of TNF mRNA in the lungs in NFATp2/2 mice was transiently elevated during the acute phase of TB infection, reaching a peak 4 weeks post-infection and decreasing 6 weeks post-infection, while in WT mice the peak occurred approximately 8 weeks post-infection. Serum TNF levels were also higher at 4 weeks post-TB infection in NFATp2/2 mice relative to WT mice, and for both WT and NFATp2/2 mice serum TNF levels rose at later time points, starting at 16 weeks post-infection, in the premortal stage of TB. While there is thus a correlation between increased mortality of MTb-infected NFATp-deficient mice and increased levels of NFATp-independent TNF in the early stages of infection, for both NFATp2/2 and WT mice treatment with the TNF blocking agent Enbrel starting at 6 weeks post-infection significantly decreased survival, while injection at 12 weeks post- infection, when NFATp2/2 and WT mice had comparable and relatively low serum levels of TNF, did not affect survival, indicating that the contribution of TNF against susceptibility to TB occurs in the early, acute phase of MTb infection. Furthermore, the increase in mortality upon blocking TNF in the early phase of TB infection was more pronounced for NFATp2/2 mice, consistent with a protective effect of NFATp- dependent genes, particularly IFN-c, in the host response to infection. The T cell-specific defect in production of both IFN-c and TNF in NFATp2/2 mice, and the lack of a robust Th1 immune response to MTb infection, underlie the increased susceptibility of NFATp2/2 mice to TB infection. Consistent with this idea, bacterial proliferation in the lungs of NFATp2/2 mice was markedly higher than in WT mice, and this increased bacterial burden, consistent with their relatively sicker state, could be expected to activate the monocytic compartment to produce high References 1. Kaufmann SH (2001) How can immunology contribute to the control of tuberculosis? Nat Rev Immunol 1: 20–30. 2. Russell DG (2001) Mycobacterium tuberculosis: here today, and here tomorrow. Nat Rev Mol Cell Biol 2: 569–577. 3. North RJ, Jung YJ (2004) Immunity to tuberculosis. Annu Rev Immunol 22: 599–623. 4. Chan J, Flynn J (2004) The immunological aspects of latency in tuberculosis. Clin Immunol 110: 2–12. 5. Ottenhoff THM, Verreck FAW, Hoeve MA, van de Vosse E (2005) Control of human host immunity to mycobacteria. Tuberculosis (Edinb) 85: 53–64. 6. Korbel DS, Schneider BE, Schaible UE (2008) Innate immunity in tuberculosis: myths and truth. Microbes Infect 10: 995–1004. 7. Harris J, Keane J (2010) How tumour necrosis factor blockers interfere with tuberculosis immunity. Clinical and experimental immunology 161: 1–9. 8. Orme I (2004) Adaptive immunity to mycobacteria. Curr Opin Microbiol 7: 58– 61. 9. Rivas-Santiago B, Vieyra-Reyes P, Araujo Z (2005) [Cell immunity response in human pulmonary tuberculosis. Review]. Invest Clin 46: 391–412. 10. Harding CV, Boom WH (2010) Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors. Nat Rev Microbiol 8: 296–307. 11. Broderick A (1996) Tuberculosis and HIV - deadly co-infection. AIDSlink: 1, 3. 12. Goldfeld AE, Ranjbar S, Tsitsikov EN (2008) Tuberculosis/human immuno- deficiency virus coinfection and the host immune response. In: Kaufmann SHE, Britton WJ, editors. Handbook of Tuberculosis: Immunology and Cell Biology. Weinheim: Wiley-VCH. 432 p. 13. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC (1999) Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA 282: 677–686. 14. Dye C, Watt CJ, Bleed DM, Hosseini SM, Raviglione MC (2005) Evolution of tuberculosis control and prospects for reducing tuberculosis incidence, prevalence, and deaths globally. JAMA 293: 2767–2775. 15. World Health Organization (2011) Global tuberculosis control: WHO report 2011. Geneva: World Health Organization. 16. Goldfeld AE, Corbett EL (2009) TB/AIDS coinfection: an integrated clinical and research response. In: Kaufmann SHE, Walker BD, editors. AIDS and Tuberculosis: a Deadly Liaison. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA. pp. 209–252. NFATp and TB Infection levels of TNF mRNA in lungs and protein in serum. This would likely include alveolar macrophages, which serve as the first line of defense from MTb pulmonary invasion [2]. While the cellular source and associated signal transduction pathways underlying the increase in TNF in the lungs of NFATp2/2 mice in the early phase of MTb infection remain to be elucidated, we have demonstrated that NFATp2/2 mice display a defect in IFN-c expression in Th1 cells as well as in the lungs, while TNF expression is reduced in Th1 and, to a relatively greater extent, Th2 cells, but is not affected in DCs. More broadly, this study has pinpointed a specific transcription factor involved in susceptibility to TB infection and has highlighted the important role of T cell-derived TNF and IFN- c. The results presented here thus provide a framework for dissecting the cell type-specific role of NFATp and its upstream signal transduction pathways in the regulation of inflammatory response genes in general, and TNF in particular, involved in MTb infection. Acknowledgments We thank Margarida Saraiva and Anne O’Garra for providing key protocols and reagents used in this study, Laurie Glimcher for the gift of the NFATp-deficient mice, Jacqueline Gonzales (TRS, NIH) for excellent technical assistance, and Renate Hellmiss for assistance with preparation of the figures. Author Contributions Conceived and designed the experiments: LEV AVT RR AEG. Performed the experiments: LEV AVT RR. Analyzed the data: LEV AVT RR JVF AEG. Wrote the paper: LEV AVT JVF AEG. 17. Jiao X, Lo-Man R, Guermonprez P, Fiette L, Deriaud E, et al. (2002) Dendritic cells are host cells for mycobacteria in vivo that trigger innate and acquired immunity. J Immunol 168: 1294–1301. 18. Serbina NV, Lazarevic V, Flynn JL (2001) CD4+ T cells are required for the development of cytotoxic CD8+ T cells during Mycobacterium tuberculosis infection. J Immunol 167: 6991–7000. 19. Cooper AM, Khader SA (2008) The role of cytokines in the initiation, expansion, and control of cellular immunity to tuberculosis. Immunol Rev 226: 191–204. 20. Do¨ffinger R, Altare F, Casanova JL (2000) Genetic heterogeneity of Mendelian susceptibility to mycobacterial infection. Microbes Infect 2: 1553–1557. 21. Remus N, Reichenbach J, Picard C, Rietschel C, Wood P, et al. (2001) Impaired interferon c-mediated immunity and susceptibility to mycobacterial infection in childhood. Pediatr Res 50: 8–13. 22. Picard C, Fieschi C, Altare F, Al-Jumaah S, Al-Hajjar S, et al. (2002) Inherited interleukin-12 deficiency: IL12B genotype and clinical phenotype of 13 patients from six kindreds. Am J Hum Genet 70: 336–348. 23. Rosenzweig S, Dorman SE, Roesler J, Palacios J, Zelazko M, et al. (2002) 561del4 defines a novel small deletion hotspot in the interferon-c receptor 1 chain. Clin Immunol 102: 25–27. 24. Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, et al. (2000) A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655–669. 25. Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR, Sleckman BP, et al. (2002) Distinct effects of T-bet in Th1 lineage commitment and IFN-c production in CD4 and CD8 T cells. Science 295: 338–342. 26. Sullivan BM, Jobe O, Lazarevic V, Vasquez K, Bronson R, et al. (2005) Increased susceptibility of mice lacking T-bet to infection with Mycobacterium tuberculosis correlates with increased IL-10 and decreased IFN-c production. J Immunol 175: 4593–4602. 27. Falvo JV, Tsytsykova AV, Goldfeld AE (2010) Transcriptional control of the TNF gene. Curr Dir Autoimmun 11: 27–60. 28. Falvo JV, Ranjbar S, Jasenosky LD, Goldfeld AE (2011) Arc of a vicious circle: pathways activated by Mycobacterium tuberculosis that target the HIV-1 LTR. Am J Respir Cell Mol Biol. 29. Falvo JV, Uglialoro AM, Brinkman BM, Merika M, Parekh BS, et al. (2000) Stimulus-specific assembly of enhancer complexes on the tumor necrosis factor alpha gene promoter. Mol Cell Biol 20: 2239–2247. 30. Falvo JV, Brinkman BMN, Tsytsykova AV, Tsai EY, Yao T-P, et al. (2000) A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor- PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e41427