The Transcription Factor NFATp Plays a Key Role in Susceptibility to TB in Mice Laura E. Via1., Alla V. Tsytsykova2., Ricardo Rajsbaum2.¤, James V. Falvo2, Anne E. Goldfeld2* 1 Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Program in Cellular and Molecular Medicine, Children’s Hospital Boston and Immune Disease Institute, Harvard Medical School, Boston, Massachusetts, United States of America Abstract In T cells, the transcription factor nuclear factor of activated T cells p (NFATp) is a key regulator of the cytokine genes tumor necrosis factor (TNF) and interferon-c (IFN-c). Here, we show that NFATp-deficient (NFATp2/2) mice have a dramatic and highly significant increase in mortality after Mycobacterium tuberculosis (MTb) infection as compared to mortality of control animals after MTb infection. Animals deficient in NFATp have significantly impaired levels of TNF and IFN-c transcription and protein expression in na¨ıve or total CD4+ T cells, but display wild-type levels of TNF mRNA or protein from MTb-stimulated dendritic cells (DC). The rapid mortality and disease severity observed in MTb-infected NFATp2/2 mice is associated with dysregulated production of TNF and IFN-c in the lungs, as well as with increased levels of TNF, in their serum. Furthermore, global blocking of TNF production by injection of a TNF neutralizaing agent at 6 weeks, but not 12 weeks, post-MTb- infection further decreased the survival rate of both wild-type and NFATp2/2 mice, indicating an early role for TNF derived from cells from the monocyte lineage in containment of infection. These results thus demonstrate that NFATp plays a critical role in immune containment of TB disease in vivo, through the NFATp-dependent expression of TNF and IFN-c in T cells. Citation: Via LE, Tsytsykova AV, Rajsbaum R, Falvo JV, Goldfeld AE (2012) The Transcription Factor NFATp Plays a Key Role in Susceptibility to TB in Mice. PLoS ONE 7(7): e41427. doi:10.1371/journal.pone.0041427 Editor: Jean Kanellopoulos, University Paris Sud, France Received July 28, 2011; Accepted June 27, 2012; Published July 23, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by grants from the NIH to A.E.G. (HL-059838 and GM076685). In addition, the work was partly supported by the Intramural Research Program of the NIH, NIAID and the Comparative Medicine Branch of NIAID is thankfully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no completing interests exist. * E-mail: goldfeld@idi.harvard.edu . These authors contributed equally to this work. ¤ Current address: Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America Introduction Mycobacterium tuberculosis (MTb) is an intracellular bacterium that causes tuberculosis (TB) infection in humans. Since a healthy immune system can usually effectively keep the bacilli in check, ,90% of infected humans do not develop active disease in their lifetime unless they are immunosuppressed by drugs or disease [1,2,3,4,5,6,7]. A strong multicellular immune response is required to maintain TB in its latent state after infection [1,4,6,8,9,10]. In the case of a weakened immune system, latent TB reactivates into active disease with a significant increase in frequency. HIV infection is a significant risk factor for the development of active TB [11,12]. By compromising the immune system, HIV greatly facilitates the activation of latent TB. TB remains the world’s deadliest infectious killer next to HIV, claiming approximately 1.5 million human lives annually, and remains the largest cause of death in HIV co-infected individuals [6,13,14,15,16]. Resistance to TB involves macrophages [2,4], dendritic cells (DC) [17,18], and T cells [18], and multiple studies in humans and mice have shown that maintenance of TB latency requires a Th1 T cell immune response and IFN-c, which is a signature cytokine of this response. Also required are the participation of alveolar macrophages and T lymphocytes, and the production of cytokines such as IL-2, IL-12, IL-18, and TNF, as well as chemokines such as RANTES, MCP-1, MIP-1a, and IL-8, which play an important role in the migration of different cell populations to the site of granuloma formation [6,9,10,19]. Humans with genetic deficien- cies in IFN-c as well as IL-12, which is produced by the monocyte/macrophage lineage, and/or their receptors are par- ticularly susceptible to uncontrollable infection by MTb and by otherwise non-pathogenic strains of atypical mycobacteria and by Mycobacterium bovis bacillus Calmette-Guerin (BCG) [5,20,21,22,23]. Consistent with these observations, mice deficient in the transcription factor T-bet, which is required for IFN-c production and establishing a general program of Th1 gene expression [24,25], are particularly susceptible to MTb infection, which is correlated with relatively decreased IFN-c production as compared to wild type mice [26]. TNF is produced by a number of cell types, including T cells, B cells, macrophages, and DCs [27,28]. Regulation of TNF gene transcription is inducer- and cell type-specific and invovles the formation of higher-order enhancer complexes, or enhanceo- somes, at the TNF promoter [27,29,30,31,32,33], as well as distal regulatory sequences [27,34,35]. TNF plays a critical protective role in the innate and adaptive response to tuberculosis in humans and mice [36,37]. For example, active TB was observed to develop at a high frequency in individuals latently infected with TB who were treated with a broadly neutralizing antibody against TNF for Crohn’s disease or rheumatoid arthritis [38]. In mice, TNF has PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41427