The Transcription Factor NFATp Plays a Key Role in Susceptibility to TB in Mice

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-γ (IFN-γ). Here, we show that NFATp-deficient (NFATp^(−/−)) 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-γ transcription and protein expression in naïve 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 NFATp^(−/−) mice is associated with dysregulated production of TNF and IFN-γ 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 NFATp^(−/−) 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-γ in T cells

NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, and activates TNF transcription during hypertonic stress alone

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays an important role in a variety of infectious and autoimmune disorders. Its transcription is regulated in a stimulus- and cell-type-specific manner via the recruitment of distinct DNA/activator complexes forming secondary structures or enhanceosomes. NFATp, a member of the nuclear factor of activated T cells (NFAT) family of transcription factors, plays a critical role in TNF gene regulation under a variety of conditions. In this study, we show that NFAT5, the most recently described NFAT family member, binds to the TNF promoter in a manner distinct from other NFAT proteins and is a key mediator in the activation of TNF gene transcription during hypertonic stress alone

NFAT5 Regulates HIV-1 in Primary Monocytes via a Highly Conserved Long Terminal Repeat Site

To replicate, HIV-1 capitalizes on endogenous cellular activation pathways resulting in recruitment of key host transcription factors to its viral enhancer. RNA interference has been a powerful tool for blocking key checkpoints in HIV-1 entry into cells. Here we apply RNA interference to HIV-1 transcription in primary macrophages, a major reservoir of the virus, and specifically target the transcription factor NFAT5 (nuclear factor of activated T cells 5), which is the most evolutionarily divergent NFAT protein. By molecularly cloning and sequencing isolates from multiple viral subtypes, and performing DNase I footprinting, electrophoretic mobility shift, and promoter mutagenesis transfection assays, we demonstrate that NFAT5 functionally interacts with a specific enhancer binding site conserved in HIV-1, HIV-2, and multiple simian immunodeficiency viruses. Using small interfering RNA to ablate expression of endogenous NFAT5 protein, we show that the replication of three major HIV-1 viral subtypes (B, C, and E) is dependent upon NFAT5 in human primary differentiated macrophages. Our results define a novel host factor–viral enhancer interaction that reveals a new regulatory role for NFAT5 and defines a functional DNA motif conserved across HIV-1 subtypes and representative simian immunodeficiency viruses. Inhibition of the NFAT5–LTR interaction may thus present a novel therapeutic target to suppress HIV-1 replication and progression of AIDS

Primate TNF Promoters Reveal Markers of Phylogeny and Evolution of Innate Immunity

Background. Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry. Methodology/Principal findings. Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages. Conclusions/significance. Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF

Inducer-Specific Enhanceosome Formation Controls Tumor Necrosis Factor Alpha Gene Expression in T Lymphocytes

We present evidence that the inducer-specific regulation of the human tumor necrosis factor alpha (TNF-α) gene in T cells involves the assembly of distinct higher-order transcription enhancer complexes (enhanceosomes), which is dependent upon inducer-specific helical phasing relationships between transcription factor binding sites. While ATF-2, c-Jun, and the coactivator proteins CBP/p300 play a central role in TNF-α gene activation stimulated by virus infection or intracellular calcium flux, different sets of activators including NFATp, Sp1, and Ets/Elk are recruited to a shared set of transcription factor binding sites depending upon the particular stimulus. Thus, these studies demonstrate that the inducer-specific assembly of unique enhanceosomes is a general mechanism by which a single gene is controlled in response to different extracellular stimuli

Varying NFATp requirement for cytokine expression in different primary cell populations.

<p><b>A and B.</b> TNF expression in BM-derived dendritic cells is not NFATp-dependent. Primary BM-DC were derived from WT and NFATp^−/− mice. After 7 days cells were stimulated with MTb lysates. At indicated time points the cells were lysed to prepare total RNA. (<b>A</b>) TNF mRNA was measured by real-time PCR. (<b>B</b>) TNF protein levels in the supernatants were measured by ELISA. <b>C and D.</b> TNF and IFN-γ expression in murine primary T cells is NFATp-dependent. Purified primary total CD4⁺ and naïve (CD4⁺CD62L⁺CD45RB^high) T cells from uninfected WT and NFATp^−/− mice were stimulated immediately with anti-CD3/CD28 antibody and then left in culture in neutral conditions for another six days and re-stimulated with anti-CD3/CD28 antibodies. Aliquots of cells were harvested at different time points post-induction as indicated. Purified total RNA was analyzed by real-time PCR with TNF and IFN-γ-specific primers, and cellular supernatants were used to measure TNF and IFN-γ protein levels by ELISA.</p

Increased susceptibility of NFATp^−/− mice to aerosolized <i>M. tuberculosis</i>.

<p><b>A.</b> Survival of BALB/c (WT) vs. NFATp^−/− mice after infection with MTb. 20 WT and 16 NFATp^−/− mice were infected with the aerosolized clinical MTb strain HN878 (∼149 CFU) and monitored for survival. <b>B</b> and <b>C.</b> Lung pathology. (<b>B</b>) NFATp^−/− and (<b>C</b>) WT mice were infected with MTb, and lungs taken from premortal NFATp^−/− mice and from the WT mice sacrificed at the same time were fixed in 10% neutral buffered Formalin. Fixed tissues were embedded in paraffin, and 6–10 µm sections were H&E or Ziehl-Neelsen acid-fast stained as indicated.</p

TNF, IFN-γ, and IL-4 production by CD4⁺ Th1 and Th2 cells from NFATp-deficient and WT mice.

<p>Primary CD4⁺ T cells from NFATp^−/− 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-γ (<b>A</b>) or TNF and IFN-γ (<b>B</b>). NFATp-dependent production of IFN-γ 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 NFATp^−/− mice.</p

Infiltration of leukocytes into the lungs after MTb infection is similar in NFATp^−/− and wild-type mice.

<p>Age-matched NFATp^−/− and BALB/c (WT) mice were infected with aerosolized HN878 MTb strain at ∼30 CFU/mouse as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041427#pone.0041427-Barczak1" target="_blank">[50]</a>. Lung cell samples from MTb-infected WT (•) and NFATp^−/− (▪) mice were obtained at 2, 4, and 6 weeks post-infection, and from uninfected WT (○) and NFATp^−/− (□) mice at times corresponding to 1, 2, 4, and 6 weeks post-infection, and the number of cells per lung for each indicated cell population (mean of 3 to 4 samples ±SD) was determined by FACS for samples at the indicated time points (<b>A–F</b>). The number of cells recruited to the lung during the course of MTb infection was determined for total leukocytes (<b>A</b>), CD11b⁺ cells, i.e., monocytes (<b>B</b>), total lymphocytes (<b>C</b>), CD3⁺ T cells (<b>D</b>), CD4⁺ T cells (<b>E</b>) and CD8⁺ T cells (<b>F</b>).</p