Jyothi Rengarajan

Transcriptional regulation of Th1/Th2 polarization

Abstract The two polarized T helper (Th) subsets Th1 and Th2 are identified by their signature cytokines, interferon γ (IFN-γ) and interleukin 4 (IL-4) respectively. Understanding the transcriptional regulation of cytokine expression is therefore critical for elucidating the process of Th cell differentiation. Ubiquitous and tissue-specific transcription factors, as well as chromatin remodeling of genomic loci have been implicated in IL-4 and IFN-γ regulation. We propose a model of Th1/Th2 polarization based on the balance between Th1- and Th2-specific transcription factors.

Inhibitory Function of Two NFAT Family Members in Lymphoid Homeostasis and Th2 Development

Nuclear factor of activated T cells (NFAT) is a critical regulator of early gene transcription in response to TCR-mediated signals. Here, we show that mice lacking both NFATp and NFAT4 develop a profound lymphoproliferative disorder likely due to a lowered threshold for TCR signaling coupled with increased resistance to apoptosis secondary to defective FasL expression. NFAT mutant mice also have allergic blepharitis, interstitial pneumonitis, and a 10(3) to 10(4) fold increase in serum IgG1 and IgE levels, secondary to a dramatic and selective increase in Th2 cytokines. This phenotype may be ascribed to unopposed occupancy of the IL-4 promoter by NFATc. Our data demonstrate that lymphoid homeostasis and Th2 activation require a critical balance among NFAT family members.

Interferon Regulatory Factor 4 (IRF4) Interacts with NFATc2 to Modulate Interleukin 4 Gene Expression

Proteins of the nuclear factor of activated T cells (NFAT) family of transcription factors are critical for lymphocyte activation in the immune system. In particular, NFATs are important regulators of inducible IL-4 gene expression. Interferon regulatory factor 4 (IRF4) is an immune system–restricted interferon regulatory factor that is required for lymphocyte activation, but its molecular functions in the T lineage remain to be elucidated. We demonstrate that IRF4 potently synergizes with NFATc2 to specifically enhance NFATc2-driven transcriptional activation of the IL-4 promoter. This function is dependent on the physical interaction of IRF4 with NFATc2. IRF4 synergizes with NFATc2 and the IL-4–inducing transcription factor, c-maf, to augment IL-4 promoter activity as well as to elicit significant levels of endogenous IL-4 production. Furthermore, naïve T helper cells from mice lacking IRF4 are compromised severely for the production of IL-4 and other Th2 cytokines. The identification of IRF4 as a partner for NFATc2 in IL-4 gene regulation provides an important molecular function for IRF4 in T helper cell differentiation.

Sequential involvement of NFAT and Egr transcription factors in FasL regulation.

The critical function of NFAT proteins in maintaining lymphoid homeostasis was revealed in mice lacking both NFATp and NFAT4 (DKO). DKO mice exhibit increased lymphoproliferation, decreased activation-induced cell death, and impaired induction of FasL. The transcription factors Egr2 and Egr3 are potent activators of FasL expression. Here we find that Egr2 and Egr3 are NFAT target genes. Activation of FasL occurs via the NFAT-dependent induction of Egr3, as demonstrated by the ability of exogenously provided NFATp to restore Egr-dependent FasL promoter activity in DKO lymph node cells. Further, Egr3 expression is enriched in Th1 cells, suggesting a molecular basis for the known preferential expression of FasL in the Th1 versus Th2 subset.

NFATc2 and NFATc3 regulate T(H)2 differentiation and modulate TCR-responsiveness of naïve T(H)cells.

The NFAT family of transcription factors are key regulators of inducible gene expression in the immune system. We examined the function of two NFAT proteins after naïve T helper (TH) cell activation. We found that naïve TH precursors that are doubly deficient in NFATc2 and NFATc3 intrinsically differentiate into TH2-secreting cells, even in the absence of interleukin 4 (IL-4) production. We also found that lack of NFATc2 and NFATc3 obviates the necessity for engagement of CD28 on naïve cells and controls the time required to reach the first cell division upon activation. These results demonstrate a key role for NFATc2 and NFATc3 in modulating T cell receptor responsiveness and regulating subsequent cell division and TH2 differentiation.

NF-AT-driven interleukin-4 transcription potentiated by NIP45.

The induction of cytokine gene transcription is mediated in part by the nuclear factor of activated T cells (NF-AT). Factors involved in the mechanisms of NF-AT-mediated transcription are not well understood. A nuclear factor that interacted with the Rel homology domain (RHD) of NF-ATp was identified with the use of a two-hybrid interaction trap. Designated NIP45 (NF-AT interacting protein), it has minimal similarity to any known genes. Transcripts encoding this factor were enriched in lymphoid tissues and testes. NIP45 synergized with NF-ATp and the proto-oncogene c-Maf to activate the interleukin-4 (IL-4) cytokine promoter; transient overexpression of NIP45 with NF-ATp and c-maf in B lymphoma cells induced measurable endogenous IL-4 protein production. The identification of NIP45 advances our understanding of gene activation of cytokines, critical mediators of the immune response.

The folate pathway is a target for resistance to the drug para‐aminosalicylic acid (PAS) in mycobacteria

The increasing rate of multidrug‐resistant tuberculosis has led to more use of second‐line antibiotics such as para‐aminosalicylic acid (PAS). The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined. We have isolated PAS‐resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels. BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway. We also find that mutations in thyA are associated with clinical PAS resistance. We have identified PAS‐resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme. Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.

Distinct Effector Memory CD4+ T Cell Signatures in Latent Mycobacterium tuberculosis Infection, BCG Vaccination and Clinically Resolved Tuberculosis

Two billion people worldwide are estimated to be latently infected with Mycobacterium tuberculosis (Mtb) and are at risk for developing active tuberculosis since Mtb can reactivate to cause TB disease in immune-compromised hosts. Individuals with latent Mtb infection (LTBI) and BCG-vaccinated individuals who are uninfected with Mtb, harbor antigen-specific memory CD4+ T cells. However, the differences between long-lived memory CD4+ T cells induced by latent Mtb infection (LTBI) versus BCG vaccination are unclear. In this study, we characterized the immune phenotype and functionality of antigen-specific memory CD4+ T cells in healthy BCG-vaccinated individuals who were either infected (LTBI) or uninfected (BCG) with Mtb. Individuals were classified into LTBI and BCG groups based on IFN-γ ELISPOT using cell wall antigens and ESAT-6/CFP-10 peptides. We show that LTBI individuals harbored high frequencies of late-stage differentiated (CD45RA−CD27−) antigen-specific effector memory CD4+ T cells that expressed PD-1. In contrast, BCG individuals had primarily early-stage (CD45RA−CD27+) cells with low PD-1 expression. CD27+ and CD27− as well as PD-1+ and PD-1− antigen-specific subsets were polyfunctional, suggesting that loss of CD27 expression and up-regulation of PD-1 did not compromise their capacity to produce IFN-γ, TNF-α and IL-2. PD-1 was preferentially expressed on CD27− antigen-specific CD4+ T cells, indicating that PD-1 is associated with the stage of differentiation. Using statistical models, we determined that CD27 and PD-1 predicted LTBI versus BCG status in healthy individuals and distinguished LTBI individuals from those who had clinically resolved Mtb infection after anti-tuberculosis treatment. This study shows that CD4+ memory responses induced by latent Mtb infection, BCG vaccination and clinically resolved Mtb infection are immunologically distinct. Our data suggest that differentiation into CD27−PD-1+ subsets in LTBI is driven by Mtb antigenic stimulation in vivo and that CD27 and PD-1 have the potential to improve our ability to evaluate true LTBI status.

Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis

Tuberculosis (TB) is a global pandaemic, partially due to the failure of vaccination approaches. Novel anti-TB vaccines are therefore urgently required. Here we show that aerosol immunization of macaques with the Mtb mutant in SigH (MtbΔsigH) results in significant recruitment of inducible bronchus-associated lymphoid tissue (iBALT) as well as CD4+ and CD8+ T cells expressing activation and proliferation markers to the lungs. Further, the findings indicate that pulmonary vaccination with MtbΔsigH elicited strong central memory CD4+ and CD8+ T-cell responses in the lung. Vaccination with MtbΔsigH results in significant protection against a lethal TB challenge, as evidenced by an approximately three log reduction in bacterial burdens, significantly diminished clinical manifestations and granulomatous pathology and characterized by the presence of profound iBALT. This highly protective response is virtually absent in unvaccinated and BCG-vaccinated animals after challenge. These results suggest that future TB vaccine candidates can be developed on the basis of MtbΔsigH.

The Intracellular Environment of Human Macrophages that Produce Nitric Oxide Promotes Growth of Mycobacteria

ABSTRACT Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-γ), l-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-γ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.