Phosphorylation of mitogen-activated protein kinases contributes to interferon γ production in response to Mycobacterium tuberculosis.

Immune control of Mycobacterium tuberculosis depends on interferon γ (IFN-γ)-producing CD4(+) lymphocytes. Previous studies have shown that T cells from patients with tuberculosis produce less IFN-γ, compared with healthy donors, in response to mycobacterial antigens, although IFN-γ responses to mitogens are preserved. In this work, we found that M. tuberculosis-induced IFN-γ production by human T cells correlated with phosphorylation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), and p38. Moreover, the majority of IFN-γ-producing T cells expressed signaling lymphocyte activation molecule (SLAM), and SLAM activation further increased ERK phosphorylation. Interestingly, patients with tuberculosis had delayed activation of ERK and p38, and this was most marked in patients with the poorest IFN-γ responses (ie, low responders). Besides, SLAM signaling failed to phosphorylate ERK in low responders. Our findings suggest that activation of p38 and ERK, in part through SLAM, mediates T-cell IFN-γ production in response to M. tuberculosis, a pathway that is defective in patients with tuberculosis.

Early secreted antigenic target of 6-kDa protein of Mycobacterium tuberculosis primes dendritic cells to stimulate Th17 and inhibit Th1 immune responses.

Early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis is a T cell Ag that is a potential vaccine candidate, but it is also a virulence factor that mediates pathogenicity. To better understand the effects of ESAT-6 on the immune response, we studied the effect of ESAT-6 on human dendritic cells (DCs). Peripheral blood monocytes were treated with GM-CSF and IL-4 to yield immature DCs, which were matured by addition of LPS and CD40 ligand (CD40L), with or without ESAT-6. ESAT-6 inhibited LPS/CD40L-induced DC expression of costimulatory molecules, reduced DC-stimulated allogeneic T cell proliferation and IL-2 and IFN-γ production, and enhanced IL-17 production. ESAT-6-treated DCs also increased IL-17 and reduced IFN-γ production by M. tuberculosis-specific autologous T cells. ESAT-6 inhibited LPS/CD40L-induced DC production of IL-12 and enhanced that of IL-23 and IL-1ß, without affecting secretion of TNF-α, IL-6, or IL-8 through specific interaction with immature DCs. The effects of ESAT-6 were not mediated through cAMP or p38 MAPK. Medium from ESAT-6-conditioned DCs increased IL-17 and reduced IFN-γ production by T cells stimulated with anti-CD3 plus anti-CD28, and ESAT-6-induced IL-17 production was blocked by neutralizing both IL-23 and IL-1ß. ESAT-6 reduced LPS/CD40L-stimulated transcription of IL-12p35 and enhanced that of IL-23p19 through inhibition of IFN regulatory factor-1 and upregulation of activating transcription factor-2 and c-Jun, transcriptional regulators of IL-12p35 and IL-23p19, respectively. We conclude that ESAT-6 increases DC production of IL-23 and IL-1ß while inhibiting that of IL-12, thus enhancing Th17 at the expense of protective Th1 responses.

IL-17 and IFN-γ expression in lymphocytes from patients with active tuberculosis correlates with the severity of the disease.

Th1 lymphocytes are crucial in the immune response against Mycobacterium tuberculosis. Nevertheless, IFN-γ alone is not sufficient in the complete eradication of the bacteria, suggesting that other cytokines might be required for pathogen removal. Th17 cells have been associated with M. tuberculosis infection, but the role of IL-17-producing cells in human TB remains to be understood. Therefore, we investigated the induction and regulation of IFN-γ and IL-17 during the active disease. TB patients were classified as High and Low Responder individuals according to their T cell responses against the antigen, and cytokine expression upon M. tuberculosis stimulation was investigated in peripheral blood and pleural fluid. Afterwards, the potential correlation among the proportions of cytokine-producing cells and clinical parameters was analyzed. In TB patients, M. tuberculosis induced IFN-γ and IL-17, but in comparison with BCG-vaccinated healthy donors, IFN-γ results were reduced significantly, and IL-17 was markedly augmented. Moreover, the main source of IL-17 was represented by CD4(+)IFN-γ(+)IL-17(+) lymphocytes, a Th1/Th17 subset regulated by IFN-γ. Interestingly, the ratio of antigen-expanded CD4(+)IFN-γ(+)IL-17(+) lymphocytes, in peripheral blood and pleural fluid from TB patients, was correlated directly with clinical parameters associated with disease severity. Indeed, the highest proportion of CD4(+)IFN-γ(+)IL-17(+) cells was detected in Low Responder TB patients, individuals displaying severe pulmonary lesions, and longest length of disease evolution. Taken together, the present findings suggest that analysis of the expansion of CD4(+)IFN-γ(+)IL-17(+) T lymphocytes in peripheral blood of TB patients might be used as an indicator of the clinical outcome in active TB.

CD137 differentially regulates innate and adaptive immunity against Mycobacterium tuberculosis.

Protective immunity against Mycobacterium tuberculosis is primarily mediated by the interaction of antigen-specific T cells and antigen presenting cells, which often depends on the interplay of cytokines produced by these cells. Costimulatory signals represent a complex network of receptor-ligand interactions that qualitatively and quantitatively influence immune responses. Thus, here we investigated the function of CD137 and CD137L, molecules known to have a central role in immune regulation, during human tuberculosis (TB). We demonstrated that M. tuberculosis antigen stimulation increased both CD137 and CD137L expression on monocytes and NK cells from TB patients and healthy donors, but only up-regulated CD137 on T lymphocytes. Blockage of the CD137 pathway enhanced the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α produced by monocytes and NK against M. tuberculosis. In contrast, CD137 blockage significantly decreased the specific degranulation of CD8(+) T cells and the percentage of specific IFN-γ and TNF-α producing lymphocytes against the pathogen. Furthermore, inhibition of the CD137 pathway markedly increased T-cell apoptosis. Taken together, our results demonstrate that CD137:CD137L interactions regulate the innate and adaptive immune response of the host against M. tuberculosis.

Role played by the programmed death-1-programmed death ligand pathway during innate immunity against Mycobacterium tuberculosis.

Tuberculous pleurisy allows the study of specific cells at the site of Mycobacterium tuberculosis infection. Among pleural lymphocytes, natural killer (NK) cells are a major source of interferon gamma (IFN-gamma), and their functions are regulated by activating and inhibitory receptors. Programmed death-1 (PD-1), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) are recognized inhibitory receptors in adaptive immunity, but their role during innate immunity remains poorly understood. We investigated the PD-1:PD-L1/PD-L2 pathway on NK cell effector functions in peripheral blood and pleural fluid from patients with tuberculosis. M. tuberculosis stimulation significantly up-regulated PD-1, PD-L1, and PD-L2 levels on NK cells. Interestingly, a direct correlation between PD-1 and IFN-gamma expression on NK cells was observed. Moreover, blockade of the PD-1 pathway markedly augmented lytic degranulation and IFN-gamma production of NK cells against M. tuberculosis. Furthermore, PD-1(+) NK cells displayed a diminished IFN-gamma mean fluorescence intensity, denoting the relevance of PD-1 on IFN-gamma regulation. Together, we described a novel inhibitory role played by PD-1:PD-L interactions in innate immunity in tuberculosis.

IFN-gamma production during active tuberculosis is regulated by mechanisms that involve IL-17, SLAM, and CREB.

Interferon-gamma (IFN-gamma) is crucial for protection against Mycobacterium tuberculosis, and the transcription factor cAMP response element binding protein (CREB) increases IFN-gamma transcription. We determined whether the transmembrane receptor signaling lymphocyte activation molecule (SLAM) and interleukin-17 (IL-17) affect CREB phosphorylation and IFN-gamma production in persons with tuberculosis. When T cells from patients with tuberculosis were activated with M. tuberculosis, 80% of SLAM(+) T cells expressed phosphorylated CREB, and SLAM activation increased CREB phosphorylation and IFN-gamma production. In contrast, IL-17 down-regulated SLAM expression, CREB phosphorylation, and IFN-gamma production. Therefore, IL-17 and SLAM have opposing effects on IFN-gamma production through CREB activation in persons with tuberculosis.

Programmed death (PD)-1:PD-ligand 1/PD-ligand 2 pathway inhibits T cell effector functions during human tuberculosis.

Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, molecules known to modulate T cell activation, in the regulation of IFN-gamma production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-gamma production from these individuals. Moreover, M. tuberculosis-induced IFN-gamma participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-gamma-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-gamma responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.