u-0126 and Tuberculosis

Crucial to the pathogenesis of the tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis is its ability to subvert host immune defenses to promote its intracellular survival. The mammalian cell entry protein 3E (Mce3E), located in the region of difference 15 of the M. tuberculosis genome and absent in Mycobacterium bovis bacillus Calmette-Guérin, has an essential role in facilitating the internalization of mammalian cells by mycobacteria. However, relatively little is known about the role of Mce3E in modulation of host innate immune responses. In this study, we demonstrate that Mce3E inhibits the activation of the ERK1/2 signaling pathway, leading to the suppression of Tnf and Il6 expression, and the promotion of mycobacterial survival within macrophages. Mce3E interacts and colocalizes with ERK1/2 at the endoplasmic reticulum in a DEF motif (an ERK-docking motif)-dependent manner, relocates ERK1/2 from cytoplasm to the endoplasmic reticulum, and finally reduces the association of ERK1/2 with MEK1 and blocks the nuclear translocation of phospho-ERK1/2. A DEF motif mutant form of Mce3E (F294A) loses its ability to suppress Tnf and Il6 expression and to promote intracellular survival of mycobacteria. Inhibition of the ERK1/2 pathway in macrophages using U0126, a specific inhibitor of the ERK pathway, also leads to the suppressed Tnf and Il6 expression and the enhanced intracellular survival of mycobacteria. Taken together, these results suggest that M. tuberculosis Mce3E exploits the ERK1/2 signaling pathway to suppress host innate immune responses, providing a potential Mce3E-ERK1/2 interface-based drug target against M. tuberculosis.

Topics: Active Transport, Cell Nucleus; Animals; Bacterial Proteins; Butadienes; Cell Line; Cell Nucleus; Drug Delivery Systems; Gene Expression Regulation; Humans; Immunity, Innate; Interleukin-6; Macrophages; MAP Kinase Signaling System; Mice; Microbial Viability; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mycobacterium bovis; Mycobacterium tuberculosis; Nitriles; Tuberculosis; Tumor Necrosis Factor-alpha

To investigate the effect of interleukin (IL)-17 on the apoptosis of neutrophils (PMNs) from peripheral blood of patients with pulmonary tuberculosis (TB) and explore the possible involved signaling pathway.. The fresh isolated PMNs from peripheral blood of TB patients and healthy adults were cultured for 0-24 hours, and then stained with annexin V. The flow cytometry was used to measure cell apoptosis of PMNs. The fresh isolated PMNs were cultured with different concentrations of IL-17 for 24 hours, with or without pretreatment of MAPK inhibitor U0126 for 30 minutes, the apoptosis of PMNs was detected by flow cytometry.. The apoptosis of PMNs in both TB patients and healthy adults increased with the culture time going on (P<0.05). The spontaneous apoptotic (annexin V⁺) rates of PMNs at 0, 6, 12 and 24 hours of culture time in TB patients were (4.49 ± 1.39)%, (21.89 ± 2.90)%, (39.96 ± 4.15)%, and (68.35 ± 7 .01)%, respectively, which were significantly higher than the corresponding ones in normal healthy groups, (2.65 ± 0.75)%, (11.00 ± 1.72)%, (25.84 ± 3.90)%, and (45.59 ± 4.10)%, respectively (P<0.05). After cultured with IL-17 for 24 hours, the apoptotic rates of PMNs in TB patients and healthy adults were respectively (59.81 ± 7.19)% and (34.65 ± 4.79)% in the group of IL-17 at 0.5 μg/L, and (51.62 ± 6.91)% and (29.04 ± 3.62)% in the group of IL-17 at 5 μg/L; they were significantly lower than those in control groups without IL-17, (68.35 ± 7.01)% and (45.59 ± 4.10)%, respectively, (P<0.05). However, in the group of IL-17 at 50 μg/L, the apoptosis rates of PMNs in TB patients and healthy adults were (76.04 ± 5.59)% and (53.24 ± 4.62)%, respectively, which were obviously higher than those of the control groups without IL-17 (P<0.05). Pretreatment of PMNs with U0126 mostly inhibited the anti-apoptosis effect of IL-17 at 5 μg/L on PMNs of TB patients and normal subjects.. The apoptosis of PMNs in both TB patients and healthy adults increases along with the culture time, and the apoptosis rate of PMNs in TB patients is higher than that of healthy adults. The lower concentrations of IL-17 can delay, but the higher concentration of Il-17 can accelerate the apoptosis of PMNs in TB patients. The inhibitory role of IL-17 in apoptosis of PMNs may be involved in the ERK pathway.

Topics: Adult; Apoptosis; Butadienes; Cells, Cultured; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Interleukin-17; Male; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Neutrophils; Nitriles; Time Factors; Tuberculosis; Young Adult

Interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha are both thought to be critical factors in the defence against mycobacteria but are known to play different roles. In this study, we investigated the regulatory pathways for IL-12 and TNF-alpha expression in human monocyte-derived macrophages (MDMs) after treatment with Mycobacterium tuberculosis H37Rv or the Triton X-100 solubilized proteins (TSP) purified from M. tuberculosis. We found a rapid phosphorylation of Akt and extracellular signal-regulated kinase (ERK), albeit with differential activation kinetics, in human MDMs treated with M. tuberculosis or TSP. Studies using inhibitors selective for phosphatidylinositol 3-kinase (PI 3-K) and ERK 1/2 show that both pathway plays an essential role in the induction of TNF-alpha at both the transcriptional and translational levels in human MDMs. In contrast, blockade of the PI 3-K/Akt or ERK 1/2 pathways significantly increased M. tuberculosis- or TSP-induced IL-12 p40 and p35 mRNA and bioactive p70 protein. The enhancement of IL-12 levels by inhibition of PI 3-K and ERK 1/2 was not reversed by neutralization of TNF-alpha or addition of rhTNF-alpha, suggesting that the negative regulation of IL-12 is not mediated by concomitant TNF-alpha suppression. Further, PI 3-K activity is required for the M. tuberculosis- or TSP-induced phosphorylation of ERK 1/2 activation. TSP from M. tuberculosis shows a similar dependency on the PI 3-K and ERK 1/2 pathways to those by M. tuberculosis. Collectively, these data suggest that the Th1-driving cytokine IL-12 and proinflammatory cytokine TNF-alpha are differentially regulated by PI 3-K and ERK 1/2 pathways in human MDMs during mycobacterial infection. These results may provide therapeutic targets for precise and specific fine-tuning of cytokine responses.

Topics: Analysis of Variance; Androstadienes; Antigens, Bacterial; Butadienes; Cells, Cultured; Chromones; Enzyme-Linked Immunosorbent Assay; Flavonoids; Humans; Interleukin-12; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Morpholines; Mycobacterium tuberculosis; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Tuberculosis; Tumor Necrosis Factor-alpha; Wortmannin