We examined the distribution of single nucleotide polymorphisms (SNPs) in nitric oxide synthase 2A, monocyte chemoattractant protein–1 (MCP-1), regulated on activation, normal T cell expressed and secreted, and macrophage inflammatory protein–1 α genes in tuberculosis patients and healthy controls from Mexico. The odds of developing tuberculosis were 2.3- and 5.4-fold higher in carriers of MCP-1 genotypes AG and GG than in homozygous AA. Cases of homozygous GG had the highest plasma levels of MCP-1 and the lowest plasma levels of IL-12p40, and these values were negatively correlated. Furthermore, stimulation of monocytes from healthy carriers of the genotype GG with Mycobacterium tuberculosis antigens yielded higher MCP-1 and lower IL-12p40 concentrations than parallel experiments with monocytes from homozygous AA. Addition of anti–MCP-1 increased IL-12p40 levels in cultures of M. tuberculosis–stimulated monocytes from homozygous GG, and addition of exogenous MCP-1 reduced IL-12p40 production by M. tuberculosis–stimulated monocytes from homozygous AA. Furthermore, we could replicate our results in Korean subjects, in whom the odds of developing tuberculosis were 2.8- and 6.9-fold higher in carriers of MCP-1 genotypes AG and GG than in homozygous AA. Our findings suggest that persons bearing the MCP-1 genotype GG produce high concentrations of MCP-1, which inhibits production of IL-12p40 in response to M. tuberculosis and increases the likelihood that M. tuberculosis infection will progress to active pulmonary tuberculosis.

The “enhanced intracellular survival” (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

Although the 38-kDa glycolipoprotein of Mycobacterium tuberculosis H37Rv is known to evoke prominent cellular and humoral immune responses in human tuberculosis (TB), little information is known about intracellular regulatory mechanisms involved in 38-kDa antigen (Ag)-induced host responses. In this study, we found that purified 38-kDa glycolipoprotein activates mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38) and induces tumor necrosis factor alpha (TNF-␣) and interleukin 6 (IL-6) in human monocytes. When the 38-kDa Ag was applied to monocytes from TB patients and healthy controls, the activation of ERK1/2 and p38 MAPK and the subsequent cytokine secretion were greater in the monocytes from the active pulmonary TB patients than in monocytes from the healthy controls. Additionally, neutralizing antibodies for Toll-like receptor 2 (TLR2) or TLR4 significantly reduced the ERK1/2 and p38 activation induced by the 38-kDa protein when the antibodies were applied to HEK293 cells overexpressing TLR2 or TLR4 as well as human primary monocytes. Furthermore, the inhibition of TLR2 significantly, and that of TLR4 partially, decreased the 38-kDa Ag-induced secretion of TNF-␣ and IL-6 in human monocytes. The intact protein moieties of the 38-kDa protein were responsible for biologic activities by this Ag. These data collectively demonstrate that the 38-kDa glycolipoprotein, acting through both TLR2 and TLR4, induces the activation of the ERK1/2 and p38 MAPK pathways, which in turn play an essential role in TNF-␣ and IL-6 expression during mycobacterial infection.

a b s t r a c tMycobacterium tuberculosis (Mtb) infection leads to the induction of the apoptotic response, which is associated with bacilli killing. The early secreted mycobacterial antigen ESAT-6 of Mtb has been shown to induce apoptosis in human macrophages and epithelial cells. In the present study, we demonstrate that the stimulation of human epithelial A549 cells by ESAT-6 induces the endoplasmic reticulum (ER) stress response. We observed that ESAT-6 treatment increases intracellular Ca 2+ concentration, which results in ROS accumulation, and therefore induces the onset of ER stress-induced apoptosis. Our results uncover a novel apoptotic mechanism of ESAT-6 through ER stress responses in pathologic conditions such as tuberculosis.

Alzheimer's disease (AD) occurs when neurons in the memory and cognition regions of the brain are accompanied by an accumulation of the long amyloid beta-proteins of the 39 to 43 amino acids derived from the amyloid precursor protein (APP) by cleavage with beta- and gamma-secretase. An increased production of Abeta-42 by mutation of PS2 genes promotes caspase expression and is associated with the Cox-2 found in the brain of AD patients. To address this question in vivo, we expressed the human mutant PS2 (hPS2m) (N141I) as well as wild PS2 (hPS2w) as a control in transgenic (Tg) mice under control of the neuron-specific enolase (NSE) promoter. Water maze tests were used to demonstrate the behavioral defect; dot blot, Western blot, and immunohistochemical analyses were performed on the brain with the hPS2, Abeta-42, caspase-3, and Cox-2 antibody. We concluded that 1) Tg mice showed a behavioral dysfunction in the water maze test, 2) levels of hPS2, Abeta-42, caspase-3, and Cox-2 expression were modulated in the brains of both Tg mice, 3) dense staining with antibody to hPS2, Abeta-42, caspase-3, and Cox-2 was visible in the brains of Tg mice compared with age-matched control mice, and 4) distinguishable AD phenotypes between hPS2w- and hPS2m-Tg mice did not appear. These results suggest that an elevation of Abeta-42 by overexpression of hPS2 and mutation of hPS2m might induce the behavioral deficit and caspase-3 and Cox-2 induction, which could be useful in the therapeutic testing of compounds to have considerable clinical effects.

BackgroundApoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages.Methodology/Principal FindingsHere, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response.Conclusion/SignificanceThese findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria.

Mammalian 2-Cys peroxiredoxin II (Prx II) is a cellular peroxidase that eliminates endogenous H2O2. The involvement of Prx II in the regulation of lipopolysaccharide (LPS) signaling is poorly understood. In this report, we show that LPS induces substantially enhanced inflammatory events, which include the signaling molecules nuclear factor κB and mitogen-activated protein kinase (MAPK), in Prx II–deficient macrophages. This effect of LPS was mediated by the robust up-regulation of the reactive oxygen species (ROS)–generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the phosphorylation of p47phox. Furthermore, challenge with LPS induced greater sensitivity to LPS-induced lethal shock in Prx II–deficient mice than in wild-type mice. Intravenous injection of Prx II–deficient mice with the adenovirus-encoding Prx II gene significantly rescued mice from LPS-induced lethal shock as compared with the injection of a control virus. The administration of catalase mimicked the reversal effects of Prx II on LPS-induced inflammatory responses in Prx II–deficient cells, which suggests that intracellular H2O2 is attributable, at least in part, to the enhanced sensitivity to LPS. These results indicate that Prx II is an essential negative regulator of LPS-induced inflammatory signaling through modulation of ROS synthesis via NADPH oxidase activities and, therefore, is crucial for the prevention of excessive host responses to microbial products.

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