Jeong-Kyu Park

The Mycobacterial 38-Kilodalton Glycolipoprotein Antigen Activates the Mitogen-Activated Protein Kinase Pathway and Release of Proinflammatory Cytokines through Toll-Like Receptors 2 and 4 in Human Monocytes

ABSTRACT 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-α) 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-α 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-α and IL-6 expression during mycobacterial infection.

Role of mitogen-activated protein kinase pathways in the production of tumor necrosis factor-alpha, interleukin-10, and monocyte chemotactic protein-1 by Mycobacterium tuberculosis H37Rv-infected human monocytes.

The role of mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-8, and monocyte chemotactic protein-1 (MCP-1) was investigated in human monocytes that were infected with Mycobacterium tuberculosis H37Rv. Analysis of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase showed rapid phosphorylation of both subfamilies in response to M. tuberculosis H37Rv. Using highly specific inhibitors of p38 (SB203580) and of MAPK kinase-1 (U0126 and PD98059), we found that both p38 and ERK were essential for M. tuberculosis H37Rv-induced TNF-α production, whereas activation of the p38 pathway, but not that of ERK, was essential for M. tuberculosis H37Rv-induced IL-10 production. Interestingly, the ERK pathway, but not that of p38, was critical for MCP-1 secretion from human monocytes that were infected with M. tuberculosis H37Rv. However, IL-8 secretion was not regulated by ERK1/2 or p38 MAPK. Collectively, these results suggest that induction of the MAPK pathway is required for the expression of TNF-α, IL-10, and MCP-1 by human monocytes during M. tuberculosis H37Rv infection.

Intracellular network of phosphatidylinositol 3-kinase, mammalian target of the rapamycin/70 kDa ribosomal S6 kinase 1, and mitogen-activated protein kinases pathways for regulating mycobacteria-induced IL-23 expression in human macrophages.

We previously demonstrated that Mycobacterium tuberculosis (M. tbc)‐induced interleukin (IL)‐12 expression is negatively regulated by the phosphatidylinositol 3‐kinase (PI3K) and extracellular signal‐regulated kinase (ERK) 1/2 pathways in human monocyte‐derived macrophages (MDMs). To extend these studies, we examined the nature of the involvement of toll‐like receptors (TLRs) and intracellular signalling pathways downstream from PI3K in M. tbc‐induced IL‐23 expression in human MDMs. M. tbc‐induced Akt activation and IL‐23 expression were essentially dependent on TLR2. Blockade of the mammalian targets of rapamycin (mTOR)/70 kDa ribosomal S6 kinase 1 (S6K1) pathway by the specific inhibitor rapamycin greatly enhanced M. tbc‐induced IL‐12/IL‐23 p40 (p40) and IL‐23 p19 (p19) mRNA and IL‐23 protein expression. In sharp contrast, p38 mitogen‐activated protein kinase (MAPK) inhibition abrogated the p40 and p19 mRNA and IL‐23 protein expression induced by M. tbc. Furthermore, the inhibition of PI3K‐Akt, but not ERK 1/2 pathway, attenuated M. tbc‐induced S6K1 phosphorylation, whereas PI3K inhibition enhanced p38 phosphorylation and apoptosis signal‐regulating kinase 1 activity during exposure to M. tbc. Although the negative or positive regulation of IL‐23 was not reversed by neutralization of IL‐10, it was significantly modulated by blocking TLR2. Collectively, these findings provide new insight into the homeostatic mechanism controlling type 1 immune responses during mycobacterial infection involving the intracellular network of PI3K, S6K1, ERK 1/2 and p38 MAPK pathways in a TLR2‐dependent manner.

Endoplasmic reticulum stress response is involved in Mycobacterium tuberculosis protein ESAT‐6‐mediated apoptosis

Mycobacterium 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 Ca2+ 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.

Depressed Interleukin-12 (IL-12), but not IL-18, Production in Response to a 30- or 32-Kilodalton Mycobacterial Antigen in Patients with Active Pulmonary Tuberculosis

ABSTRACT The secreted 30-kDa antigen (Ag) of Mycobacterium tuberculosis directly stimulates Th1-type protective cytokine responses in healthy tuberculin reactors but not in patients with active tuberculosis (TB). To examine the cytokine profiles attributable to Th1 suppression associated with active TB, interleukin-12 (IL-12), IL-18, and IL-10 production in response to a 30- or 32-kDa Ag in 16 patients with active pulmonary TB and 24 healthy controls was investigated by enzyme-linked immunosorbent assay. In TB patients, production of IL-12 p40, as well as gamma interferon (IFN-γ), by 30- or 32-kDa Ag-stimulated peripheral blood mononuclear cells (PBMC) was significantly decreased compared with that in healthy tuberculin reactors. There were no significant differences in IL-18 production between patients and controls early during stimulation (16 h). However, PBMC from patients showed significantly enhanced IL-18 proteins after 96 h of stimulation. Similarly, higher IL-10 production was observed in the TB patients than in healthy tuberculin reactors. After 2 months of anti-TB therapy, the mean IFN-γ and IL-12 p40 production and the mean blastogenic responses were significantly increased in PBMC in the 10 TB patients who were followed up. Our findings provide evidence that depressed IL-12 in response to the 30- or 32-kDa Ag is involved in the immunopathogenesis of human active pulmonary TB.

Polymorphisms of interleukin-10 and tumour necrosis factor-alpha genes are associated with newly diagnosed and recurrent pulmonary tuberculosis.

Background and objectives:  The genetic determinants for developing TB or having recurrent TB are unknown. The present study investigated the relationship between susceptibility to tuberculosis and human tumour necrosis factor‐α (TNF‐α) and interleukin‐10 genes (IL‐10).

Endoplasmic Reticulum Stress Pathway-Mediated Apoptosis in Macrophages Contributes to the Survival of Mycobacterium tuberculosis

Background Apoptosis 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 Findings Here, 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/Significance These 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.

Mycobacterium tuberculosis Rv0652 stimulates production of tumour necrosis factor and monocytes chemoattractant protein‐1 in macrophages through the Toll‐like receptor 4 pathway

Mycobacterial proteins interact with host macrophages and modulate their functions and cytokine gene expression profile. The protein Rv0652 is abundant in culture filtrates of Mycobacterium tuberculosis K‐strain, which belongs to the Beijing family, compared with levels in the H37Rv and CDC1551 strains. Rv0652 induces strong antibody responses in patients with active tuberculosis. We investigated pro‐inflammatory cytokine production induced by Rv0652 in murine macrophages and the roles of signalling pathways. In RAW264.7 cells and bone marrow‐derived macrophages, recombinant Rv0652 induced predominantly tumour necrosis factor (TNF) and monocyte chemoattractant protein (MCP)‐1 production, which was dependent on mitogen‐activated protein kinases and nuclear factor‐κB. Specific signalling pathway inhibitors revealed that the extracellular signal‐regulated kinase 1/2 (ERK1/2), p38 and phosphatidylinositol 3‐kinase (PI3K) pathways were essential for Rv0652‐induced TNF production, whereas the ERK1/2 and PI3K pathways, but not the p38 pathway, were critical for MCP‐1 production in macrophages. Rv0652‐stimulated TNF and MCP‐1 secretion by macrophages occurred in a Toll‐like receptor 4‐dependent and MyD88‐dependent manner. In addition, Rv0652 significantly up‐regulated the expression of the mannose receptor, CD80, CD86 and MHC class II molecules. These results suggest that Rv0652 can induce a protective immunity against M. tuberculosis through the macrophage activation.

Mycobacterium tuberculosis HBHA Protein Reacts Strongly with the Serum Immunoglobulin M of Tuberculosis Patients

ABSTRACT Identification and characterization of serologically active mycobacterial antigens are prerequisites for the development of diagnostic reagents. We examined the humoral immune responses of active tuberculosis (TB) patients against Triton-soluble proteins extracted from Mycobacterium tuberculosis by immunoblotting. A 29-kDa protein reacted with immunoglobulin M (IgM) in the pooled sera of the patients, and its N-terminal amino acid sequence matched that of the heparin-binding hemagglutinin (HBHA). Recombinant full-length HBHA was expressed in Escherichia coli (rEC-HBHA) and M. smegmatis (rMS-HBHA). In immunoblot analysis, the IgM antibodies of the TB patients reacted strongly with rMS-HBHA but not with rEC-HBHA, whereas the IgG antibodies of these patients reacted weakly with both recombinant HBHA proteins. In enzyme-linked immunosorbent assay analysis using rMS-HBHA and 85B as antigens, the mean levels and sensitivities of the anti-HBHA IgM antibodies of the TB patients were significantly higher than those of the anti-antigen 85B IgM antibodies, while the IgG antibodies showed the opposite results. Of interest in this respect, the pooled sera from the TB patients that contained anti-HBHA IgM antibodies neutralized the entry of M. tuberculosis into epithelial cells. These findings suggest that IgM antibody to HBHA may play a role in protection against extrapulmonary dissemination.

Targeting of Mycobacterium tuberculosis Heparin-Binding Hemagglutinin to Mitochondria in Macrophages

Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA), a virulence factor involved in extrapulmonary dissemination and a strong diagnostic antigen against tuberculosis, is both surface-associated and secreted. The role of HBHA in macrophages during M. tuberculosis infection, however, is less well known. Here, we show that recombinant HBHA produced by Mycobacterium smegmatis effectively induces apoptosis in murine macrophages. DNA fragmentation, nuclear condensation, caspase activation, and poly (ADP-ribose) polymerase cleavage were observed in apoptotic macrophages treated with HBHA. Enhanced reactive oxygen species (ROS) production and Bax activation were essential for HBHA-induced apoptosis, as evidenced by a restoration of the viability of macrophages pretreated with N-acetylcysteine, a potent ROS scavenger, or transfected with Bax siRNA. HBHA is targeted to the mitochondrial compartment of HBHA-treated and M. tuberculosis-infected macrophages. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in both macrophages and isolated mitochondria treated with HBHA. Disruption of HBHA gene led to the restoration of ΔΨm impairment in infected macrophages, resulting in reduced apoptosis. Taken together, our data suggest that HBHA may act as a strong pathogenic factor to cause apoptosis of professional phagocytes infected with M. tuberculosis.