Tae-Hyun Paik

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.

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.

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.

Protein kinase C zeta plays an essential role for Mycobacterium tuberculosis-induced extracellular signal-regulated kinase 1/2 activation in monocytes/macrophages via Toll-like receptor 2.

This study characterized the upstream signalling molecules involved in extracellular signal‐regulated kinase (ERK) 1/2 activation and determined their effects on differential tumour necrosis factor (TNF)‐α expression by monocytes/macrophages infected with virulent or avirulent mycobacteria. The avirulent Mycobacterium tuberculosis (MTB) strain H37Ra (MTBRa) induced higher levels of activation of ERK 1/2 and the upstream MAPK kinase (MEK)1 and, subsequently, higher levels of TNF‐α expression in human primary monocytes and monocyte‐derived macrophages, as compared with MTB strain H37Rv (MTBRv). The MTB‐induced activation of ERK 1/2 was not dependent on Ras or Raf. However, inhibition of the activity of atypical protein kinase C (PKC) ζ decreased the in vitro phosphorylation of MEK, ERK 1/2 activation and subsequent TNF‐α induction caused by MTBRv or MTBRa. Toll‐like receptor (TLR) 2 was found to play a major role in MTB‐induced TNF‐α expression and PKCζ phosphorylation. Co‐immunoprecipitation experiments showed that PKCζ interacts physically with TLR2 after MTB stimulation. Moreover, PKCζ phosphorylation was increased more in macrophages following MTBRa, versus MTBRv, infection. This is the first demonstration that PKCζ interacts with TLR2 to play an essential role in MTB‐induced ERK 1/2 activation and subsequent TNF‐α expression in monocytes/macrophages.

Bacillus calmette-guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy.

The cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guerin (BCG/CWS) is an effective anti-tumor immunotherapy agent. Here, we demonstrate that BCG/CWS has a radiosensitizing effect on colon cancer cells through the induction of autophagic cell death. Exposure of HCT116 colon cancer cells to BCG/CWS before ionizing radiation (IR) resulted in increased cell death in a caspase-independent manner. Treatment with BCG/CWS plus IR resulted in the induction of autophagy in colon cancer cells. Either the autophagy inhibitor 3-methyladenine or knockdown of Beclin-1 or Atg7 significantly reduced tumor cell death induced by BCG/CWS plus IR, whereas the caspase inhibitor z-VAD-fmk failed to do so. BCG/CWS plus IR-mediated autophagy and cell death was mediated predominantly by the generation of reactive oxygen species (ROS). The c-Jun NH2 terminal kinase pathway functioned upstream of ROS generation in the induction of autophagy and cell death in HCT116 cells after co-treatment with BCG/CWS and IR. Furthermore, Toll-like receptor (TLR) 2, and in part, TLR4, were responsible for BCG/CWS-induced radiosensitization. In vivo studies revealed that BCG/CWS-mediated radiosensitization of HCT116 xenograft growth is accompanied predominantly by autophagy. Our data suggest that BCG/CWS in combination with IR is a promising therapeutic strategy for enhancing radiation therapy in colon cancer cells through the induction of autophagy.

Profiles of IFN-γ and its regulatory cytokines (IL-12, IL-18 and IL-10) in peripheral blood mononuclear cells from patients with multidrug-resistant tuberculosis

This study investigated the profiles of IFN‐γ and its regulatory cytokines (IL‐12, IL‐18 and IL‐10) in response to a purified protein derivative (PPD) antigen in peripheral blood mononuclear cells (PBMC) from 18 HIV‐negative patients with multidrug‐resistant tuberculosis (MDRTB), and compared them with those from 19 healthy tuberculin reactors (HTR). ELISA results showed that following stimulation with PPD, IFN‐γ production was significantly reduced, whereas production of both IL‐18 and IL‐10 was significantly elevated in MDRTB patients compared with HTR. Three out of 18 patients with MDRTB of greater than 4 years duration showed significantly elevated IL‐12 p70 production, induced by in vitro PPD stimulation of their PBMC, when compared with data from HTR. However, when taken as a group, MDRTB patients were similar to HTR in their IL‐12 p70‐producing capacity. IL‐12 p70 protein paralleled IL‐12 p40 protein expression. In addition, the production of IL‐12 p40 was significantly correlated with IL‐10 in all patients, but was not correlated with IFN‐γ. Neutralization of IL‐10 increased IL‐12 p40 about twofold, but did not significantly alter IFN‐γ induction in MDRTB. IFN‐γ in MDRTB was highly correlated with lymphoproliferation and CD4 counts, but was not correlated with IL‐12, IL‐18 or IL‐10 production. Our findings suggest that patients with MDRTB have dysregulated IL‐12, IL‐18 and IL‐10 production during Mycobacterium tuberculosis infection, and the cytokine profiles are similar to those in patients with drug‐sensitive advanced TB previously reported in the literature. In addition, IL‐10 may not have a dominant role in defective IFN‐γ production in patients with MDRTB.

The production of tumour necrosis factor‐alpha is decreased in peripheral blood mononuclear cells from multidrug‐resistant tuberculosis patients following stimulation with the 30‐kDa antigen of Mycobacterium tuberculosis

The clearance of intracellular bacteria requires the appropriate induction of proinflammatory cytokines and chemokines to recruit macrophages and T cells to the site of infection. In this study, we investigated the production of tumour necrosis factor (TNF)‐α, interleukin (IL)‐8 and interferon (IFN)‐γ by the peripheral blood mononuclear cells (PBMC) of patients with multidrug‐resistant tuberculosis (MDR‐TB) in response to in vitro stimulation with the 30‐kDa antigen of Mycobacterium tuberculosis. The results were compared with those from cases of newly diagnosed TB (N‐TB) and TB with treatment failure (TF‐TB), and healthy tuberculin reactors (HTR). The most significantly depressed TNF‐α levels were found in MDR‐TB patients. IFN‐γ production was depressed significantly in all groups of TB patients compared with the HTR group. TNF‐α secretion in response to the 30‐kDa antigen was unchanged by coculturing with recombinant human interferon (rhIFN)‐γ, and was increased dramatically following IL‐10 neutralization with an anti‐human IL‐10 antibody. The IL‐8 levels were depressed significantly in MDR‐TB patients compared with N‐TB patients, but were similar to the IL‐8 levels in TF‐TB patients. Furthermore, rhTNF‐α directly increased IL‐8 secretion, and neutralizing antibody to TNF‐α inhibited IL‐8 production by the PBMC of MDR‐TB patients that were stimulated with the 30‐kDa antigen. Taken together, these data suggest that the PBMC of MDR‐TB patients typically show TNF‐α depression in response to the 30‐kDa antigen, and this effect is modulated by IL‐10. In addition, we highlight the role of TNF‐α in IL‐8 secretion in MDR‐TB patients.

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.

Roles of Reactive Oxygen Species in CXCL8 and CCL2 Expression in Response to the 30-kDa Antigen of Mycobacterium tuberculosis

BackgroundThe 30-kDa antigen (Ag) of Mycobacterium tuberculosis (M. tuberculosis) is a strong inducer of innate and adaptive immune responses in human tuberculosis. The generation of reactive oxygen species (ROS) plays an important role in inflammatory signaling as well as antimicrobial defense.Materials and MethodsIn this study, we investigated the role of ROS in the activation of mitogen-activated protein kinases (MAPKs) and secretion of the CXC chemokine ligand 8 (CXCL8) and CC chemokine ligand 2 (CCL2) by human monocytes stimulated with the 30-kDa Ag of M. tuberculosis H37Rv.ResultsTreatment of human monocytes with the 30-kDa Ag activated rapid superoxide generation. In addition, the 30-kDa Ag activated mRNA and protein expression of CXCL8 and CCL2 in human primary monocytes through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent ROS generation. Analysis of MAPK activation (extracellular signal-regulated kinase (ERK) 1/2 and p38) showed rapid phosphorylation of both subfamilies in response to the 30-kDa Ag. In addition, 30-kDa-induced MAPK activation was inhibited in a dose-dependent manner by pretreatment with ROS scavengers. Toll-like receptor (TLR) 2 was required for ROS generation, chemokine production, and MAPK activation following stimulation with the 30-kDa Ag. Using highly specific signaling pathway inhibitors, we found that both p38 and ERK1/2 activation are essential for 30-kDa Ag-induced CCL2 but not CXCL8 production in human monocytes.ConclusionThese results indicate that TLR2–ROS signaling plays a crucial role in the 30-kDa Ag-mediated expression of CXCL8 and CCL2 in human monocytes. In addition, both p38 and ERK1/2 activation are essential for 30-kDa Ag-stimulated CCL2 production by monocytes.

Differential regulation of interleukin-12 and tumour necrosis factor-α by phosphatidylinositol 3-kinase and ERK 1/2 pathways during Mycobacterium tuberculosis infection

Interleukin (IL)‐12 and tumour necrosis factor (TNF)‐α 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‐α 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‐α 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‐α or addition of rhTNF‐α, suggesting that the negative regulation of IL‐12 is not mediated by concomitant TNF‐α 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‐α 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.