A-Rum Shin

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.

Improved sensitivity of diagnosis of tuberculosis in patients in Korea via a cocktail enzyme-linked immunosorbent assay containing the abundantly expressed antigens of the K strain of Mycobacterium tuberculosis.

ABSTRACT Tuberculosis (TB) is the leading cause of death from a single infectious agent in Korea. In this study, we compared the proteins present in culture filtrates from Mycobacterium tuberculosis strain K, which is the dominant clinical isolate in Korea, with those present in culture filtrates from M. tuberculosis H37Rv. Several differences in expression were detected between the two strains for those proteins with a molecular mass of <20 kDa. ESAT-6, HSP-X, and CFP-10 were found to be abundantly expressed in the strain K culture filtrates by liquid chromatography-electrospray ionization-time of flight mass spectrometry. The serodiagnostic potentials of recombinant antigens rESAT-6, rHSP-X, and rCFP-10 and two native antigens (Ag85 and PstS1) were evaluated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera collected from 46 TB patients with active disease and 46 healthy controls. As for our ELISA results, HSP-X was superior to the other antigens in terms of sensitivity when a single antigen was employed. The results of a receiver operator characteristic analysis revealed that a cocktail ELISA using all five antigens was significantly more sensitive (77.8%) than the use of a single antigen and offered equivalent specificity; moreover, it produced the largest area under the curve (0.91 versus 0.55 to 0.87). Therefore, a cocktail ELISA containing abundantly expressed antigens enhances the sensitivity of a single antigen and can be a useful diagnostic tool for the detection of active TB.

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.

Rv0315, a novel immunostimulatory antigen of Mycobacterium tuberculosis , activates dendritic cells and drives Th1 immune responses

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the most deadly infectious diseases, with approximately two million people dying of TB annually. An effective therapeutic method for activating dendritic cells (DCs) and driving Th1 immune responses would improve host defenses and further the development of a TB vaccine. Given the importance of DC maturation in eliciting protective immunity against TB, we investigated whether Rv0315, a newly identified Mtb antigen, can prompt DC maturation. We found that Rv0315 functionally activated DCs by augmenting the expression of the co-stimulatory molecules CD80 and CD86 as well as MHC class I/II molecules. Moreover, it increased DC secretion of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. Unlike LPS, however, Rv0315 induced the secretion of IL-12p70, but not IL-10. In addition, Rv0315-treated DCs accelerated the proliferation of CD4+ and CD8+ splenic T cells from Mtb-infected mice, with increased levels of IFN-γ, in syngeneic and allogeneic mixed lymphocyte reactions, indicating that Rv0315 contributes to Th1 polarization of the immune response. Importantly, both mitogen-activated protein kinases and nuclear factor κB signaling mediated the expression of DC surface markers and cytokines. Taken together, our results indicate that Rv0315 is a novel DC maturation-inducing antigen that drives T cell immune responses toward Th1 polarization, suggesting that Rv0315 plays a key role in determining the nature of the immune response to TB.

In vitro and ex vivo activity of new derivatives of acetohydroxyacid synthase inhibitors against Mycobacterium tuberculosis and non-tuberculous mycobacteria

Sulfometuron methyl (SM) is an inhibitor of acetohydroxyacid synthase (AHAS), the first common enzyme in the branched-chain amino acid biosynthetic pathway, and shows activity against Mycobacterium tuberculosis both in vitro and in vivo. To develop AHAS inhibitor derivatives with more potent activity, 100 sulfonylurea analogues were screened for antimycobacterial activity against M. tuberculosis and non-tuberculous mycobacteria (NTM), and then evaluated for intracellular activity using mouse macrophages. Three new compounds with antimycobacterial activity comparable with that of SM were identified. These compounds exhibit significant activity against intracellular M. tuberculosis (including the drug-resistant M. tuberculosis strains), and NTM Mycobacterium abscessus and Mycobacterium kansasii, respectively.

Identification of seroreactive proteins in the culture filtrate antigen of Mycobacterium avium ssp. paratuberculosis human isolates to sera from Crohn's disease patients.

The etiology of Crohns disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA (P<0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.

Diagnosis of pulmonary tuberculosis using MTB12 and 38-kDa antigens.

Background and objective:  Mycobacterium tuberculosis MTB12 protein plays an essential role in pro‐inflammatory responses during the early stages of human pulmonary tuberculosis (TB), even though the T‐cell immunoreactivity of MTB12 is weaker than that of the 30‐kDa antigen (Ag). The objective of this study was to evaluate the humoral immune responses induced by MTB12 Ag during human TB.

Role of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in the secretion of tumor necrosis factor-alpha and interleukin-10 by the PPD antigen of Mycobacterium tuberculosis.

Here we investigated the role of the phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-10 in human primary monocytes after stimulation with the PPD antigen of Mycobacterium tuberculosis. MAPK [extracellular signal-regulated kinase (ERK) 1/2 and p38] and Akt are rapidly phosphorylated in human monocytes stimulated with PPD. We found that the PI 3-K-Akt pathway stimulated by PPD is essential for both IL-10 and TNF-α production, although the inhibition of IL-10 production was more pronounced. The analysis of cytokine production using specific inhibitors of the MAPK pathway revealed that both p38 and ERK activation are essential for PPD-induced TNF-α production, whereas p38, but not ERK, activation is essential for IL-10 secretion. The inhibition of PI 3-K did not significantly activate p38 MAPK or ERK 1/2 in PPD-stimulated human monocytes. Further, the Src inhibitor PP2 inhibited the release of TNF-α but enhanced IL-10 release, suggesting the differential regulation of Src kinase in upstream signaling. Collectively, these data suggest that the PI 3-K and MAPK pathways play a central role in the regulation of both pro- and anti-inflammatory cytokines by the PPD antigen of M. tuberculosis.

Mycobacterial Heparin-binding Hemagglutinin Antigen Activates Inflammatory Responses through PI3-K/Akt, NF-κB, and MAPK Pathways

Background Mycobacterium tuberculosis (Mtb) heparin binding hemagglutinin (HBHA) is an Ag known to evoke effective host immune responses during tuberculosis infection. However, the molecular basis of the host immune response to HBHA has not been fully characterized. In this study, we examined the molecular mechanisms by which HBHA can induce the expression of proinflammatory cytokines in macrophages. Methods HBHA-induced mRNA and protein levels of proinflammatory cytokines were determined in bone marrow-derived macrophages (BMDMs) using RT-PCR and ELISA analysis. The roles of intracellular signaling pathways for NF-κB, PI3-K/Akt, and MAPKs were investigated in macrophage proinflammatory responses after stimulation with HBHA. Results HBHA robustly activated the expression of mRNA and protein of both TNF-α and IL-6, and induced phosphorylation of NF-κB, Akt, and MAPKs in BMDMs. Both TNF-α and IL-6 production by HBHA was regulated by the NF-κB, PI3-K, and MAPK pathways. Furthermore, PI3-K activity was required for the HBHA-induced activation of ERK1/2 and p38 MAPK, but not JNK, pathways. Conclusion These data suggest that mycobacterial HBHA significantly induces proinflammatory responses through crosstalk between the PI3-K and MAPK pathways in macrophages.

Lithium inhibits growth of intracellular Mycobacterium kansasii through enhancement of macrophage apoptosis

Mycobacterium kansasii (Mk) is an emerging pathogen that causes a pulmonary disease similar to tuberculosis. Macrophage apoptosis contributes to innate host defense against mycobacterial infection. Recent studies have suggested that lithium significantly enhances the cytotoxic activity of death stimuli in many cell types. We examined the effect of lithium on the viability of host cells and intracellular Mk in infected macrophages. Lithium treatment resulted in a substantial reduction in the viability of intracellular Mk in macrophages. Macrophage cell death was significantly enhanced after adding lithium to Mk-infected cells but not after adding to uninfected macrophages. Lithium-enhanced cell death was due to an apoptotic response, as evidenced by augmented DNA fragmentation and caspase activation. Reactive oxygen species were essential for lithium-induced apoptosis. Intracellular scavenging by N-acetylcysteine abrogated the lithium-mediated decrease in intracellular Mk growth as well as apoptosis. These data suggest that lithium is associated with control of intracellular Mk growth through modulation of the apoptotic response in infected macrophages.