Han-Gyu Choi

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.

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.

Mycobacterium tuberculosis RpfB drives Th1-type T cell immunity via a TLR4-dependent activation of dendritic cells

The failure of Mycobacterium bovis BCG as a TB vaccine against TB reactivation suggests that latency‐associated proteins should be included in alternative TB vaccine development. Further, antigens known to generate protective immunity against the strong Th1 stimulatory response to reactivated TB should be included in novel vaccine design. Recent studies have emphasized the importance of Rpfs from Mycobacterium tuberculosis in the reactivation process and cellular immunity. However, little is known about how RpfB mediates protective immunity against M. tuberculosis. Here, we investigated the functional roles and signaling mechanisms of RpfB in DCs and its implications in the development of T cell immunity. DCs treated with RpfB displayed features of mature and functional status, with elevated expression of cell surface molecules (CD80, CD86, and MHC class I and II) and proinflammatory cytokine production (TNF‐α, IL‐1β, IL‐6, and IL‐12p70). Activation of DCs was mediated by direct binding of RpfB to TLR4, followed by MyD88/TRIF‐dependent signaling to MAPKs and NF‐κB signaling pathways. Specifically, we found that the RpfB G5 domain is the most important part in RpfB binding to TLR4. RpfB‐treated DCs effectively polarized naïve CD4+ and CD8+ T cells to secrete IFN‐γ and IL‐2. Importantly, RpfB induced the expansion of memory CD4+/CD8+CD44highCD62Llow T cells in the spleen of M. tuberculosis‐infected mice. Our data suggest that RpfB regulates innate immunity and activates adaptive immunity through TLR4, a finding that may help in the design of more effective vaccines.

Mycobacterium tuberculosis RpfE promotes simultaneous Th1- and Th17-type T-cell immunity via TLR4-dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4+ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4+ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4−/− mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.

Mycobacterium paratuberculosis CobT Activates Dendritic Cells via Engagement of Toll-like Receptor 4 Resulting in Th1 Cell Expansion

Background: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne and Crohn diseases. Results: CobT contributes to the development of T cell immunity through the activation of dendritic cells (DC). Conclusion: CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion. Significance: CobT can be an important candidate for the development of vaccine and a IFN-γ-based diagnostic tool. Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease in animals and MAP involvement in human Crohn disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, and induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-κB. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-γ and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate that MAP CobT contributes to T helper (Th) 1 polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4+/CD8+CD44highCD62Llow memory T cells in the mesenteric lymph node of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP.

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.

Mycobacterium avium MAV2054 protein induces macrophage apoptosis by targeting mitochondria and reduces intracellular bacterial growth

Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.

Mycobacterium tuberculosis Rv2882c Protein Induces Activation of Macrophages through TLR4 and Exhibits Vaccine Potential

Macrophages constitute the first line of defense against Mycobacterium tuberculosis and are critical in linking innate and adaptive immunity. Therefore, the identification and characterization of mycobacterial proteins that modulate macrophage function are essential for understanding tuberculosis pathogenesis. In this study, we identified the novel macrophage-activating protein, Rv2882c, from M. tuberculosis culture filtrate proteins. Recombinant Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and express co-stimulatory and major histocompatibility complex molecules via Toll-like receptor 4, myeloid differentiation primary response protein 88, and Toll/IL-1 receptor-domain-containing adaptor inducing IFN-beta. Mitogen-activated protein kinases and NF-κB signaling pathways were involved in Rv2882c-induced macrophage activation. Further, Rv2882c-treated macrophages induced expansion of the effector/memory T cell population and Th1 immune responses. In addition, boosting Bacillus Calmette-Guerin vaccination with Rv2882c improved protective efficacy against M. tuberculosis in our model system. These results suggest that Rv2882c is an antigen that could be used for tuberculosis vaccine development.

Mycobacterium tuberculosis Protein Rv3841 Activates Dendritic Cells and Contributes to a T Helper 1 Immune Response

The attenuated vaccine Mycobacterium bovis BCG (Bacille Calmette Guerin) has limited protective efficacy against TB. The development of more effective TB vaccines has focused on the mycobacterial antigens that cause strong T helper 1 (Th1) responses. Mtb protein Rv3841 (bacterioferritin B; BfrB) is known to play a crucial role in the growth of Mtb. Nonetheless, it is unclear whether Rv3841 can induce protective immunity against Mtb. Here, we studied the action of Rv3841 in maturation of dendritic cells (DCs) and its engagement in the development of T-cell immunity. We found that Rv3841 functionally activated DCs by upregulating costimulatory molecules and increased secretion of proinflammatory cytokines. Activation of DCs by Rv3841 was mediated by Toll-like receptor 4 (TLR4), followed by triggering of mitogen-activated protein kinase and nuclear factor-κB signaling pathways. In addition, Rv3841-matured DCs effectively proliferated and polarized Th1 immune response of naïve CD4+ and CD8+ T-cells. Moreover, Rv3841 specifically caused the expansion of CD4+CD44highCD62Llow T-cells from Mtb-infected mice; besides, the T-cells activated by Rv3841-matured DCs inhibited intracellular mycobacterial growth. Our data suggest that Rv3841 induces DC maturation and protective immune responses, a finding that may provide candidate of effective TB vaccines.

Mycobacterium tuberculosis protein Rv2220 induces maturation and activation of dendritic cells

Tuberculosis remains a serious health problem worldwide. Characterization of the dendritic cell (DC)-activating mycobacterial proteins has driven the development of effective TB vaccine candidates besides improving the understanding of immune responses. Some studies have emphasized the essential role of protein Rv2220 from M. tuberculosis in mycobacterial growth. Nonetheless, little is known about cellular immune responses to Rv2220. In this study, our aim was to test whether protein Rv2220 induces maturation and activation of DCs. Rv2220-activated DCs appeared to be in a mature state with elevated expression of relevant surface molecules and proinflammatory cytokines. DC maturation caused by Rv2220 was mediated by MAPK and NF-κB signaling pathways. Specifically, Rv2220-matured DCs induced the expansion of memory CD62LlowCD44highCD4+ T cells in the spleen of mycobacteria-infected mice. Our results suggest that Rv2220 regulates host immune responses through maturation of DCs, a finding that points to a new vaccine candidate against tuberculosis.