In vitro and in vivo anti-pigmentation effects of 2-mercaptobenzimidazoles as nanomolar tyrosinase inhibitors on mammalian cells and zebrafish embryos: Preparation of pigment-free zebrafish embryos.

Recently, 10 2-mercaptobenzo[d]imidazole (2-MBI) compounds (1-10) were synthesized. Although all 2-MBI compounds are tyrosinase inhibitors that inhibit mushroom tyrosinase at extremely low concentrations (IC50 values: 20-740 nM) and effectively inhibit the browning of apples, to our knowledge, no studies have determined whether 2-MBI compounds inhibit mammalian tyrosinase. Mammalian tyrosinase is different from mushroom tyrosinase in its distribution within the cell and has structural characteristics that are different from mushroom tyrosinase in amino acid sequence and in the presence of a quaternary structure. Thus, the effect of the 10 2-MBI compounds on mammalian tyrosinase activity was investigated in B16F10 cells. Six compounds (1-6) exhibited stronger intracellular tyrosinase inhibition than that of kojic acid and phenylthiourea (PTU), which are known to be the most potent tyrosinase inhibitors; their strong tyrosinase inhibitory activity robustly inhibited intracellular melanin production in B16F10 cells. None of the tested 2-MBI compounds exhibited appreciable cytotoxicity in HaCaT and B16F10 cells. To confirm the anti-melanogenic efficacy of the 2-MBI compounds in vivo, a zebrafish embryo model was used. At concentrations 100 times lower than kojic acid, most 2-MBI compounds demonstrated much stronger depigmentation efficacy than that of kojic acid, and three 2-MBI compounds (2-4) showed depigmentation activity similar to or more potent than that of PTU, resulting in nearly pigment-free zebrafish embryos. These results suggest that 2-MBI compounds may be potential therapeutic agents for hyperpigmentation-related disorders.

Anti-Browning Effect of 2-Mercaptobenzo[d]imidazole Analogs with Antioxidant Activity on Freshly-Cut Apple Slices and Their Highly Potent Tyrosinase Inhibitory Activity.

Ten 2-mercaptobenzimidazole (2-MBI) analogs were synthesized as potential tyrosinase inhibitors because mercapto-containing compounds can bind to copper ions at the active site of tyrosinase to inhibit enzyme activity. Nine 2-MBI analogs showed sub-micromolar IC50 values for mushroom tyrosinase monophenolase activity; analog 4 was 280-fold more potent than kojic acid, and in diphenolase activity, 6 was 970-fold more potent than kojic acid. The inhibition mode of the 2-MBI analogs was investigated using kinetic studies supported by docking simulations. Benzimidazoles without the 2-mercapto substituent of the 2-MBI analogs lost their tyrosinase inhibitory activity, implying that the 2-mercapto substituent plays an important role in tyrosinase inhibition. The 2-MBI analogs exerted potent antioxidant effects against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and reactive oxygen species (ROS). The results obtained from apple slices and human embryonic kidney cells (HEK-293) suggest that most 2-MBI analogs are sufficiently safe candidates to delay the browning of apple slices effectively. Thus, these results support the potential use of 2-MBI analogs as anti-browning agents in foods such as mushrooms, vegetables, and fruits.

Identification and molecular mechanism of novel 5-alkenyl-2-benzylaminothiazol-4(5H)-one analogs as anti-melanogenic and antioxidant agents.

Mushroom tyrosinase is a tetramer, whereas mammalian tyrosinase is a monomeric glycoprotein. In addition, the amino acid sequence of mushroom tyrosinases differs from that of mammalian tyrosinases. MHY2081 exhibits potent inhibitory activity against both mushroom and mammalian tyrosinases. Accordingly, based on the MHY2081 structure, 5-alkenyl-2-benzylaminothiazol-4(5H)-one analogs were designed as a novel anti-tyrosinase agent and synthesized using 2-((3,4-dimethoxybenzyl)amino)thiazol-4(5H)-one (16), a key intermediate obtained via the rearrangement of a benzylamino group. Compounds 6 and 9 (IC50 = 1.5-4.6 µM) exhibited higher mushroom tyrosinase inhibitory activity than kojic acid (IC50 = 20-21 µM) in the presence of l-tyrosine and/or l-dopa. Based on kinetic analysis using Lineweaver-Burk plots, 6 was a mixed inhibitor, whereas 9 was a competitive inhibitor, and docking simulation results supported that these compounds could bind to the active site of mushroom tyrosinase. Using B16F10 mammalian cells, we demonstrated that these compounds inhibited melanogenesis more potently than kojic acid, and their anti-melanogenic effects could be attributed to tyrosinase inhibition. All synthesized compounds could scavenge reactive oxygen species (ROS), with five compounds exhibiting mild-to-strong ABTS+ and DPPH radical-scavenging abilities. Compounds 6 and 9 were potent tyrosinase inhibitors with strong antioxidant activities against ROS, ABTS+, and DPPH radicals. Moreover, the compounds significantly suppressed tyrosinase expression in a dose-dependent manner. Collectively, these results suggest that the novel 5-alkenyl-2-benzylaminothiazol-4(5H)-one analogs, especially 6 and 9, are potential anti-melanogenic agents with antioxidant activity.

A Dendritic Cell-Activating Rv1876 Protein Elicits Mycobacterium Bovis BCG-Prime Effect via Th1-Immune Response.

The widely administered tuberculosis (TB) vaccine, Bacillus Calmette-Guerin (BCG), is the only licensed vaccine, but has highly variable efficiency against childhood and pulmonary TB. Therefore, the BCG prime-boost strategy is a rational solution for the development of new TB vaccines. Studies have shown that Mycobacterium tuberculosis (Mtb) culture filtrates contain proteins that have promising vaccine potential. In this study, Rv1876 bacterioferritin was identified from the culture filtrate fraction with strong immunoreactivity. Its immunobiological potential has not been reported previously. We found that recombinant Rv1876 protein induced dendritic cells' (DCs) maturation by MAPK and NF-κB signaling activation, induced a T helper type 1 cell-immune response, and expanded the population of the effector/memory T cell. Boosting BCG with Rv1876 protein enhanced the BCG-primed Th1 immune response and reduced the bacterial load in the lung compared to those of BCG alone. Thus, Rv1876 is a good target for the prime-boost strategy.

Mycobacterium tuberculosis RpfE-Induced Prostaglandin E2 in Dendritic Cells Induces Th1/Th17 Cell Differentiation.

Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Currently, there are no reports on the mycobacterial components that regulate PGE2 production. Previously, we have reported that RpfE-treated dendritic cells (DCs) effectively expanded the Th1 and Th17 cell responses simultaneously; however, the mechanism underlying Th1 and Th17 cell differentiation is unclear. Here, we show that PGE2 produced by RpfE-activated DCs via the MAPK and cyclooxygenase 2 signaling pathways induces Th1 and Th17 cell responses mainly via the EP4 receptor. Furthermore, mice administered intranasally with PGE2 displayed RpfE-induced antigen-specific Th1 and Th17 responses with a significant reduction in bacterial load in the lungs. Furthermore, the addition of optimal PGE2 amount to IL-2-IL-6-IL-23p19-IL-1ß was essential for promoting differentiation into Th1/Th17 cells with strong bactericidal activity. These results suggest that RpfE-matured DCs produce PGE2 that induces Th1 and Th17 cell differentiation with potent anti-mycobacterial activity.

Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling.

Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.

Recombinant Rv1654 protein of Mycobacterium tuberculosis induces mitochondria-mediated apoptosis in macrophage.

Mycobacterium tuberculosis contains diverse immunologically active components. This study investigated the biological function of a newly identified component, Rv1654, with the potential to induce apoptosis in macrophages. Recombinant Rv1654 induced macrophage apoptosis in a caspase-9/3-dependent manner through the production of reactive oxygen species (ROS) and interaction with Toll-like receptor 4. In addition, Rv1654 induced the production of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 through the mitogen-activated protein kinase pathway. Furthermore, Rv1654-induced c-Jun N-terminal kinase (JNK) activation was inhibited by the ROS scavenger and Rv1654-induced apoptosis was inhibited by the JNK inhibitor. Moreover, it was found that treatment of macrophages with Rv1654 led to the loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, and translocation of Bax into the mitochondria. Finally, Rv1654-mediated apoptosis was inhibited in macrophages transfected with Bax siRNA. These results suggest that Rv1654 induces macrophage apoptosis through a mitochondrial-dependent pathway and ROS-mediated JNK activation.

Mycobacterium tuberculosis Rv2005c Induces Dendritic Cell Maturation and Th1 Responses and Exhibits Immunotherapeutic Activity by Fusion with the Rv2882c Protein.

Immunotherapy represents a promising approach for improving current antibiotic treatments through the engagement of the host's immune system. Latency-associated antigens have been included as components of multistage subunit tuberculosis vaccines. We first identified Rv2005c, a DosR regulon-encoded protein, as a seroreactive protein. In this study, we found that Rv2005c induced dendritic cell (DC) maturation and Th1 responses, and its expression by Mycobacterium tuberculosis (Mtb) within macrophages was enhanced by treatment with CoCl2, a hypoxia-mimetic agent. T cells activated by Rv2005c-matured DCs induced antimycobacterial activity in macrophages under hypoxic conditions but not under normoxic conditions. However, Rv2005c alone did not exhibit any significant vaccine efficacy in our mouse model. The fusion of Rv2005c to the macrophage-activating protein Rv2882c resulted in significant activation of DCs and antimycobacterial activity in macrophages, which were enhanced under hypoxic conditions. Furthermore, the Rv2882c-Rv2005c fusion protein showed significant adjunctive immunotherapeutic effects and led to the generation of long-lasting, antigen-specific, multifunctional CD4+ T cells that coproduced TNF-α, IFN-γ and IL-2 in the lungs of our established mouse model. Overall, these results provide a novel fusion protein with immunotherapeutic potential as adjunctive chemotherapy for tuberculosis.

Antigen-Specific IFN-γ/IL-17-Co-Producing CD4+ T-Cells Are the Determinants for Protective Efficacy of Tuberculosis Subunit Vaccine.

The antigen-specific Th17 responses in the lungs for improved immunity against Mycobacterium tuberculosis (Mtb) infection are incompletely understood. Tuberculosis (TB) vaccine candidate HSP90-ESAT-6 (E6), given as a Bacillus Calmette-Guérin (BCG)-prime boost regimen, confers superior long-term protection against the hypervirulent Mtb HN878 infection, compared to BCG or BCG-E6. Taking advantage of protective efficacy lead-out, we found that ESAT-6-specific multifunctional CD4+IFN-γ+IL-17+ T-cells optimally correlated with protection level against Mtb infection both pre-and post-challenge. Macrophages treated with the supernatant of re-stimulated lung cells from HSP90-E6-immunised mice significantly restricted Mtb growth, and this phenomenon was abrogated by neutralising anti-IFN-γ and/or anti-IL-17 antibodies. We identified a previously unrecognised role for IFN-γ/IL-17 synergism in linking anti-mycobacterial phagosomal activity to enhance host control against Mtb infection. The implications of our findings highlight the fundamental rationale for why and how Th17 responses are essential in the control of Mtb, and for the development of novel anti-TB subunit vaccines.

Recombinant Rv3261 protein of Mycobacterium tuberculosis induces apoptosis through a mitochondrion-dependent pathway in macrophages and inhibits intracellular bacterial growth.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen known to persist in host cells. The apoptotic response of macrophages serves as a defense mechanism to inhibit the growth of intracellular bacteria, the failure of which can favor the spread of the pathogen to new cells. However, the mycobacterial components that regulate cell death and the related underlying mechanisms remain poorly understood. In this study, we investigated protein Rv3261, isolated from an Mtb culture filtrate, for its apoptotic potential using multidimensional fractionation. Rv3261 was found to induce macrophage apoptosis through the caspase-3/-9-dependent pathway. Furthermore, the ROS-dependent JNK activation pathway was found to be critical in Rv3261-mediated apoptosis. Rv3261 inhibited the growth of intracellular Mtb, which was significantly abrogated by pre-treatment with the ROS scavenger N-acetylcysteine (NAC), suggesting that Rv3261-mediated apoptosis may act as a host defense response. These findings suggest that Rv3261 is involved in the apoptotic modulation of Mtb-infected macrophages.

Mycobacterium avium subsp. paratuberculosis MAP1889c Protein Induces Maturation of Dendritic Cells and Drives Th2-biased Immune Responses.

:Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of chronic granulomatous bowel disease in animals and is associated with various autoimmune diseases in humans including Crohn's disease. A good understanding of the host-protective immune response and antibacterial immunity controlled by MAP and its components may contribute to the development of effective control strategies. MAP1889c was identified as a seroreactive antigen in Crohn's disease patients. In this study, we investigated the immunological function of MAP1889c in dendritic cells (DCs). MAP1889c stimulated DCs to increase expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) class molecules and to secret higher interleukin (IL)-10 and moderate IL-6, tumor necrosis factor (TNF)-α, and IL-12p70 levels through the Toll-like receptor (TLR) 4 pathway. MAP1889c-induced DC activation was mediated by mitogen-activated protein kinases (MAPKs), cAMPp-response element binding protein (CREB), and nuclear factor kappa B (NF-κB). In particular, the CREB signal was essential for MAP1889c-mediated IL-10 production but not TNF-α and IL-12p70. In addition, MAP1889c-matured DCs induced T cell proliferation and drove the Th2 response. Production of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokines and anti-inflammatory cytokines was suppressed and enhanced respectively by MAP1889c pretreatment in DCs and T cells. Furthermore, treatment of MAP1889c in M. avium-infected macrophages promoted intracellular bacterial growth and IL-10 production. These findings suggest that MAP1889c modulates the host antimycobacterial response and may be a potential virulence factor during MAP infection.

Mycobacterium tuberculosis Rv3463 induces mycobactericidal activity in macrophages by enhancing phagolysosomal fusion and exhibits therapeutic potential.

Macrophages are responsible for innate and adaptive immune response activation necessary for eliminating infections. Optimal activation of macrophages to phagocytize Mycobacterium tuberculosis is critical in anti-mycobacterial defense. Here, we identified a novel Rv3463 hypothetical protein that induces macrophage activation in Mtb culture filtrate. Recombinant Rv3463 activated mouse bone marrow-derived macrophages to induce the expression of surface molecules and secrete pro-inflammatory cytokines via the TLR2 and TLR4 pathways. Mitogen activated protein kinase, phospatidylinositol-4,5-bisphosphate 3-kinases, and the NF-κB signaling pathways are involved in Rv3463-mediated macrophage activation. Furthermore, Rv3463 induced bactericidal effects in Mtb-infected macrophages through phagosome maturation and phagolysosomal fusion enhanced by phospatidylinositol-4,5-bisphosphate 3-kinases and Ca2+ signaling pathways and exhibited therapeutic effects in a short-term Mtb-infection mouse model. Overexpression of Rv3463 in M. smegmatis caused rapid clearance of bacteria in macrophages and mice. Our study suggests that Rv3463 is a promising target for the development of post-exposure tuberculosis vaccines or adjunct immune-therapy.

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.

Rv2299c, a novel dendritic cell-activating antigen of Mycobacterium tuberculosis, fused-ESAT-6 subunit vaccine confers improved and durable protection against the hypervirulent strain HN878 in mice.

Understanding functional interactions between DCs and antigens is necessary for achieving an optimal and desired immune response during vaccine development. Here, we identified and characterized protein Rv2299c (heat-shock protein 90 family), which effectively induced DC maturation. The Rv2299c-maturated DCs showed increased expression of surface molecules and production of proinflammatory cytokines. Rv2299c induced these effects by binding to TLR4 and stimulating the downstream MyD88-, MAPK- and NF-κB-dependent signaling pathways. The Rv2299c-maturated DCs also showed an induced Th1 cell response with bactericidal activity and expansion of effector/memory T cells. The Rv2299c-ESAT-6 fused protein had greater immunoreactivity than ESAT-6. Furthermore, boosting BCG with the fused protein significantly reduced hypervirulent Mycobacterium tuberculosis HN878 burdens post-challenge. The pathological study of the lung from the challenged mice assured the efficacy of the fused protein. The fused protein boosting also induced Rv2299c-ESAT-6-specific multifunctional CD4+ T-cell response in the lungs of the challenged mice. Our findings suggest that Rv2299c is an excellent candidate for the rational design of an effective multiantigenic TB vaccine.

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 avium MAV2052 protein induces apoptosis in murine macrophage cells through Toll-like receptor 4.

Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.