Immunogenicity and protective efficacy of RipA, a peptidoglycan hydrolase, against Mycobacterium tuberculosis Beijing outbreak strains.

Given that individuals with latent tuberculosis (TB) infection represent the major reservoir of TB infection, latency-associated antigens may be promising options for development of improved multi-antigenic TB subunit vaccine. Thus, we selected RipA, a peptidoglycan hydrolase required for efficient cell division of Mycobacterium tuberculosis (Mtb), as vaccine candidate. We found that RipA elicited activation of dendritic cells (DCs) by induction of phenotypic maturation, increased production of inflammatory cytokines, and prompt stimulation of MAPK and NF-κB signaling pathways. In addition, RipA-treated DCs promoted Th1-polarzied immune responses of naïve CD4+ T cells with increased proliferation and activated T cells from Mtb-infected mice, which conferred enhanced control of mycobacterial growth inside macrophages. Moreover, mice immunized with RipA formulated in GLA-SE adjuvant displayed remarkable generation of Ag-specific polyfunctional CD4+ T cells in both lung and spleen. Following an either conventional or ultra-low dose aerosol challenges with 2 Mtb Beijing clinical strains, RipA/GLA-SE-immunization was not inferior to BCG by mediating protection as single Ag. Collectively, our findings highlighted that RipA could be a novel candidate as a component of multi-antigenic TB subunit vaccines.

Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells.

Chronic viral infection impairs systemic immunity in the host; however, the mechanism underlying the dysfunction of immune cells in chronic viral infection is incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic viral infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone marrow (BM) to elucidate the modulation of immune cell differentiation and found that the populations highly restrained in their differentiation were common myeloid progenitors (CMPs) and common dendritic cell progenitors (CDPs). Of interest, the main immune cells infected with LCMV Clone 13 (CL13) in the BM were CD11b/c+ myeloid DCs. We next characterized CD11b+ DCs that differentiated during chronic LCMV infection. These DCs displayed a less immunogenic phenotype than DCs in naive or acutely infected mice, showing low expression of CD80 but high expression of PD-L1, B7-H4, IDO, TGF-ß, and IL-10. Consequently, these CD11b+ DCs induced less effective CD8+ T cells and more Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during CL13 infection could not induce effective CD8+ T cells specific to the antigens of newly invading pathogens. Our findings demonstrate that DCs generated from the BM during chronic viral infection cannot activate fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause of the functional alterations in the T cell immune response during chronic viral infection.

3D Imaging of the Transparent Mycobacterium tuberculosis-Infected Lung Verifies the Localization of Innate Immune Cells With Granuloma.

Using a novel tissue-clearing method, we aimed to visualize the three-dimensional (3D) distribution of immune cells within Mycobacterium tuberculosis (Mtb)-infected mice lungs. Ethyl cinnamate-based tissue clearing of Mtb-infected mice lungs was performed to obtain transparent lung samples, which were then imaged using a light sheet fluorescence microscope. Using the 3D images, we performed quantitative analysis of the immune cell population within multiple granulomas. In addition, to compare the data from the tissue clearing method, we performed histopathological and immunofluorescence analyses, and flow cytometry. We then created 3D images of the Mtb-infected lung that successfully demonstrated the distribution of blood vessels, immune cells, and granulomas. Since the immune cells within a granuloma could be separately selected and counted, the immune cell population within a specific lesion could be quantified. In addition, macroscopic analysis, e.g., the size or shape of a granuloma, as well as microscopic analysis could be performed as intact lung samples were used. The use of the tissue clearing method in infected lungs could be a novel modality for understanding the role of the immune system in the pathogenesis of tuberculosis.

Toll-like receptor 4 signaling-mediated responses are critically engaged in optimal host protection against highly virulent Mycobacterium tuberculosis K infection.

Toll-like receptors (TLRs) play critical roles in the innate recognition of Mycobacterium tuberculosis (Mtb) by host immune cells. However, controversy has arisen regarding the role of TLR4 in determining the outcomes of Mtb infection. To address this controversy, the function of TLR4 in the induction of an optimal protective immune response against the highly virulent Mtb K-infection was comparatively investigated in C3 H/HeJ (TLR4-deficient mutant) and C3 H/HeN (TLR4-competent wild-type) mice. Interestingly, following Mtb infection, C3 H/HeJ mice showed a more severe disease phenotype than C3 H/HeN mice, exhibiting reduced weight and a marked increase in bacterial burden along with necrotic lung inflammation. Analysis of the immune cell composition revealed significantly increased neutrophils in the lung and significant production of IL-10 accompanied by the impairment of the protective Th1 response in C3 H/HeJ mice. Reducing the neutrophil numbers by treating C3 H/HeJ mice with an anti-Ly6 G monoclonal antibody (mAb) and blocking IL-10 signaling with an anti-IL-10 receptor mAb reduced the excessive lung inflammation and bacterial burden in C3 H/HeJ mice. Therefore, abundant IL-10 signaling and neutrophils have detrimental effects in TLR4-deficient mice during Mtb infection. However, the blockade of IL-10 signaling produced an increase in the CD11bhiLy6 Ghi neutrophil population, but the phenotypes of these neutrophils were different from those of the CD11bintLy6 Gint neutrophils from mice with controlled infections. Collectively, these results show that TLR4 positively contributes to the generation of an optimal protective immunity against Mtb infection. Furthermore, investigating the TLR4-mediated response will provide insight for the development of effective control measures against tuberculosis.

PPE39 of the Mycobacterium tuberculosis strain Beijing/K induces Th1-cell polarization through dendritic cell maturation.

In a previous study, we have identified MTBK_24820, the complete protein form of PPE39 in the hypervirulent Mycobacterium tuberculosis (Mtb) strain Beijing/K by using comparative genomic analysis. PPE39 exhibited vaccine potential against Mtb challenge in a murine model. Thus, in this present study, we characterize PPE39-induced immunological features by investigating the interaction of PPE39 with dendritic cells (DCs). PPE39-treated DCs display reduced dextran uptake and enhanced MHC-I, MHC-II, CD80 and CD86 expression, indicating that this PPE protein induces phenotypic DC maturation. In addition, PPE39-treated DCs produce TNF-α, IL-6 and IL-12p70 to a similar and/or greater extent than lipopolysaccharide-treated DCs in a dose-dependent manner. The activating effect of PPE39 on DCs was mediated by TLR4 through downstream MAPK and NF-κB signaling pathways. Moreover, PPE39-treated DCs promoted naïve CD4+ T-cell proliferation accompanied by remarkable increases of IFN-γ and IL-2 secretion levels, and an increase in the Th1-related transcription factor T-bet but not in Th2-associated expression of GATA-3, suggesting that PPE39 induces Th1-type T-cell responses through DC activation. Collectively, the results indicate that the complete form of PPE39 is a so-far-unknown TLR4 agonist that induces Th1-cell biased immune responses by interacting with DCs.This article has an associated First Person interview with the first author of the paper.

Vaccine efficacy of a Mycobacterium tuberculosis Beijing-specific proline-glutamic acid (PE) antigen against highly virulent outbreak isolates.

Bacillus Calmette-Guerin vaccine confers insufficient pulmonary protection against tuberculosis (TB), particularly the Mycobacterium tuberculosis (Mtb) Beijing strain infection. Identification of vaccine antigens (Ags) by considering Mtb genetic diversity is crucial for the development of improved TB vaccine. MTBK_20640, a new Beijing genotype-specific proline-glutamic acid-family Ag, was identified by comparative genomic analysis. Its immunologic features were characterized by evaluating interactions with dendritic cells (DCs), and immunogenicity and vaccine efficacy were determined against highly virulent Mtb Beijing outbreak Korean Beijing (K) strain and HN878 strain in murine infection model. MTBK_20640 induced DCs via TLR2 and downstream MAPK and NF-κB signaling pathways, effectively promoting naive CD4-positive (CD4+) T-cell proliferation and IFN-γ production. Different IFN-γ response was observed in mice infected with Mtb K or reference H37Rv strain. Significant induction of T helper type 1 cell-polarized Ag-specific multifunctional CD4+ T cells and a marked Ag-specific IgG2c response were observed in mice immunized with MTBK_20640/glucopyranosyl lipid adjuvant-stable emulsion. The immunization conferred long-term protection against 2 Mtb Beijing outbreak strains, as evidenced by a significant reduction in colony-forming units in the lung and spleen and reduced lung inflammation. MTBK_20640 vaccination conferred long-term protection against highly virulent Mtb Beijing strains. MTBK_20640 may be developed into a novel Ag component in multisubunit TB vaccines in the future.-Kwon, K. W., Choi, H.-H., Han, S. J., Kim, J.-S., Kim, W. S., Kim, H., Kim, L.-H., Kang, S. M., Park, J., Shin, S. J. Vaccine efficacy of a Mycobacterium tuberculosis Beijing-specific proline-glutamic acid (PE) antigen against highly virulent outbreak isolates.

Vaccine potential of ESAT-6 protein fused with consensus CD4+ T-cell epitopes of PE/PPE proteins against highly pathogenic Mycobacterium tuberculosis strain HN878.

Pro-Glu/Pro-Pro-Glu (PE/PPE) family proteins in Mycobacterium tuberculosis (Mtb) are contributors to pathogenesis and immune evasion. These proteins have a unique structure in which the sequence is conserved. We investigated the vaccine potential of ESAT-6 fused with consensus CD4+ T-cell epitopes of PE/PPE proteins against highly pathogenic Mtb strain HN878 in a murine model. We selected consensus CD4+ T-cell epitopes of PE/PPE proteins by multiple alignments, investigated their IFN-γ response during Mtb infection, and produced their fused ESAT-6 vaccine antigens. Our results showed an increased immune response in PE/PPE peptide -ESAT-6 fusion protein immunization group compared to ESAT-6 only immunization group. After challenge with Mtb strain HN878, we observed that induced CD4+ T-cells secreted double-positive cytokine IL-2+/IFN-γ+, which is considered to be associated with protective T-cell immunity. Additionally, lower numbers of colony-forming units were observed in the spleen of fusion protein immunization groups than in those of single ESAT-6 group. Therefore, conjugation of consensus CD4+ T-cell epitopes in N terminus of PE/PPE to vaccine antigens could potentially increase the protective efficacy of subunit vaccine.

A Novel Therapeutic Approach Using Mesenchymal Stem Cells to Protect Against Mycobacterium abscessus.

Recent studies have demonstrated the therapeutic potential of mesenchymal stem cells (MSCs) for the treatment of acute inflammatory injury and bacterial pneumonia, but their therapeutic applications in mycobacterial infections have not been investigated. In this study, we demonstrated the use of MSCs as a novel therapeutic strategy against Mycobacterium abscessus (M. abscessus), which is the most drug-resistant and difficult-to-treat mycobacterial pathogen. The systemic intravenous injection of MSCs not only improved mouse survival but also enhanced bacterial clearance in the lungs and spleen. Additionally, MSCs enhanced IFN-γ, TNF-α, IL-6, MCP-1, nitric oxide (NO) and PGE2 production and facilitated CD4(+) /CD8(+) T cell, CD11b(high) macrophage, and monocyte recruitment in the lungs of M. abscessus-infected mice. To precisely elucidate the functions of MSCs in M. abscessus infection, an in vitro macrophage infection system was used. MSCs caused markedly increased NO production via NF-κB activation in M. abscessus-infected macrophages cultured in the presence of IFN-γ. Inhibiting NO or NF-κB signaling using specific inhibitors reduced the antimycobacterial activity of MSCs. Furthermore, the cellular crosstalk between TNF-α released from IFN-γ-stimulated M. abscessus-infected macrophages and PGE2 produced by MSCs was necessary for the mycobacterial-killing activity of the macrophages. Finally, the importance of increased NO production in response to MSC administration was confirmed in the mouse M. abscessus infection model. Our results suggest that MSCs may offer a novel therapeutic strategy for treating this drug-resistant mycobacterial infection by enhancing the bacterial-killing power of macrophages. Stem Cells 2016;34:1957-1970.

Mycobacterium tuberculosis MmsA, a novel immunostimulatory antigen, induces dendritic cell activation and promotes Th1 cell-type immune responses.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is an outstanding pathogen that modulates the host immune response. This inconvenient truth drives the continual identification of antigens that generate protective immunity, including Th1-type T cell immunity. Here, the contribution of methylmalonate semialdehyde dehydrogenase (MmsA, Rv0753c) of Mtb to immune responses was examined in the context of dendritic cell (DC) activation and T cell immunity both in vitro and in vivo. The results showed that MmsA induced DC activation by activating the MAPK and NF-κB signaling pathways. Additionally, MmsA-treated DCs activated naïve T cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2, and induced T cell proliferation. These results indicate that MmsA is a novel DC maturation-inducing antigen that drives the Th1 immune response. Thus, MmsA was found to potentially regulate immune responses via DC activation toward Th1-type T cell immunity, enhancing our understanding of Mtb pathogenesis.

Mycobacterium kansasii-induced death of murine macrophages involves endoplasmic reticulum stress responses mediated by reactive oxygen species generation or calpain activation.

Although pathogenic mechanisms of tuberculosis have been extensively studied, little is known about the pathogenic mechanisms of Mycobacterium kansasii. In this work the influence of virulence and ER-stress mediated apoptosis of macrophages during two different strains of M. kansasii infection was investigated. We show that M. kansasii infection is associated with ER stress-mediated apoptosis in the murine macrophage cell line RAW 264.7. Infection of RAW 264.7 cells in vitro with apoptosis-inducing a clinical isolate of M. kansasii SM-1 (SM-1) resulted in strong induction of ER stress responses compared with M. kansasii type strain (ATCC 12478)-infected RAW 264.7 cells. Interestingly, inhibition of calpain prevented the induction of CHOP and Bip in ATCC 12478-infected RAW 264.7 cells but not in RAW 264.7 cells infected with SM-1. In contrast, reactive oxygen species (ROS) were significantly increased only in RAW 264.7 cells infected with SM-1. We propose that ROS generation is important for triggering ER stress-mediated apoptosis during SM-1 infection, whereas ATCC 12478-induced, ER stress-mediated apoptosis is associated with calpain activation. Our results demonstrate that the ER stress pathway plays important roles in the pathogenesis of M. kansasii infections, and that different strains of M. kansasii induce different patterns of ER stress-mediated apoptosis.

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.

Effects of mycobacterial infection on proliferation of hematopoietic precursor cells.

Bacterial infection can affect hematopoietic precursor cells in bone marrow, because the infected tissues produce various cytokines and chemokines. Little is known about hematopoietic precursor cells, including hematopoietic stem cells and their progenitors, during mycobacterial infection. Here, we showed that mycobacterial infections result in the expansion of not only the lin-c-kit+sca-1+ (LKS+) cell population, but also granulocyte-monocyte progenitor cells in a chronic murine tuberculosis model. Interestingly, stimulation of LKS+ cells with attenuated Mycobacterium tuberculosis H37Ra culture filtrate (RaCF) was significantly stronger than that by virulent H37Rv culture filtrate (RvCF). Lower TNF-α and IL-6 levels were observed in RvCF-stimulated bone marrow cells. Neutralization of TNF-α or IL-6 in RaCF-stimulated bone marrow cells markedly suppressed LKS+ cell clonal expansion. Additionally, numbers of LKS+ cells were lower in TLR2(-/-) and MyD88(-/-) mice after mycobacterial infection. Taken together, LKS+ cell proliferation related to mycobacterial virulence may be related to the secretion of TNF-α and IL-6 associated with TLR signaling. Expansion of hematopoietic progenitor cells may, therefore, play an important role during mycobacterial infection.

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 Ca(2+) 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.

Mycobacterium tuberculosis lipoprotein-induced association of TLR2 with protein kinase C zeta in lipid rafts contributes to reactive oxygen species-dependent inflammatory signalling in macrophages.

Membrane lipid rafts are enriched in cholesterol and play an important role as signalling platforms. However, the roles of lipid rafts and associated signalling molecules in the innate immune responses to mycobacteria remain unknown. Here we show that stimulation with Mycobacterium tuberculosis 19 kDa lipoprotein, a TLR2/1 agonist, results in translocation of TLR2 to lipid rafts, coalescence of lipid rafts and production of reactive oxygen species (ROS) that drive pro-inflammatory responses. Disruption of lipid raft organization markedly reduced lipoprotein-induced ROS and inflammatory responses. Remarkably, the atypical protein kinase C (PKC) zeta was specifically recruited into detergent-resistant membrane fractions and associated with TLR2. PKCzeta activity was critical for lipoprotein-dependent ROS generation, raft coalescence and the pro-inflammatory responses by macrophages. Moreover, lipid raft organization was required for 19 kDa mediated PKCzeta activation. These results demonstrate that TLR2 trafficking and raft coalescence play an essential role for the initiation of lipoprotein-induced innate immune responses via TLR2 and ROS signalling. In addition, PKCzeta targets to lipid rafts and may act as a critical adaptor molecule to regulate lipid raft dynamics during TLR2 signalling.