ABSTRACT
Accumulating evidence suggests that drug repurposing has drawn attention as an anticipative strategy for controlling tuberculosis (TB), considering the dwindling drug discovery and development pipeline. In this study, we explored the antigout drug febuxostat and evaluated its antibacterial activity against Mycobacterium species. Based on MIC evaluation, we found that febuxostat treatment significantly inhibited mycobacterial growth, especially that of Mycobacterium tuberculosis (Mtb) and its phylogenetically close neighbors, M. bovis, M. kansasii, and M. shinjukuense, but these microorganisms were not affected by allopurinol and topiroxostat, which belong to a similar category of antigout drugs. Febuxostat concentration-dependently affected Mtb and durably mediated inhibitory functions (duration, 10 weeks maximum), as evidenced by resazurin microtiter assay, time-kill curve analysis, phenotypic susceptibility test, and the Bactec MGIT 960 system. Based on these results, we determined whether the drug shows antimycobacterial activity against Mtb inside murine bone marrow-derived macrophages (BMDMs). Notably, febuxostat markedly suppressed the intracellular growth of Mtb in a dose-dependent manner without affecting the viability of BMDMs. Moreover, orally administered febuxostat was efficacious in a murine model of TB with reduced bacterial loads in both the lung and spleen without the exacerbation of lung inflammation, which highlights the drug potency. Taken together, unexpectedly, our data demonstrated that febuxostat has the potential for treating TB.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Allopurinol , Animals , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Febuxostat/pharmacology , Febuxostat/therapeutic use , Mice , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
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.
Subject(s)
Antigens, Bacterial/immunology , Dendritic Cells/immunology , Mycobacterium tuberculosis/immunology , Th1 Cells/immunology , Animals , Bacterial Proteins/immunology , Cell Differentiation/immunology , Cell Polarity/immunology , Cell Proliferation , Dendritic Cells/microbiology , Humans , Lipopolysaccharides/pharmacology , Mice , Mycobacterium tuberculosis/genetics , Signal Transduction , Th1 Cells/microbiology , Tuberculosis Vaccines/immunologyABSTRACT
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.
Subject(s)
Antigens, Bacterial , Disease Outbreaks/prevention & control , Mycobacterium tuberculosis , Tuberculosis Vaccines , Tuberculosis, Pulmonary , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Disease Models, Animal , Female , Humans , Male , Mice , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/immunology , Mycobacterium tuberculosis/pathogenicity , Th1 Cells/immunology , Th1 Cells/pathology , Tuberculosis Vaccines/genetics , Tuberculosis Vaccines/immunology , Tuberculosis, Pulmonary/epidemiology , Tuberculosis, Pulmonary/genetics , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/prevention & controlABSTRACT
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.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Epitopes, T-Lymphocyte/immunology , Mycobacterium tuberculosis/immunology , Vaccines, Subunit/chemical synthesis , Amino Acid Sequence , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/therapeutic use , Bacterial Proteins/genetics , Bacterial Proteins/therapeutic use , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/therapeutic use , Interferon-gamma/drug effects , Interferon-gamma/metabolism , Interleukin-2/metabolism , Mice , Mycobacterium tuberculosis/drug effects , Protein Engineering , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , Vaccines, Subunit/pharmacologyABSTRACT
Mycobacterium tuberculosis inhibits optimal T helper type 1 (Th1) responses during infection. However, the precise mechanisms by which virulent M. tuberculosis limits Th1 responses remain unclear. Here, we infected dendritic cells (DCs) with the virulent M. tuberculosis strain H37Rv or the attenuated strain H37Ra to investigate the phenotypic and functional alterations in DCs and resultant T-cell responses. H37Rv-infected DCs suppressed Th1 responses more strongly than H37Ra-infected DCs. Interestingly, H37Rv, but not H37Ra, impaired DC surface molecule expression (CD80, CD86 and MHC class II) due to prominent interleukin-10 (IL-10) production while augmenting the expression of tolerogenic molecules including PD-L1, CD103, Tim-3 and indoleamine 2,3-dioxygenase on DCs in a multiplicity-of-infection (MOI) -dependent manner. These results indicate that virulent M. tuberculosis drives immature DCs toward a tolerogenic phenotype. Notably, the tolerogenic phenotype of H37Rv-infected DCs was blocked in DCs generated from IL-10-/- mice or DCs treated with an IL-10-neutralizing monoclonal antibody, leading to restoration of Th1 polarization. These findings suggest that IL-10 induces a tolerogenic DC phenotype. Interestingly, p38 mitogen-activated protein kinase (MAPK) activation predominantly mediates IL-10 production; hence, H37Rv tends to induce a tolerogenic DC phenotype through expression of tolerogenic molecules in the p38 MAPK-IL-10 axis. Therefore, suppressing the tolerogenic cascade in DCs is a novel strategy for stimulating optimal protective T-cell responses against M. tuberculosis infection.
Subject(s)
Dendritic Cells/immunology , Interleukin-10/biosynthesis , Mycobacterium tuberculosis/immunology , Th1 Cells/cytology , Th1 Cells/immunology , Animals , Cell Proliferation , Dendritic Cells/metabolism , Interleukin-10/deficiency , Interleukin-10/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Virulence/immunologyABSTRACT
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.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium Infections, Nontuberculous/therapy , Mycobacterium abscessus/physiology , Animals , Cell Communication/drug effects , Cytokines/biosynthesis , Dinoprostone/metabolism , Guanidines/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Mice, Inbred C57BL , Mycobacterium Infections, Nontuberculous/pathology , Mycobacterium abscessus/drug effects , Mycobacterium abscessus/growth & development , NF-kappa B/metabolism , Nitric Oxide/biosynthesis , Signal Transduction/drug effects , Survival Analysis , Up-Regulation/drug effectsABSTRACT
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-1ß, 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.
Subject(s)
Bacterial Proteins/immunology , Cytokines/immunology , Dendritic Cells/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Toll-Like Receptor 4/immunology , Tuberculosis/immunology , Animals , Cell Differentiation/immunology , Cell Separation , Female , Flow Cytometry , Immunoblotting , Immunoprecipitation , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Mycobacterium tuberculosis/immunologyABSTRACT
Although Mycobacterium abscessus (M. abscessus) is becoming more prevalent in patients without overt immunodeficiency, little is known about the factors that contribute to disease susceptibility. This study was undertaken to investigate how Toll-like receptor 2 (TLR2) functionally contributes to the generation of protective immunity against M. abscessus in a morphotype-specific manner. We found that Tlr2-/- mice were extremely susceptible to an intravenous (i.v.) model of infection by M. abscessus rough variants, displaying uncontrolled infection in the lungs and a significantly lower survival rate than with wild-type (WT) mice. This uncontrolled infection resulted from failures in the following processes: (i) production of the crucial cytokines gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 12p70 (IL-12p70); (ii) early infiltration of neutrophils, monocytes, and dendritic cells (DCs) in the lungs of Tlr2-/- mice; (iii) rapid influx of CD4+ and CD8+ T cells; and (iv) the expansion of memory/effector T cells. Notably, systemic administration of M. abscessus culture filtrate-treated syngeneic DCs from WT mice greatly strengthened immune priming in vivo, resulting in a dramatic reduction in bacterial growth and improved long-term survival in Tlr2-/- mice, with a recovery of protective immunity. Our findings demonstrate that TLR2 is an essential contributor to instructive and effector immunity during M. abscessus infection in a morphotype-specific manner.
Subject(s)
Mycobacterium/immunology , Th1 Cells/immunology , Toll-Like Receptor 2/immunology , Animals , Bone Marrow Cells/immunology , CD8-Positive T-Lymphocytes/immunology , Cells, Cultured , Dendritic Cells/immunology , Immunologic Memory/immunology , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Lung/immunology , Lung/microbiology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Toll-Like Receptor 2/genetics , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
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.
Subject(s)
Dendritic Cells/immunology , Methylmalonate-Semialdehyde Dehydrogenase (Acylating)/immunology , Mitogen-Activated Protein Kinases/metabolism , Mycobacterium tuberculosis/immunology , NF-kappa B/metabolism , Animals , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , Female , Interferon-gamma/metabolism , Interleukin-2/metabolism , Lymphocyte Activation/immunology , MAP Kinase Signaling System/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Th1 Cells/immunology , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/microbiologyABSTRACT
Tuberculosis (TB) treatment requires a long therapeutic duration and induces adverse effects such as hepatotoxicity, causing discontinuation of treatment. Reduced adherence to TB medications elevates the risk of recurrence and the development of drug resistance. Additionally, severe cavitary TB with a high burden of Mycobacterium tuberculosis (Mtb) and inflammation-mediated tissue damage may need an extended treatment duration, resulting in a higher tendency of drug-induced toxicity. We previously reported that the administration of Lactobacillus sakei CVL-001 (L. sakei CVL-001) regulates inflammation and improves mucosal barrier function in a murine colitis model. Since accumulating evidence has reported the functional roles of probiotics in drug-induced liver injury and pulmonary inflammation, we employed a parabiotic form of the L. sakei CVL-001 to investigate whether this supplement may provide beneficial effects on the reduction in drug-induced liver damage and pulmonary inflammation during chemotherapy. Intriguingly, L. sakei CVL-001 administration slightly reduced Mtb burden without affecting lung inflammation and weight loss in both Mtb-resistant and -susceptible mice. Moreover, L. sakei CVL-001 decreased T cell-mediated inflammatory responses and increased regulatory T cells along with an elevated antigen-specific IL-10 production, suggesting that this parabiotic may restrain excessive inflammation during antibiotic treatment. Furthermore, the parabiotic intervention significantly reduced levels of alanine aminotransferase, an indicator of hepatotoxicity, and cell death in liver tissues. Collectively, our data suggest that L. sakei CVL-001 administration has the potential to be an adjunctive therapy by reducing pulmonary inflammation and liver damage during anti-TB drug treatment and may benefit adherence to TB medication in lengthy treatment.
Subject(s)
Latilactobacillus sakei , Mycobacterium tuberculosis , Probiotics , Animals , Probiotics/therapeutic use , Probiotics/administration & dosage , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/immunology , Mice , Pneumonia/drug therapy , Pneumonia/immunology , Antitubercular Agents/therapeutic use , Antitubercular Agents/adverse effects , Female , Tuberculosis/drug therapy , Tuberculosis/immunology , Mice, Inbred C57BL , Chemical and Drug Induced Liver Injury/drug therapy , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/etiology , Humans , Lung/pathology , Lung/drug effects , Lung/immunology , Lung/microbiology , Interleukin-10/metabolism , Mice, Inbred BALB C , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Liver/drug effects , Liver/pathology , Liver/immunologyABSTRACT
The quest for effective and enhanced multiantigenic tuberculosis (TB) subunit vaccine necessitates the induction of a protective pathogen-specific immune response while circumventing detrimental inflammation within the lung milieu. In line with this goal, we engineered a modified iteration of the quadrivalent vaccine, namely HSP90-ESAT-6-HspX-RipA (HEHR), which was coupled with the TLR4 adjuvant, CIA09A. The ensuing formulation was subjected to comprehensive assessment to gauge its protective efficacy against the hypervirulent Mycobacterium tuberculosis (Mtb) Haarlem clinical strain M2, following a BCG-prime boost regimen. Regardless of vaccination route, both intramuscular and subcutaneous administration with the HEHR vaccine exhibited remarkable protective efficacy in significantly reducing the Mtb bacterial burden and pulmonary inflammation. This underscores its notably superior protective potential compared to the BCG vaccine alone or a former prototype, the HSP90-E6 subunit vaccine. In addition, this superior protective efficacy was confirmed when testing a tag-free version of the HEHR vaccine. Furthermore, the protective immune determinant, represented by durable antigen-specific CD4+IFN-γ+IL-17A+ T-cells expressing a CXCR3+KLRG1- cell surface phenotype in the lung, was robustly induced in HEHR-boosted mice at 12 weeks post-challenge. Collectively, our data suggest that the BCG-prime HEHR boost vaccine regimen conferred improved and long-term protection against hypervirulent Mtb strain with robust antigen-specific Th1/Th17 responses.
ABSTRACT
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.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis Vaccines , Tuberculosis , Animals , Mice , N-Acetylmuramoyl-L-alanine Amidase , Beijing , Tuberculosis/prevention & control , Disease Outbreaks , Antigens, Bacterial , BCG VaccineABSTRACT
Bacillus Calmette-Guerin (BCG) vaccine is the only licensed vaccine for tuberculosis (TB) prevention. Previously, our group demonstrated the vaccine potential of Rv0351 and Rv3628 against Mycobacterium tuberculosis (Mtb) infection by directing Th1-biased CD4+ T cells co-expressing IFN-γ, TNF-α, and IL-2 in the lungs. Here, we assessed immunogenicity and vaccine potential of the combined Ags (Rv0351/Rv3628) formulated in different adjuvants as subunit booster in BCG-primed mice against hypervirulent clinical Mtb strain K (Mtb K). Compared to BCG-only or subunit-only vaccine, BCG prime and subunit boost regimen exhibited significantly enhanced Th1 response. Next, we evaluated the immunogenicity to the combined Ags when formulated with four different types of monophosphoryl lipid A (MPL)-based adjuvants: 1) dimethyldioctadecylammonium bromide (DDA), MPL, and trehalose dicorynomycolate (TDM) in liposome form (DMT), 2) MPL and Poly I:C in liposome form (MP), 3) MPL, Poly I:C, and QS21 in liposome form (MPQ), and 4) MPL and Poly I:C in squalene emulsion form (MPS). MPQ and MPS displayed greater adjuvancity in Th1 induction than DMT or MP did. Especially, BCG prime and subunit-MPS boost regimen significantly reduced the bacterial loads and pulmonary inflammation against Mtb K infection when compared to BCG-only vaccine at a chronic stage of TB disease. Collectively, our findings highlighted the importance of adjuvant components and formulation to induce the enhanced protection with an optimal Th1 response.
ABSTRACT
BACKGROUND AND PURPOSE: To diversify and expand possible tuberculosis (TB) drug candidates and maximize limited global resources, we investigated the effect of colchicine, an FDA-approved anti-gout drug, against Mycobacterium tuberculosis (Mtb) infection because of its immune-modulating effects. EXPERIMENTAL APPROACH: We evaluated the intracellular anti-Mtb activity of different concentrations of colchicine in murine bone marrow-derived macrophages (BMDMs). To elucidate the underlying mechanism, RNA sequencing, biological and chemical inhibition assays, and Western blot, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical analyses were employed. Finally, type I interferon-dependent highly TB-susceptible A/J mice were challenged with virulent Mtb H37Rv, and the host-directed therapeutic effect of oral colchicine administration on bacterial burdens and lung inflammation was assessed 30 days post-infection (2.5 mg·kg-1 every 2 days). KEY RESULTS: Colchicine reinforced the anti-Mtb activity of BMDMs without affecting cell viability, indicating that colchicine facilitated macrophage immune activation upon Mtb infection. The results from RNA sequencing, NLRP3 knockout BMDM, IL-1 receptor blockade, and immunohistochemistry analyses revealed that this unexpected intracellular anti-Mtb activity of colchicine was mediated through NLRP3-dependent IL-1ß signalling and Cox-2-regulated PGE2 production in macrophages. Consequently, the TB-susceptible A/J mouse model showed remarkable protection, with decreased bacterial loads in both the lungs and spleens of oral colchicine-treated mice, with significantly elevated Cox-2 expression at infection sites. CONCLUSIONS AND IMPLICATIONS: The repurposing of colchicine against Mtb infection in this study highlights its unique function in macrophages upon Mtb infection and its novel potential use in treating TB as host-directed or adjunctive therapy.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Animals , Colchicine/metabolism , Colchicine/pharmacology , Cyclooxygenase 2/metabolism , Dinoprostone/pharmacology , Mice , NLR Family, Pyrin Domain-Containing 3 Protein/metabolismABSTRACT
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is often exacerbated upon coinfection, but the underlying immunological mechanisms remain unclear. Here, to elucidate these mechanisms, we use an Mtb and lymphocytic choriomeningitis virus coinfection model. Viral coinfection significantly suppresses Mtb-specific IFN-γ production, with elevated bacterial loads and hyperinflammation in the lungs. Type I IFN signaling blockade rescues the Mtb-specific IFN-γ response and ameliorates lung immunopathology. Single-cell sequencing, tissue immunofluorescence staining, and adoptive transfer experiments indicate that viral infection-induced type I IFN signaling could inhibit CXCL9/10 production in myeloid cells, ultimately impairing pulmonary migration of Mtb-specific CD4+ T cells. Thus, our study suggests that augmented and sustained type I IFNs by virus coinfection prior to the pulmonary localization of Mtb-specific Th1 cells exacerbates TB immunopathogenesis by impeding the Mtb-specific Th1 cell influx. Our study highlights a negative function of viral coinfection-induced type I IFN responses in delaying Mtb-specific Th1 responses in the lung.
Subject(s)
Coinfection , Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Tuberculosis , CD4-Positive T-Lymphocytes , Humans , Lung/pathology , Th1 CellsABSTRACT
Previously, we reported that a hygromycin resistant version of the BCGΔBCG1419c vaccine candidate reduced tuberculosis (TB) disease in BALB/c, C57BL/6, and B6D2F1 mice infected with Mycobacterium tuberculosis (Mtb) H37Rv. Here, the second-generation version of BCGΔBCG1419c (based on BCG Pasteur ATCC 35734, without antibiotic resistance markers, and a complete deletion of BCG1419c) was compared to its parental BCG for immunogenicity and protective efficacy against the Mtb clinical isolate M2 in C57BL/6 mice. Both BCG and BCGΔBCG1419c induced production of IFN-γ, TNF-α, and/or IL-2 by effector memory (CD44+CD62L-), PPD-specific, CD4+ T cells, and only BCGΔBCG1419c increased effector memory, PPD-specific CD8+ T cell responses in the lungs and spleens compared with unvaccinated mice before challenge. BCGΔBCG1419c increased levels of central memory (CD62L+CD44+) T CD4+ and CD8+ cells compared to those of BCG-vaccinated mice. Both BCG strains elicited Th1-biased antigen-specific polyfunctional effector memory CD4+/CD8+ T cell responses at 10 weeks post-infection, and both vaccines controlled Mtb M2 growth in the lung and spleen. Only BCGΔBCG1419c significantly ameliorated pulmonary inflammation and decreased neutrophil infiltration into the lung compared to BCG-vaccinated and unvaccinated mice. Both BCG strains reduced pulmonary TNF-α, IFN-γ, and IL-10 levels. Taken together, BCGΔBCG1419c increased memory CD8+T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG.
Subject(s)
Mycobacterium tuberculosis , Pneumonia , Tuberculosis , Animals , BCG Vaccine , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Interleukin-10 , Interleukin-2 , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Tuberculin , Tuberculosis/microbiology , Tumor Necrosis Factor-alphaABSTRACT
Mycobacterium avium complex (MAC) causing pulmonary disease in humanshas emerged worldwide. Thus, effective strategies simultaneously aiming to prevent MAC infection and accelerate therapeutic efficacy are required. To this end, subunit vaccine-induced protection against a well-defined virulent Mycobacterium avium (Mav) isolate was assessed as a preventative and therapeutic modality in murine models. Mav-derived culture filtrate antigen (CFA) was used as a vaccine antigen with glucopyranosyl lipid A stable emulsion (GLA-SE) or GLA-SE plus cyclic-di-GMP (GLA-SE/CDG), and we compared the immunogenicities, protective efficacies and immune correlates. Interestingly, CFA+GLA-SE/CDG immunization induced greater CFA-specific Th1/Th17 responses in both the lung and spleen than among the tested groups. Consequently, protective efficacy was optimally achieved with CFA+GLA-SE/CDG by significantly reducing bacterial loads along with long-lasting maintenance of antigen-specific Th1/Th17 cytokine-producing multifunctional T cell responses and relevant cytokine productions. Thus, we employed this subunit vaccine as an adjunct to antibiotic treatment. However, this vaccine was ineffective in further reducing bacterial loads. Collectively, our study demonstrates that strong Mav CFA-specific Th1/Th17 responses are critical for preventative protection against Mav infection but may be ineffective or even detrimental in an established and progressive chronic disease, indicating that different approaches to combating Mav infection are necessary according to vaccination purposes.
Subject(s)
Mycobacterium Infections, Nontuberculous , Mycobacterium tuberculosis , Tuberculosis Vaccines , Tuberculosis , Adjuvants, Immunologic/pharmacology , Animals , Anti-Bacterial Agents/therapeutic use , Chronic Disease , Cytokines , Immunity , Mice , Mycobacterium avium , Th1 Cells , Th17 Cells , Tuberculosis/microbiology , Tuberculosis Vaccines/pharmacology , Vaccination , Vaccines, SubunitABSTRACT
Accumulating evidence suggests that two chronic respiratory diseases, nontuberculous mycobacterium (NTM)-pulmonary disease (PD) and allergic asthma, are frequently present together and that they likely influence the disease development and progression of each other. However, their precise interactions regarding the pathogenesis of comorbid diseases versus that of individual diseases are not well understood. In this study, comorbid diseases (i.e., Mycobacteria avium (Mav) pulmonary infection (PI) (Mav-PI) and ovalbumin-induced allergic asthma) were established in mice in different orders and at different time periods. Individual disease-specific characteristics, including alterations in immune cell populations and antigen-specific immune responses, were analyzed and compared. To assess Mav-PI pathogenesis, lung inflammation and bacterial burden levels were also determined. Allergic asthma induction in the presence of Mav-PI markedly aggravated Mav-PI pathogenesis by increasing the bacterial burden and the severity of lung inflammation. Interestingly, the general outcome of allergic asthma with goblet cell hyperplasia was alleviated at a chronic stage in the comorbid mouse model. Overall, the increase in the number of Mav CFUs was inversely correlated with the Mav-specific Th17 response, as confirmed by comparing BALB/c and C57BL/6J mice. Overall, the pathogenesis of existing Mav-PI is more severely affected by allergen exposure than vice versa. This Mav-PI exacerbation is associated with disruption of Mav-specific Th17 responses. This study provides the first evidence that the Mav-specific Th17 response plays an important role in the control of Mav pathogenesis in the presence of allergic asthma, indicating that targeting the Th17 response has therapeutic potential for NTM-PD accompanied by allergic asthma.
Subject(s)
Asthma , Mycobacterium Infections, Nontuberculous , Mycobacterium , Animals , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mycobacterium avium , Th17 CellsABSTRACT
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.
Subject(s)
Signal Transduction/immunology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Tuberculosis/immunology , Animals , Bacterial Load , Cytokines/immunology , Immunity, Innate , Lung/microbiology , Mice , Mice, Inbred C57BL , Specific Pathogen-Free OrganismsABSTRACT
PURPOSE: Simple and reliable animal models of human diseases contribute to the understanding of disease pathogenesis as well as the development of therapeutic interventions. Although several murine models to mimic human asthma have been established, most of them require anesthesia, resulting in variability among test individuals, and do not mimic asthmatic responses accompanied by T-helper (Th) 17 and neutrophils. As dendritic cells (DCs) are known to play an important role in initiating and maintaining asthmatic inflammation, we developed an asthma model via adoptive transfer of allergen-loaded DCs. METHODS: Ovalbumin (OVA)-loaded bone marrow-derived DCs (BMDCs) (OVA-BMDCs) were injected intravenously 3 times into non-anesthetized C57BL/6 mice after intraperitoneal OVA-sensitization. RESULTS: OVA-BMDC-transferred mice developed severe asthmatic immune responses when compared with mice receiving conventional OVA challenge intranasally. Notably, remarkable increases in systemic immunoglobulin (Ig) E and IgG1 responses, Th2/Th17-associated cytokines (interleukin [IL]-5, IL-13 and IL-17), Th2/Th17-skewed T-cell responses, and cellular components, including eosinophils, neutrophils, and goblet cells, were observed in the lungs of OVA-BMDC-transferred mice. Moreover, the asthmatic immune responses and severity of inflammation were correlated with the number of OVA-BMDCs transferred, indicating that the disease severity and asthma type may be adjusted according to the experimental purpose by this method. Furthermore, this model exhibited less variation among the test individuals than the conventional model. In addition, this DCs-based asthma model was partially resistant to steroid treatment. CONCLUSIONS: A reliable murine model of asthma by intravenous (i.v.) transfer of OVA-BMDCs was successfully established without anesthesia. This model more accurately reflects heterogeneous human asthma, exhibiting a robust Th2/Th17-skewed response and eosinophilic/neutrophilic infiltration with good reproducibility and low variation among individuals. This model will be useful for understanding the pathogenesis of asthma and would serve as an alternative tool for immunological studies on the function of DCs, T-cell responses and new drugs.