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1.
PLoS Biol ; 21(8): e3002231, 2023 08.
Article in English | MEDLINE | ID: mdl-37590294

ABSTRACT

Mycobacterium tuberculosis (Mtb) defends host-mediated killing by repressing the autophagolysosome machinery. For the first time, we report NCoR1 co-repressor as a crucial host factor, controlling Mtb growth in myeloid cells by regulating both autophagosome maturation and lysosome biogenesis. We found that the dynamic expression of NCoR1 is compromised in human peripheral blood mononuclear cells (PBMCs) during active Mtb infection, which is rescued upon prolonged anti-mycobacterial therapy. In addition, a loss of function in myeloid-specific NCoR1 considerably exacerbates the growth of M. tuberculosis in vitro in THP1 differentiated macrophages, ex vivo in bone marrow-derived macrophages (BMDMs), and in vivo in NCoR1MyeKO mice. We showed that NCoR1 depletion controls the AMPK-mTOR-TFEB signalling axis by fine-tuning cellular adenosine triphosphate (ATP) homeostasis, which in turn changes the expression of proteins involved in autophagy and lysosomal biogenesis. Moreover, we also showed that the treatment of NCoR1 depleted cells by Rapamycin, Antimycin-A, or Metformin rescued the TFEB activity and LC3 levels, resulting in enhanced Mtb clearance. Similarly, expressing NCoR1 exogenously rescued the AMPK-mTOR-TFEB signalling axis and Mtb killing. Overall, our data revealed a central role of NCoR1 in Mtb pathogenesis in myeloid cells.


Subject(s)
Mycobacterium tuberculosis , Nuclear Receptor Co-Repressor 1 , Animals , Humans , Mice , AMP-Activated Protein Kinases , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Leukocytes, Mononuclear , Myeloid Cells , TOR Serine-Threonine Kinases , Nuclear Receptor Co-Repressor 1/metabolism
2.
Redox Biol ; 59: 102575, 2023 02.
Article in English | MEDLINE | ID: mdl-36565644

ABSTRACT

Dendritic cells (DCs) undergo rapid metabolic reprogramming to generate signal-specific immune responses. The fine control of cellular metabolism underlying DC immune tolerance remains elusive. We have recently reported that NCoR1 ablation generates immune-tolerant DCs through enhanced IL-10, IL-27 and SOCS3 expression. In this study, we did comprehensive metabolic profiling of these tolerogenic DCs and identified that they meet their energy requirements through enhanced glycolysis and oxidative phosphorylation (OXPHOS), supported by fatty acid oxidation-driven oxygen consumption. In addition, the reduced pyruvate and glutamine oxidation with a broken TCA cycle maintains the tolerogenic state of the cells. Mechanistically, the AKT-mTOR-HIF-1α-axis mediated glycolysis and CPT1a-driven ß-oxidation were enhanced in these tolerogenic DCs. To confirm these observations, we used synthetic metabolic inhibitors and found that the combined inhibition of HIF-1α and CPT1a using KC7F2 and etomoxir, respectively, compromised the overall transcriptional signature of immunological tolerance including the regulatory cytokines IL-10 and IL-27. Functionally, treatment of tolerogenic DCs with dual KC7F2 and etomoxir treatment perturbed the polarization of co-cultured naïve CD4+ T helper (Th) cells towards Th1 than Tregs, ex vivo and in vivo. Physiologically, the Mycobacterium tuberculosis (Mtb) infection model depicted significantly reduced bacterial burden in BMcDC1 ex vivo and in CD103+ lung DCs in Mtb infected NCoR1DC-/-mice. The spleen of these infected animals also showed increased Th1-mediated responses in the inhibitor-treated group. These findings suggested strong involvement of NCoR1 in immune tolerance. Our validation in primary human monocyte-derived DCs (moDCs) showed diminished NCOR1 expression in dexamethasone-derived tolerogenic moDCs along with suppression of CD4+T cell proliferation and Th1 polarization. Furthermore, the combined KC7F2 and etomoxir treatment rescued the decreased T cell proliferative capacity and the Th1 phenotype. Overall, for the first time, we demonstrated here that NCoR1 mediated control of glycolysis and fatty acid oxidation fine-tunes immune tolerance versus inflammation balance in murine and human DCs.


Subject(s)
Interleukin-10 , Interleukin-27 , Humans , Mice , Animals , Interleukin-10/metabolism , Interleukin-27/metabolism , Dendritic Cells/metabolism , Immune Tolerance , Glycolysis , Fatty Acids/metabolism , Cell Differentiation , Cells, Cultured , Nuclear Receptor Co-Repressor 1/metabolism
3.
Front Immunol ; 9: 2224, 2018.
Article in English | MEDLINE | ID: mdl-30337923

ABSTRACT

Due to emergence of new variants of pathogenic micro-organisms the treatment and immunization of infectious diseases have become a great challenge in the past few years. In the context of vaccine development remarkable efforts have been made to develop new vaccines and also to improve the efficacy of existing vaccines against specific diseases. To date, some vaccines are developed from protein subunits or killed pathogens, whilst several vaccines are based on live-attenuated organisms, which carry the risk of regaining their pathogenicity under certain immunocompromised conditions. To avoid this, the development of risk-free effective vaccines in conjunction with adequate delivery systems are considered as an imperative need to obtain desired humoral and cell-mediated immunity against infectious diseases. In the last several years, the use of nanoparticle-based vaccines has received a great attention to improve vaccine efficacy, immunization strategies, and targeted delivery to achieve desired immune responses at the cellular level. To improve vaccine efficacy, these nanocarriers should protect the antigens from premature proteolytic degradation, facilitate antigen uptake and processing by antigen presenting cells, control release, and should be safe for human use. Nanocarriers composed of lipids, proteins, metals or polymers have already been used to attain some of these attributes. In this context, several physico-chemical properties of nanoparticles play an important role in the determination of vaccine efficacy. This review article focuses on the applications of nanocarrier-based vaccine formulations and the strategies used for the functionalization of nanoparticles to accomplish efficient delivery of vaccines in order to induce desired host immunity against infectious diseases.


Subject(s)
Antigen-Presenting Cells/immunology , Communicable Diseases/immunology , Immunity, Cellular/immunology , Nanoparticles/administration & dosage , Vaccines/immunology , Communicable Disease Control/methods , Communicable Diseases/therapy , Drug Delivery Systems/methods , Humans , Models, Immunological , Nanoparticles/chemistry , Vaccination/methods , Vaccines/chemistry
4.
Sci Rep ; 6: 24184, 2016 04 26.
Article in English | MEDLINE | ID: mdl-27113139

ABSTRACT

In order to improve the chemotherapy of tuberculosis, there is an urgent need to enhance the efficacy of existing agents and also to develop more efficient drug delivery systems. Here, we synthesized a novel anti-TB drug complex consisting of zinc and rifampicin (Zn-RIF), and encapsulated it into transferrin-conjugated silver quantum-dots (Zn-RIF-Tf-QD) to improve delivery in macrophages. Successful synthesis of Zn-RIF and Zn-RIF-Tf-QD was confirmed by UV/Vis-spectroscopy, TEM, FTIR, photoluminescence, XRD, XPS, and NMR. The sizes of silver QDs and transferrin-conjugated QDs were found to be in the range of 5-20 nm. Activity assays showed that Zn-RIF-Tf-QD exhibited 10-fold higher antibacterial activity against Mycobacterium smegmatis and Mycobacterium bovis-BCG as compared to Zn-RIF, RIF and Zn. Immunofluorescence studies showed that Zn-RIF-Tf-QD-conjugates were actively endocytosed by macrophages and dendritic cells, but not by lung epithelial cells. Treatment with Zn-RIF-Tf-QD efficiently killed mycobacteria residing inside macrophages without exhibiting cytotoxicity and genotoxicity. Moreover, the conjugates remained stable for upto 48 h, were taken up into the late endosomal compartment of macrophages, and released the drug in a sustainable manner. Our data demonstrate that Zn-RIF-Tf-QDs have a great potential as anti-TB drugs. In addition, transferrin-conjugated QDs may constitute an effective drug delivery system for tuberculosis therapy.


Subject(s)
Antitubercular Agents/chemistry , Drug Carriers/chemistry , Quantum Dots/chemistry , Rifampin/chemistry , Silver/chemistry , Transferrin/chemistry , Zinc/chemistry , A549 Cells , Animals , Antitubercular Agents/metabolism , Antitubercular Agents/pharmacology , Cell Survival/drug effects , Endocytosis , Humans , Macrophages/cytology , Macrophages/metabolism , Macrophages/microbiology , Magnetic Resonance Spectroscopy , Mice , Microscopy, Fluorescence , Mycobacterium bovis/drug effects , Mycobacterium smegmatis/drug effects , Particle Size , Photoelectron Spectroscopy , Rifampin/metabolism , Rifampin/pharmacology , Spectroscopy, Fourier Transform Infrared
5.
Toxicol Sci ; 150(2): 454-72, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26794139

ABSTRACT

Zinc oxide nanoparticles (ZnO-NPs) have wide biological applications, which have raised serious concerns about their impact on the health and environment. Although, various studies have shown ZnO-NP toxicity on different cells underin vitroconditions, sufficient information is lacking regarding toxicity and underlying mechanisms underin vivoconditions. In this work, we investigated genotoxic, clastogenic, and cytotoxic effects of ZnO-NPs on macrophages and in adult mice. ZnO-NP-treated mice showed signs of toxicity such as loss in body weight, passive behavior and reduced survival. Further mechanistic studies revealed that administration of higher dose caused severe DNA damage in peripheral blood and bone marrow cells as evident by the formation of COMET tail, micronuclei, chromosomal fragmentation, and phosphorylation of H2A histone family member X. Moreover, ZnO-NPs inhibited DNA repair mechanism by downregulating the expression offen-1andpolBproteins. Histopathological examinations showed severe inflammation and damage to liver, lungs, and kidneys. Cell viability and wound healing assays revealed that ZnO-NPs killed macrophages in a dose-dependent manner, caused severe wounds and inhibited cellular migration by irreversible actin depolymerization and degradation. Reduction in the viability of macrophages was due to the arrest of the cell cycle at the G0/G1 phase, inhibition of superoxide dismutase and catalase and eventually reactive oxygen species. Furthermore, treatment with an antioxidant drug N-acetyl cysteine significantly reduced the ZnO-NP induced genotoxicity bothin vitroandin vivo Altogether, this study gives detailed pathological insights of ZnO-NP that impair cellular functions, thus will enable to arbitrate their biological applications.


Subject(s)
Actin Depolymerizing Factors/genetics , DNA Damage , Macrophages/drug effects , Mutagens/toxicity , Nanoparticles/toxicity , Oxidative Stress/drug effects , Zinc Oxide/toxicity , Animals , Cell Culture Techniques , Cell Cycle/drug effects , Cell Line , Cell Movement/drug effects , Cell Survival/drug effects , Chromosomal Instability/drug effects , Comet Assay , Dose-Response Relationship, Drug , Macrophages/enzymology , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Inbred BALB C , Micronuclei, Chromosome-Defective/chemically induced , Nanoparticles/chemistry , Oxidative Stress/genetics , Zinc Oxide/chemistry
6.
Antimicrob Agents Chemother ; 59(2): 763-71, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25403677

ABSTRACT

N-acylated homoserine lactonases are known to inhibit the signaling molecules of the biofilm-forming pathogens. In this study, gold nanoparticles were coated with N-acylated homoserine lactonase proteins (AiiA AuNPs) purified from Bacillus licheniformis. The AiiA AuNPs were characterized by UV-visible spectra, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and X-ray diffraction (XRD). The synthesized AiiA AuNPs were found to be spherical in shape and 10 to 30 nm in size. Treatment with AiiA protein-coated AuNPs showed maximum reduction in exopolysaccharide production, metabolic activities, and cell surface hydrophobicity and potent antibiofilm activity against multidrug-resistant Proteus species compared to treatment with AiiA protein alone. AiiA AuNPs exhibited potent antibiofilm activity at 2 to 8 µM concentrations without being harmful to the macrophages. We conclude that at a specific dose, AuNPs coated with AiiA can kill bacteria without harming the host cells, thus representing a potential template for the design of novel antibiofilm and antibacterial protein drugs to decrease bacterial colonization and to overcome the problem of drug resistance. In summary, our data suggest that the combined effect of the lactonase and the gold nanoparticles of the AiiA AuNPs has promising antibiofilm activity against biofilm-forming and multidrug-resistant Proteus species.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacillus/enzymology , Biofilms/drug effects , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/pharmacology , Gold/chemistry , Metal Nanoparticles/chemistry , Proteus/drug effects , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/metabolism , Proteus/growth & development
7.
Nanomedicine ; 10(6): 1195-208, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24607937

ABSTRACT

Here we studied immunological and antibacterial mechanisms of zinc oxide nanoparticles (ZnO-NPs) against human pathogens. ZnO-NPs showed more activity against Staphylococcus aureus and least against Mycobacterium bovis-BCG. However, BCG killing was significantly increased in synergy with antituberculous-drug rifampicin. Antibacterial mechanistic studies showed that ZnO-NPs disrupt bacterial cell membrane integrity, reduce cell surface hydrophobicity and down-regulate the transcription of oxidative stress-resistance genes in bacteria. ZnO-NP treatment also augmented the intracellular bacterial killing by inducing reactive oxygen species production and co-localization with Mycobacterium smegmatis-GFP in macrophages. Moreover, ZnO-NPs disrupted biofilm formation and inhibited hemolysis by hemolysin toxin producing S. aureus. Intradermal administration of ZnO-NPs significantly reduced the skin infection, bacterial load and inflammation in mice, and also improved infected skin architecture. We envision that this study offers novel insights into antimicrobial actions of ZnO-NPs and also demonstrates ZnO-NPs as a novel class of topical anti-infective agent for the treatment of skin infections. FROM THE CLINICAL EDITOR: This in-depth study demonstrates properties of ZnO nanoparticles in infection prevention and treatment in several skin infection models, dissecting the potential mechanisms of action of these nanoparticles and paving the way to human applications.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Mycobacterium/drug effects , Nanoparticles/therapeutic use , Staphylococcal Skin Infections/drug therapy , Staphylococcus aureus/drug effects , Zinc Oxide/therapeutic use , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Female , Humans , Macrophages/drug effects , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Monocytes/drug effects , Monocytes/microbiology , Mycobacterium/physiology , Mycobacterium Infections/drug therapy , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Oxidative Stress/drug effects , Skin/microbiology , Staphylococcal Infections/drug therapy , Staphylococcal Skin Infections/microbiology , Staphylococcus aureus/physiology , Zinc Oxide/chemistry , Zinc Oxide/pharmacology
8.
BMC Complement Altern Med ; 14: 87, 2014 Mar 05.
Article in English | MEDLINE | ID: mdl-24597853

ABSTRACT

BACKGROUND: Artemisia nilagirica (Asteraceae) and Murraya koenigii (Rutaceae) are widely distributed in eastern region of India. Leaves of Artemisia nilagirica plant are used to treat cold and cough by the local tribal population in east India. Murraya koenigii is an edible plant previously reported to have an antibacterial activity. Pathogenic strains of mycobacteria are resistant to most of the conventional antibiotics. Therefore, it is imperative to identify novel antimycobacterial molecules to treat mycobacterial infection. METHODS: In this study, ethanol, petroleum ether and water extracts of Artemisia nilagirica and Murraya koenigii were tested for antibacterial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG in synergy with first line anti-tuberculosis (TB) drugs, and for cytotoxic activities on mouse macrophage RAW264.7 cells. Antibacterial activity was determined by colony forming unit (CFU) assay. Intracellular survival assay was performed by infecting RAW264.7 cells with M. smegmatis before and after treatment with plant extracts. Cytotoxity was checked by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. Genotoxicity was studied by DAPI staining and COMET assay using mouse macrophage RAW264.7 cell line. Cell apoptosis was checked by Annexin-V/FITC dual staining method. Reactive oxygen species and nitric oxide production was checked by DCFH staining and Griess reagent, respectively. RESULTS: Ethanol extracts of A. nilagirica (IC50 300 µg/ml) and M. koenigii (IC50 400 µg/ml) were found to be more effective against Mycobacterium smegmatis as compared to petroleum ether and water extracts. M. koenigii extract showed maximum activity against M. bovis BCG in combination with a first line anti-TB drug rifampicin. M. koenigii leaf extract also exerted more cytototoxic (IC50 20 µg/ml), genotoxic and apoptosis in mouse macrophage RAW 264.7 cell line. Treatment of mouse macrophages with A. nilagirica extract increased intracellular killing of M. smegmatis by inducing production of reactive oxygen species and nitric oxide. CONCLUSIONS: Ethanol extracts of A. nilagirica and M. koenigii were found to be more effective against mycobacteria. As compared to A. nilagirica, M. koenigii ethanol extract exhibited significant synergistic antibacterial activity against M. smegmatis and M. bovis BCG in combination with anti-tuberculosis drug rifampicin, and also showed increased cytotoxicity, DNA damage and apoptosis in mouse macrophages.


Subject(s)
Artemisia/chemistry , Macrophages/drug effects , Murraya/chemistry , Mycobacterium/drug effects , Plant Extracts/pharmacology , Plant Leaves/chemistry , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/toxicity , Antitubercular Agents/pharmacology , Apoptosis/drug effects , Cell Line , Drug Synergism , Mice , Plant Extracts/toxicity
9.
J Biol Chem ; 289(6): 3555-70, 2014 Feb 07.
Article in English | MEDLINE | ID: mdl-24297177

ABSTRACT

L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared with WT-EcA. Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagine and also down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.


Subject(s)
Antineoplastic Agents , Asparaginase , Epitopes, B-Lymphocyte , Escherichia coli Proteins , Mutation, Missense , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Amino Acid Substitution , Animals , Antineoplastic Agents/immunology , Antineoplastic Agents/pharmacology , Asparaginase/genetics , Asparaginase/immunology , Asparaginase/pharmacology , Caspase 3/genetics , Caspase 3/immunology , Caspase 3/metabolism , Cell Line, Tumor , Cytochromes c/genetics , Cytochromes c/immunology , Cytochromes c/metabolism , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Epitopes, B-Lymphocyte/pharmacology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/immunology , Escherichia coli Proteins/pharmacology , Female , Humans , Male , Mice , Mice, Inbred BALB C , Mutagenesis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , bcl-X Protein/genetics , bcl-X Protein/immunology , bcl-X Protein/metabolism
10.
Antimicrob Agents Chemother ; 57(8): 3688-98, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23689720

ABSTRACT

With the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs compared to M. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose against M. smegmatis. In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G1-phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , DNA Damage/drug effects , Macrophages/drug effects , Metal Nanoparticles/administration & dosage , Mycobacterium smegmatis/drug effects , Silver/pharmacology , Alstonia/chemistry , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/administration & dosage , Antitubercular Agents/administration & dosage , Antitubercular Agents/pharmacology , Apoptosis , Cell Line, Tumor , Drug Combinations , Drug Evaluation, Preclinical , G1 Phase Cell Cycle Checkpoints , Metal Nanoparticles/ultrastructure , Mice , Micronucleus Tests , Microscopy, Electron, Transmission , Molecular Sequence Data , Particle Size , Rifampin/pharmacology , Spores, Fungal/chemistry , Trichoderma/chemistry
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