RESUMO
BACKGROUND: Multidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis. METHODS: A clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (nâ =â 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, ß-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression). RESULTS: VitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and LC3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA-treated macrophages. The combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a >125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid. CONCLUSIONS: Immunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis.
Assuntos
Peptídeos Antimicrobianos/imunologia , Colecalciferol/farmacologia , Agentes de Imunomodulação/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos , Antibióticos Antituberculose/farmacologia , Células Cultivadas , Humanos , Isoniazida/farmacologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/imunologiaRESUMO
BACKGROUND: Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. RESULTS: Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. CONCLUSIONS: Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.
Assuntos
Antituberculosos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Testes de Sensibilidade Microbiana/métodos , Microscopia/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/microbiologia , Automação , Humanos , Isoniazida/farmacologia , Linezolida/farmacologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fenótipo , Rifampina/farmacologiaRESUMO
The effect of corticosteroids on human physiology is complex and their use in tuberculosis patients remains controversial. In a high-throughput screening approach designed to discover virulence inhibitors, several corticosteroids were found to prevent cytolysis of fibroblasts infected with mycobacteria. Further experiments with Mycobacterium tuberculosis showed anti-cytolytic activity in the 10â¯nM range, but no effect on bacterial growth or survival in the absence of host cells at 20⯵M. The results from a panel of corticosteroids with various affinities to the glucocorticoid- and mineralocorticoid receptors indicate that the inhibition of cytolysis most likely is mediated through the glucocorticoid receptor. Using live-imaging of M. tuberculosis-infected human monocyte-derived macrophages, we also show that corticosteroids to some extent control intracellular bacteria. In vitro systems with reduced complexity are to further study and understand the interactions between bacterial infection, immune defense and cell signaling.
Assuntos
Corticosteroides/farmacologia , Fibroblastos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Tuberculose/tratamento farmacológico , Antituberculosos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Tuberculose/metabolismo , Tuberculose/microbiologiaRESUMO
Drug resistance in Mycobacterium tuberculosis is an emerging threat that makes the discovery of new candidate drugs a priority. In particular, drugs with high sterilizing activity within host cells are needed to improve efficacy and reduce treatment duration. We aimed to develope and validate a High Content Screening assay based on Mycobacterium tuberculosis-infected primary human monocyte-derived macrophages as its natural reservoir. Infected primary human monocyte-derived macrophages were exposed to control antibiotics or tested compounds on 384 well plates. Intracellular bacterial growth and macrophage numbers were evaluated using an ImageXpress High Content Screening system and Z'-factor was calculated to assess the reproducibility. The combination of isoniazid and rifampicin as a positive control rendered a Z'-factor above 0.4, demonstrating suitability of the assay for screening and compound profiling purposes. In a validation experiment, isoniazid, rifampicin, moxifloxacin and levofloxacin all effectively inhibited intracellular growth as expected. Finally, a pilot screening campaign including 5700 compounds from diverse libraries resulted in the identification of three compounds with confirmed antimycobacterial activity in the low micromolar range and low host cell toxicity. The assay represents an attractive screening platform for both academic research on host-pathogen mechanisms in tuberculosis and for the identification and characterization of novel antimycobacterial compounds.
Assuntos
Mycobacterium tuberculosis , Tuberculose dos Linfonodos , Antituberculosos/farmacologia , Humanos , Isoniazida/farmacologia , Macrófagos/microbiologia , Reprodutibilidade dos Testes , Rifampina/farmacologiaRESUMO
Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of tuberculosis (TB). We have established an experimental protocol to follow immune polarization of myeloid-derived cells into M1 (classically activated) or M2 (alternatively activated) macrophage-like cells through assessment with a 10-color flow cytometry panel that allows visualization and deep-characterization of green-fluorescent-protein (GFP)-labeled Mtb in diverse macrophages subsets. Monocytes obtained from healthy blood donors were polarized into M1 or M2 cells using differentiation with granulocyte macrophage-colony-stimulating factor (GM-CSF) or macrophage-colony stimulating factor (M-CSF) followed by polarization with IFN-γ and lipopolysaccharide (LPS) or IL-4, respectively. Fully polarized M1 and M2 cells were infected with Mtb-GFP for 4 hours before detached Mtb-infected macrophages were stained with flow cytometry at 4- or 24-hours post-infection. Sample acquisition was performed with flow cytometry and the data was analyzed using a flow cytometry analysis software. Manual gating as well as dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph analysis was performed. This protocol resulted in effective M1/M2 polarization characterized by elevated levels of CD64, CD86, TLR2, HLA-DR and CCR7 on uninfected M1 cells, while uninfected M2 cells exhibited a strong up-regulation of the M2 phenotype markers CD163, CD200R, CD206 and CD80. M1-polarized cells typically contained fewer bacteria compared to M2-polarized cells. Several M1/M2 markers were downregulated after Mtb infection, which suggests that Mtb can modulate macrophage polarization. In addition, 24 different cell clusters of different sizes were found to be uniquely distributed among the M1 and M2 uninfected and Mtb-infected cells at 24-hours post-infection. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs in different areas of research.
Assuntos
Polaridade Celular , Citometria de Fluxo/métodos , Macrófagos/citologia , Monócitos/citologia , Tuberculose/imunologia , Biomarcadores/metabolismo , Células Cultivadas , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Tuberculose/metabolismoRESUMO
BACKGROUND: Intracellular delivery of antimicrobial agents by nanoparticles, such as mesoporous silica particles (MSPs), offers an interesting strategy to treat intracellular infections. In tuberculosis (TB), Mycobacterium tuberculosis avoids components of the immune system by residing primarily inside alveolar macrophages, which are the desired target for TB therapy. METHODS AND FINDINGS: We have previously identified a peptide, called NZX, capable of inhibiting both clinical and multi-drug resistant strains of M. tuberculosis at therapeutic concentrations. In this study we analysed the potential of MSPs containing NZX for the treatment of tuberculosis. The MSPs released functional NZX gradually into simulated lung fluid and the peptide filled MSPs were easily taken up by primary macrophages. In an intracellular infection model, the peptide containing particles showed increased mycobacterial killing compared to free peptide. The therapeutic potential of peptide containing MSPs was investigated in a murine infection model, showing that MSPs preserved the effect to eliminate M. tuberculosis in vivo. CONCLUSIONS: In this study we found that loading the antimicrobial peptide NZX into MSPs increased the inhibition of intracellular mycobacteria in primary macrophages and preserved the ability to eliminate M. tuberculosis in vivo in a murine model. Our studies provide evidence for the feasibility of using MSPs for treatment of tuberculosis.
Assuntos
Antibacterianos , Peptídeos Catiônicos Antimicrobianos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Nanopartículas , Dióxido de Silício , Tuberculose Pulmonar/tratamento farmacológico , Animais , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Porosidade , Dióxido de Silício/química , Dióxido de Silício/farmacocinética , Dióxido de Silício/farmacologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
Tuberculosis has been reaffirmed as the infectious disease causing most deaths in the world. Co-infection with HIV and the increase in multi-drug resistant Mycobacterium tuberculosis strains complicate treatment and increases mortality rates, making the development of new drugs an urgent priority. In this study we have identified a promising candidate by screening antimicrobial peptides for their capacity to inhibit mycobacterial growth. This non-toxic peptide, NZX, is capable of inhibiting both clinical strains of M. tuberculosis and an MDR strain at therapeutic concentrations. The therapeutic potential of NZX is further supported in vivo where NZX significantly lowered the bacterial load with only five days of treatment, comparable to rifampicin treatment over the same period. NZX possesses intracellular inhibitory capacity and co-localizes with intracellular bacteria in infected murine lungs. In conclusion, the data presented strongly supports the therapeutic potential of NZX in future anti-TB treatment.
Assuntos
Antituberculosos/farmacologia , Pulmão/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Animais , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana Múltipla , Feminino , Humanos , Pulmão/microbiologia , Pulmão/ultraestrutura , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fatores de Tempo , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
The causative agent of tuberculosis, Mycobacterium tuberculosis, shares several characteristics with organisms that produce biofilms during infections. One of these is the ability to form tight bundles also known as cords. However, little is known of the physiological relevance of the cording phenotype. In this study, we investigated whether cord-forming M. tuberculosis induce the formation of macrophage extracellular traps (METs) in human monocyte-derived macrophages. Macrophages have previously been shown to produce extracellular traps in response to various stimuli. We optimized bacterial culturing conditions that favored the formation of the cord-forming phenotype as verified by scanning electron microscopy. Microscopy analysis of METs formation during experimental infection of macrophages with M. tuberculosis revealed that cord-forming M. tuberculosis induced significantly more METs compared to the non-cording phenotype. Deletion of early secreted antigenic target-6 which is an important virulence factor of M. tuberculosis, abrogated the ability of the bacteria to induce METs. The release of extracellular DNA from host cells during infection may represent a defense mechanism against pathogens that are difficult to internalize, including cord-forming M. tuberculosis.
Assuntos
Armadilhas Extracelulares/microbiologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/microbiologia , Linhagem Celular , DNA/genética , DNA/metabolismo , Armadilhas Extracelulares/metabolismo , Histonas/metabolismo , Humanos , Macrófagos/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fenótipo , Tuberculose/genética , Tuberculose/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need for continuous development of new and efficient methods to determine the susceptibility of isolates of Mycobacterium tuberculosis in the search for novel antimycobacterial agents. Natural products constitute an important source of new drugs, and design and implementation of antimycobacterial susceptibility testing methods are necessary to evaluate the different extracts and compounds. In this study we have explored the antimycobacterial properties of 50 ethanolic extracts from different parts of 46 selected medicinal plants traditionally used in Sudan to treat infectious diseases. MATERIALS AND METHODS: Plants were harvested and ethanolic extracts were prepared. For selected extracts, fractionation with hydrophilic and hydrophobic solvents was undertaken. A luminometry-based assay was used for determination of mycobacterial growth in broth cultures and inside primary human macrophages in the presence or absence of plant extracts and fractions of extracts. Cytotoxicity was also assessed for active fractions of plant extracts. RESULTS: Of the tested extracts, three exhibited a significant inhibitory effect on an avirulent strain of Mycobacterium tubercluosis (H37Ra) at the initial screening doses (125 and 6.25µg/ml). These were bark and leaf extracts of Khaya senegalensis and the leaf extract of Rosmarinus officinalis L. Further fractions of these plant extracts were prepared with n-hexane, chloroform, ethyl acetate, n-butanol, ethanol and water, and the activity of these extracts was retained in hydrophobic fractions. Cytotoxicity assays revealed that the chloroform fraction of Khaya senegalensis bark was non-toxic to human monocyte-derived macrophages and other cell types at the concentrations used and hence, further analysis, including assessment of IC50 and intracellular activity was done with this fraction. CONCLUSION: These results encourage further investigations to identify the active compound(s) within the chloroform fraction of Khaya senegalensis bark.