RESUMEN
Mycobacterium tuberculosis is the main causal agent of pulmonary tuberculosis (TB); the treatment of this disease is long and involves a mix of at least four different antibiotics that frequently lead to abandonment, favoring the surge of drug-resistant mycobacteria (MDR-TB), whose treatment becomes more aggressive, being longer and more toxic. Thus, the search for novel strategies for treatment that improves time or efficiency is of relevance. In this work, we used a murine model of pulmonary TB produced by the MDR-TB strain to test the efficiency of gene therapy with adenoviral vectors codifying TNF (AdTNF), a pro-inflammatory cytokine that has protective functions in TB by inducing apoptosis, granuloma formation and expression of other Th1-like cytokines. When compared to the control group that received an adenoviral vector that codifies for the green fluorescent protein (AdGFP), a single dose of AdTNF at the chronic active stage of the disease produced total survival, decreasing bacterial load and tissue damage (pneumonia), which correlated with an increase in cells expressing IFN-γ, iNOS and TNF in pneumonic areas and larger granulomas that efficiently contain and eliminate mycobacteria. Second-line antibiotic treatment against MDR-TB plus AdTNF gene therapy reduced bacterial load faster within a week of treatment compared to empty vector plus antibiotics or antibiotics alone, suggesting that AdTNF is a new potential type of treatment against MDR-TB that can shorten second-line chemotherapy but which requires further experimentation in other animal models (non-human primates) that develop a more similar disease to human pulmonary TB.
RESUMEN
Purpose of the study: During the early and progressive (late) stages of murine experimental pulmonary tuberculosis, the differential activation of macrophages contributes to disease development by controlling bacterial growth and immune regulation. Mycobacterial proteins P27 and PE_PGRS33 can target the mitochondria of macrophages. This study aims to evaluate the effect of both proteins on macrophage activation during mycobacterial infection. Materials and methods: We assess both proteins for mitochondrial oxygen consumption, and morphological changes, as well as bactericide activity, production of metabolites, cytokines, and activation markers in infected MQs. The cell line MH-S was used for all the experiments. Results: We show that P27 and PE_PGRS33 proteins modified mitochondrial dynamics, oxygen consumption, bacilli growth, cytokine production, and some genes that contribute to macrophage alternative activation and mycobacterial intracellular survival. Conclusions: Our findings showed that these bacterial proteins partially contribute to promoting M2 differentiation by altering mitochondrial metabolic activity.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Ratones , Animales , Activación de Macrófagos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Macrófagos Alveolares/metabolismo , MitocondriasRESUMEN
Tuberculosis (TB) is the leading cause of death from a single bacterial infectious agent and is one of the most relevant issues of public health. Another pandemic disease is type II diabetes mellitus (T2D) that is estimated to affect half a billion people in the world. T2D is directly associated with obesity and a sedentary lifestyle and is frequently associated with immunosuppression. Immune dysfunction induced by hyperglycemia increases infection frequency and severity. Thus, in developing countries the T2D/TB co-morbidity is frequent and represents one of the most significant challenges for the health-care systems. Several immunoendocrine abnormalities are occurring during the chronic phase of both diseases, such as high extra-adrenal production of active glucocorticoids (GCs) by the activity of 11-ß-hydroxysteroid dehydrogenase type 1 (11-ßHSD1). 11-ßHSD1 catalyzes the conversion of inactive cortisone to active cortisol or corticosterone in lungs and liver, while 11-ß-hydroxysteroid dehydrogenase type 2 (11-ßHSD2) has the opposite effect. Active GCs have been related to insulin resistance and suppression of Th1 responses, which are deleterious factors in both T2D and TB. The anabolic adrenal hormone dehydroepiandrosterone (DHEA) exerts antagonistic effects on GC signaling in immune cells and metabolic tissues; however, its anabolic effects prohibit its use to treat immunoendocrine diseases. 16α-bromoepiandrosterone (BEA) is a water miscible synthetic sterol related to DHEA that lacks an anabolic effect while amplifying the immune and metabolic properties with important potential therapeutic uses. In this work, we compared the expression of 11-ßHSD1 and the therapeutic efficacy of BEA in diabetic mice infected with tuberculosis (TB) (T2D/TB) with respect to non-diabetic TB-infected mice (TB). T2D was induced by feeding mice with a high-fat diet and administering a single low-dose of streptozotocin. After 4 weeks of T2D establishment, mice were infected intratracheally with a high-dose of Mycobacterium tuberculosis strain H37Rv. Then, mice were treated with BEA three times a week by subcutaneous and intratracheal routes. Infection with TB increased the expression of 11-ßHSD1 and corticosterone in the lungs and liver of both T2D/TB and TB mice; however, T2D/TB mice developed a more severe lung disease than TB mice. In comparison with untreated animals, BEA decreased GC and 11-ßHSD1 expression while increasing 11-ßHSD2 expression. These molecular effects of BEA were associated with a reduction in hyperglycemia and liver steatosis, lower lung bacillary loads and pneumonia. These results uphold BEA as a promising effective therapy for the T2D/TB co-morbidity.
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Androsterona/farmacología , Antituberculosos/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/farmacología , Tuberculosis/tratamiento farmacológico , 11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Comorbilidad , Corticosterona/farmacología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Hidrocortisona/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/metabolismoRESUMEN
As components of the innate immune response, antimicrobial peptides (AMPs) efficiently contribute to infection control and maintenance of a latent state in pulmonary tuberculosis (TB). As a therapeutic strategy, the administration of recombinant AMPs could be limited by enzymatic degradation and high production costs. Likewise, strategies based on the induction of AMPs have generated controversial results. In this study, 2 recombinant type-5 adenoviruses (Ad) expressing the human ß-defensin 3 (HßD3) or cathelicidin (LL37) were assessed in a murine pulmonary TB model. Mice infected with either a high dose of a drug-sensitive (H37Rv) or a multidrug-resistant (MDR) strain of Mycobacterium tuberculosis (Mtb) were treated with a single administration of AdHßD3, AdLL37, AdGFP (control vector expressing a green fluorescent protein), or saline solution (SS). Lungs were obtained to determine the bacterial burden, histologic damage, and cytokine expression at different time points. Mice treated with AdHßD3 or AdLL37 showed significantly lower bacterial load and pneumonia, and higher proinflammatory cytokine expression than the control groups AdGFP and SS. A synergistic therapeutic effect could be observed when first- or second-line antibiotics (ABs) were administered with adenoviral therapy in animals infected with H37Rv or MDR strains, respectively. Adenovirus-delivered AMP's administration constitutes a promising adjuvant therapy for current anti-TB drugs by enhancing a protective immune response and potentially reducing current AB regimes' duration.
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Péptidos Catiónicos Antimicrobianos/administración & dosificación , Antituberculosos/administración & dosificación , Tuberculosis Pulmonar/patología , beta-Defensinas/administración & dosificación , Adenoviridae , Animales , Quimioterapia Combinada/métodos , Vectores Genéticos , Humanos , Ratones , Tuberculosis Resistente a Múltiples Medicamentos/patología , CatelicidinasRESUMEN
The global control of Tuberculosis remains elusive, and Bacillus Calmette-Guérin (BCG) -the most widely used vaccine in history-has proven insufficient for reversing this epidemic. Several authors have suggested that the mass presence of vaccinated hosts might have affected the Mycobacterium tuberculosis (MTB) population structure, and this could in turn be reflected in a prevalence of strains with higher ability to circumvent BCG-induced immunity, such as the recent Beijing genotype. The effect of vaccination on vaccine-escape variants has been well-documented in several bacterial pathogens; however the effect of the interaction between MTB strains and vaccinated hosts has never been previously described. In this study we show for the first time the interaction between MTB Beijing-genotype strains and BCG-vaccinated hosts. Using a well-controlled murine model of progressive pulmonary tuberculosis, we vaccinated BALB/c mice with two different sub-strains of BCG (BCG-Phipps and BCG-Vietnam). Following vaccination, the mice were infected with either one of three selected MTB strains. Strains were selected based on lineage, and included two Beijing-family clinical isolates (strains 46 and 48) and a well-characterized laboratory strain (H37Rv). Two months after infection, mice were euthanized and the bacteria extracted from their lungs. We characterized the genomic composite of the bacteria before and after exposure to vaccinated hosts, and also characterized the local response to the bacteria by sequencing the lung transcriptome in animals during the infection. Results from this study show that the interaction within the lungs of the vaccinated hosts results in the selection of higher-virulence bacteria, specifically for the Beijing genotype strains 46 and 48. After exposure to the BCG-induced immune response, strains 46 and 48 acquire genomic mutations associated with several virulence factors. As a result, the bacteria collected from these vaccinated hosts have an increased ability for immune evasion, as shown in both the host transcriptome and the histopathology studies, and replicates far more efficiently compared to bacteria collected from unvaccinated hosts or to the original-stock strain. Further research is warranted to ascertain the pathways associated with the genomic alterations. However, our results highlight novel host-pathogen interactions induced by exposure of MTB to BCG vaccinated hosts.
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Interacciones Huésped-Patógeno/inmunología , Pulmón/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Vacunación , Animales , Vacuna BCG/inmunología , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Genoma Bacteriano , Genotipo , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Mycobacterium tuberculosis/patogenicidad , VirulenciaRESUMEN
Tuberculosis (TB), although a curable disease, remains a major cause of morbidity and mortality worldwide. It is necessary to develop a short-term therapy with reduced drug toxicity in order to improve adherence rate and control disease burden. Granulocyte-macrophage colony-stimulating factor (GM-CSF) may be a key cytokine in the treatment of pulmonary TB since it primes the activation and differentiation of myeloid and non-myeloid precursor cells, inducing the release of protective Th1 cytokines. In this work, we administrated by intratracheal route recombinant adenoviruses encoding GM-CSF (AdGM-CSF). This treatment produced significant bacterial elimination when administered in a single dose at 60 days of infection with drug sensitive or drug resistant Mtb strains in a murine model of progressive disease. Moreover, AdGM-CSF combined with primary antibiotics produced more rapid elimination of pulmonary bacterial burdens than conventional chemotherapy suggesting that this form of treatment could shorten the conventional treatment.
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Terapia Genética/métodos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Tuberculosis Resistente a Múltiples Medicamentos/terapia , Tuberculosis Pulmonar/terapia , Adenoviridae/genética , Animales , Antibióticos Antituberculosos/uso terapéutico , Recuento de Colonia Microbiana , Terapia Combinada , Citocinas/biosíntesis , Citocinas/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Inmunoterapia/métodos , Masculino , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/crecimiento & desarrollo , ARN Mensajero/genética , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiologíaRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is often used to treat leucopenia. Other haematopoietins may increase the number of circulating leucocytes with higher efficiency, but GM-CSF has additional effects that may be far more relevant than its haematopoietic activity. GM-CSF induces differentiation, proliferation and activation of macrophages and dendritic cells which are necessary for the subsequent T helper cell type 1 and cytotoxic T lymphocyte activation. GM-CSF haematopoietic and non-haematopoietic functions have pro-inflammatory and immune regulatory potential to treat a variety of autoimmune diseases and tumours. On the other hand, GM-CSF deficiency leads to various immune dysfunctions and the current utilization of GM-CSF as haematopoietic factor might be an accurate but very incomplete indication for a cytokine with vast clinical potential.