16α‐Bromoepiandrosterone as a new candidate for experimental diabetes‐tuberculosis comorbidity treatment

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-β-hydroxysteroid dehydrogenase type 1 (11-βHSD1). 11-βHSD1 catalyzes the conversion of inactive cortisone to active cortisol or corticosterone in lungs and liver, while 11-β-hydroxysteroid dehydrogenase type 2 (11-βHSD2) 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-βHSD1 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-βHSD1 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-βHSD1 expression while increasing 11-βHSD2 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.Fil: López Torres, Manuel Othoniel. Instituto Nacional de la Nutrición Salvador Zubiran; MéxicoFil: Marquina Castillo, Brenda. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Ramos Espinosa, Octavio. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Mata Espinosa, Dulce. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Barrios Payan, Jorge A.. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Baay Guzman, Guillermina. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Huerta Yepez, Sara. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Bini, Estela Isabel. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Torre Villalvazo, Ivan. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Torres, Nimbe. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Tovar, Armando. Instittuto de Ciencias Médicas y Nutrición; MéxicoFil: Chamberlin, William. No especifíca;Fil: Ge, Yu. No especifíca;Fil: Carranza, Maria Andrea. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto Alberto C. Taquini de Investigaciones En Medicina Traslacional. - Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiologicas "prof. Dr. Alberto C. Taquini". Instituto Alberto C. Taquini de Investigaciones En Medicina Traslacional.; ArgentinaFil: Hernández Pando, Rogelio. Instituto Nacional de Ciencias Medicas y Nutricion; Méxic

Inmunidad y esteroides sexuales en la tuberculosis estudios clínicos y experimentales

La relación entre hombres y mujeres en cuanto al padecimiento de la tuberculosis pulmonar es 7/3 a favor de los primeros. Las hormonas sexuales pueden ser un factor a tener en cuenta en esta diferencia, dado los efectos inhibitorios de la testosterona sobre la activación de macrófagos y la producción de citocinas pro-inflamatorias, mientras que los estrógenos son más proclives a inducir la producción de estos mediadores. En función de estas cuestiones, en una primera etapa el presente trabajo de tesis estuvo orientado a comparar la evolución de la tuberculosis en ratones machos y hembras en base a la utilización de un modelo de enfermedad progresiva. Ratones BALB/c, machos y hembras se asignaron al azar en dos grupos: castrados o con operación simulada y se infectaron por vía intratraqueal con una alta dosis de la cepa de Mycobacterium tuberculosis H37Rv. Los ratones fueron sacrificados a diferentes momentos y en sus pulmones se determinaron la carga bacilar, la reacción inflamatoria, expresión de ARN mensajeros para diferentes citocinas, la supervivencia y niveles de testosterona en suero. Los ratones machos no castrados mostraron mayor mortalidad y carga bacilar durante la enfermedad avanzada respecto de las hembras y machos castrados. En comparación con los machos sin manipular, las hembras y machos castrados mostraron una mayor inflamación en todos los compartimentos de pulmón, como así también una reacción granulomatosa y desarrollo de neumonía más precoz, mientras que entre los orquitectomizados y las hembras no hubo diferencias significativas. Las hembras y machos castrados mostraron una mayor expresión de mARN para TNFα, IFNγ, IL-12, iNOS e IL-17 respecto de los machos no castrados, durante el primer mes de la infección. Los niveles de testosterona de los machos se hallaron aumentados durante la infección tardía. La orquitectomía al día 60 post-infección produjo una disminución significativa de la carga bacilar en coexistencia con una mayor expresión de TNFα, IL-12 e IFNγ. Resulta claro que en el modelo analizado, los ratones machos resultaron más susceptibles a la tuberculosis y este fenómeno fue revertido por la castración lo cual habla del rol de la testosterona en este déficit de la respuesta defensiva. La lógica para el estudio en pacientes tuberculosos se contextualizó en torno a la naturaleza crónica de la tuberculosis y la reacción inflamatoria acompañante la cual está implicada en una serie de cambios metabólicos e inmuno-endócrinos típicos de la enfermedad. Para profundizar en torno a esta problemática nos propusimos explorar en primer término las variaciones en cuanto a los componentes del eje hipotálamo-hipofisario- gonadal y su relación con las citocinas claramente inmunopatológicas en tuberculosis, en pacientes varones con enfermedad activa. Se utilizaron muestras de plasma de 36 casos no tratados con TB pulmonar, HIV negativos en quienes se determinaron los niveles de TNFα, IFNγ, TGF-β, IL-6, cortisol, dehidroepiandrosterona, testosterona, progesterona, estradiol, hormona luteinizante (LH) y la hormona folículo-estimulante (por ELISA), en simultáneo con 21 voluntarios sanos sin contacto con pacientes tuberculosos y edad similar (media general 40 ± 16.8 años, desvío estándar). Los pacientes mostraron disminución de los niveles de testosterona en presencia de altas cantidades de LH, junto con concentraciones aumentadas de IFNγ, IL-6 y TGF-β. Dado que esta disociación entre LH elevada y testosterona descendida sugería la existencia de algún fenómeno inhibitorio a nivel gonadal, se procesaron muestras histológicas testiculares de necropsias de pacientes que mueren de tuberculosis para la inmunomarcación de IL-1β, TNFα, IL-6 e IFNγ, habida cuenta que estas citocinas podría interferir con la síntesis de esteroides. Secciones histológicas testiculares mostraron abundante presencia de IL-1β, TNFα, IL-6 e IFNγ en macrófagos intersticiales, células de Sertoli y algunas espermatogonias. A partir de esta evidencia se decidió trabajar con células de Leydig murinas (línea celular TM3) la cual fue expuesta a diferentes concentraciones de citocinas de ratón relevantes en la inmunopatología de la tuberculosis como TNFα, IFNγ TGF-β. En el tratamiento in vitro de células de Leydig con estas citocinas condujo a una notable reducción de la producción de testosterona. Ello pone de relieve las implicancias de la respuesta inflamatoria sobre la síntesis de esteroides gonadales con todas las eventuales repercusiones que esto podría tener en torno a las acciones de este andrógeno sea como hormona anti-inflamatoria, inmunomoduladora, anabólica y obviamente de la reproducción

The Role of High Mobility Group Box 1 Protein (HMGB1) in the Immunopathology of Experimental Pulmonary Tuberculosis

Background The high mobility group box 1 (HMGB1) is the prototype of alarmin protein released by stressed or dying cells. The redox state of this protein confers different functions in the regulation of inflammation and immune response. Aim Determine the kinetics, cellular sources and function of HMGB1 in experimental tuberculosis. Methods BALB/c mice were infected with Mycobacterium tuberculosis strain H37Rv. At different time points, HMGB1 was quantified in bronchial lavage fluid (BALF) and in lungs was determined its cellular sources by immunohistochemistry. HMGB1 was blocked with specific antibodies or recombinant HMGB1 was administered during early or late infection. Bacilli burdens, inflammation and cytokines expression were determined. Results The maximal concentration of HMGB1 in BALF was at day one of infection. Bronchial epithelium and macrophages were the most important sources. At day 7 to 21 the oxidized HMGB1 was predominant, while during late infection only the reduced form was seen. Blocking HMGB1 during early infection produced significant decrease of bacilli burdens and high production of pro-inflammatory cytokines, while the opposite was seen when HMGB1 was administered. Blocking HMGB1 activity or administrated it in high amounts during late infection worsening the disease. Conclusions HMGB1 is liberated during experimental tuberculosis and promotes or suppress the immune response and inflammation depending on the redox state.Fil: Bottasso, Oscar. Institute of Experimental and Clinic Immunology, Rosario, IDICER, CONICET, School of Medical Sciences. UNR. Rosario; ArgentinaFil: Bini, Estela Isabel. Institute of Experimental and Clinic Immunology, Rosario, IDICER, CONICET, School of Medical Sciences. UNR. Rosario; Argentin

The influence of sex steroid hormones in the immunopathology of experimental pulmonary tuberculosis.

The relation between men and women suffering pulmonary tuberculosis is 7/3 in favor to males. Sex hormones could be a significant factor for this difference, considering that testosterone impairs macrophage activation and pro-inflammatory cytokines production, while estrogens are proinflammatory mediator's inducer. The aim of this work was to compare the evolution of tuberculosis in male and female mice using a model of progressive disease. BALB/c mice, male and female were randomized into two groups: castrated or sham-operated, and infected by the intratracheal route with a high dose of Mycobacterium tuberculosis strain H37Rv. Mice were euthanized at different time points and in their lungs were determined bacilli loads, inflammation, cytokines expression, survival and testosterone levels in serum. Non-castrated male mice showed significant higher mortality and bacilli burdens during late disease than female and castrated male animals. Compared to males, females and castrated males exhibited significant higher inflammation in all lung compartments, earlier formation of granulomas and pneumonia, while between castrated and non-castrated females there were not significant differences. Females and castrated males expressed significant higher TNF-α, IFN γ, IL12, iNOS and IL17 than non-castrated males during the first month of infection. Serum Testosterone of males showed higher concentration during late infection. Orchidectomy at day 60 post-infection produced a significant decrease of bacilli burdens in coexistence with higher expression of TNFα, IL-12 and IFNγ. Thus, male mice are more susceptible to tuberculosis than females and this was prevented by castration suggesting that testosterone could be a tuberculosis susceptibility factor

Cellular sources of HMGB1 determined by immunohistochemistry.

<p>(A) Lung section from a mouse after one day of infection, there is strong HMGB1 immunostaining in the cytoplasm of bronchial epithelial cells. (B) After 60 days of infection, the bronchial epithelium show scarce immunereactivity (asterisk), while macrophages in the pneumonic areas show strong immunostaining (arrows). (C) The morphometric study shows a progressive increase of macrophages with HMGB1 immunostaining. (D) Immunoelectronmicroscopy micrograph from a bronchial epithelial cell after one day of infection, there are several submembranal vesicles (V) with HMGB1 detected with antibodies labeled with colloidal gold (arrows). (E) An apoptotic macrophage from day seven of infection shows many black dots that correspond to HMGB1 detected with specific antibodies labelled with colloidal gold (arrows).</p

Effect of administrate recombinant HMGB1 during early infection on bacilli burdens and inflammation.

<p>(A) Groups of infected BALB/c mice with <i>M</i>.<i>tuberculosis</i> strain H37Rv were treated with recombinant human HMGB1 administered each other day by intratracheal route since the first day of infection and during three weeks (solid symbols); control animals received human albumin (white symbols). (B) The lungs from three mice per each sacrifice day were used to determine the area in square microns occupied by inflammatory cells in the indicated lung compartment in mice that received by intratracheal route recombinant human HMGB1(black bars) or human albumin (control group,white bars). Asterisks represent statistical significance (p<0.05 student T test). Representative micrographs of lungs from mice treated with recombinant HMGB1 or control animals: (C) After 28 days of infection, treated mouse with recombinant HMGB1 show areas of pneumonia (asterisk). (D) In contrast, control mouse at the same time of infection show perivascular inflammation (arrow), without pneumonic areas. (E) Ocassional cells show foxP3 immunostaining around blood vessels (arrows) in control mice at 7 days of infection. (F) In comparison, more inflammation and foxP3 immunostained cells (arrows) are seen in mice treated with recombinant HMGB1 at 14 days of infection. (G) Control mouse show some foxP3 immunostained cells in the pneumonic areas after 60 days of infection (arrows). (H) The same areas show some IL-10 immunostained cells (arrows). (I) The lung of a mouse treated with recombinant HMGB1 during late infection show more foxP3 immunostained cells around blood vessel and in the pneumonia areas (J).</p

Effect of blocking HMGB1 during early infection on bacilli loads and inflammation.

<p>(A) Groups of infected BALB/c mice with <i>M</i>.<i>tuberculosis</i> strain H37Rv were treated with blocking specific antibodies against HMGB1 administered each other day since the first day of infection and during three weeks (solid symbols); control animals received isotype irrelevant chicken antibodies (white symbols). (B) The lungs from three mice per each sacrifice day were used to determine the area in square microns occupied by inflammatory cells in the indicated lung compartment, in mice treated with HMGB1 blocking antibodies (black bars) and control group (white bars). Asterisks represent statistical significance (p<0.05 student T test). (C) Representative lung micrograph from a mouse treated with blocking HMGB1 antibodies after one week of infection, there are numerous lymphocytes around blood vessels (BV), airways (AW) and alveolar-capillary interstitium (arrow). (D) In contrast, control mouse show lower inflammation in the same lung compartments after 7 days of infection. (E) After one month of infection, the lung of mouse treated with blocking antibodies shows high inflammatory response around blood vessels (arrows) and in the interstitium (asterisk) but there is not pneumonia. (F) In comparison, the lung of control mouse show focal patches of pneumonia (asterisk). (All figures H/E staining, scale bar = 100μ).</p

Close Related Drug-Resistance Beijing Isolates of Mycobacterium tuberculosis Reveal a Different Transcriptomic Signature in a Murine Disease Progression Model

Mycobacterium tuberculosis (MTB) lineage 2/Beijing is associated with high virulence and drug resistance worldwide. In Colombia, the Beijing genotype has circulated since 1997, predominantly on the pacific coast, with the Beijing-Like SIT-190 being more prevalent. This genotype conforms to a drug-resistant cluster and shows a fatal outcome in patients. To better understand virulence determinants, we performed a transcriptomic analysis with a Beijing-Like SIT-190 isolate (BL-323), and Beijing-Classic SIT-1 isolate (BC-391) in progressive tuberculosis (TB) murine model. Bacterial RNA was extracted from mice lungs on days 3, 14, 28, and 60. On average, 0.6% of the total reads mapped against MTB genomes and of those, 90% against coding genes. The strains were independently associated as determined by hierarchical cluster and multidimensional scaling analysis. Gene ontology showed that in strain BL-323 enriched functions were related to host immune response and hypoxia, while proteolysis and protein folding were enriched in the BC-391 strain. Altogether, our results suggested a differential bacterial transcriptional program when evaluating these two closely related strains. The data presented here could potentially impact the control of this emerging, highly virulent, and drug-resistant genotype