miR-30c is specifically repressed in patients with active pulmonary tuberculosis.

Tuberculous pleurisy (PLTB) is a common form of extrapulmonary tuberculosis. It often resolves without chemotherapy being hence considered a rather benign manifestation of the disease. Patients with PLTB mount an effective anti-mycobacterial response, unlike those with active pulmonary TB (pTB) that were shown to present an imbalance in plasma immune and endocrine mediators. In this work, we explored whether expression of the active isoform of the glucocorticoid receptor (hGRα) in the context of the inflammatory-anti-inflammatory responses of TB patients may be associated to microRNA levels. As expected, the inflammatory response triggered in patients coexists with increased circulating cortisol and altered hGRα levels in the peripheral blood mononuclear cells. However, while hGRα expression is significantly downregulated in PLTB, its levels in pTB patients are higher within the control values. These results point out to the existence of an additional mechanism tending to preserve hGRα levels probably to deal with the chronic inflammation observed in pTB. In this regard, we found that miR-30c is strongly downregulated in mononuclear cells of pTB patients compared to PLTB cases, showing an expression profile opposite to that seen with hGRα. Interestingly, low levels of miR-30c are specific for this active form of TB, as its expression is not altered in mononuclear cells from either healthy controls or patients with tuberculous or non-tuberculous pleurisy. Moreover, miR-30c and hGRα also showed an inverse expression pattern in M. tuberculosis-stimulated THP-1 macrophage cultures. In sum, our studies identify miR-30c as a specific correlate of pulmonary manifestations of TB, potentially involved in the altered glucocorticoid sensitivity observed in these patients.

Levels of inflammatory cytokines, adrenal steroids, and mRNA for GRα, GRß and 11ßHSD1 in TB pleurisy.

Our previous work on the immune-endocrine features of patients with pulmonary tuberculosis (TB) showed markedly decreased plasma levels of dehydroepiandrosterone (DHEA) together with augmented concentrations of Cortisol and pro- and anti-inflammatory cytokines. Studies in peripheral blood mononuclear cells (PBMC) indicated a lower mRNA α/ß ratio of glucocorticoid receptors -GR- together with a higher 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) mRNA expression in cases with severe pulmonary TB. Since Pleural TB (PLTB) is a rather benign manifestation of TB, we now analyzed the systemic and local immune-endocrine profile as well as the GRα, GRß, 11ßHSD1 and 11ßHSD2 transcripts in PBMC and pleural effusion mononuclear cells (PEMC) of patients with PLTB. PLTB patients had increased levels of IL-1ß, IL-6 and IFNγ together with reduced Cortisol and DHEA concentrations in pleural fluids. Also, a significantly increased expression of 11ßHSD1 and GRα was found in PEMC compared to PBMC. Findings point out to an appropriate immune response and a substantial inflammatory reaction, wherein the low Cortisol concentrations may be equally effective, because of the increased expression of GRα and 11ßHSD1 transcripts which may optimize the immunomodulatory properties of Cortisol.

Altered microRNA expression levels in mononuclear cells of patients with pulmonary and pleural tuberculosis and their relation with components of the immune response.

Different lines of evidence demonstrate that microRNAs (miRNAs) play an important role in host-pathogen interactions. In this study we investigated the expression patterns of several miRNAs, most of them involved in regulating inflammatory responses, in patients with tuberculosis (TB). In order to understand the events occurring at the site of infection, we employed mononuclear cells obtained from both peripheral blood (PBMC) and pleural fluids (PFMC) of patients. Interestingly, we found that the miRNA signature of each compartment is different, with a strong down-regulation in PFMCs of miR-223, miR-144* and miR-421. In addition, we observed that miR-146a expression is also down-regulated in tuberculosis patients, both in PBMCs and PFMCs while miR-424 levels are elevated only in the peripheral compartments. We also showed that systemic expression of these miRNAs changes upon specific treatment and is associated with IL-6 levels, a cytokine playing a substantial role in TB immunopathology. Present results contribute to a better knowledge of the host responses in TB pathogenesis, pointing out the role of miRNAs in this disease.

Changes in the immune and endocrine responses of patients with pulmonary tuberculosis undergoing specific treatment.

We evaluated immune and endocrine status following antituberculosis treatment in HIV-negative patients with newly diagnosed tuberculosis (TB). Treatment led to a decrease in IL-6, IL-1ß, and C-reactive protein levels. Cortisol levels decreased throughout the anti-TB treatment, particularly after 4 months, but changes were less pronounced than those seen in proinflammatory mediators. Specific therapy resulted in increased dehydroepiandrosterone (DHEA) levels, which peaked after 4 months and started to decline after 6 months of treatment, reaching levels below those detected at inclusion. In contrast, in most patients, dehydroepiandrosterone sulfate (DHEAS) levels remained unchanged, although a trend toward increased concentrations was observed in a few cases 3 months after the treatment was finished. Specific therapy also resulted in more balanced cortisol/DHEA and cortisol/DHEAS ratios. Etiologic treatment involves favorable immune and endocrine changes, which may account for its beneficial effects.

Studies on the activating capacity of heat killed M. tuberculosis and its culture filtrate proteins on polymorphonuclear neutrophils and peripheral mononuclear cells from tuberculosis patients

En este estudio se investiga la eficacia de M. tuberculosis muerto porcalor (Mtbi) y las Proteinas del Filtrado del Cultivo (PFC) en la activación de las células mononucleares (MC) y polimorfonucleares neutrolilos (PMN)de sangre periférica de pacientes tuberculosos. Se evalua en 16 pacientes tuberculosos, HIV- y 12 controles sanos el Estallido Respiratorio, los metabolitos derivados del NO y la producción de IL-2, IL-12 y TNFeÁ por las células estimuladas. Se detecta un incremento en la concentración de TNFeÁ en el sobrenadante de cultivo (s.c.) de PMN al comparar con los valoresbasales y en la evaluada en s.c. de MC y PMN estimulados, al ser comparadas con las del grupo control, excepto para los neutrófilos estimuladoscon PFC. Se mostraron niveles aumentados de IL-12 e IL-2 en s.c. de ambas células, MC y PMN estimuladas por en PTB, mientras que no se hallarondiferencias en los s.c. de los controles. Los valores basales de Estallido Respiratorio (RB) detectada en MC y PMN de pacientes no difirieron significantivamente de los correspondientes al grupo control. La expresión del Estallido Respiratorio en ambos tipos celulares fue menor en los pacientes que en los controles, independientemente del estímulo empleado. Sedeterminaron concentraciones de nitritos más elevadas en los sobrenadantesde las MC estimuladas con Mtbi y PFC provenientes de pacientes, comparadas con las de los controles. Los datos obtenidos relacionados al estímulo de la respuesta celular, nos proporcionan información sobre la inmunidad protectiva contra el M. tuberculosis y, a la vez, aportan algunos recursos útiles para una terapia anti-tuberculosa más eficiente (AU)

The efficacy of heat-killed Mycobacterium tuberculosis (HKMtb) andits culture filtrate proteins (CFP) to activate blood mononuclear cells (MC)and polymorphonuclear neutrophils (PMN) from tuberculosis patientswas investigated. Respiratory burst, NO-derived metabolites, IL-2, IL-12and TNF-¦Á production of stimulated cells from 16 HIV- tuberculosispatients and 12 healthy controls were analyzed. Increased amounts ofTNF-¦Á in supernatants from baseline and stimulated polymorphonuclear and mononuclear cells of tuberculosis patients were detected whencompared with controls, except for CFP stimulated neutrophils. Augmented IL-2 and IL-12 levels were observed in supernatants of both stimulated MC and PMN from TBP while no differences were found in control supernatants. The patients had a lower respiratory burst responsethan the controls, for both cell types, regardless of the stimulus employed. Higher nitrite concentrations were found in HKMtb- and CFP-stimulated mononuclear supernatants from patients, compared with controls. The obtained data of the stimulated cellular responses provides usinformation about the protective immunity against Mycobacterium tuberculosis and some resources to obtain a more efficient anti-tuberculous therapy (AU)1

The adrenal steroid response during tuberculosis and its effects on the mycobacterial-driven IFN-gamma production of patients and their household contacts.

Earlier studies revealed that patients with tuberculosis (TB) have imbalanced immunoendocrine responses and that adrenal steroids [cortisol and dehydroepiandrosterone (DHEA)] can modify their specific cell-mediated immune response. Because most household contacts (HHCs) of contagious TB patients develop a subclinical and self-controlled process (latent TB), we studied some features of their immune and endocrine responses, particularly those related to the hypothalamic-pituitary-adrenal axis. Nineteen HHCs, 24 untreated TB patients (15 moderate, 9 advanced), and 18 healthy controls of similar age were studied. Patients had increased and reduced levels of cortisol and DHEA, respectively. DHEA levels were also reduced in HHCs. Stimulation of peripheral blood mononuclear cells (PBMC) with Mycobacterium tuberculosis sonicate resulted in increased in vitro lymphoproliferation in HHCs, while advanced patients showed the lowest response. Significantly higher amounts of interferon (IFN)-gamma were detected in supernatants from stimulated PBMC of HHCs when compared to controls and TB patients. Addition of cortisol to the cultures inhibited mycobacterial antigen-driven IFN-gamma production in all groups, although HHC supernatant contained significantly higher concentrations. In contrast, addition of DHEA to cultures of cells from HHCs resulted in increased IFN-gamma levels. These results suggest the existence of a particular immunoendocrine relation assuring a preserved IFN-gamma production in healthy housemates of TB patients.

Functional characteristics of neutrophils and mononuclear cells from tuberculosis patients stimulated in vitro with heat killed M. tuberculosis.

BACKGROUND: The major protective immune response against intracellular bacteria, such as Mycobacterium tuberculosis, is a cell-mediated immunity involving neutrophils (PMNs) and peripheral mononuclear cells (MCs), contributing to the clearance of this microorganism and the resolution of the infection. This study was addressed to evaluate PMNs and MCs for their bactericidal function. METHODS: The sample comprised 14 tuberculosis (TB) inpatients (HIV-), and 10 healthy controls (HCo). Peripheral PMNs and MCs were separated by Ficoll-Hypaque and cultured in RPMI with or without heat-killed Mycobacterium tuberculosis (HK Mtb). Respiratory burst (RB), CD11b, IL-8 and TNFalpha receptor expression were assessed by flow cytometry in cells undergoing stimulation or not. Presence of IL-8 and TNFalpha in cell culture supernatants was determined by ELISA. RESULTS: TB patients had a lower RB response than HCo for both cell types (MCs, p <0.05, PMNs, p <0.01) regardless of HK Mtb stimulation. Compared to HCo, PMNs and MCs from TB patients presented a reduced CD11b expression, with the two subject groups showing a decrease in this marker expression following HK. Mtb was added to both cell cultures. Whereas fewer IL-8 and TNFalpha receptors were found when studying MCs and PMNs from TB patients, antigen stimulation significantly raised the expression for both cytokine receptors. Culture supernatants from MCs and PMNs of TB patients contained increased amounts of IL-8 and TNFalpha. CONCLUSIONS: The present findings may provide some explanation as to the different roles played by PMNs and MCs in TB immunopathology.

Endocrine and cytokine responses in humans with pulmonary tuberculosis.

Endocrine responses during chronic infections such as lung tuberculosis are poorly characterized. Hormonal changes are likely to occur since some of the cytokines produced during this disease could affect endocrine mechanisms that, in turn, influence the course of infectious/inflammatory processes. A main purpose of this work was to study endocrine responses involving pituitary, adrenal, gonadal, and thyroid hormones in parallel to IFN-gamma, IL-10, and IL-6 levels in tuberculosis patients with different degree of pulmonary involvement. We have also studied whether products derived from peripheral immune cells obtained from the patients can affect the in vitro production of adrenal steroids. The population studied comprised HIV-negative newly diagnosed, untreated male patients with mild, moderate, and advanced lung tuberculosis, and matched, healthy controls. IFN-gamma, IL-10, and IL-6 levels were elevated in patients with tuberculosis. Dehydroepiandrosterone and testosterone levels were profoundly decreased and growth hormone levels were markedly elevated in patients, in parallel to modest increases in cortisol, estradiol, prolactin, and thyroid hormone concentrations. Supernatants of peripheral blood mononuclear cells obtained from the patients and stimulated in vitro with Mycobacterium tuberculosis antigens significantly inhibited dehydroepiandrosterone secretion by the human adrenal cell line NCI-H295-R. These results support the hypothesis that at least some of the endocrine changes observed in the patients may be mediated by endogenous cytokines. The endocrine profile of tuberculosis patients would favor a reduction of protective cell-mediated immunity and an exacerbation of inflammation leading to perpetuation of the lung injury and to the hypercatabolic condition that characterizes this disease.

TNF-alpha, TGF-beta and NO relationship in sera from tuberculosis (TB) patients of different severity.

Tuberculosis (TB) is the main cause of death by infection diseases worldwide. Considering that NO, TNF-alpha and TGF-beta participate a great deal in TB immunopathogenesis, we wished to analyse whether these mediators showed some relationship with the degree of pulmonary affectation. The sample comprised 29 TB (HIV-), inpatients with mild-moderate (n = 10) or advanced (n = 19) newly-diagnosed disease, together with 12 healthy controls HCo. Serum nitrite was assessed by reducing nitrate to nitrite, and further measured by the Griess reaction. Levels of TNF-alpha and TGF-beta were determined by ELISA (R&D Systems). Serum levels of TNF-alpha were significantly higher in the advanced TB cases if compared with HCo, (p < 0.05 ) and from values of Mild-Moderate TB patients (p < 0.05). Serum levels of TGF-beta from advanced TB patients have increased values if compared with Hco (p < 0.005) and Mild-Moderate patients (p < 0.05). These values were also significantly different from Mild-Moderate cases + HCo (p = 0.01) Advanced TB patients had significantly reduced nitrite levels compared with those of Mild-Moderate patients and HCo (p < 0.002). Taken as a whole NO-derived metabolites in TB patients (M-M and Advanced cases) remained lower than values in HCo (p = 0.005) A negative correlation was found when comparing the two cytokines with nitrites(r = -0.44 ).TGF-beta and TNF-alpha were positively correlated (r = 0.44, p < 0.01), 0.44, p < 0.01. In synthesis, the inverse correlation found between both cytokines concentrations and NO levels in TB patients may be viewed as a consequence of a more predominant TGF-beta effect.