Tuberculosis Killing Research Articles

Steroid hormone modulates the production of cathelicidin and human β-defensins in lung epithelial cells and macrophages promoting Mycobacterium tuberculosis killing

Several studies have documented the interaction between the immune and endocrine systems as an effective defense strategy against tuberculosis, involving the production of several molecules and immunological processes. In this study, we determined the effect of cortisol and dehydroepiandrosterone (DHEA) on the production of antimicrobial peptides such as cathelicidin and human β-defensin (HBD) −2, and HBD-3 and their effect on intracellular growth of Mycobacterium tuberculosis (Mtb) in lung epithelial cells and macrophages. Our results showed that DHEA promotes the production of these antimicrobial peptides in infected cells, correlating with the decrease of Mtb bacilli loads. These results suggest the use of exogenous DHEA as an adjuvant for tuberculosis therapy.

Effect of specimen processing, growth supplement, and different metabolic population on Mycobacterium tuberculosis laboratory diagnosis.

IntroductionSputum specimen decontamination steps are essential due to the presence of other saprophytic and infectious organisms. However, they negatively affect the mycobacterial recovery. In addition, little is known about the Mycobacterium tuberculosis killing efficacy of the PANTA (polymyxin-B, amphotericin-B, nalidixic acid, trimethoprim, azilocillin) antibiotics. Moreover, M. tuberculosis can be present in more than one metabolic population, but the effect of different growth characteristics on the mycobacterial growth indicator tube (MGIT) based time-to-positive (TTP) is not well studied.MethodsWe performed—(1) experiments using the solid agar and MGIT method to determine the effect of the NALC-NaOH decontamination method, (2) concentration-response studies with each individual antibiotic in the PANTA, and (3) the effect of the M. tuberculosis metabolic population on the TTP. TTP was recorded using the Epicenter software and exponential growth equation was used to calculate the doubling time of the bacteria, whereas, CFU/mL was analyzed using the Inhibitory Sigmoid Emax model for each antibiotic.ResultsDecontamination resulted in 4.36+0.13 log10 CFU/mL difference in cultures treated with NALC-NaOH versus no decontamination process and the limit of detection decreased from 1.47 log10 CFU/mL to the 0.42 log10 CFU/mL following NALC-NaOH treatment. PANTA at currently used antibiotic concentrations, did not had negative effect on mycobacterial recovery. Exponential growth model estimated doubling time for the log-phase growth M. tuberculosis as 2.04 days, for the semi-dormant bacilli as 2.80 days, and 6.37 days for the anaerobic cultures.ConclusionSpecimen decontamination method negatively affect the laboratory diagnosis of M. tuberculosis, polymyxin-B and nalidixic acid have anti-tuberculosis efficacy at high concentrations, and the doubling time of different metabolic population should be considered when deciding the time-in-protocol for the MGIT system.

Metformin promotes Mycobacterium tuberculosis killing and increases the production of human β-defensins in lung epithelial cells and macrophages

Diabetes has been associated with an increased risk of developing tuberculosis. The reasons related to the increased susceptibility to develop TB in type 2 diabetes mellitus (T2DM) individuals, has not been completely elucidated. However, this susceptibility has been attributed to several factors including failures and misfunctioning of the immune system. In the present study, we aimed to determine the role of anti-hyperglycemic drugs such as glyburide, insulin, and metformin to promote the killing of mycobacteria through the regulation of innate immune molecules such as host defense peptides (HDP) in lung epithelial cells and macrophages. Our results showed that metformin reduces bacillary loads in macrophages and lung epithelial cells which correlates with higher production of β-defensin-2, -3 and -4. Since β-defensins are crucial molecules for controlling Mycobacteriumtuberculosis growth, the present results suggest that the use of metformin would be the first choice in the treatment for T2DM2, in patients within tuberculosis-endemic areas.

CD157 Confers Host Resistance to Mycobacterium tuberculosis via TLR2-CD157-PKCzeta-Induced Reactive Oxygen Species Production.

Recruitment of monocytes to the infection site is critical for host resistance against Mycobacterium tuberculosis CD157 has a crucial role in neutrophil and monocyte transendothelial migration and adhesion, but its role in tuberculosis (TB) is unclear. Here, we show that both mRNA and protein levels of Cd157 are significantly increased during M. tuberculosis infection. Deficiency of Cd157 impaired host response to M. tuberculosis infection by increasing bacterial burden and inflammation in the lung in the murine TB model. In vitro experiments show that the bactericidal ability was compromised in Cd157 knockout (KO) macrophages, which was due to impaired M. tuberculosis-induced reactive oxygen species (ROS) production. We further reveal that CD157 interacts with TLR2 and PKCzeta and facilitates M. tuberculosis-induced ROS production in Cd157 KO macrophages, which resulted in enhanced M. tuberculosis killing. For the clinic aspect, we observe that the expression of CD157 decreases after effective anti-TB chemotherapy. CD157 is specifically increased in pleural fluid in tuberculous pleurisy patients compared to pneumonia and lung cancer patients. Interestingly, the levels of soluble CD157 (sCD157) correlate with human peripheral monocyte-derived macrophage bactericidal activity. Exogenous application of sCD157 could compensate for macrophage bactericidal ability and restore ROS production. In conclusion, we have identified a novel protective immune function of CD157 during M. tuberculosis infection via TLR2-dependent ROS production. Application of sCD157 might be an effective strategy for host-directed therapy against TB in those with insufficient CD157 production.IMPORTANCE Tuberculosis, a chronic bacterial disease caused by Mycobacterium tuberculosis, remains a major global health problem. CD157, a dual-function receptor and β-NAD+-metabolizing ectoenzyme, promotes cell polarization, regulates chemotaxis induced through the high-affinity fMLP receptor, and controls transendothelial migration. The role of CD157 in TB pathogenesis remains unknown. In this study, we find that both mRNA and protein levels of CD157 are significantly increased in TB. Deficiency of CD157 impaired host defense against M. tuberculosis infection both in vivo and in vitro, which is mediated by an interaction among CD157, TLR2, and PKCzeta. This interaction facilitates M. tuberculosis-induced macrophagic ROS production, which enhances macrophage bactericidal activity. Interestingly, the sCD157 level in plasma is reversibly associated with MDM M. tuberculosis killing activity. By uncovering the role of CD157 in pathogenesis of TB for the first time, our work demonstrated that application of soluble CD157 might be an effective strategy for host-directed therapy against TB.

In vitro antimycobacterial activity of 2-(((2-hydroxyphenyl)amino)methylene)-5,5-dimethylcyclohexane-1,3-dione: a new chemical entity against Mycobacterium tuberculosis

This study reports on the in vitro antituberculosis potential of 2-(((2-hydroxyphenyl) amino)methylene)-5,5-dimethylcyclohexane-1,3-dione (PAMCHD) against Mycobacterium tuberculosis H37Rv. PAMCHD has been proven to be a tuberculostatic as well as a tuberculocidal agent by agar and broth dilution methods with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values equivalent to some standard antituberculosis drugs (ATDs). The dynamics of M. tuberculosis killing revealed the time- as well as concentration-dependent antituberculosis activity of PAMCHD and it sterilized M. tuberculosis culture at or above 10.0 µg/mL. PAMCHD acts either synergistically or additively with ATDs. Isoniazid (INH) and PAMCHD post-antibiotic effects increased with concentration from 16.18 ± 13.30 and 31.64 ± 13.30 to 127.9 ± 27.60 and 138.71 ± 16.42 h, respectively, from 1 × MIC to 8 × MIC; no significant difference was observed between INH and PAMCHD post-antibiotic effects. M. tuberculosis mutation frequency against PAMCHD is lower than that of INH. Mutant prevention concentration (MPC) of INH, rifampin (RIF) and PAMCHD were observed to be 40, 160 and 160 µg/mL, respectively, and their MPC/MIC values were 128, 2051 and 64, respectively; this lowest MPC/MIC highlights the advantage of PAMCHD over RIF and INH.

Amikacin Optimal Exposure Targets in the Hollow-Fiber System Model of Tuberculosis.

Aminoglycosides such as amikacin are currently used for the treatment of multidrug-resistant tuberculosis (MDR-TB). However, formal pharmacokinetic/pharmacodynamic (PK/PD) studies to identify amikacin exposures and dosing schedules that optimize Mycobacterium tuberculosis killing have not been performed. It is believed that aminoglycosides do not work well under acidic conditions, which, if true, would mean poor sterilizing activity against semidormant bacilli at low pH. We performed time-kill studies to compare the bactericidal effect of amikacin in log-phase-growth bacilli with the sterilizing effect in semidormant bacilli at pH 5.8 in broth. In log-phase M. tuberculosis at normal pH versus semidormant M. tuberculosis at pH 5.8, the maximal kill (Emax) estimate and 95% confidence interval (CI) were 5.39 (95% CI, 4.91 to 5.63) versus 4.88 (CI, 4.46 to 5.22) log10 CFU/ml, while the concentration mediating 50% of Emax (EC50) was 1.0 (CI, 0. 0.86 to 1.12) versus 0.60 (CI, 0.50 to 0.66) times the MIC, respectively. Thus, the optimal exposures and kill rates identified for log-phase M. tuberculosis will be optimal even for semidormant bacilli. Next, we performed exposure-response and dose-scheduling studies in the hollow-fiber system model of tuberculosis using log-phase M. tuberculosis. We recapitulated the amikacin concentration-time profiles observed in lungs of patients treated over 28 days. The PK/PD index linked to M. tuberculosis kill was the peak concentration (Cmax)-to-MIC ratio (r2 > 0.99), closely followed by the area under the concentration-time curve from 0 to 24 h (AUC0–24)-to-MIC ratio (r2 = 0.98). The EC90 was a Cmax/MIC ratio of 10.13 (95% CI, 7.73 to 12.48). The EC90 is the dosing target for intermittent therapy that optimizes cure in TB programs for MDR-TB patients.

Dietary Vitamin D3 Suppresses Pulmonary Immunopathology Associated with Late-Stage Tuberculosis in C3HeB/FeJ Mice.

Tuberculosis (TB) is a significant human disease caused by inhalation of Mycobacterium tuberculosis. Left untreated, TB mortality is associated with a failure to resolve pulmonary immunopathology. There is currently widespread interest in using vitamin D3 (VitD3) as an adjunct therapy for TB because numerous in vitro studies have shown that VitD3 has direct and indirect mycobactericidal activities. However, to date, there have been no in vivo studies addressing whether VitD3 affects experimental TB outcome. In this study, we used C3HeB/FeJ mice to determine whether dietary VitD3 influences the outcome of experimental TB. We observed that although M. tuberculosis burdens did not differ between mice on a VitD3-replete diet (VitD(HI) mice) and mice on a VitD3-deficient diet (VitD(LO) mice), the inflammatory response in VitD(HI) mice was significantly attenuated relative to VitD(LO) controls. Specifically, the expression of multiple inflammatory pathways was reduced in the lungs at later disease stages as were splenocyte IL12/23p40 and IFN-γ levels following ex vivo restimulation. Dietary VitD3 also suppressed the accumulation of T cells in the mediastinal lymph nodes and lung granulomatous regions while concomitantly accelerating the accumulation of F4/80(+) and Ly6C/Ly6G(+) lineages. The altered inflammatory profile of VitD(HI) mice also associated with reductions in pulmonary immunopathology. VitD receptor-deficient (vdr(-/-)) radiation bone marrow chimeras demonstrate that reductions in pulmonary TB immunopathology are dependent on hematopoietic VitD responsiveness. Collectively, our data support a model wherein the in vivo role of VitD3 during TB is not to promote M. tuberculosis killing but rather to function through hematopoietic cells to reduce M. tuberculosis-elicited immunopathology.

The effect of hyperglycaemia on in vitro cytokine production and macrophage infection with Mycobacterium tuberculosis.

Type 2 diabetes mellitus is an established risk factor for tuberculosis but the underlying mechanisms are largely unknown. We examined the effects of hyperglycaemia, a hallmark of diabetes, on the cytokine response to and macrophage infection with Mycobacterium tuberculosis. Increasing in vitro glucose concentrations from 5 to 25 mmol/L had marginal effects on cytokine production following stimulation of peripheral blood mononuclear cells (PBMCs) with M. tuberculosis lysate, LPS or Candida albicans, while 40 mmol/L glucose increased production of TNF-α, IL-1β, IL-6 and IL-10, but not of IFN-γ, IL-17A and IL-22. Macrophage differentiation under hyperglycaemic conditions of 25 mmol/L glucose was also associated with increased cytokine production upon stimulation with M. tuberculosis lysate and LPS but in infection experiments no differences in M. tuberculosis killing or outgrowth was observed. The phagocytic capacity of these hyperglycaemic macrophages also remained unaltered. The fact that only very high glucose concentrations were able to significantly influence cytokine production by macrophages suggests that hyperglycaemia alone cannot fully explain the increased susceptibility of diabetes mellitus patients to tuberculosis.

Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis.

The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·(-), with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·(-)-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.

Type I IFN Induces IL-10 Production in an IL-27–Independent Manner and Blocks Responsiveness to IFN-γ for Production of IL-12 and Bacterial Killing in Mycobacterium tuberculosis–Infected Macrophages

Tuberculosis, caused by the intracellular bacterium Mycobacterium tuberculosis, currently causes ∼1.4 million deaths per year, and it therefore remains a leading global health problem. The immune response during tuberculosis remains incompletely understood, particularly regarding immune factors that are harmful rather than protective to the host. Overproduction of the type I IFN family of cytokines is associated with exacerbated tuberculosis in both mouse models and in humans, although the mechanisms by which type I IFN promotes disease are not well understood. We have investigated the effect of type I IFN on M. tuberculosis–infected macrophages and found that production of host-protective cytokines such as TNF-α, IL-12, and IL-1β is inhibited by exogenous type I IFN, whereas production of immunosuppressive IL-10 is promoted in an IL-27–independent manner. Furthermore, much of the ability of type I IFN to inhibit cytokine production was mediated by IL-10. Additionally, type I IFN compromised macrophage activation by the lymphoid immune response through severely disrupting responsiveness to IFN-γ, including M. tuberculosis killing. These findings describe important mechanisms by which type I IFN inhibits the immune response during tuberculosis.

Correlates of protective immunity to Mycobacterium tuberculosis in humans.

Correlates of protective immunity to Mycobacterium tuberculosis in humans are desirable for identifying protective antigens, demonstrating the immunogenicity of a vaccine candidate and its potential efficacy, and permitting optimization of the dose, vehicle, adjuvant, and schedule of immunization. Potential correlates can be proposed on the basis of animal models and ex vivo/in vitro studies in humans. Most critical is their validation; ultimate validation will require correlation with protection in a phase III efficacy trial of an effective vaccine. Other approaches, however, can allow selection of the most promising correlates for inclusion in phase I and II and, ultimately, phase III vaccine trials. Current data from experimental models and studies of patients with pulmonary tuberculosis and their household contacts indicate that Mycobacterium tuberculosis-stimulated whole-blood production of interferon-gamma, although imperfect, is the best available correlate. Nonetheless, further refinement of this assay and additional studies of more complex assays that model M. tuberculosis killing and cytotoxic T lymphocyte activity are warranted. During planning of a vaccine trial, the best available correlates of immunity can be selected for inclusion.