Mycobacterium Tuberculosis Infection In Mice Research Articles

Local pulmonary immunotherapy with siRNA targeting TGFβ1 enhances antimicrobial capacity in Mycobacterium tuberculosis infected mice

In this study we demonstrate that it is possible to shift the immune system during a chronic infection with Mycobacterium tuberculosis. TGFβ and IL10 cytokines inhibit the Th1 response during chronic pulmonary infection with M. tuberculosis. We show that intrapulmonary delivery of siRNA targeting TGFβ1 is able to reduce the pulmonary bacillary load in mice chronically infected with M. tuberculosis: an effect that appears to be partly dependent on IL10 expression. To demonstrate this, we induced gene silencing of tgfβ1 in the lungs of wild type and IL10 knockout mice using a non-invasive aerosolized intrapulmonary delivery of siRNA targeting TGFβ1. Five days after the last treatment with siRNA, the levels of tgfb1 transcripts and TGFβ1 protein were reduced when compared with control groups treated with RNase-free water or non-targeting siRNA. Mice treated with siRNA also had increased expression of the antimicrobial mediators (NO and iNOS) which effectively reduced the bacterial load by 0.17 and 0.47 log(10) in C57BL/6 and IL-10 KO mice respectively when compared with their respective control mice. More importantly, the bacterial load in siRNA treated IL-10 KO mice four weeks after the last treatment remained 0.32 log(10) lower than in control mice.

Blocking IL-1α but not IL-1β increases susceptibility to chronic Mycobacterium tuberculosis infection in mice

IL-1α and IL-1β are potent inflammatory cytokines and important mediators of immune responses to intracellular pathogens such as Mycobacterium tuberculosis (Mtb). Here, we investigated the role of IL-1α and IL-1β during chronic Mtb infection and spontaneous reactivation in mice. For long-term neutralization of IL-1α, IL-1β or both, mice were immunized with virus-like particles (VLPs) displaying either of the cytokines, inducing strong and long-lasting neutralizing IgG responses. Blocking of IL-1α but not of IL-1β resulted in increased susceptibility to chronic infection with Mtb. Neutralizing either IL-1α or IL-1β alone did not lead to increased reactivation of latent tuberculosis. The generation of antibodies neutralizing both IL-1α and IL-1β simultaneously, did not influence weight gain during Mtb reactivation and the slight increase in pulmonary bacillary counts were not significant when compared to control-immunized group. Thus, the results suggest that IL-1α is the major mediator of the IL-1RI-dependent and protective innate immune responses to Mtb in mice.

Global Reemergence of Tuberculosis: Are Host Defense Peptides an Option to Ameliorate Disease Burden?

Tuberculosis is the most relevant infectious disease worldwide according to the estimates of the World Health Organization, and despite being a curable disease, it requires a 6-9-month therapy with multiple antibiotics. Intermittent drug therapy due to noncompliance or poor delivery of therapy promotes the emergence of bacterial strains showing resistance to multiple drugs and the rise of extremely drug-resistant strains. Moreover, increased antibiotic resistance has been observed for several microorganisms, including extremely drug-resistant tuberculosis, vancomycin-resistant Enterococcus faecalis, or methicillin-resistant Staphylococcus aureus. In vitro, cathelicidin induction results in enhanced mycobacterial clearance, and synthetic human neutrophil peptides had a rather modest bactericidal effect in Mycobacterium tuberculosis-infected mice. In vivo therapeutic efficacy of improved molecules that show enhanced bactericidal action in vitro remains to be tested.

Characterization of murine T-cell epitopes on mycobacterial DNA-binding protein 1 (MDP1) using DNA vaccination

Mycobacterial DNA-binding protein 1 (MDP1) is a major protein antigen in mycobacteria and induces protective immunity against Mycobacterium tuberculosis infection in mice. In this study we determined murine T-cell epitopes on MDP1 with MDP1 DNA immunization in mice. We analyzed interferon-γ production from the MDP1 DNA-immune splenocytes in response to 20-mer overlapping peptides covering MDP1 protein. We identified several CD4+ T-cell epitopes in three inbred mouse strains and one CD8+ T-cell epitope in C57BL/6 mice. These T-cell epitopes would be feasible for analysis of the role of MDP1-specific T-cells in protective immunity and for future vaccine design against M. tuberculosis infection.

OL-061 A subunit vaccine consisting of fusion protein Ag85B-MPT64190-198-Mtb8.4 and adjuvant of dimethyl-dioctyldecyl ammonium and BCG polysaccharide nucleic acid enhances BCG-primed protection against Mycobacterium tuberculosis infection in mice

OL-061 A subunit vaccine consisting of fusion protein Ag85B-MPT64190-198-Mtb8.4 and adjuvant of dimethyl-dioctyldecyl ammonium and BCG polysaccharide nucleic acid enhances BCG-primed protection against Mycobacterium tuberculosis infection in mice

Synthetic EthR inhibitors boost antituberculous activity of ethionamide

The side effects associated with tuberculosis therapy bring with them the risk of noncompliance and subsequent drug resistance. Increasing the therapeutic index of antituberculosis drugs should thus improve treatment effectiveness. Several antituberculosis compounds require in situ metabolic activation to become inhibitory. Various thiocarbamide-containing drugs, including ethionamide, are activated by the mycobacterial monooxygenase EthA, the production of which is controlled by the transcriptional repressor EthR. Here we identify drug-like inhibitors of EthR that boost the bioactivation of ethionamide. Compounds designed and screened for their capacity to inhibit EthR-DNA interaction were co-crystallized with EthR. We exploited the three-dimensional structures of the complexes for the synthesis of improved analogs that boosted the ethionamide potency in culture more than tenfold. In Mycobacterium tuberculosis-infected mice, one of these analogs, BDM31343, enabled a substantially reduced dose of ethionamide to lessen the mycobacterial load as efficiently as the conventional higher-dose treatment. This provides proof of concept that inhibiting EthR improves the therapeutic index of thiocarbamide derivatives, which should prompt reconsideration of their use as first-line drugs.

A novel recombinant human lactoferrin augments the BCG vaccine and protects alveolar integrity upon infection with Mycobacterium tuberculosis in mice

Lactoferrin, an iron binding glycoprotein, possesses multiple immune modulatory activities, including the ability to promote antigen specific cell-mediated immunity. Previous studies showed that adding bovine lactoferrin to the BCG vaccine (an attenuated strain of Mycobacterium bovis Bacillus Calmette Guerin) resulted in increased host protective responses upon subsequent challenge with virulent Erdman Mycobacterium tuberculosis (MTB) in mice. The studies outlined here investigate utility of a novel recombinant human lactoferrin to enhance the BCG vaccine and protect against alveolar injury during experimental MTB infection in mice. Sialylated and non-sialylated forms of the recombinant human lactoferrin (rhLF), glycoengineered in yeast ( Pichia pastoris) and expressing humanized N-glycosylation patterns, were examined for their ability to enhance efficacy of the BCG vaccine in a murine TB model system. Results indicated that the sialylated form of the recombinant human lactoferrin generated increased antigen specific recall responses to BCG antigens. Furthermore, augmented protection was demonstrated using the sialylated lactoferrin adjuvant with BCG, resulting in significant reduction in associated pathology following challenge with virulent organisms.

RP105 Facilitates Macrophage Activation by Mycobacterium tuberculosis Lipoproteins

RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

Tumor Necrosis Factor Signaling Mediates Resistance to Mycobacteria by Inhibiting Bacterial Growth and Macrophage Death

Tumor necrosis factor (TNF), a key effector in controlling tuberculosis, is thought to exert protection by directing formation of granulomas, organized aggregates of macrophages and other immune cells. Loss of TNF signaling causes progression of tuberculosis in humans, and the increased mortality of Mycobacterium tuberculosis-infected mice is associated with disorganized necrotic granulomas, although the precise roles of TNF signaling preceding this endpoint remain undefined. We monitored transparent Mycobacterium marinum-infected zebrafish live to conduct a stepwise dissection of how TNF signaling operates in mycobacterial pathogenesis. We found that loss of TNF signaling caused increased mortality even when only innate immunity was operant. In the absence of TNF, intracellular bacterial growth and granuloma formation were accelerated and was followed by necrotic death of overladen macrophages and granuloma breakdown. Thus, TNF is not required for tuberculous granuloma formation, but maintains granuloma integrity indirectly by restricting mycobacterial growth within macrophages and preventing their necrosis.

Fcγ Receptors Regulate Immune Activation and Susceptibility during Mycobacterium tuberculosis Infection

The critical role of cellular immunity during tuberculosis (TB) has been extensively studied, but the impact of Abs upon this infection remains poorly defined. Previously, we demonstrated that B cells are required for optimal protection in Mycobacterium tuberculosis-infected mice. FcgammaR modulate immunity by engaging Igs produced by B cells. We report that C57BL/6 mice deficient in inhibitory FcgammaRIIB (RIIB-/-) manifested enhanced mycobacterial containment and diminished immunopathology compared with wild-type controls. These findings corresponded with enhanced pulmonary Th1 responses, evidenced by increased IFN-gamma-producing CD4+ T cells, and elevated expression of MHC class II and costimulatory molecules B7-1 and B7-2 in the lungs. Upon M. tuberculosis infection and immune complex engagement, RIIB-/- macrophages produced more of the p40 component of the Th1-promoting cytokine IL-12. These data strongly suggest that FcgammaRIIB engagement can dampen the TB Th1 response by attenuating IL-12p40 production or activation of APCs. Conversely, C57BL/6 mice lacking the gamma-chain shared by activating FcgammaR had enhanced susceptibility and exacerbated immunopathology upon M. tuberculosis challenge, associated with increased production of the immunosuppressive cytokine IL-10. Thus, engagement of distinct FcgammaR can divergently affect cytokine production and susceptibility during M. tuberculosis infection.

Multiple Mycobacterium antigens induce interferon-gamma production from sarcoidosis peripheral blood mononuclear cells.

Studies of sarcoidosis immunology have noted oligoclonal T cell populations, suggesting cell-mediated immunity that is antigen-specific. Sarcoidosis immunology and pathology are most similar to mycobacterial infections. Mycobacterium tuberculosis infection in mice and humans reflects T helper 1 (Th1) immune responses to multiple cell wall and secreted antigens. We investigated if the oligoclonal immune response in individual sarcoidosis subjects could be elicited by multiple secreted mycobacterial antigens by performing ex vivo enzyme-linked immunospot assay (ELISPOT) on peripheral blood mononuclear cells (PBMC) from 30 sarcoidosis, 26 purified protein derivative negative (PPD-) control and 10 latent tuberculosis subjects (PPD+) to assess Th1 responses to mycobacterial superoxide dismutase A (sodA), catalase-peroxidase (katG) and early secreted antigenic target protein (ESAT-6). A significant difference was noted among the sarcoidosis and PPD- control subjects to ESAT-6 [12 of 30 versus one of 26 (P = 0.0014)], katG [nine of 30 versus none of 26 (P = 0.002)] and sodA [12 of 30 versus none of 26 (P = 0.002)]. There was no significant difference between sarcoidosis and PPD+ subjects. Twelve sarcoidosis subjects recognized two or more mycobacterial proteins, as well as multiple distinct epitopes within individual proteins. One sarcoidosis subject on whom we collected bronchoalveolar lavage (BAL) fluid and PBMC had no recognition of mycobacterial antigens using PBMC, but BAL fluid demonstrated strong Th1 immune responses to ESAT-6 and katG. Individual sarcoidosis subjects recognized not only multiple mycobacterial proteins, but multiple distinct peptides within a specific protein, thus demonstrating that multiple mycobacterial epitopes elicit the Th1 immune response observed. Immune responses by sarcoidosis T cells to mycobacterial proteins may have an important role in sarcoidosis pathogenesis.

Inhibition of leukotriene biosynthesis abrogates the host control of Mycobacterium tuberculosis

Leukotrienes produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. Recently, it has been demonstrated that leukotrienes also play an important role in host defense against microorganisms. In vitro studies have shown that leukotrienes augmented the anti-mycobacterial activity of neutrophils. In this study, we examined the role of leukotrienes in regulating host response and cytokine generation in a murine model of tuberculosis. Administration of the 5-LO pathway inhibitor MK 886, which reduced lung levels of both the leukotriene B 4 and the anti-inflammatory substance lipoxin A 4 by ∼50%, increased 60-day mortality from 14% to ∼57% in Mycobacterium tuberculosis-infected mice, and increased lung bacterial burden by ∼15-fold. Although MK 886-treated animals exhibited no reduction in pulmonary leukocyte accumulation, they did manifest reduced levels of nitric oxide generation and of the protective type 1 cytokines interleukin-12 and gamma interferon. Together our results demonstrate that 5-LO pathway product(s) – presumably leukotrienes – positively regulate protective Th1 responses against mycobacterial infection in vivo. Moreover, the immunosuppressive phenotype in infected mice observed with MK 886 is most consistent with inhibition of an activator (LTB 4) rather than a suppressor (LXA 4) of antimicrobial defense, suggesting the major effect of leukotrienes.

Mycobacterium hsp65 DNA entrapped into TDM-loaded PLGA microspheres induces protection in mice against Leishmania (Leishmania) major infection

Heat shock proteins (HSPs) are highly conserved among different organisms. A mycobacterial HSP65 DNA vaccine was previously shown to have prophylactic and immunotherapeutic effects against Mycobacterium tuberculosis infection in mice. Here, BALB/c mice were immunized with mycobacterial DNA-hsp65 or with DNA-hsp65 and trehalose dymicolate (TDM), both carried by biodegradable microspheres (MHSP/TDM), and challenged with Leishmania (Leishmania) major. MHSP/TDM conferred protection against L. major infection, as indicated by a significant reduction of edema and parasite loads in infected tissues. Although high levels of interferon-gamma and low levels of interleukin (IL)-4 and IL-10 were detected in mice immunized with DNA-hsp65 or MHSP/TDM, only animals immunized with MHSP/TDM displayed a consistent Th1 immune response, i.e., significantly higher levels of anti-soluble Leishmania antigen (SLA) immunoglobulin G (IgG)2a and low anti-SLA IgG1 antibodies. These findings indicate that encapsulated MHSP/TDM is more immunogenic than naked hsp65 DNA, and has great potential to improve vaccine effectiveness against leishmaniasis and tuberculosis.

An oily, sustained counter-regulatory response to TB

Lipoxins are potent antiinflammatory lipid mediators that restrain and promote the resolution of a wide variety of inflammatory processes. Recent studies implicating deficient lipoxin production in the pathogenesis of diverse inflammatory diseases, along with numerous reports of the beneficial effects of lipoxin analog administration in animal models of inflammatory pathology, have suggested that harnessing the pleiotropic activities of the lipoxins is a strategy with considerable therapeutic promise. In this issue of the JCI, Bafica et al. address the other side of the coin, reporting that endogenous lipoxins compromise immune-mediated control of Mycobacterium tuberculosis infection in mice. In addition to providing novel insight into the mechanisms that interfere with the development of protective immune responses to M. tuberculosis, the study raises the possibility that pharmacological inhibition of lipoxin synthesis may provide a method of augmenting inefficient immune responses in TB and other important chronic infectious diseases.

A polyvalent DNA vaccine expressing an ESAT6–Ag85B fusion protein protects mice against a primary infection with Mycobacterium tuberculosis and boosts BCG-induced protective immunity

In this study, we evaluated the protective efficacy of a DNA vaccine (pE6/85) expressing an ESAT6–Ag85B fusion protein against a primary Mycobacterium tuberculosis infection in mice. In short-term studies, vaccination with pE6/85 protected as well as Mycobacterium bovis BCG immunization with similar lung pathology and bacterial burdens detected 28 days after a low dose aerogenic challenge (>1.0 log 10 reduction relative to naïves). In a survival experiment, the protection induced by pE6/85 immunization was also not significantly different than that elicited by BCG vaccination with the mean-times-to-death (±standard error of the mean) being 102 ± 20, 271 ± 32 and 299 ± 14 days for naïve, pE6/85 and BCG-vaccinated mice, respectively. Furthermore, boosting with pE6/85 but not BCG or a DNA vaccine cocktail at 1 year after an initial BCG immunization (when BCG-induced protection was declining), augmented protection in the lung at 15 and 18 months to levels detected at 3 months post-BCG vaccination.

Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8+ cells, interferon-gamma recovery and reduction of lung injury.

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-gamma recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-alpha. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-gamma and to restrict the growth of bacilli.

Subcutaneous nanoparticle-based antitubercular chemotherapy in an experimental model.

Poly (DL-lactide-co-glycolide) (PLG) nanoparticles encapsulating three front-line antitubercular drugs, i.e. rifampicin, isoniazid and pyrazinamide, were prepared by the multiple emulsion technique and administered subcutaneously to mice for pharmacokinetic/chemotherapeutic study. A single subcutaneous dose of drug-loaded PLG nanoparticles resulted in sustained therapeutic drug levels in the plasma for 32 days and in the lungs/spleen for 36 days. The mean residence time and absolute bioavailability were increased several-fold as compared with unencapsulated drugs. Further, drug-loaded PLG nanoparticles resulted in undetectable bacterial counts in the lungs and spleen of Mycobacterium tuberculosis-infected mice, thereby demonstrating a better chemotherapeutic efficacy, as compared with daily free drug treatment. Hence, injectable PLG nanoparticles hold promise for increasing drug bioavailability and reducing dosing frequency for better management of tuberculosis.

Nanoparticle encapsulated antitubercular drugs as a potential oral drug delivery system against murine tuberculosis

Patient non-compliance is the major drawback associated with the long-duration chemotherapy of tuberculosis (TB); hence, reduction in dosing frequency forms an important therapeutic strategy. The present study reports the formulation of three frontline antitubercular drugs (ATD), i.e. rifampicin (RIF), isoniazid (INH) and pyrazinamide (PZA) encapsulated in poly ( dl-lactide-co-glycolide) (PLG) nanoparticles. Drug encapsulation efficiencies were 56.9±2.7% for RIF, 66.3±5.8% for INH and 68±5.6% for PZA. Following a single oral administration of these preparations to mice, the drugs could be detected in the circulation for 6 days (RIF) and 9 days (INH/PZA), whereas therapeutic concentrations in the tissues were maintained for 9–11 days. Further, on oral administration of drug-loaded nanoparticles to Mycobacterium tuberculosis-infected mice at every 10th day, no tubercle bacilli could be detected in the tissues after 5 oral doses of treatment. Therefore, nanoparticle-based ATD therapy forms a sound basis for reduction in dosing frequency for better management of TB.

In vivo IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice.

The production of immunosuppressive cytokines, such as IL-10 and TGF-beta, has been documented in individuals diagnosed with active tuberculosis. In addition, IL-10 production is increased within the lungs of mice that have chronic mycobacterial infection. Therefore, we hypothesized that the down-regulatory properties of IL-10 might contribute to the reactivation of chronic Mycobacterium tuberculosis infection in mice. To determine the influence of IL-10 on the course of infection, transgenic mice producing increased amounts of IL-10 under the control of the IL-2 promotor were infected with M. tuberculosis via the respiratory route. Mice that overexpressed IL-10 showed no increase in susceptibility during the early stages of infection, but during the chronic phase of the infection showed evidence of reactivation tuberculosis with a highly significant increase in bacterial numbers within the lungs. Reactivation was associated with the formation of macrophage-dominated lesions, decreased mRNA production for TNF and IL-12p40, and a decrease in Ag-specific IFN-gamma secretion. These data support the hypothesis that IL-10 plays a pivotal role during the chronic/latent stage of pulmonary tuberculosis, with increased production playing a potentially central role in promoting reactivation tuberculosis.

Toll-like receptor 4 expression is required to control chronic Mycobacterium tuberculosis infection in mice.

Endotoxin from Gram-negative bacteria bound to CD14 signals through Toll-like receptor (TLR) 4, while components of Gram-positive bacteria, fungi, and Mycobacterium tuberculosis (M.tb.) preferentially use TLR2 signaling. We asked whether TLR4 plays any role in host resistance to M.tb. infection in vivo. Therefore, we infected the TLR4 mutant C3H/HeJ mice and their controls, C3H/HeN mice, with M.tb. by aerosol. TLR4 mutant mice had a reduced capacity to eliminate mycobacteria from the lungs, spread the infection to spleen and liver, with 10-100 times higher CFU organ levels than the wild-type mice and succumbed within 5-7 mo, whereas most of the wild-type mice controlled infection and survived the duration of the experiment. The lungs of TLR4 mutant mice showed chronic pneumonia with increased neutrophil infiltration, reduced macrophages recruitment, and abundant acid-fast bacilli. Furthermore, the pulmonary expression of TNF-alpha, IL-12p40, and monocyte chemoattractant protein 1 was significantly lower in C3H/HeJ mice when compared with the wild-type controls. C3H/HeJ-derived macrophages infected in vitro with M.tb. produced lower levels of TNF-alpha. Finally, the purified mycobacterial glycolipid, phosphatidylinositol mannosides, induced signaling in both a TLR2- and TLR4-dependent manner, thus suggesting that recognition of phosphatidylinositol mannosides in vivo may influence the development of protective immunity. In summary, macrophage recruitment and the proinflammatory response to M.tb. are impaired in TLR4 mutant mice, resulting in chronic infection with impaired elimination of mycobacteria. Therefore, TLR4 signaling is required to mount a protective response during chronic M.tb. infection.