The balance between protective and pathogenic immune responses in the TB-infected lung.

Tuberculosis is a disease of the lung, and efficient transmission is dependent on the generation of a lesion in the lung, which results in a bacterium-laden cough. Mycobacterium tuberculosis (Mtb) is able to manipulate both the innate and acquired immune response of the host. This manipulation results in an effective CD4(+) T cell response that limits disease throughout the body but can also promote the development of progressively destructive lesions in the lung. In this way Mtb infection can result in an ambulatory individual who has a lesion in the lung capable of transmitting Mtb. The inflammatory environment within the lung lesion is manipulated by Mtb throughout infection and can limit the expression of acquired immunity by a variety of pathways.

Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens.

Toll-like receptor (TLR) signaling in macrophages is required for antipathogen responses, including the biosynthesis of nitric oxide from arginine, and is essential for immunity to Mycobacterium tuberculosis, Toxoplasma gondii and other intracellular pathogens. Here we report a 'loophole' in the TLR pathway that is advantageous to these pathogens. Intracellular pathogens induced expression of the arginine hydrolytic enzyme arginase 1 (Arg1) in mouse macrophages through the TLR pathway. In contrast to diseases dominated by T helper type 2 responses in which Arg1 expression is greatly increased by interleukin 4 and 13 signaling through the transcription factor STAT6, TLR-mediated Arg1 induction was independent of the STAT6 pathway. Specific elimination of Arg1 in macrophages favored host survival during T. gondii infection and decreased lung bacterial load during tuberculosis infection.

The onset of adaptive immunity in the mouse model of tuberculosis and the factors that compromise its expression.

Mycobacterium tuberculosis (Mtb) has been evolving with its human host for over 50 000 years and is an exquisite manipulator of the human immune response. It induces both a strong inflammatory and a strong acquired immune response, and Mtb then actively regulates these responses to create an infectious lesion in the lung while maintaining a relatively ambulatory host. The CD4(+) T cell plays a critical yet contradictory role in this process by both controlling disseminated disease while promoting the development of the lesion in the lung that mediates transmission. In light of this manipulative relationship between Mtb and the human immune response, it is not surprising that our ability to vaccinate against tuberculosis (TB) has not been totally successful. To overcome the current impasse in vaccine development, we need to define the phenotype of CD4(+) T cells that mediate protection and to determine those bacterial and host factors that regulate the effective function of these cells. In this review, we describe the initiation and expression of T cells during TB as well as the fulminant inflammatory response that can compromise T-cell function and survival.

The formation of the granuloma in tuberculosis infection.

The development of the granuloma and its subsequent degeneration and necrosis, is the hallmark of infection caused by Mycobacterium tuberculosis. These structures probably evolved as primitive particle responses, but in mammals they are facilitated by the emerging acquired immune response, in which cytokines and chemokines help control their formation and integrity. In this brief review we discuss the pathology of these lesions in the two most widely used animal models (mice and guinea pigs). In addition, we argue against the idea that there is a balance between host immunity and bacterial survival, and that the latter possess mechanisms that control this, as some currently believe, and moreover discuss newer information regarding the ability of bacilli to persist in these structures long enough to eventually escape and become retransmitted.

bis-Molybdopterin guanine dinucleotide is required for persistence of Mycobacterium tuberculosis in guinea pigs.

Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.

Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox) from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox) was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox) interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to Mtb virulence via attenuation of NADPH oxidase-mediated host innate immunity.

Increased severity of tuberculosis in Guinea pigs with type 2 diabetes: a model of diabetes-tuberculosis comorbidity.

Impaired glucose tolerance and type 2 diabetes were induced in guinea pigs to model the emerging comorbidity of Mycobacterium tuberculosis infection in diabetic patients. Type 2 diabetes mellitus was induced by low-dose streptozotocin in guinea pigs rendered glucose intolerant by first feeding a high-fat, high-carbohydrate diet before M. tuberculosis exposure. M. tuberculosis infection of diabetic guinea pigs resulted in severe and rapidly progressive tuberculosis (TB) with a shortened survival interval, more severe pulmonary and extrapulmonary pathology, and a higher bacterial burden compared with glucose-intolerant and nondiabetic controls. Compared with nondiabetics, diabetic guinea pigs with TB had an exacerbated proinflammatory response with more severe granulocytic inflammation and higher gene expression for the cytokines/chemokines interferon-γ, IL-17A, IL-8, and IL-10 in the lung and for interferon-γ, tumor necrosis factor-α, IL-8, and monocyte chemoattractant protein-1 in the spleen. TB disease progression in guinea pigs with impaired glucose tolerance was similar to that of nondiabetic controls in the early stages of infection but was more severe by day 90. The guinea pig model of type 2 diabetes-TB comorbidity mimics important features of the naturally occurring disease in humans. This model will be beneficial in understanding the complex pathogenesis of TB in diabetic patients and to test new strategies to improve TB and diabetes control when the two diseases occur together.

Host response to nontuberculous mycobacterial infections of current clinical importance.

The nontuberculous mycobacteria are a large group of acid-fast bacteria that are very widely distributed in the environment. While Mycobacterium avium was once regarded as innocuous, its high frequency as a cause of disseminated disease in HIV-positive individuals illustrated its potential as a pathogen. Much more recently, there is growing evidence that the incidence of M. avium and related nontuberculous species is increasing in immunocompetent individuals. The same has been observed for M. abscessus infections, which are very difficult to treat; accordingly, this review focuses primarily on these two important pathogens. Like the host response to M. tuberculosis infections, the host response to these infections is of the TH1 type but there are some subtle and as-yet-unexplained differences.

The endothelin system has a significant role in the pathogenesis and progression of Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains a major global health problem, and although multiple studies have addressed the relationship between Mycobacterium tuberculosis and the host on an immunological level, few studies have addressed the impact of host physiological responses. Proteases produced by bacteria have been associated with important alterations in the host tissues, and a limited number of these enzymes have been characterized in mycobacterial species. M. tuberculosis produces a protease called Zmp1, which appears to be associated with virulence and has a putative action as an endothelin-converting enzyme. Endothelins are a family of vasoactive peptides, of which 3 distinct isoforms exist, and endothelin 1 (ET-1) is the most abundant and the best-characterized isoform. The aim of this work was to characterize the Zmp1 protease and evaluate its role in pathogenicity. Here, we have shown that M. tuberculosis produces and secretes an enzyme with ET-1 cleavage activity. These data demonstrate a possible role of Zmp1 for mycobacterium-host interactions and highlights its potential as a drug target. Moreover, the results suggest that endothelin pathways have a role in the pathogenesis of M. tuberculosis infections, and ETA or ETB receptor signaling can modulate the host response to the infection. We hypothesize that a balance between Zmp1 control of ET-1 levels and ETA/ETB signaling can allow M. tuberculosis adaptation and survival in the lung tissues.

Metabolomic signatures in guinea pigs infected with epidemic-associated W-Beijing strains of Mycobacterium tuberculosis.

With the understanding that the laboratory propagated strain of Mycobacterium tuberculosis H37Rv is of modest virulence and is drug susceptible, in the present study, we performed a nuclear magnetic resonance-based metabolomic analysis of lung tissues and serum obtained from guinea pigs infected by low dose aerosol exposure to clinical isolates of Mycobacterium tuberculosis. High Resolution Magic Angle Spinning NMR coupled with multivariate statistical analysis of 159 lung tissues obtained from multiple locations of age-matched naïve and 30 and 60 days of infected guinea pig lungs revealed a wide dispersal of metabolic patterns, but within these, distinct clusters of signatures could be seen that differentiated between naive control and infected animals. Several metabolites were identified that changed in concert with the progression of each infection. Major metabolites that could be interpreted as indicating host glutaminolysis were consistent with activated host immune cells encountering increasingly hypoxic conditions in the necrotic lung lesions. Moreover, glutathione levels were constantly elevated, probably in response to oxygen radical production in these lesions. Additional distinct signatures were also seen in infected serum, with altered levels of several metabolites. Multivariate statistical analysis clearly differentiated the infected from the uninfected sera; in addition, Receiver Operator Characteristic curve generated with principal component 1 scores showed an area under the curve of 0.908. These data raise optimism that discrete metabolomic signatures can be defined that can predict the progression of the tuberculosis disease process, and form the basis of an innovative and rapid diagnostic process.

Importance of confirming data on the in vivo efficacy of novel antibacterial drug regimens against various strains of Mycobacterium tuberculosis.

In preclinical testing of antituberculosis drugs, laboratory-adapted strains of Mycobacterium tuberculosis are usually used both for in vitro and in vivo studies. However, it is unknown whether the heterogeneity of M. tuberculosis stocks used by various laboratories can result in different outcomes in tests of antituberculosis drug regimens in animal infection models. In head-to-head studies, we investigated whether bactericidal efficacy results in BALB/c mice infected by inhalation with the laboratory-adapted strains H37Rv and Erdman differ from each other and from those obtained with clinical tuberculosis strains. Treatment of mice consisted of dual and triple drug combinations of isoniazid (H), rifampin (R), and pyrazinamide (Z). The results showed that not all strains gave the same in vivo efficacy results for the drug combinations tested. Moreover, the ranking of HRZ and RZ efficacy results was not the same for the two H37Rv strains evaluated. The magnitude of this strain difference also varied between experiments, emphasizing the risk of drawing firm conclusions for human trials based on single animal studies. The results also confirmed that the antagonism seen within the standard HRZ regimen by some investigators appears to be an M. tuberculosis strain-specific phenomenon. In conclusion, the specific identity of M. tuberculosis strain used was found to be an important variable that can change the apparent outcome of in vivo efficacy studies in mice. We highly recommend confirmation of efficacy results in late preclinical testing against a different M. tuberculosis strain than the one used in the initial mouse efficacy study, thereby increasing confidence to advance potent drug regimens to clinical trials.

Defined tuberculosis vaccine, Mtb72F/AS02A, evidence of protection in cynomolgus monkeys.

The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette-Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-gamma, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters.

Cigarette smoke increases susceptibility to tuberculosis--evidence from in vivo and in vitro models.

BACKGROUND: Cigarette smoke (CS) exposure is an epidemiological risk factor for tuberculosis, although the biological basis has not been elucidated. METHODS: We exposed C57BL/6 mice to CS for 14 weeks and examined their ability to control an aerosol infection of Mycobacterium tuberculosis Erdman. RESULTS: CS-exposed mice had more M. tuberculosis isolated from the lungs and spleens after 14 and 30 d, compared with control mice. The CS-exposed mice had worse lung lesions and less lung and splenic macrophages and dendritic cells (DCs) producing interleukin12 and tumor necrosis factor α (TNF-α). There were significantly more interleukin 10-producing macrophages and DCs in the spleens of infected CS-exposed mice than in non-CS-exposed controls. CS-exposed mice also showed a diminished influx of interferon γ-producing and TNF-α-producing CD4(+) and CD8(+) effector and memory T cells into the lungs and spleens. There was a trend toward an increased number of viable intracellular M. tuberculosis in macrophages isolated from humans who smoke compared with nonsmokers. THP-1 human macrophages and primary human alveolar macrophages exposed to CS extract, nicotine, or acrolein showed an increased burden of intracellular M. tuberculosis. CONCLUSION: CS suppresses the protective immune response to M. tuberculosis in mice, human THP-1 cells, and primary human alveolar macrophages.

Activities of TMC207, rifampin, and pyrazinamide against Mycobacterium tuberculosis infection in guinea pigs.

The experimental compound TMC207 is showing promise against infections caused by Mycobacterium tuberculosis both in a variety of animal studies and in the field. In this study, we used the guinea pig model, a species that shows several similarities to human tuberculosis, including the hallmark of primary granuloma necrosis, to determine the efficacy of a combination regimen combining TMC207 with rifampin and pyrazinamide. This drug regimen rapidly reduced the bacterial load in the lungs to undetectable levels by 8 weeks of treatment. This reduction was associated with a substantial improvement in lung pathology, but despite this effect areas of residual necrosis still remained. In the draining lymph nodes, however, tissue damage was rapid and not significantly reversed by the drug treatment. Approximately 10 to 11 months after the treatment had ended, the animals began to trigger a Karnovsky scale indicating bacterial regrowth and potential relapse, an event confirmed by the new development of both pulmonary and extrapulmonary granulomatous lesions. Interestingly, a similar rate of relapse was also seen in animals receiving 24 weeks of rifampin, pyrazinamide, and isoniazid standard chemotherapy. These data indicate that TMC207 could be a useful addition to current treatment regimens for tuberculosis.

Comparative studies evaluating mouse models used for efficacy testing of experimental drugs against Mycobacterium tuberculosis.

Methodologies for preclinical animal model testing of drugs against Mycobacterium tuberculosis vary from laboratory to laboratory; however, it is unknown if these variations result in different outcomes. Thus, a series of head-to-head comparisons of drug regimens in three commonly used mouse models (intravenous, a low-dose aerosol, and a high-dose aerosol infection model) and in two strains of mice are reported here. Treatment with standard tuberculosis (TB) drugs resulted in similar efficacies in two mouse species after a low-dose aerosol infection. When comparing the three different infection models, the efficacies in mice of rifampin and pyrazinamide were similar when administered with either isoniazid or moxifloxacin. Relapse studies revealed that the standard drug regimen showed a significantly higher relapse rate than the moxifloxacin-containing regimen. In fact, 4 months of the moxifloxacin-containing combination regimen showed similar relapse rates as 6 months of the standard regimen. The intravenous model showed slower bactericidal killing kinetics with the combination regimens tested and a higher relapse of infection than either aerosol infection models. All three models showed similar outcomes for in vivo efficacy and relapse of infection for the drug combinations tested, regardless of the mouse infection model used. Efficacy data for the drug combinations used also showed similar results, regardless of the formulation used for rifampin or timing of the drugs administered in combination. In all three infection models, the dual combination of rifampin and pyrazinamide was less sterilizing than the standard three-drug regimen, and therefore the results do not support the previously reported antagonism between standard TB agents.

Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i) M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii) Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii) Rv2623 binds ATP; and iv) the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

Host immune response to rapidly growing mycobacteria, an emerging cause of chronic lung disease.

Rapidly growing mycobacteria (RGM) are environmental organisms classified under the broader category of nontuberculous mycobacteria. The most common RGM to cause human diseases are Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium massiliense. Infections due to the RGM are an emerging health problem in the United States. Chronic pulmonary disease and skin/soft-tissue infections are the two most common disorders due to these organisms. Clinical outcomes in the treatment of M. abscessus infections are generally disappointing. Because less is known about the nature of the immune response to M. abscessus than for tuberculosis, we herein highlight the major clinical features associated with infections due to M. abscessus and other RGM, and review the known host immune response to RGM, drawing from experimental animal and clinical studies. Based on in vitro and in vivo murine models, Toll-like receptor 2, dectin-1, tumor necrosis factor (TNF)-α, IFN-γ, leptin, T cells, and possibly neutrophils are important components in the host defense against RGM infections. However, excessive induction of TNF-α by the R morphotype of M. abscessus may allow it to be more pathogenic than the S morphotype. Clinical observations and/or genetic studies in humans corroborate many of the findings in animals in that those with cell-mediated immunodeficiency, genetic defects in IFN-γ-IL-12 axis, and those individuals on TNF-α blockers are at increased risk for nontuberculous mycobacteria infections, including the RGM. However, much remains to be discovered on why seemingly healthy individuals, particularly slender postmenopausal women with thoracic cage anomalies, appear to be at increased risk.

Evaluation of standard chemotherapy in the guinea pig model of tuberculosis.

The purpose of this study was 2-fold. First, we evaluated standard chemotherapy in the guinea pig model of tuberculosis to determine if this animal species could productively be used for this purpose. Second, given the similarities of the pathology of disease in guinea pigs and humans, we wished to evaluate additional parameters, including magnetic resonance imaging, microscopy, and cytokine expression and lymphocyte phenotypes, in response to an infection treated with drug therapy. This study shows that conventional rifampin-isoniazid-pyrazinamide chemotherapy significantly decreased the numbers of the highly virulent Erdman K01 strain of Mycobacterium tuberculosis, with most of the bacilli being eliminated in a month. Despite this result, bacteria could still be detected in the lungs and other tissues for at least another 3 to 4 months. Resolution of the nonnecrotic granulomas in the lungs and lymph nodes could be clearly visualized by magnetic resonance imaging at the macroscopic level. Microscopically, the majority of the pulmonary and extrapulmonary inflammation resolved spontaneously, leaving residual lesions composed of dystrophic calcification and fibrosis marking the site of necrosis of the primary lesion. Residual calcified lesions, which were also associated with pulmonary lymphangitis, contained acid-fast bacilli even following aggressive chemotherapy. The presence of intact extracellular bacilli within these lesions suggests that these could serve as the primary sites of disease reactivation. The chemotherapy reduced the level of T-cell influx into infected tissues and was accompanied by a large and sustained increase in TH1 cytokine expression. Chemotherapy also prevented the emergence in lung tissues of high levels of interleukin-10 and Foxp3-positive cells, known markers of regulatory T cells.

Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis.

The inhibition of apoptosis of infected host cells is a well-known but poorly understood function of pathogenic mycobacteria. We show that inactivation of the secA2 gene in Mycobacterium tuberculosis, which encodes a component of a virulence-associated protein secretion system, enhanced the apoptosis of infected macrophages by diminishing secretion of mycobacterial superoxide dismutase. Deletion of secA2 markedly increased priming of antigen-specific CD8(+) T cells in vivo, and vaccination of mice and guinea pigs with a secA2 mutant significantly increased resistance to M. tuberculosis challenge compared with standard M. bovis bacille Calmette-Guérin vaccination. Our results define a mechanism for a key immune evasion strategy of M. tuberculosis and provide what we believe to be a novel approach for improving mycobacterial vaccines.