Search

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 [alpha] (TNF-[alpha]). 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 [gamma]-producing and TNF-[alpha]-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. [ABSTRACT FROM AUTHOR]

Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (T CM ), follicular helper T cells (T FH ) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

The purpose of vaccination against tuberculosis and other diseases is to establish a heightened state of acquired specific resistance in which the memory immune response is capable of mediating an accelerated and magnified expression of protection to the pathogen when this is encountered at a later time. In the earliest studies in mice infected with Mycobacterium tuberculosis , memory immunity and the cells that express this were definable both in terms of kinetics of emergence, and soon thereafter by the levels of expression of markers including CD44, CD62L, and the chemokine receptor CCR7, allowing the identification of effector memory and central memory T cell subsets. Despite these initial advances in knowledge, more recent information has not revealed more clarity, but instead, has created a morass of complications-complications that, if not resolved, could harm correct vaccine design. Here, we discuss two central issues. The first is that we have always assumed that memory is induced in the same way, and consists of the same T cells, regardless of whether that immunity is generated by BCG vaccination, or by exposure to M. tuberculosis followed by effective chemotherapy. This assumption is almost certainly incorrect. Second, a myriad of additional memory subsets have now been described, such as resident, stem cell-like, tissue specific, among others, but as yet we know nothing about the relative importance of each, or whether if a new vaccine needs to induce all of these, or just some, to be fully effective.

Transmission of Mycobacterium tuberculosis bacilli from one individual to another is the basis of the disease process. While considerable emphasis has been placed on the role of host mechanisms of resistance in establishing or preventing new infection, far less has been expended on understanding possible factors operative at the bacterial level. In this study we established a panel of clinical isolates of M. tuberculosis strains obtained from the Western Cape region of South Africa, each of which had been carefully tracked in terms of their degree of transmission in the community. Each of the panel were used to infect guinea pigs with 15-20 bacilli by aerosol exposure and the course of the infection then determined. Strains with different degrees of transmission could not be distinguished in terms of their capacity to grow in the main target organs of infected animals. However, rather surprisingly, while strains with no evidence of transmission [NOT] in general caused moderate to severe lung damage, this parameter in animals infected with highly transmitted [HT] strains was mostly mild. In terms of TH1 immunity these signals were strongest in these latter animals, as was IL-17 gene expression, whereas minimal signals for regulatory molecules including IL-10 and FoxP3 were seen across the entire panel. In terms of T cell numbers, responses of both CD4 and CD8 were both far faster and far higher in animals infected with the HT strains. At the gene expression level we observed a major three-fold difference [both up and down] between NOT and HT strains, but in terms of proteins of key interest only a few [including PD-L1 and HIF-3] showed major differences between the two groups. Overall, it was apparent that NOT strains were far more inflammatory that HT strains, and appeared to trigger a much larger number of genes, possibly explaining the observed damage to the lungs and progressive pathology. In contrast, the HT strains, while equally virulent, were more immunogenic and developed much stronger T cell responses, while keeping lung damage to a minimum. Hence, in terms of trying to explain the capacity of these strains to cause transmission, these results are clearly paradoxical., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As yet, very few vaccine candidates with activity in animals against Mycobacterium tuberculosis infection have been tested as therapeutic postexposure vaccines. We recently described two pools of mycobacterial proteins with this activity, and here we describe further studies in which four of these proteins (Rv1738, Rv2032, Rv3130, and Rv3841) were generated as a fusion polypeptide and then delivered in a novel yeast-based platform (Tarmogen) which itself has immunostimulatory properties, including activation of Toll-like receptors. This platform can deliver antigens into both the class I and class II antigen presentation pathways and stimulate strong Th1 and Th17 responses. In mice this fusion vaccine, designated GI-19007, was immunogenic and elicited strong gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses; despite this, they displayed minimal prophylactic activity in mice that were subsequently infected with a virulent clinical strain. In contrast, in a therapeutic model in the guinea pig, GI-19007 significantly reduced the lung bacterial load and reduced lung pathology, particularly in terms of secondary lesion development, while significantly improving survival in one-third of these animals. In further studies in which guinea pigs were vaccinated with BCG before challenge, therapeutic vaccination with GI-19007 initially improved survival versus that of animals given BCG alone, although this protective effect was gradually lost at around 400 days after challenge. Given its apparent ability to substantially limit bacterial dissemination within and from the lungs, GI-19007 potentially can be used to limit lung damage as well as facilitating chemotherapeutic regimens in infected individuals., (Copyright © 2017 King et al.)

There is increasing enthusiasm and optimism that a vaccine could be developed that prevents infection rather than disease. In this article I discuss the fact that despite this optimism nothing has been produced so far that seems to have this capability, and moreover even the borderline between when infection ends and disease begins has not even been defined. To be effective such a vaccine, or at least the immunity it would generate, would have to work within the confines of the pulmonary physiological systems, which are complex. To date much of the emphasis here has turned away from T cell mediated immunity and towards establishing specific antibodies in the lungs. Here, I argue that with the exception of a possible exclusionary function, most claims of a protective role of antibody are completely over-blown. Finally, even if we had a potential "anti-infection" vaccine, how would we test and validate it?, (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This article describes the nature of the host response to Mycobacterium tuberculosis in the mouse and guinea pig models of infection. It describes the great wealth of information obtained from the mouse model, reflecting the general availability of immunological reagents, as well as genetic manipulations of the mouse strains themselves. This has led to a good understanding of the nature of the T-cell response to the infection, as well as an appreciation of the complexity of the response involving multiple cytokine- and chemokine-mediated systems. As described here and elsewhere, we have a growing understanding of how multiple CD4-positive T-cell subsets are involved, including regulatory T cells, TH17 cells, as well as the subsequent emergence of effector and central memory T-cell subsets. While, in contrast, our understanding of the host response in the guinea pig model is less advanced, considerable strides have been made in the past decade in terms of defining the basis of the immune response, as well as a better understanding of the immunopathologic process. This model has long been the gold standard for vaccine testing, and more recently is being revisited as a model for testing new drug regimens (bedaquiline being the latest example).

This article provides an overview of the animal models currently used in tuberculosis research, both for understanding the basic science of the disease process and also for practical issues such as testing new vaccine candidates and evaluating the activity of potential new drugs. Animals range in size, from zebrafish to cattle, and in degrees of similarity to the human disease from both an immunological and pathologic perspective. These models have provided a great wealth of information (impossible to obtain simply from observing infected humans), but we emphasize here that one must use care in interpreting or applying this information, and indeed the true art of animal modeling is in deciding what is pertinent information and what might not be. These ideas are discussed in the context of current approaches in vaccine and drug development, including a discussion of certain limitations the field is currently facing in such studies.

Increasing information has shown that many newly emerging strains of Mycobacterium tuberculosis, including the highly prevalent and troublesome Beijing family of strains, can potently induce the emergence of Foxp3(+)CD4 Tregs Although the significance of this is still not fully understood, we have previously provided evidence that the emergence of this population can significantly ablate the protective effect of BCG vaccination, causing progressive fatal disease in the mouse model. However, whether the purpose of this response is to control inflammation or to directly dampen the acquired immune response is still unclear. In the present study, we have shown, using both cell depletion and adoptive transfer strategies, that Tregs can have either properties. Cell depletion resulted in a rapid, but transient, decrease in the lung bacterial load, suggesting release or temporary re-expansion of effector immunity. Transfer of Tregs into Rag2(-/-)or marked congenic mice worsened the disease course and depressed cellular influx of effector T cells into the lungs. Tregs from infected donors seemed to preferentially depress the inflammatory response and granulocytic influx. In contrast, those from BCG-vaccinated and then challenged donors seemed more focused on depression of acquired immunity. These qualitative differences might be related to increasing knowledge reflecting the plasticity of the Treg response., (© Society for Leukocyte Biology.)

To date, most new vaccines against Mycobacterium tuberculosis, including new recombinant versions of the current BCG vaccine, have usually been screened against the laboratory strains H37Rv or Erdman. In this study we took advantage of our recent work in characterizing an increasingly large panel of newly emerging clinical isolates [from the United States or from the Western Cape region of South Africa], to determine to what extent vaccines would protect against these [mostly high virulence] strains. We show here that both BCG Pasteur and recombinant BCG Aeras-422 [used here as a good example of the new generation BCG vaccines] protected well in both mouse and guinea pig low dose aerosol infection models against the majority of clinical isolates tested. However, Aeras-422 was not effective in a long term survival assay compared to BCG Pasteur. Protection was very strongly expressed against all of the Western Cape strains tested, reinforcing our viewpoint that any attempt at boosting BCG would be very difficult to achieve statistically. This observation is discussed in the context of the growing argument made by others that the failure of a recent vaccine trial disqualifies the further use of animal models to predict vaccine efficacy. This viewpoint is in our opinion completely erroneous, and that it is the fitness of prevalent strains in the trial site area that is the centrally important factor, an issue that is not being addressed by the field.

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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Traditionally, the design of new vaccines directed against Mycobacterium tuberculosis, the most successful bacterial pathogen on the planet, has focused on prophylactic candidates that would be given to individuals while they are still young. It is becoming more apparent, however, that there are several types of vaccine candidates now under development that could be used under various conditions. Thus, in addition to prophylactic vaccines, such as recombinant Mycobacterium bovis BCG or BCG-boosting vaccines, other applications include vaccines that could prevent infection, vaccines that could be given in emergency situations as postexposure vaccines, vaccines that could be used to facilitate chemotherapy, and vaccines that could be used to reduce or prevent relapse and reactivation disease. These approaches are discussed here, including the type of immunity we are trying to specifically target, as well as the limitations of these approaches., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

In this study we conducted a microarray-based whole genomic analysis of gene expression in the lungs after exposure of guinea pigs to a low dose aerosol of the Atypical Beijing Western Cape TT372 strain of Mycobacterium tuberculosis, after harvesting lung tissues three weeks after infection at a time that effector immunity is starting to peak. The infection resulted in a very large up-regulation of multiple genes at this time, particularly in the context of a "chemokine storm" in the lungs. Overall gene expression was considerably reduced in animals that had been vaccinated with BCG two months earlier, but in both cases strong signatures featuring gamma interferon [IFNγ] and tumor necrosis factor [TNFα] were observed indicating the potent TH1 response in these animals. Even though their effects are not seen until later in the infection, even at this early time point gene expression patterns associated with the potential emergence of regulatory T cells were observed. Genes involving lung repair, response to oxidative stress, and cell trafficking were strongly expressed, but interesting these gene patterns differed substantially between the infected and vaccinated/infected groups of animals. Given the importance of this species as a relevant and cost-effective small animal model of tuberculosis, this approach has the potential to provide new information regarding the effects of vaccination on control of the disease process.

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The purpose of vaccination is to establish a stable population of long lived memory T cells. In the context of tuberculosis, the BCG vaccine has been widely used for well over 60 years, but during that time its weaknesses, particularly its ineffectiveness in adults, has been increasingly recognized. In this commentary we review what is known about memory T cells, both in general and in the context of their role in expressing specific acquired resistance to tuberculosis. Current knowledge indicates that both effector memory and central memory can be generated, depending on the experimental conditions, but both in animal models and in clinical studies it is clear that effector memory T cells are the predominant subset. These issues are of importance, given the concerted effort to make new TB vaccines, not all of which may work in precisely the same manner. At the present time whether a TB vaccine would work better if it targeted one specific T cell subset rather than another is as yet completely unknown, and this is now further complicated by new evidence that suggests other subsets such as IL-17 secreting CD4 T cells and cells with stem cell-like qualities may also play important roles., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

There is an urgent need to improve methods used to screen antituberculosis drugs. An in vitro assay was developed to test drug treatment strategies that specifically target drug-tolerant Mycobacterium tuberculosis. The H37Rv strain of M. tuberculosis survived antimicrobial treatment as attached microbial communities when maintained in tissue culture media (RPMI-1640) with or without lysed human peripheral blood leukocytes. When cultured planktonically in the presence of Tween-80, bacilli failed to form microbial communities or reach logarithmic phase growth yet remained highly susceptible to antimicrobial drugs. In the absence of Tween, bacilli tolerated drug therapy by forming complex microbial communities attached to untreated well surfaces or to the extracellular matrix derived from lysed human leukocytes. Treatment of microbial communities with DNase I or Tween effectively dispersed bacilli and restored drug susceptibility. These data demonstrate that in vitro expression of drug tolerance by M. tuberculosis is linked to the establishment of attached microbial communities and that dispersion of bacilli targeting the extracellular matrix including DNA restores drug susceptibility. Modifications of this in vitro assay may prove beneficial in a high-throughput platform to screen new antituberculosis drugs especially those that target drug-tolerant bacilli., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The purpose of this study was to attempt to develop therapeutic or post-exposure vaccines that could slow progressive disease in guinea pigs infected by low dose aerosol with very high virulence Beijing isolates of Mycobacterium tuberculosis, currently classified as Category C biodefense pathogens by the NIH and CDC. After screening several candidates we focused on the use of three candidates; these were a pool of bacterial iron acquisition proteins, a pool of antigens recognized by T cells from chronically infected mice thought to represent proteins made by the bacillus in response to decreases in local oxygen tension, and a bacterial lipoprotein that is a potent TLR2 agonist. When delivered in a potent GLA-based adjuvant [targeting TLR4 and TLR9], in most cases we were unable to reduce the bacterial load in the lungs. However, the pathologic appearance of lungs from these animals showed that, while primary lesions were most unaffected and had become necrotic, the development of large, lung consolidating secondary lesions seemed to have been mostly prevented. In animals given both a priming vaccination and a boost the effects were prominent, and almost certainly contributed to significantly prolonged survival in these animals. In a biodefense situation, this prolonged survival would be potentially long enough to allow for the organism to be identified and a drug susceptibility profile determined., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

It is set in stone that Mycobacterium tuberculosis is a facultative intracellular bacterial parasite. This axiom drives our knowledge of the host response, the way we design vaccines against the organism by generating protective T cells, and to a lesser extent, the way we try to target anti-microbial drugs. The purpose of this article is to commit total heresy. I believe that M. tuberculosis can equally well be regarded as an extracellular pathogen and may in fact spend a large percentage of its human lung "life-cycle" in this environment. It is of course intracellular as well, but this may well be little more than a brief interlude after infection of a new host during which the bacterium must replicate to increase its chances of transmission and physiologically adapt prior to moving back to an extracellular phase. As a result, by focusing almost completely on just the intracellular phase, we may be making serious strategic errors in the way we try to intervene in this pathogenic process. It is my opinion that when a TB bacillus enters the lungs and starts to reside inside an alveolar macrophage, its central driving force is to switch on a process leading to lung necrosis, since it is only by this process that the local lung tissue can be destroyed and the bacillus can be exhaled and transmitted. I present here a new model of the pathogenesis of the disease that attempts to unify the pathogenic process of infection, disease, persistence [rather than latency], and reactivation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The purpose of this simple study was to characterize a panel of clinical isolates of Mycobacterium tuberculosis obtained from the Western Cape region of South Africa where new clinical vaccine trials are beginning, in the low dose aerosol guinea pig infection model. Most of the strains tested grew well in the lungs and other organs of these animals, and in most cases gave rise to moderate to very severe lung damage. We further observed that the current BCG vaccine was highly protective against two randomly selected strains, giving rise to significantly prolonged survival., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Tuberculosis is one of the world's leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1(int)CD11b(+) cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design., Methodology/principal Findings: We compared the bacterial burden, lung pathology and Gr1(int)CD11b(+) myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1(+) cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1(+)CD11b(+) cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1(hi) and Gr1(int) populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1(int) populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1(hi) and Gr1(int) cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4(+) T to Gr1(+) cells increased. Our results illustrate a yet unrecognized interplay between Gr1(+) cells and CD4(+) T cells in tuberculosis.

Very substantial efforts have been made over the past decade or more to develop vaccines against tuberculosis. Historically, this began with a view to replace the current vaccine, Bacillus Calmette Guérin (BCG), but more recently most candidates are either new forms of this bacillus, or are designed to boost immunity in children given BCG as infants. Good progress is being made, but very few have, as yet, progressed into clinical trials. The leading candidate has advanced to phase IIb efficacy testing, with disappointing results. This article discusses the various types of vaccines, including those designed to be used in a prophylactic setting, either alone or BCG-boosting, true therapeutic (post-exposure) vaccines, and therapeutic vaccines designed to augment chemotherapy. While there is no doubt that progress is still being made, we have a growing awareness of the limitations of our animal model screening processes, further amplified by the fact that we still do not have a clear picture of the immunological responses involved, and the precise type of long-lived immunity that effective new vaccines will need to induce.

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.

In drug development, there are typically a series of preclinical studies that must be completed with new compounds or regimens before use in humans. A sequence of in vitro assays followed by in vivo testing in validated animal models to assess the activity against Mycobacterium tuberculosis, pharmacology and toxicity is generally used for advancing compounds against tuberculosis in a preclinical stage. A plethora of different assay systems and conditions are used to study the effect of drug candidates on the growth of M. tuberculosis, making it difficult to compare data from one laboratory to another. The Bill and Melinda Gates Foundation recognized the scientific gap to delineate the spectrum of variables in experimental protocols, identify which of these are biologically significant, and converge towards a rationally derived standard set of optimized assays for evaluating compounds. The goals of this document are to recommend protocols and hence accelerate the process of TB drug discovery and testing. Data gathered from preclinical in vitro and in vivo assays during personal visits to laboratories and an electronic survey of methodologies sent to investigators is reported. Comments, opinions, experiences as well as final recommendations from those currently engaged in such preclinical studies for TB drug testing are being presented. Certain in vitro assays and mouse efficacy models were re-evaluated in the laboratory as head-to-head experiments and a summary is provided on the results obtained. It is our hope that this information will be a valuable resource for investigators in the field to move forward in an efficient way and that key variables of assays are included to ensure accuracy of results which can then be used for designing human clinical trials. This document then concludes with remaining questions and critical gaps that are in need of further validation and experimentation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

The Beijing family of Mycobacterium tuberculosis strains is part of lineage 2 (also known as the East Asian lineage). In clinical studies, we have observed that isolates from the sublineage RD207 of lineage 2 were more readily transmitted among humans. To investigate the basis for this difference, we tested representative strains with the characteristic Beijing spoligotype from four of the five sublineages of lineage 2 in the guinea pig model and subjected these strains to comparative whole-genome sequencing. The results of these studies showed that all of the clinical strains were capable of growing and causing lung pathology in guinea pigs after low-dose aerosol exposure. Differences between the abilities of the four sublineages to grow in the lungs of these animals were not overt, but members of RD207 were significantly more pathogenic, resulting in severe lung damage. The RD207 strains also induced much higher levels of markers associated with regulatory T cells and showed a significant loss of activated T cells in the lungs over the course of the infections. Whole-genome sequencing of the strains revealed mutations specific for RD207 which may explain this difference. Based on these data, we hypothesize that the sublineages of M. tuberculosis are associated with distinct pathological and clinical phenotypes and that these differences influence the transmissibility of particular M. tuberculosis strains in human populations.

Recent clinical observations shows that individuals treated with chemotherapy for tuberculosis who live in endemic areas are four times more likely to develop secondary disease, often as not caused by exogenous reinfection. In a mouse model described here, we show that mice infected with the virulent W-Beijing Mycobacterium tuberculosis strain HN878, then given chemotherapy to clear the infection, were resistant to re-challenge with the same organism thereafter. This resistance, which was mediated by rapid expression of CD4 T cells expressing markers consistent with both central and effector memory immunity, was only transient however. After 20-30 days of the reinfection the numbers of these cells steadily declined, the bacterial load in the lungs surged up, and the lung tissues became increasingly consolidated. No evidence was found for a regulatory T cell response in these mice, but many T cells harvested from the lungs showed evidence of increased PD-1 expression, indicating exhaustion. These data indicate that the memory T cell response to reinfection may not be as stable and long lived as previously thought, a finding with obvious implications for vaccine development., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Bacillus-Calmette-Guerin (BCG), the only human tuberculosis vaccine, primes a partially protective immune response against Mycobacterium tuberculosis infection in humans and animals. In guinea pigs, BCG vaccination slows the progression of disease and reduces the severity of necrotic granulomas, which harbor a population of drug-tolerant bacilli. The objective of this study was to determine if reducing disease severity by BCG vaccination of guinea pigs prior to M. tuberculosis challenge enhanced the efficacy of combination drug therapy. At 20 days of infection, treatment of vaccinated and non-vaccinated animals with rifampin, isoniazid, and pyrizinamide (RHZ) was initiated for 4 or 8 weeks. On days 50, 80 and 190 of infection (10 weeks after drug were withdrawn), treatment efficacy was evaluated by quantifying clinical condition, bacterial loads, lesion severity, and dynamic changes in peripheral blood and lung leukocyte numbers by flow cytometry. In a separate, long-term survival study, treatment efficacy was evaluated by determining disease reactivation frequency post-mortem. BCG vaccination alone delayed pulmonary and extra-pulmonary disease progression, but failed to prevent dissemination of bacilli and the formation of necrotic granulomas. Drug therapy either alone or in combination with BCG, was more effective at lessening clinical disease and lesion severity compared to control animals or those receiving BCG alone. Fewer residual lesions in BCG vaccinated and drug treated animals, equated to a reduced frequency of reactivation disease and improvement in survival even out to 500 days of infection. The combining of BCG vaccination and drug therapy was more effective at resolving granulomas such that fewer animals had evidence of residual infection and thus less reactivation disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence.

Toll-like receptor (TLR) agonists are currently being examined as adjuvants for vaccines, with several lead candidates now in licensed products or in late-stage clinical development. Guinea pigs are widely used for preclinical testing of drugs and vaccines; however, evaluation of TLR agonists in this model is hindered by the limited availability of immunological tools and reagents. In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-α, IL-1β, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potential to be used as adjuvant components in vaccines against various pathogens., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4(+) memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4(+) cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.

There is increasing evidence that clinical isolates of Mycobacterium tuberculosis that belong to the W-Beijing genotype of newly emerging strains are often of very high virulence when tested in small animal models, including the mouse and guinea pig. In this report we provide further evidence to support this contention, and show that two W-Beijing strains are of very high virulence when introduced by low dose aerosol into outbred guinea pigs. In addition to severe lung pathology, each of these infections was associated with large influxes of activated CD4 and CD8 T cells into the lungs. Large influxes of macrophages were also observed, but the fraction of these showing evidence of activation by Class-II expression was relatively low. A progressive increase in neutrophils was also seen, with highest levels accumulating in the lungs of the W-Beijing infected animals. In the case of these two infections mRNA levels for TH1 cytokines was elevated early, but these then declined, and were replaced by increasing levels of message encoding for Foxp3, IL-10, and TGFβ. These observations support the hypothesis that W-Beijing strains are potent inducers of regulatory T cells, and that this event may enhance survival and transmission of these bacilli., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Despite issues relating to variable efficacy in the past, the Mycobacterium bovis BCG vaccine remains the basis for new-generation recombinant vaccines currently in clinical trials. To date, vaccines have been tested mostly against laboratory strains and not against the newly emerging clinical strains. In this study, we evaluated the ability of BCG Pasteur to protect mice from aerosol infections with two highly virulent W-Beijing clinical strains, HN878 and SA161. In a conventional 30-day protection assay, BCG was highly protective against both strains, but by day 60 of the assay, this protection was diminished. Histological examination of the lungs of vaccinated animals showed reduced lung consolidation and smaller and more-organized granulomas in the vaccinated mice after 30 days, but in both cases, these tissues demonstrated worsening pathology over time. Effector T cell responses were increased in the vaccinated mice infected with HN878, but these diminished in number after day 30 of the infections concomitant with increased CD4(+) Foxp3(+) T cells in the lungs, draining lymph nodes, and the spleen. Given the concomitant decrease in effector immunity and continued expansion of regulatory Foxp3(+) cells observed here, it is reasonable to hypothesize that downregulation of effector immunity by these cells may be a serious impediment to the efficacy of BCG-based vaccines.

This unit describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non-tuberculosis mycobacterial species., (© 2011 by John Wiley & Sons, Inc.)

A natural TB infection model using guinea pigs may provide useful information for investigating differences in transmission efficiency and establishment of active disease by clinical TB strains in a highly susceptible host under controlled environmental conditions. We sought to examine the capacity of naturally transmitted multidrug-resistant Mycobacterium tuberculosis to establish infection and produce active disease in guinea pigs. Guinea pigs were continuously exposed for 4 months to the exhaust air of a 6-bed multidrug-resistant tuberculosis inpatient hospital ward in South Africa. Serial tuberculin skin test reactions were measured to determine infection. All animals were subsequently evaluated for histologic disease progression at necropsy. Although 75% of the 362 exposed guinea pigs had positive skin test reactions [≥6 mm], only 12% had histopathologic evidence of active disease. Reversions (≥6 mm change) in skin test reactivity were seen in 22% of animals, exclusively among those with reactions of 6-13 mm. Only two of 86 guinea pigs with reversion had histological evidence of disease compared to 47% (31/66) of guinea pigs with large, non-reverting reactions. Immunosuppression of half the guinea pigs across all skin test categories did not significantly accelerate disease progression. In guinea pigs that reverted a skin test, a second positive reaction in 27 (33%) of them strongly suggested re-infection due to ongoing exposure. These results show that a large majority of guinea pigs naturally exposed to human-source strains of multidrug-resistant tuberculosis became infected, but that many resolved their infection and a large majority failed to progress to detectable disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Predictive biomarkers illustrating the effectiveness of chemotherapeutic regimens for tuberculosis still remain elusive. To date, most are predicated on assays using sputum or serum; as a result, if not predictive, treatment failure in patients may not be evident for some time. We report here the results of a simple screening study in which T cell surface markers were examined in mice infected with Mycobacterium tuberculosis and then treated with drugs. These studies identified certain markers, the exhaustion markers PD-1 and TIM-3, as well as the marker KLRG-1, particularly on CD8 T cells, that changed in concert with reduction of the bacterial load in the lungs. While there is no guarantee these changes would also be seen on T cells in the blood, this approach should be further investigated., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The limited efficacy of the BCG vaccine for tuberculosis, coupled with emerging information suggesting that it is poorly protective against newly emerging strains of Mycobacterium tuberculosis such as the W-Beijing isolates, makes it paramount to search for more potent alternatives. One such class of candidates is attenuated mutants derived from M. tuberculosis itself. We demonstrate here, in an initial short term assay, that mutants derived from disruption of the mce genes of the bacillus were highly protective in guinea pigs exposed by low dose aerosol infection with the virulent W-Beijing isolate SA161. This protection was demonstrated by a significant reduction in the numbers of bacilli harvested from the lungs, and dramatic improvements in lung histopathology., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

The concomitant HIV and TB epidemics pose an enormous threat to humanity. After invading the host Mycobacterium tuberculosis initially behaves as an intracellular pathogen, which elicits the emergence of acquired specific resistance in the form of a T-helper-1 T-cell response, and involves the secretion of a myriad of cytokines and chemokines to drive protective immunity and granuloma formation. However, after that, a second phase of the disease process involves survival of bacilli in an extracellular state that is still poorly understood. This article briefly reviews the various strategies currently being used to improve both vaccination and drug therapy of TB, and attempts to make the argument that current viewpoints that dominate [both the field and the current literature] may be seriously flawed. This includes both the choice of new vaccine and drug candidates, and also the ways these are being tested in animal models, which in the opinion of the author run the risk of driving the field backwards rather than forward.

Background: Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in the world and exacerbates disease caused by the human immunodeficiency virus (HIV). HIV infected individuals are prone to lung infections by a variety of microbial pathogens, including M. tuberculosis. While the destruction of the adaptive immune response by HIV is well understood, the actual pathogenesis of tuberculosis in co-infected individuals remains unclear. Tat is an HIV protein essential for efficient viral gene transcription, is secreted from infected cells, and is known to influence a variety of host inflammatory responses. We hypothesize Tat contributes to pathophysiological changes in the lung microenvironment, resulting in impaired host immune responses to infection by M. tuberculosis., Results: Herein, we show transgenic mice that express Tat by lung alveolar cells are more susceptible than non-transgenic control littermates to a low-dose aerosol infection of M. tuberculosis W-Beijing SA161. Survival assays demonstrate accelerated mortality rates of the Tat transgenic mice compared to non-transgenics. Tat transgenic mice also showed poorly organized lung granulomata-like lesions. Analysis of the host immune response using quantitative RT-PCR, flow cytometry for surface markers, and intracellular cytokine staining showed increased expression of pro-inflammatory cytokines in the lungs, increased numbers of cells expressing ICAM1, increased numbers of CD4+CD25+Foxp3+ T regulatory cells, and IL-4 producing CD4+ T cells in the Tat transgenics compared to infected non-tg mice., Conclusions: Our data show quantitative differences in the inflammatory response to the SA161 clinical isolate of M. tuberculosis W-Beijing between Tat transgenic and non-transgenic mice, suggesting Tat contributes to the pathogenesis of tuberculosis.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)