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Host protective immunity against pathogenic Mycobacterium tuberculosis (Mtb) infection is variable and poorly understood. Both prior Mtb infection and BCG vaccination have been reported to confer some protection against subsequent infection and/or disease. However, the immune correlates of host protection with or without BCG vaccination remain poorly understood. Similarly, the host response to concomitant infection with mixed Mtb strains is unclear. In this study, we used the rabbit model to examine the host response to various infectious doses of virulent Mtb HN878 with and without prior BCG vaccination, as well as simultaneous infection with a mixture of two Mtb clinical isolates HN878 and CDC1551. We demonstrate that both the ability of host immunity to control pulmonary Mtb infection and the protective efficacy of BCG vaccination against the progression of Mtb infection to disease is dependent on the infectious inoculum. The host response to infection with mixed Mtb strains mirrors the differential responses seen during infection with each of the strains alone. The protective response mounted against a hyperimmunogenic Mtb strain has a limited impact on the control of disease caused by a hypervirulent strain. This preclinical study will aid in predicting the success of any vaccination strategy and in optimizing TB vaccines., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has co-evolved with the human immune system and utilizes multiple strategies to persist within infected cells, to hijack several immune mechanisms, and to cause severe pathology and tissue damage in the host. This delays the efficacy of current antibiotic therapy and contributes to the evolution of multi-drug-resistant strains. These challenges led to the development of the novel approach in TB treatment that involves therapeutic targeting of host immune response to control disease pathogenesis and pathogen growth, namely, host-directed therapies (HDTs). Such HDT approaches can (1) enhance the effect of antibiotics, (2) shorten treatment duration for any clinical form of TB, (3) promote development of immunological memory that could protect against relapse, and (4) ameliorate the immunopathology including matrix destruction and fibrosis associated with TB. In this review we discuss TB-HDT candidates shown to be of clinical relevance that thus could be developed to reduce pathology, tissue damage, and subsequent impairment of pulmonary function. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

NLRP10 is a nucleotide-binding oligomerization domain-like receptor that functions as an intracellular pattern recognition receptor for microbial products. Here, we generated a Nlrp10 -/- mouse to delineate the role of NLRP10 in the host immune response and found that Nlrp10 -/- dendritic cells (DCs) elicited sub-optimal IFNγ production by antigen-specific CD4 + T cells compared to wild-type (WT) DCs. In response to T-cell encounter, CD40 ligation or Toll-like receptor 9 stimulation, Nlrp10 -/- DCs produced low levels of IL-12, due to a substantial decrease in NF-κB activation. Defective IL-12 production was also evident in vivo and affected IFNγ production by CD4 + T cells. Upon Mycobacterium tuberculosis ( Mtb ) infection, Nlrp10 -/- mice displayed diminished T helper 1-cell responses and increased bacterial growth compared to WT mice. These data indicate that NLRP10-mediated IL-12 production by DCs is critical for IFNγ induction in T cells and contributes to promote the host defense against Mtb .

Mycobacterium tuberculosis ( Mtb ) executes a plethora of immune-evasive mechanisms, which contribute to its pathogenesis, limited efficacy of current therapy, and the emergence of drug-resistant strains. This has led to resurgence in attempts to develop new therapeutic strategies/targets against tuberculosis (TB). We show that Mtb down-regulates sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase, in monocytes/macrophages, TB animal models, and TB patients with active disease. Activation of SIRT1 reduced intracellular growth of drug-susceptible and drug-resistant strains of Mtb and induced phagosome-lysosome fusion and autophagy in a SIRT1-dependent manner. SIRT1 activation dampened Mtb -mediated persistent inflammatory responses via deacetylation of RelA/p65, leading to impaired binding of RelA/p65 on the promoter of inflammatory genes. In Mtb -infected mice, the use of SIRT1 activators ameliorated lung pathology, reduced chronic inflammation, and enhanced efficacy of anti-TB drug. Mass cytometry-based high-dimensional analysis revealed that SIRT1 activation mediated modulation of lung myeloid cells in Mtb -infected mice. Myeloid cell-specific SIRT1 knockout mice display increased inflammatory responses and susceptibility to Mtb infection. Collectively, these results provide a link between SIRT1 activation and TB pathogenesis and indicate a potential of SIRT1 activators in designing an effective and clinically relevant host-directed therapies for TB.

The lengthy duration of multidrug therapy needed to cure tuberculosis (TB) poses significant challenges for global control of the disease. Moreover, chronic inflammation associated with TB leads to pulmonary damage that can remain even after successful cure. Thus, there is a great need for the development of effective shorter drug regimens to improve clinical outcome and strengthen TB control. Host-directed therapy (HDT) is emerging as a novel adjunctive strategy to enhance the efficacy and shorten the duration of TB treatment. Previously, we showed that the administration of CC-3052, a phosphodiesterase-4 inhibitor (PDE4i), reduced the host inflammatory response during Mycobacterium tuberculosis (Mtb) infection and improved the antimicrobial efficacy of isoniazid (INH) in both the mouse and rabbit models. In the present study, we evaluated the pharmacokinetics and explored the mechanism underlying the efficacy of a more potent PDE4i, CC-11050, as adjunct to INH treatment in a mouse model of pulmonary Mtb infection. Genome-wide lung transcriptome analysis confirmed the dampening of inflammation and associated network genes that we previously reported with CC-3052. Consistent with the reduction in inflammation, a significant improvement in Mtb control and pathology was observed in the lungs of mice treated with CC-11050 plus INH, compared to INH alone. This important confirmatory study will be used to help design upcoming human clinical trials with CC-11050 as an HDT for TB treatment.

Objectives: Adjunctive host-directed therapy is emerging as a new potential approach to improve the outcome of conventional antimicrobial treatment for tuberculosis (TB). We tested the ability of a phosphodiesterase-4 inhibitor (PDE4i) CC-11050, co-administered with the first-line anti-TB drug isoniazid (INH), to accelerate bacillary killing and reduce chronic inflammation in the lungs of rabbits with experimental Mycobacterium tuberculosis (Mtb) infection., Methods: A rabbit model of pulmonary TB that recapitulates the pathologic manifestations seen in humans was used. Rabbits were infected with virulent Mtb by aerosol exposure and treated for eight weeks with INH with or without CC-11050, starting at four weeks post infection. The effect of CC-11050 treatment on disease severity, pathology, bacillary load, T cell proliferation and global lung transcriptome profiles were analyzed., Results: Significant improvement in bacillary clearance and reduced lung pathology and fibrosis were noted in the rabbits treated for eight weeks with INH + CC-11050, compared to those treated with INH or CC-11050 only. In addition, expression of host genes associated with tissue remodeling, tumor necrosis factor alpha (TNF-α) regulation, macrophage activation and lung inflammation networks was dampened in CC-11050-treated, compared to the untreated rabbits., Conclusions: Adjunctive CC-11050 therapy significantly improves the response of rabbits with experimental pulmonary TB to INH treatment. We propose that CC-11050 may be a promising candidate for host directed therapy of patients with pulmonary TB, reducing the duration and improving clinical outcome of antibiotic treatment.

Bdellovibrio spp. and Micavibrio spp. are Gram-negative predators that feed on other Gram-negative bacteria, making predatory bacteria potential alternatives to antibiotics for treating multi-drug resistant infections. While the ability of predatory bacteria to control bacterial infections in vitro is well documented, the in vivo effect of predators on a living host has yet to be extensively examined. In this study, respiratory and intravenous inoculations were used to determine the effects of predatory bacteria in mice. We found no reduction in mouse viability after intranasal or intravenous inoculation of B. bacteriovorus 109J, HD100 or M. aeruginosavorus. Introducing predators into the respiratory tract of mice provoked a modest inflammatory response at 1 hour post-exposure, but was not sustained at 24 hours, as measured by RT-qPCR and ELISA. Intravenous injection caused an increase of IL-6 in the kidney and spleen, TNF in the liver and CXCL-1/KC in the blood at 3 hours post-exposure, returning to baseline levels by 18 hours. Histological analysis of tissues showed no pathological changes due to predatory bacteria. Furthermore, qPCR detected predators were cleared from the host quickly and efficiently. This work addresses some of the safety concerns regarding the potential use of predatory bacteria as a live antibiotic.

The formation and maintenance of granulomas is central to the host response to Mycobacterium tuberculosis (Mtb) infection. It is widely accepted that the lungs of patients with tuberculosis (TB) usually contain multiple infection foci, and that the granulomas evolve and differentiate independently, resulting in considerable heterogeneity. Although gene expression profiles of human blood cells have been proposed as biomarkers of Mtb infection and/or active disease, the immune profiles of discrete lesion types has not been studied extensively. Using histology, immunopathology and genome-wide transcriptome analysis, we explored the immunological profile of human lung TB granulomas. We show that although the different granulomas share core similarities in their immunological/inflammatory characteristics, they also exhibit significant divergence. Despite similar numbers of CD68+ macrophages in the different lesions, the extent of immune reactivity, as determined by the density of CD3+ T cells in the macrophage rich areas, and the extent of fibrosis, shows considerable variation. Both quantitative and qualitative differences among significantly differentially expressed genes (SDEG) were noted in each of the lesion types studied. Further, network/pathway analysis of SDEG revealed differential regulation of inflammatory response, immune cell trafficking, and cell mediated immune response in the different lesions. Our data highlight the formidable challenges facing ongoing efforts to identify peripheral blood biomarkers due to the diversity of lesion types and complexity of local immune responses in the lung.

The global burden of tuberculosis (TB) morbidity and mortality remains immense. A potential new approach to TB therapy is to augment protective host immune responses. We report that the antidiabetic drug metformin (MET) reduces the intracellular growth of Mycobacterium tuberculosis (Mtb) in an AMPK (adenosine monophosphate-activated protein kinase)-dependent manner. MET controls the growth of drug-resistant Mtb strains, increases production of mitochondrial reactive oxygen species, and facilitates phagosome-lysosome fusion. In Mtb-infected mice, use of MET ameliorated lung pathology, reduced chronic inflammation, and enhanced the specific immune response and the efficacy of conventional TB drugs. Moreover, in two separate human cohorts, MET treatment was associated with improved control of Mtb infection and decreased disease severity. Collectively, these data indicate that MET is a promising candidate host-adjunctive therapy for improving the effective treatment of TB., (Copyright © 2014, American Association for the Advancement of Science.)

Treatment of chronic inflammatory diseases with tumor necrosis factor alpha (TNF-α) antagonists has been associated with increased risk of tuberculosis (TB). We examined the usefulness of the rabbit model of active pulmonary TB for studying the impact of the human immune modulatory reagent etanercept on the host immune response. Control of Mycobacterium tuberculosis (Mtb) infection, disease pathology, and the global transcriptional response in Mtb-infected lungs of rabbits were studied. Etanercept treatment exacerbated disease pathology and reduced bacillary control in the lungs, compared with infected untreated animals. Reduced collagen and fibrin deposition in the granulomas was associated with significant downregulation of the collagen metabolism and fibrosis network genes and upregulation of genes in the inflammatory response and cell recruitment networks in the lungs of etanercept treated, compared with untreated rabbits. Our results suggest that targeting the TNF-α signaling pathway disrupts the tissue remodeling process, which is required for the formation and maintenance of well-differentiated granulomas and for control of Mtb growth in the lungs. These results validate the use of the rabbit model for investigating the impact of selected human immune modulatory drugs, such as a TNF-α antagonist, on the host immune response and pathogenesis in TB.

Background: Pulmonary infection of humans by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), results in active disease in 5-10% of individuals, while asymptomatic latent Mtb infection (LTBI) is established in the remainder. The host immune responses that determine this differential outcome following Mtb infection are not fully understood. Using a rabbit model of pulmonary TB, we have shown that infection with the Mtb clinical isolate HN878 (a hyper-virulent W-Beijing lineage strain) leads to progressive cavitary disease similar to what is seen in humans with active TB. In contrast, infection with Mtb CDC1551 (a hyper-immunogenic clinical isolate) is efficiently controlled in rabbit lungs, with establishment of LTBI, which can be reactivated upon treatment with immune-suppressive drugs. We hypothesize that the initial interaction of Mtb with the cells of the host response in the lungs determine later outcome of infection., Results: To test this hypothesis, we used our rabbit model of pulmonary TB and infected the animals with Mtb HN878 or CDC1551. At 3 hours, with similar lung bacillary loads, HN878 infection caused greater accumulation of mononuclear and polymorphonuclear leukocytes (PMN) in the lungs, compared to animals infected with CDC1551. Using whole-genome microarray gene expression analysis, we delineated the early transcriptional changes in the lungs of HN878- or CDC1551-infected rabbits at this time and compared them to the differential response at 4 weeks of Mtb-infection. Our gene network and pathway analysis showed that the most significantly differentially expressed genes involved in the host response to HN878, compared to CDC1551, at 3 hours of infection, were components of the inflammatory response and STAT1 activation, recruitment and activation of macrophages, PMN, and fMLP (N-formyl-Methionyl-Leucyl-Phenylalanine)-stimulation. At 4 weeks, the CDC1551 bacillary load was significantly lower and the granulomatous response reduced compared to HN878 infection. Moreover, although inflammation was dampened in both Mtb infections at 4 weeks, the majority of the differentially expressed gene networks were similar to those seen at 3 hours., Conclusions: We propose that differential regulation of the inflammation-associated innate immune response and related gene expression changes seen at 3 hours determine the long term outcome of Mtb infection in rabbit lungs.

Background: Infection of humans with Mycobacterium tuberculosis (Mtb) results in latent tuberculosis infection (LTBI) in 90-95% of immune competent individuals, with no symptoms of active disease. The World Health Organization estimates that 1.5 billion people have LTBI, which can reactivate in the setting of waning host immunity, posing a threat to global TB control. Various animal models have been used to study the pathogenesis of TB. However, besides nonhuman primates, rabbits are the only animal model that fully recapitulates the pathological features of human TB, including progressive disease with necrosis and cavitation or establishment of spontaneous latency., Results: We defined the molecular immunological correlates of LTBI establishment in a rabbit model of pulmonary infection with Mtb CDC1551. After aerosol infection, exponential bacterial growth was noted in the lungs for 4 weeks, followed by a significant decline by 12 weeks, resulting in the absence of cultivable bacilli by 24 weeks. We used rabbit whole genome microarrays to profile the lung transcriptome during the course of infection. At 2 weeks post-infection, gene networks involved in natural killer (NK) and dendritic cell (DC) activation and macrophage antimicrobial activities were highly upregulated. This was followed by upregulation of gene networks involved in macrophage and T cell activation and autophagy, peaking at 4 to 8 weeks. Concomitantly, host Th1, but not Th2 or inflammatory, immune response genes were significantly upregulated. Thus, the expression kinetics of genes involved in cross-talk between innate and adaptive immunity over the first 8 weeks post-infection were consistent with early efficient control of infection in the lungs. Interestingly, expression of many genes of the host innate and adaptive immune response pathways was downregulated at 12 weeks, suggesting that immune activation did not persist once bacilli began to clear from the infected lungs., Conclusions: Our results suggest that early activation of host innate immunity prior to efficient activation of T cell-mediated adaptive immunity but not inflammation is essential for establishment of LTBI in Mtb CDC1551-infected rabbits. We also show that T cell activation and the host adaptive immune response networks are dampened once bacterial growth is controlled, ultimately resulting in spontaneous LTBI.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune-competent individuals in the absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by a positive immunological response to Mtb antigens, with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts. Animal models of LTBI have been hampered generally by an inability to achieve complete bacillary clearance. Herein, we have characterized a rabbit model of LTBI in which, similar to most humans, complete clearance of pulmonary Mtb infection and pathological characteristics occurs spontaneously. The evidence that Mtb-CDC1551-infected rabbits achieve LTBI, rather than sterilization, is based on the ability of the bacilli to be reactivated after immune suppression. These rabbits showed early activation of T cells and macrophages and an early peak in the TNFα level, which decreased in association with clearance of bacilli from the lungs. In the absence of sustained tumor necrosis factor-α production, no necrosis was seen in the evolving lung granulomas. In addition, bacillary control was associated with down-regulation of several metalloprotease genes and an absence of lung fibrosis. This model will be used to characterize molecular markers of protective immunity and reactivation., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The molecular determinants of the immune response to Mycobacterium tuberculosis HN878 infection in a rabbit model of pulmonary cavitary tuberculosis were studied. Aerosol infection of rabbits resulted in a highly differentially expressed global transcriptome in the lungs at 2 weeks, which dropped at 4 weeks and then gradually increased. While IFNγ was progressively upregulated throughout the infection, several other genes in the IFNγ network were not. T-cell activation network genes were gradually upregulated and maximally induced at 12 weeks. Similarly, the IL4 and B-cell activation networks were progressively upregulated, many reaching high levels between 12 and 16 weeks. Delayed peak expression of genes associated with macrophage activation and Th1 type immunity was noted. Although spleen CD4(+) and CD8(+) T cells showed maximal tuberculosis antigen-specific activation by 8 weeks, macrophage activation in lungs, lymph nodes and spleen did not peak until 12 weeks. In the lungs, infecting bacilli grew exponentially up to 4 weeks, followed by a steady-state high bacillary load to 12 weeks that moderately increased during cavitation at 16 weeks. Thus, the outcome of HN878 infection of rabbits was determined early during infection by a suboptimal activation of innate immunity and delayed T-cell activation.

Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment.

Tuberculosis (TB) is responsible for significant morbidity and mortality worldwide. Even after successful microbiological cure of TB, many patients are left with residual pulmonary damage that can lead to chronic respiratory impairment and greater risk of additional TB episodes due to reinfection with Mycobacterium tuberculosis. Elevated levels of the proinflammatory cytokine tumor necrosis factor-α and several other markers of inflammation, together with expression of matrix metalloproteinases, have been associated with increased risk of pulmonary fibrosis, tissue damage, and poor treatment outcomes in TB patients. In this study, we used a rabbit model of pulmonary TB to evaluate the impact of adjunctive immune modulation, using a phosphodiesterase-4 inhibitor that dampens the innate immune response, on the outcome of treatment with the antibiotic isoniazid. Our data show that cotreatment of M. tuberculosis infected rabbits with the phosphodiesterase-4 inhibitor CC-3052 plus isoniazid significantly reduced the extent of immune pathogenesis, compared with antibiotic alone, as determined by histologic analysis of infected tissues and the expression of genes involved in inflammation, fibrosis, and wound healing in the lungs. Combined treatment with an antibiotic and CC-3052 not only lessened disease but also improved bacterial clearance from the lungs. These findings support the potential for adjunctive immune modulation to improve the treatment of pulmonary TB and reduce the risk of chronic respiratory impairment., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Despite the availability of many animal models for tuberculosis (TB) research, there still exists a need for better understanding of the quiescent stage of disease observed in many humans. Here, we explored the use of the Wistar rat model for the study of protective immunity and control of Mycobacterium tuberculosis (Mtb) infection., Methodology/principal Findings: The kinetics of bacillary growth, evaluated by the colony stimulating assay (CFU) and the extent of lung pathology in Mtb infected Wistar rats were dependent on the virulence of the strains and the size of the infecting inoculums. Bacillary growth control was associated with induction of T helper type 1 (Th1) activation, the magnitude of which was also Mtb strain and dose dependent. Histopathology analysis of the infected lungs demonstrated the formation of well organized granulomas comprising epithelioid cells, multinucleated giant cells and foamy macrophages surrounded by large numbers of lymphocytes. The late stage subclinical form of disease was reactivated by immunosuppression leading to increased lung CFU., Conclusion: The Wistar rat is a valuable model for better understanding host-pathogen interactions that result in control of Mtb infection and potentially establishment of latent TB. These properties together with the ease of manipulation, relatively low cost and well established use of rats in toxicology and pharmacokinetic analyses make the rat a good animal model for TB drug discovery.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the leading infectious disease causes of morbidity and mortality worldwide. Though current antibiotic regimens can cure the disease, treatment requires at least six months of drug therapy. One reason for the long duration of therapy is that the currently available TB drugs were selected for their ability to kill replicating organisms and are less effective against subpopulations of non-replicating persistent bacilli. Evidence from in vitro models of Mtb growth and mouse infection studies suggests that host immunity may provide some of the environmental cues that drive Mtb towards non-replicating persistence. We hypothesized that selective modulation of the host immune response to modify the environmental pressure on the bacilli may result in better bacterial clearance during TB treatment. For this proof of principal study, we compared bacillary clearance from the lungs of Mtb-infected mice treated with the anti-TB drug isoniazid (INH) in the presence and absence of an immunomodulatory phosphodiesterase 4 inhibitor (PDE4i), CC-3052. The effects of CC-3052 on host global gene expression, induction of cytokines, and T cell activation in the lungs of infected mice were evaluated. We show that CC-3052 modulates the innate immune response without causing generalized immune suppression. Immune modulation combined with INH treatment improved bacillary clearance and resulted in smaller granulomas and less lung pathology, compared to treatment with INH alone. This novel strategy of combining anti-TB drugs with an immune modulating molecule, if applied appropriately to patients, may shorten the duration of TB treatment and improve clinical outcome.

The progression of human tuberculosis (TB) to active disease and transmission involves the development of a caseous granuloma that cavitates and releases infectious Mycobacterium tuberculosis bacilli. In the current study, we exploited genome-wide microarray analysis to determine that genes for lipid sequestration and metabolism were highly expressed in caseous TB granulomas. Immunohistological analysis of these granulomas confirmed the disproportionate abundance of the proteins involved in lipid metabolism in cells surrounding the caseum; namely, adipophilin, acyl-CoA synthetase long-chain family member 1 and saposin C. Biochemical analysis of the lipid species within the caseum identified cholesterol, cholesteryl esters, triacylglycerols and lactosylceramide, which implicated low-density lipoprotein-derived lipids as the most likely source. M. tuberculosis infection in vitro induced lipid droplet formation in murine and human macrophages. Furthermore, the M. tuberculosis cell wall lipid, trehalose dimycolate, induced a strong granulomatous response in mice, which was accompanied by foam cell formation. These results provide molecular and biochemical evidence that the development of the human TB granuloma to caseation correlates with pathogen-mediated dysregulation of host lipid metabolism.

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the beta-clamp, consistent with its canonical C-terminal beta-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N(2)-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

Mycobacterium tuberculosis possesses a diversity of potential virulence factors including complex branched lipids such as the phenolic glycolipid PGL-tb. PGL-tb expression by the clinical M. tuberculosis isolate HN878 has been associated with a less efficient Th1 response and increased virulence in mice and rabbits. It has been suggested that the W-Beijing family is the only group of M. tuberculosis strains with an intact pks1-15 gene, required for the synthesis of PGL-tb and capable of producing PGL-tb. We have found that some strains with an intact pks1-15 do not produce PGL-tb while others may produce a variant of PGL-tb. We examined the early host cytokine response to infection with these strains in vitro to better understand the effect of PGL-tb synthesis on immune responses. In addition, we generated a PGL-tb-producing H37Rv in order to determine the effect of PGL-tb production on the host immune response during infection by a strain normally devoid of PGL-tb synthesis. We observed that PGL-tb production by clinical M. tuberculosis isolates affected cytokine production differently depending on the background of the strain. Importantly, while ectopic PGL-tb production by H37Rv suppressed the induction of several pro- and anti-inflammatory cytokines in vitro in human monocytes, it did not lead to increased virulence in infected mice and rabbits. Collectively, our data indicate that, while PGL-tb may play a role in the immunogenicity and/or virulence of M. tuberculosis, it probably acts in concert with other bacterial factors which seem to be dependent on the background of the strain.

Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.

Using a rabbit model of tuberculous meningitis (TBM), we compared the protective efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination against central nervous system infection with the virulent M. tuberculosis clinical isolate HN878 and the laboratory strain H37Rv. Although BCG clearly provided protection against infection with either challenge strain, protection against disease manifestations was significantly poorer in rabbits infected with HN878. BCG was less efficient in protecting against HN878 dissemination to the liver and spleen and against HN878-induced inflammation, loss of body weight, lung and brain pathology, and signs of disease. We suggest that the efficacy of newly developed vaccines should be tested in animal models not only against challenge with M. tuberculosis H37Rv but also with different clinical isolates including the highly virulent strains of the W-Beijing family.

Using a rabbit model of tuberculous meningitis, we evaluated the protective efficacy of vaccination with the recombinant polyprotein Mtb72F, which is formulated in two alternative adjuvants, AS02A and AS01B, and compared this to vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) alone or as a BCG prime/Mtb72F-boost regimen. Vaccination with Mtb72F formulated in AS02A (Mtb72F+AS02A) or Mtb72F formulated in AS01B (Mtb72F+AS01B) was protective against central nervous system (CNS) challenge with Mycobacterium tuberculosis H37Rv to an extent comparable to that of vaccination with BCG. Similar accelerated clearances of bacilli from the cerebrospinal fluid, reduced leukocytosis, and less pathology of the brain and lungs were noted. Weight loss of infected rabbits was less extensive for Mtb72F+AS02A-vaccinated rabbits. In addition, protection against M. tuberculosis H37Rv CNS infection afforded by BCG/Mtb72F in a prime-boost strategy was similar to that by BCG alone. Interestingly, Mtb72F+AS01B induced better protection against leukocytosis and weight loss, suggesting that the polyprotein in this adjuvant may boost immunity without exacerbating inflammation in previously BCG-vaccinated individuals.

The role of type I interferons (IFNs) in the host response to bacterial infections is controversial. Here, we examined the role of IFN-alpha/beta in the murine response to infection with Mycobacterium tuberculosis, using wildtype mice, mice with impaired signaling through the type I IFN receptor (IFNAR), and mice treated to reduce levels of type I IFNs. In this study, we used virulent clinical isolates of M. tuberculosis, including HN878, W4, and CDC1551. Our results indicate that higher levels of type I IFNs are induced by the HN878 and W4 strains. Induction of type I IFNs was associated with lower levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin- 12 (IL-12) and reduced T cell activation, and associated with decreased survival of the mice infected with HN878 or W4 relative to infection with CDC1551. Infection of mice with HN878 and W4 was also associated with relatively higher levels of mRNA for a number of negative regulators of the Jak-Stat signaling pathway, such as suppressors of cytokine signaling (SOCS) 1, 4, and 5, CD45, protein inhibitor of activated Stat1 (PIAS1), protein tyrosine phosphatase nonreceptor type 1 (Ptpn1), and protein tyrosine phosphatase nonreceptor type substrate 1 (Ptpns1). Taken together, these results suggest that increased type I IFNs may be deleterious for survival of M. tuberculosis-infected mice in association with reduced Th1 immunity.

Infection with Mycobacterium tuberculosis in humans results in active disease in approximately 10% of immune-competent individuals, with the most-severe clinical manifestations observed when the bacilli infect the central nervous system (CNS). Here, we use a rabbit model of tuberculous meningitis to evaluate the severity of disease caused by the M. tuberculosis clinical isolates CDC1551, a highly immunogenic strain, and HN878 or W4, 2 members of the W/Beijing family of strains. Compared with infection with CDC1551, CNS infection with HN878 or W4 resulted in higher bacillary loads in the cerebrospinal fluid and brain, increased dissemination of bacilli to other organs, persistent levels of tumor necrosis factor-alpha , higher leukocytosis, and more-severe clinical manifestations. This pathogenic process is associated with the production by HN878 of a polyketide synthase-derived phenolic glycolipid (PGL), as demonstrated by reduced virulence in rabbits infected with an HN878 mutant disrupted in the pks1-15 gene, which is required for PGL synthesis.

Pathogenetic processes that facilitate the entry, replication, and persistence of Mycobacterium tuberculosis (MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection. Identification of genes that are differentially expressed in vivo would provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited. In this study, the levels of selected MTB mRNAs were quantified in vitro in axenic culture, in vivo in the lungs of mice, and in lung specimens obtained from TB patients with active disease. We report the differential expression of MTB mRNAs associated with iron limitation, alternative carbon metabolism, and cellular hypoxia, conditions that are thought to exist within the granulomatous lesions of TB, in the lungs of wild-type C57BL/6 mice as compared with bacteria grown in vitro. Analysis of the same set of mRNAs in lung specimens obtained from TB patients revealed differences in MTB gene expression in humans as compared with mice.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a DeltanrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O(2)-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O(2) restriction. However, survival of the DeltanrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F(1) mice infected with the DeltanrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.

Tuberculous meningitis (TBM), the most severe form of Mycobacterium tuberculosis infection in humans, is associated with significant morbidity and mortality despite successful treatment with antituberculous drugs. This is due to the irreversible brain damage subsequent to the local inflammatory response of the host to M. tuberculosis. Corticosteroids have been used in conjunction with antituberculous therapy in an attempt to modulate the inflammatory response, but this strategy has been of limited success. Therefore, we examined whether combining antituberculous drugs with the immunomodulatory drug thalidomide or with a new thalidomide analog, immunomodulatory drug 3 (IMiD3), would be effective in reducing morbidity and mortality in an experimental rabbit model of TBM. Intracisternal inoculation of 5 x 10(4) CFU of Mycobacterium bovis Ravenel in rabbits induced progressive subacute meningitis characterized by high cerebrospinal fluid (CSF) leukocytosis, protein influx, release of tumor necrosis factor (TNF), substantial meningeal inflammation, and mortality by day 28. Treatment with antituberculous drugs or with antituberculous drugs plus thalidomide improved the clinical course of disease somewhat and increased survival to about 50%. In contrast, treatment with antituberculous drugs in combination with IMiD3 limited pathological neurologic changes and resulted in marked improvement (73%) in survival. IMiD3 treatment was also associated with reduced leukocytosis in the CSF and significantly lower levels of TNF in CSF and plasma. Histologically, the meningeal inflammation in animals treated with antituberculous drugs plus IMiD3 was considerably attenuated compared to that of the other treatment groups. These results suggest a potential role for IMiD3 in the management of TBM in patients.

To control tuberculosis worldwide, the burden of adult pulmonary disease must be reduced. Although widely used, Mycobacterium bovis BCG vaccination given at birth does not protect against adult pulmonary disease. Therefore, postexposure vaccination of adults with mycobacterial antigens is being considered. We examined the effect of various mycobacterial antigens on mice with prior M. tuberculosis infection. Subcutaneous administration of live or heat-treated BCG with or without lipid adjuvants to infected mice induced increased antigen-specific T-cell proliferation but did not reduce the bacterial load in the lungs and caused larger lung granulomas. Similarly, additional mycobacterial antigen delivered directly to the lungs by aerosol infection with viable M. tuberculosis mixed with heat-killed Mycobacterium tuberculosis (1:1) also did not reduce the bacillary load but caused increased expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), which was associated with larger granulomas in the lungs. When M. tuberculosis-infected mice were treated with recombinant BCG that secreted cytokines shown to reduce disease in a preinfection vaccine model, the BCG secreting TNF-alpha, and to a lesser extent, IL-2 and gamma interferon (IFN-gamma), caused a significant increase in granuloma size in the lungs. Moreover, treatment of M. tuberculosis-infected mice with recombinant murine TNF-alpha resulted in increased inflammation in the lungs and accelerated mortality without affecting the bacillary load. Taken together, these studies suggest that administration of mycobacterial antigens to mice with prior M. tuberculosis infection leads to immune activation that may exacerbate lung pathology via TNF-alpha-induced inflammation without reducing the bacillary load.

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-gamma production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and IFN-gamma mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-alpha mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-alpha/beta. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

To better understand the contribution of interferon-gamma (IFN-gamma) to the immune response during the first 60 days of mycobacterial infection in the lungs, IFN-gamma gene disrupted (IFN-gamma-/-) mice were infected via aerosol with recombinant Mycobacterium bovis Bacillus Calmette-Guerin (BCG) secreting murine IFN-gamma (BCG-IFN-gamma) and compared to mice infected with recombinant BCG containing the vector only (BCG-vector). When IFN-gamma-/- mice were infected with BCG-vector, increasing bacillary loads and large undifferentiated granulomas that did not express inducible nitric oxide synthase (iNOS) were observed in the lungs. In contrast, infection with BCG-IFN-gamma resulted in reduced bacillary load and better differentiated granulomas containing epithelioid macrophages expressing iNOS as well as reduced levels of interleukin 10 (IL-10) mRNA. However, local production of IFN-gamma by the recombinant BCG did not protect IFN-gamma-/- mice from subsequent challenge with M. tuberculosis. Infection of IFN-gamma-/- peritoneal macrophages in vitro with BCG-IFN-gamma led to induction of iNOS expression and lower IL-10 mRNA levels. Nevertheless, the growth of the intracellular BCG was unaffected. Since IFN-gamma induced-iNOS protein and reduced IL-10 production were insufficient to control mycobacterial growth in vitro, the results suggest that additional mediator(s) present in vivo are required for control of mycobacterial growth., (Copyright 2000 Academic Press.)

Mycobacterium tuberculosis CDC1551, a clinical isolate reported to be hypervirulent and to grow faster than other isolates, was compared with two other clinical isolates (HN60 and HN878) and two laboratory strains (H37Rv and Erdman). The initial (1-14 days) growth of CDC1551, HN60, HN878, and H37Rv was similar in the lungs of aerosol-infected mice, but growth of Erdman was slower. Thereafter, the growth rate of CDC1551 decreased relative to the other strains which continued to grow at comparable rates up to day 21. In the lungs of CDC1551-infected mice, small well-organized granulomas with high levels of TNF-alpha, IL-6, IL-10, IL-12, and IFN-gamma mRNA were apparent sooner than in lungs of mice infected with the other strains. CDC1551-infected mice survived significantly longer. These findings were confirmed in vitro. The growth rates of H37Rv and CDC1551 in human monocytes were the same, but higher levels of TNF-alpha, IL-10, IL-6, and IL-12 were induced in monocytes after infection with CDC1551 or by exposure of monocytes to lipid fractions from CDC1551. CD14 expression on the surface of the monocytes was up-regulated to a greater extent by exposure to the lipids of CDC1551. Thus, CDC1551 is not more virulent than other M. tuberculosis isolates in terms of growth in vivo and in vitro, but it induces a more rapid and robust host response.

The pathogenesis of tuberculous meningitis, a devastating complication of tuberculosis in man, is poorly understood. We previously reported that rabbits with experimental tuberculous meningitis were protected from death by a combination of antibiotics and thalidomide therapy. Survival was associated with inhibition of tumor necrosis factor alpha (TNF-alpha) production by thalidomide. To test whether cerebrospinal fluid (CSF) levels of TNF-alpha correlated with pathogenesis, the response of rabbits infected in the central nervous system (CNS) with various mycobacterial strains was studied. CNS infection with Mycobacterium bovis Ravenel, M. bovis bacillus Calmette-Guérin (BCG) Pasteur, and M. bovis BCG Montreal were compared. M. bovis Ravenel induced the highest levels of TNF-alpha in the CSF in association with high leukocytosis, protein accumulation, and severe meningeal inflammation. BCG Pasteur had intermediate effects, and BCG Montreal was the least virulent. In addition, M. bovis Ravenel numbers were highest in the brain and CSF and the bacilli also disseminated more efficiently to distant organs, compared with BCG Pasteur and BCG Montreal. In subsequent experiments, rabbits were infected with either recombinant M. bovis BCG Montreal (vector), or BCG Montreal expressing the murine gene for TNF-alpha (BCG mTNF-alpha). BCG Montreal was rendered virulent by the expression of murine TNF-alpha, as demonstrated by high CSF leukocytosis, high protein accumulation, severe meningeal inflammation, persistent bacillary load, and progressive clinical deterioration. Taken together, these results demonstrate that the level of TNF-alpha produced during mycobacterial CNS infection determines, at least in part, the extent of pathogenesis.

Tuberculous meningitis (TBM) is a devastating form of tuberculosis that occurs predominantly in children and in immunocompromised adults. To study the pathogenesis of TBM, a rabbit model of acute mycobacterial central nervous system infection was set up (8-day study). Inoculation of live Mycobacterium bovis Ravenel intracisternally induced leukocytosis (predominantly mononuclear cells), high protein levels, and release of tumor necrosis factor-alpha (TNF-alpha) into the cerebrospinal fluid within 1 day. Histologically, severe meningitis with thickening of the leptomeninges, prominent vasculitis, and encephalitis was apparent, and mortality was 75% by day 8. In animals treated with antituberculous antibiotics only, the inflammation and lesions of the brain persisted despite a decrease in mycobacteria; 50% of the rabbits died. When thalidomide treatment was combined with antibiotics, there was a marked reduction in TNF-alpha levels, leukocytosis, and brain pathology. With this combination treatment, 100% of the infected rabbits survived, suggesting a potential clinical use for thalidomide in TBM.