A single‐stranded DNA aptamer against mannose‐capped lipoarabinomannan enhances anti‐tuberculosis activity of macrophages through downregulation of lipid‐sensing nuclear receptor peroxisome proliferator‐activated receptor γ expression

Mannose‐capped lipoarabinomannan (ManLAM) is an immunomodulatory epitope of Mycobacterium tuberculosis (Mtb). An aptamer (ZXL1) that specifically binds to ManLAM from the virulent Mtb H37Rv strain was previously generated and it was found that ZXL1 functions as an antagonist, inhibiting the ManLAM‐induced immunosuppression of DCs. In the present study, it was found that ZXL1 inhibits Mtb entry into murine macrophages and that ZXL1 enhances IL‐1β and IL‐12 mRNA expression and cytokine production in ManLAM‐treated macrophages but decreases IL‐10 production. Inducible nitric oxide synthase expression in macrophages was upregulated in the presence of ZXL1 after stimulation with ManLAM. ZXL1 was also found to inhibit expression of lipid‐sensing nuclear receptor peroxisome proliferator‐activated receptor γ (PPAR‐γ). These results suggest that ZXL1 promotes anti‐tuberculosis activity through downregulation of PPAR‐γ expression, which may contribute to M1 macrophage polarization and Mtb killing by macrophages.     

Multiple cytokines are released when blood from patients with tuberculosis is stimulated with Mycobacterium tuberculosis antigens

Background

Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-γ) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-γ were assessed for potential to provide robust measures of Mtb infection.

Methods

Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens.

Results

Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-γ, IL-2, IL-8, MCP-1 and MIP-1β for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-γ, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, and TNF-α responses among patients compared with controls. One CCTB patient with a falsely negative IFN-γ response had elevated responses with other cytokines.

Conclusions

Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-γ alone.     

Simultaneous immunization against tuberculosis

Background

BCG, the only licensed vaccine against tuberculosis, provides some protection against disseminated disease in infants but has little effect on prevention of adult pulmonary disease. Newer parenteral immunization prime boost regimes may provide improved protection in experimental animal models but are unproven in man so that there remains a need for new and improved immunization strategies.

Methods and Findings

Mice were immunized parenterally, intranasally or simultaneously by both routes with BCG or recombinant mycobacterial antigens plus appropriate adjuvants. They were challenged with Mycobacterium tuberculosis (Mtb) and the kinetics of Mtb growth in the lungs measured. We show that simultaneous immunization (SIM) of mice by the intranasal and parenteral routes is highly effective in increasing protection over parenteral BCG administration alone. Intranasal immunization induces local pulmonary immunity capable of inhibiting the growth of Mtb in the early phase (the first week) of infection, while parenteral immunization has a later effect on Mtb growth. Importantly, these two effects are additive and do not depend on priming and boosting the immune response. The best SIM regimes reduce lung Mtb load by up to 2 logs more than BCG given by either route alone.

Conclusions

These data establish SIM as a novel and highly effective immunization strategy for Mtb that could be carried out at a single clinic visit. The efficacy of SIM does not depend on priming and boosting an immune response, but SIM is complementary to prime boost strategies and might be combined with them.     

Innate immune response to Mycobacterium tuberculosis Beijing and other genotypes

Background

As a species, Mycobacterium tuberculosis is more diverse than previously thought. In particular, the Beijing family of M. tuberculosis strains is spreading and evoluating throughout the world and this is giving rise to public health concerns. Genetic diversity within this family has recently been delineated further and a specific genotype, called Bmyc10, has been shown to represent over 60% of all Beijing clinical isolates in several parts of the world. How the host immune system senses and responds to various M. tuberculosis strains may profoundly influence clinical outcome and the relative epidemiological success of the different mycobacterial lineages. We hypothesised that the success of the Bmyc10 group may, at least in part, rely upon its ability to alter innate immune responses and the secretion of cytokines and chemokines by host phagocytes.

Methodology/Principal Findings

We infected human macrophages and dendritic cells with a collection of genetically well-defined M. tuberculosis clinical isolates belonging to various mycobacterial families, including Beijing. We analyzed cytokine and chemokine secretion on a semi-global level using antibody arrays allowing the detection of sixty-five immunity-related soluble molecules. Our data indicate that Beijing strains induce significantly less interleukin (IL)-6, tumor necrosis factor (TNF), IL-10 and GRO-α than the H37Rv reference strain, a feature that is variously shared by other modern and ancient M. tuberculosis families and which constitutes a signature of the Beijing family as a whole. However, Beijing strains did not differ relative to each other in their ability to modulate cytokine secretion.

Conclusions/Significance

Our results confirm and expand upon previous reports showing that M. tuberculosis Beijing strains in general are poor in vitro cytokine inducers in human phagocytes. The results suggest that the epidemiological success of the Beijing Bmyc10 is unlikely to rely upon any specific ability of this group of strains to impair anti-mycobacterial innate immunity.     

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

Expression of the ARPC4 Subunit of Human Arp2/3 Severely Affects Mycobacterium tuberculosis Growth and Suppresses Immunogenic Response in Murine Macrophages

Background

The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms facilitating the intra-macrophage survival of Mycobacterium tuberculosis are as yet not entirely understood. However, there is evidence showing the involvement of host cell cytoskeleton in every step of establishment and persistence of mycobacterial infection.

Methodology/Principal Findings

Here we show that expression of ARPC4, a subunit of the Actin related protein 2/3 (Arp2/3) protein complex, severely affects the pathogen’s growth. TEM studies display shedding of the mycobacterial outer-coat. Furthermore, in infected macrophages, mycobacteria expressing ARPC4 were cleared off at a much faster rate, and were unable to mount a pro-inflammatory cytokine response. The translocation of ARPC4-expressing mycobacteria to the lysosome of the infected macrophage was also impaired. Additionally, the ARPC4 subunit was shown to interact with Rv1626, an essential secretory mycobacterial protein. Real-time PCR analysis showed that upon expression of ARPC4 in mycobacteria, Rv1626 expression is downregulated as much as six-fold. Rv1626 was found to also interact with mammalian cytoskeleton protein, Arp2/3, and enhance the rate of actin polymerization.

Conclusions/Significance

With crystal structures for Rv1626 and ARPC4 subunit already known, our finding lays out the effect of a novel molecule on mycobacteria, and represents a viable starting point for developing potent peptidomimetics.     

Role of pulmonary intravascular macrophages in endotoxin-induced lung inflammation and mortality in a rat model

Background

Bile-duct ligated (BDL) rats recruit pulmonary intravascular macrophages (PIMs) and are highly susceptible to endotoxin-induced mortality. The mechanisms of this enhanced susceptibility and mortality in BDL rats, which are used as a model of hepato-pulmonary syndrome, remain unknown. We tested a hypothesis that recruited PIMs promote endotoxin-induced mortality in a rat model.

Methods

Rats were subjected to BDL to induce PIM recruitment followed by treatment with gadolinium chloride (GC) to deplete PIMs. Normal and BDL rats were treated intravenously with E. coli lipopolysaccharide (LPS) with or without GC pre-treatment followed by collection and analyses of lungs for histopathology, electron microscopy and cytokine quantification.

Results

BDL rats recruited PIMs without any change in the expression of IL-1β, TNF-α and IL-10. GC caused reduction in PIMs at 48 hours post-treatment (P < 0.05). BDL rats treated intravenously with E. coli LPS died within 3 hours of the challenge while the normal LPS-treated rats were euthanized at 6 hours after the LPS treatment. GC treatment of rats 6 hours or 48 hours before LPS challenge resulted in 80% (1/5) and 100% (0/5) survival, respectively, at 6 hours post-LPS treatment. Lungs from BDL+LPS rats showed large areas of perivascular hemorrhages compared to those pre-treated with GC. Concentrations of IL-1β, TNF-α and IL-10 were increased in lungs of BDL+LPS rats compared to BDL rats treated with GC 48 hours but not 6 hours before LPS (P < 0.05).

Conclusion

We conclude that PIMs increase susceptibility for LPS-induced lung injury and mortality in this model, which is blocked by a reduction in their numbers or their inactivation.     

Application of Hyperbranched Rolling Circle Amplification for Direct Detection of Mycobacterium Tuberculosis in Clinical Sputum Specimens

Background

Global tuberculosis (TB) control is encumbered by the lack of a rapid and simple detection method for diagnosis, especially in low-resource areas. An isothermal amplification method, hyperbranched rolling circle amplification (HRCA), was optimized to detect Mycobacterium tuberculosis (Mtb) in clinical sputum specimens.

Methods

A clinical validation study was performed to assess the diagnostic accuracy of HRCA. In order to analyze the detection limit of HRCA under optimal conditions, the method was initially used to detect purified H37Rv strain DNA and culture suspensions. Next, three strains of Mycobacterium tuberculosis complex (MTC) and eight strains of non-tuberculosis mycobacterium (NTM) were analyzed in order to evaluate specificity. Sputum specimens from 136 patients with diagnosed pulmonary TB, 38 lung cancer patients, and 34 healthy donors were tested by HRCA to validate the clinical application of HRCA for the rapid detection of Mtb.

Results

The detection limit of HRCA for purified H37Rv DNA and culture suspensions was 740 aM and 200cfu/ml, respectively. The results of all MTC strains were positive in contrast to the NTM specimens which were all negative. The detection sensitivity for the 136 sputum specimens from TB patients was 77.2% (105/136), which was slightly lower than that of quantitative real-time PCR(79.4%, 108/136) and culture (80.9%,110/136). The sensitivity of all three methods was statistically higher than smear microscopy (44.9%, 61/136). The overall specificity of HRCA was 98.6% (71/72) which was similar to that of quantitative real-time PCR (qRT-PCR) and smear/culture methods (100%, 72/72).

Conclusions

Use of the HRCA assay for detection of Mtb within clinical sputum specimens was demonstrated to be highly sensitive and specific. Moreover, the performance of HRCA is simple and cost-effective compared with qRT-PCR and is less time consuming than culture. Therefore, HRCA is a promising TB diagnostic tool that can be used routinely in low-resource clinical settings.     

Orientia tsutsugamushi Subverts Dendritic Cell Functions by Escaping from Autophagy and Impairing Their Migration

Background

Dendritic cells (DCs) are the most potent antigen-presenting cells that link innate and adaptive immune responses, playing a pivotal role in triggering antigen-specific immunity. Antigen uptake by DCs induces maturational changes that include increased surface expression of major histocompatibility complex (MHC) and costimulatory molecules. In addition, DCs actively migrate to regional lymph nodes and activate antigen-specific naive T cells after capturing antigens. We characterize the functional changes of DCs infected with Orientia tsutsugamushi, the causative agent of scrub typhus, since there is limited knowledge of the role played by DCs in O. tsutsugamushi infection.

Methodology/Principal Finding

O. tsutsugamushi efficiently infected bone marrow-derived DCs and induced surface expression of MHC II and costimulatory molecules. In addition, O. tsutsugamushi induced autophagy activation, but actively escaped from this innate defense system. Infected DCs also secreted cytokines and chemokines such as IL-6, IL-12, MCP5, MIP-1α, and RANTES. Furthermore, in vitro migration of DCs in the presence of a CCL19 gradient within a 3D collagen matrix was drastically impaired when infected with O. tsutsugamushi. The infected cells migrated much less efficiently into lymphatic vessels of ear dermis ex vivo when compared to LPS-stimulated DCs. In vivo migration of O. tsutsugamushi-infected DCs to regional lymph nodes was significantly impaired and similar to that of immature DCs. Finally, we found that MAP kinases involved in chemotactic signaling were differentially activated in O. tsutsugamushi-infected DCs.

Conclusion/Significance

These results suggest that O. tsutsugamushi can target DCs to exploit these sentinel cells as replication reservoirs and delay or impair the functional maturation of DCs during the bacterial infection in mammals.     

Variation of Mycobacterium tuberculosis Antigen-Specific IFN-γ and IL-17 Responses in Healthy Tuberculin Skin Test (TST)-Positive Human Subjects

Objective

To determine the variation of IFN-γ and IL-17 responses to M. tuberculosis antigens in healthy TST+ humans.

Methods

We isolated peripheral blood mononuclear cells from 21 TST+ healthy adults, stimulated them with phytohemagglutinin (PHA), PPD, Ag85B, ESAT-6, and live M. bovis BCG, and assayed IFN-γ and IL-17 secretion by ELISA in supernatants after 24 or 72 hours of incubation respectively.

Results

As in other studies, we found a wide range of IFN-γ responses to M. tuberculosis antigens; the variation significantly exceeded that observed in the same donors to the polyclonal T cell stimulus, phytohemagglutinin (PHA). In addition, we assayed IL-17 secretion in response to the same stimuli, and found less subject-to-subject variation. Analysis of the ratio of IFN-γ to IL-17 secretion on a subject-to-subject basis also revealed a wide range, with the majority of results distributed in a narrow range, and a minority with extreme results all of which were greater than that in the majority of subjects. The data suggest that study of exceptional responses to M. tuberculosis antigens may reveal immunologic correlates with specific outcomes of M. tuberculosis infection.

Conclusion

Variation of IFNγ and IFN-γ/IL-17 responses to mycobacterial antigens exceeds that of responses to the polyclonal stimulus, PHA, in TST positive healthy humans. This indicates a quantitative spectrum of human immune responses to infection with M. tuberculosis. Since the outcome of human infection with M. tuberculosis varies greatly, systematic study of multiple immune responses to multiple antigens is likely to reveal correlations between selected immune responses and the outcomes of infection.     

Endoplasmic reticulum stress pathway-mediated apoptosis in macrophages contributes to the survival of Mycobacterium tuberculosis

Background

Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages.

Methodology/Principal Findings

Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response.

Conclusion/Significance

These findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria.     

TLR3 regulates mycobacterial RNA-induced IL-10 production through the PI3K/AKT signaling pathway

Cytokine induction in response to Mycobacterium tuberculosis (Mtb) infection is critical for pathogen control, by (i) mediating innate immune effector functions and (ii) instructing specific adaptive immunity. IL-10 is an important anti-inflammatory cytokine involved in pathogenesis of tuberculosis (TB). Here, we show that TLR3, a sensor of extracellular viral or host RNA with stable stem structures derived from infected or damaged cells, is essential for Mtb-induced IL-10 production. Upon Mycobacterium bovis Bacillus Calmette–Guérin (BCG) infection, TLR3^−/− macrophages expressed lower IL-10 but higher IL-12p40 production, accompanied by reduced phosphorylation of AKT at Ser473. BCG-infected TLR3^−/− mice exhibited reduced IL-10 but elevated IL-12 expression compared to controls. Moreover, higher numbers of splenic Th1 cells and reduced pulmonary bacterial burden and tissue damage were observed in BCG-infected TLR3^−/− mice. Finally, BCG RNA induced IL-10 in macrophages via TLR3-mediated activation of PI3K/AKT. Our findings demonstrate a critical role of TLR3-mediated regulation in the pathogenesis of mycobacterial infection involving mycobacterial RNA, which induces IL-10 through the PI3K/AKT signaling pathway.     

CD86 and IL-12p70 Are Key Players for T Helper 1 Polarization and Natural Killer Cell Activation by Toll-Like Receptor-Induced Dendritic Cells

Background

Dendritic cells (DCs) determine the activation and polarization of T cells via expression of costimulatory molecules and secretion of cytokines. The function of DCs derived from monocytes ex vivo strongly depends on the composition of the maturation cocktail used.

Methodology/Principal Findings

We analyzed the effect of costimulatory molecule expression and cytokine secretion by DCs on T and natural killer (NK) cell activation by conducting a head-to-head comparison of a Toll-like receptor (TLR) agonist-based cocktail with the standard combination of proinflammatory cytokines or IL-10 alone. We could show that TLR-induced DCs are characterized by a predominance of costimulatory over coinhibitory molecules and by high secretion of IL-12p70, but not IL-10. Functionally, these signals translated into an increase in IFN-γ secreting Th1 cells and a decrease in regulatory T cells. T cell activation and polarization were dependent on IL-12p70 and CD86, but remarkably not on CD80 signaling. By means of IL-12p70 secretion, only TLR-induced DCs activated NK cells.

Conclusions/Significance

TLR-matured DCs are highly suitable for application in immunotherapeutic strategies that rely on strong type 1 polarization and NK cell activation. Their effects particularly depend on high CD86 expression and IL-12p70 secretion.     

Mycobacterium tuberculosis Infection of Dendritic Cells Leads to Partially Caspase-1/11-Independent IL-1β and IL-18 Secretion but Not to Pyroptosis

Background

Interleukin-1β (IL-1β) is important for host resistance against Mycobacterium tuberculosis (Mtb) infections. The response of the dendritic cell inflammasome during Mtb infections has not been investigated in detail.

Methodology/Principal Findings

Here we show that Mtb infection of bone marrow-derived dendritic cells (BMDCs) induces IL-1β secretion and that this induction is dependent upon the presence of functional ASC and NLRP3 but not NLRC4 or NOD2. The analysis of cell death induction in BMDCs derived from these knock-out mice revealed the important induction of host cell apoptosis but not necrosis, pyroptosis or pyronecrosis. Furthermore, NLRP3 inflammasome activation and apoptosis induction were both reduced in BMDCs infected with the esxA deletion mutant of Mtb demonstrating the importance of a functional ESX-1 secretion system. Surprisingly, caspase-1/11-deficient BMDCs still secreted residual levels of IL-1βand IL-18 upon Mtb infection which was abolished in cells infected with the esxA Mtb mutant.

Conclusion

Altogether we demonstrate the partially caspase-1/11-independent, but NLRP3- and ASC- dependent IL-1β secretion in Mtb-infected BMDCs. These findings point towards a potential role of DCs in the host innate immune response to mycobacterial infections via their capacity to induce IL-1β and IL-18 secretion.     

Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells

Background

Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies.

Methodology/Principal Findings

Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b⁺/CD11c⁺ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.

Conclusions/Significance

The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.     

Protective immunity to Mycobacterium tuberculosis infection by chemokine and cytokine conditioned CFP-10 differentiated dendritic cells

Background

Dendritic cells (DCs) play major roles in mediating immune responses to mycobacteria. A crucial aspect of this is the priming of T cells via chemokines and cytokines. In this study we investigated the roles of chemokines RANTES and IP-10 in regulating protective responses from Mycobacterium tuberculosis (M. tb) 10 kDa Culture Filtrate Protein-10 (CFP-10) differentiated DCs (CFP10-DCs).

Methods and Findings

Infection of CFP10-DCs with mycobacteria down-modulated RANTES and IP-10 levels. Pathway specific microarray analyses showed that in addition to RANTES and IP-10, mycobacteria infected CFP10-DCs showed reduced expression of many Th1 promoting chemokines and chemokine receptors. Importantly, T cells co-cultured with RANTES and IP-10 conditioned CFP10-DCs mediated killing of mycobacteria from infected macrophages. Similarly, T cells recruited by RANTES and IP-10 conditioned CFP10-DCs mediated significant killing of mycobacteria from infected macrophages. IFN-gamma treatment of CFP10-DCs restored RANTES and IP-10 levels and T cells activated by these DCs mediated significant killing of virulent M. tb inside macrophages. Adoptive transfer of either RANTES and IP-10 or IL-12 and IFN-gamma conditioned CFP10-DCs cleared an established M. tb infection in mice. The extent of clearance was similar to that obtained with drug treatment.

Conclusions

These results indicate that chemokine and cytokine secretion by DCs differentiated by M. tb antigens such as CFP-10 play major roles in regulating protective immune responses at sites of infection.     

Genomic diversity among Beijing and non-Beijing Mycobacterium tuberculosis isolates from Myanmar

Background

The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar.

Methodology/Principle Findings

Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates.

Conclusion

This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.     

Natural variation in immune responses to neonatal Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Vaccination in a Cohort of Gambian infants

Background

There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-γ) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN-γ responses to BCG in this age group are poorly described. Characterisation of IFN-γ responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy.

Methodology/Principal Findings

236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-γ, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89–98% depending on the antigen) made IFN-γ responses and there was significant correlation between IFN-γ responses to the different mycobacterial antigens (Spearman''s coefficient ranged from 0.340 to 0.675, p = 10⁻⁶–10⁻²²). IL-13 and IL-5 responses were generally low and there were more non-responders (33–75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens

Conclusions/Significance

Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN-γ responses.     

Modern Lineages of Mycobacterium tuberculosis Exhibit Lineage-Specific Patterns of Growth and Cytokine Induction in Human Monocyte-Derived Macrophages

Background

Strains of Mycobacterium tuberculosis vary in virulence. Strains that have caused outbreaks in the United States and United Kingdom have been shown to subvert the innate immune response as a potential immune evasion mechanism. There is, however, little information available as to whether these patterns of immune subversion are features of individual strains or characteristic of broad clonal lineages of M. tuberculosis.

Methods

Strains from two major modern lineages (lineage 2 [East-Asian] and lineage 4 [Euro-American]) circulating in the Western Cape in South Africa as well as a comparator modern lineage (lineage 3 [CAS/Delhi]) were identified. We assessed two virulence associated characteristics: mycobacterial growth (in liquid broth and monocyte derived macrophages) and early pro-inflammatory cytokine induction.

Results

In liquid culture, Lineage 4 strains grew more rapidly and reached higher plateau levels than other strains (lineage 4 vs. lineage 2 p = 0.0024; lineage 4 vs. lineage 3 p = 0.0005). Lineage 3 strains were characterized by low and early plateau levels, while lineage 2 strains showed an intermediate growth phenotype. In monocyte-derived macrophages, lineage 2 strains grew faster than lineage 3 strains (p<0.01) with lineage 4 strains having an intermediate phenotype. Lineage 2 strains induced the lowest levels of pro-inflammatory TNF and IL-12p40 as compared to other lineages (lineage 2: median TNF 362 pg/ml, IL-12p40 91 pg/ml; lineage 3: median TNF 1818 pg/ml, IL-12p40 123 pg/ml; lineage 4: median TNF 1207 pg/ml, IL-12p40 205 pg/ml;). In contrast, lineage 4 strains induced high levels of IL-12p40 and intermediate level of TNF. Lineage 3 strains induced high levels of TNF and intermediate levels of IL-12p40.

Conclusions

Strains of M. tuberculosis from the three major modern strain lineages possess distinct patterns of growth and cytokine induction. Rapid growth and immune subversion may be key characteristics to the success of these strains in different human populations.