The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membraneperforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly + rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly + rBCG (∆ureC hly + rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. ∆ureC hly + rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of ∆ureC hly + rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

Successful host defense against numerous pulmonary infections depends on bacterial clearance by polymorphonuclear leukocytes (PMNs); however, excessive PMN accumulation can result in life-threatening lung injury. Local expression of CXC chemokines is critical for PMN recruitment. The impact of chemokinedependent PMN recruitment during pulmonary Mycobacterium tuberculosis infection is not fully understood. Here, we analyzed expression of genes encoding CXC chemokines in M. tuberculosis-infected murine lung tissue and found that M. tuberculosis infection promotes upregulation of Cxcr2 and its ligand Cxcl5. To determine the contribution of CXCL5 in pulmonary PMN recruitment, we generated Cxcl5 -/-mice and analyzed their immune response against M. tuberculosis. Both Cxcr2 -/-mice and Cxcl5 -/-mice, which are deficient for only one of numerous CXCR2 ligands, exhibited enhanced survival compared with that of WT mice following high-dose M. tuberculosis infection. The resistance of Cxcl5 -/-mice to M. tuberculosis infection was not due to heightened M. tuberculosis clearance but was the result of impaired PMN recruitment, which reduced pulmonary inflammation. Lung epithelial cells were the main source of CXCL5 upon M. tuberculosis infection, and secretion of CXCL5 was reduced by blocking TLR2 signaling. Together, our data indicate that TLR2-induced epithelial-derived CXCL5 is critical for PMN-driven destructive inflammation in pulmonary tuberculosis.

Among the first cells to invade a site of infection, polymorphonuclear neutrophils (PMN) play an important role in the control of numerous infections. While PMN are considered critical for control of acute infections, their role in chronic infections remains less well understood. Here we report that PMN are essential for accurate early granuloma formation during chronic M. tuberculosis infection without influencing mycobacterial growth restriction. The PMNmediated regulation of granuloma formation depended on chemokines signaling through CXCR3, in particular MIG, as indicated by immune histochemical analysis of lung sections from C57BL/6 wild-type and CXCR3 -/-mutant mice and supported by microarray transcriptome analysis. Hence, PMN play a central role in regulating the focal granulomatous response in the lung, and this early granuloma formation can be segregated from long-term protection against pulmonary M. tuberculosis infection.

A potent Th1 immune response is critical to the control of tuberculosis. The impact of an additive Th2 response on the course of disease has so far been insufficiently characterized, despite increased morbidity after co-infection with Mycobacterium tuberculosis and Th2-eliciting helminths and possible involvement of Th2 polarization in reactivation of latent tuberculosis. Here, we describe the gene expression profile of murine bone marrow-derived macrophages alternatively activated by IL-4 in response to infection with M. tuberculosis. Comparison of transcriptional profiles of infected IL-4-and IFN-c-activated macrophages revealed delayed and partially diminished responses to intracellular bacteria in alternatively activated macrophages, characterized by reduced exposure to nitrosative stress and increased iron availability, respectively. Alternative activation of host macrophages correlated with elevated expression of the M. tuberculosis iron storage protein bacterioferritin as well as reduced expression of the mycobactin synthesis genes mbtI and mbtJ. The extracellular matrix-remodeling enzyme matrix metalloproteinase (MMP)-12 was induced in alternatively activated macrophages in vitro, and MMP-12-expressing macrophages were abundant at late, but not early, stages of tuberculosis in murine lungs. Our findings emphasize that alternative activation deprives macrophages of control mechanisms that limit mycobacterial growth in vivo, thus supporting intracellular persistence of M. tuberculosis.

The hallmark of pulmonary tuberculosis is the granuloma, which consists predominantly of lymphocytes and macrophages and promotes immune-cell interaction with the causative pathogen, Mycobacterium tuberculosis. Granuloma formation is a highly organized process, which depends on leukocyte recruitment facilitated by adhesion molecules and chemokines. Thus, during chronic experimental tuberculosis, granulomata display characteristics of lymphoid structures comprising follicular aggregation of B cells, formation of high endothelial venules, presence of follicular dendritic cells, and expression of the homeostatic chemokines CXCL13 and CCL19. CCR7-/- mice, which are deficient in CCL19 and CCL21 signaling, exhibit increased local inflammatory infiltrates but no follicular B-cell aggregation within those lymphoid structures. However, CCR7-deficient mice are fully capable to control pulmonary tuberculosis; at time points later than 6 weeks postinfection, they carry a lower bacterial load in peripheral organs. Our results show that, during chronic pulmonary tuberculosis in mice, the homeostatic chemokine signaling-network contributes to spatial organization of the granulomatous response, which participates in both containment of M. tuberculosis and the latter's dissemination to other organs.

Ziehl-Neelsen (ZN) staining is the key technique for diagnosis of mycobacterial infections; however, a high percentage of patients exhibit positive signs of tuberculosis, as indicated by pathology, culture of mycobacteria, and polymerase chain-reaction analysis, and yet show negative results on ZN staining. In this report we present evidence that such ZN-negative specimens represent Mycobacterium tuberculosis bacilli in a dormant state with distinct cell-wall alterations: the classical cell-wall composition-dependent ZN staining of M. tuberculosis in lung sections gradually discontinued with persistence of infection, both in mice and in human patients; in contrast, detection of mycobacteria by cell-wall composition-independent staining using a polyclonal anti-M. bovis Bacille-Calmette-Guérin serum continued with persistence of infection. These findings have important implications for diagnosis, as well as for both chemotherapy and development of vaccine strategies.'

Background. New vaccines against tuberculosis (TB) are urgently needed because the only available vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), fails to protect against pulmonary TB in adults. The recombinant ΔureC hly+ BCG (rBCG) is more efficient than parental BCG (pBCG) against pulmonary TB in preclinical studies and has proven safe and immunogenic in phase I clinical trials.Methods. In an attempt to identify the mechanisms underlying the superior protection of rBCG, we compared the immune responses elicited after vaccination and subsequent aerosol infection with Mycobacterium tuberculosis (MTB) in mice.Results. We demonstrate that both rBCG and pBCG induce marked type 1 cytokine responses, whereas only rBCG elicits a profound type 17 cytokine response in addition. We observed earlier recruitment of antigen-specific T lymphocytes to the lung upon MTB infection of rBCG-vaccinated mice. These T cells produced abundant type 1 cytokines after restimulation, resulting in 10-fold reduced bacterial burden 90 days after infection.Conclusions. Our findings identify a general immunologic pathway for improved vaccination strategies against TB that can also be harnessed by other vaccine candidates.