The human tuberculous granuloma provides the morphological basis for local immune processes central to the outcome of tuberculosis. Because of the scarcity of information in human patients, the aim of the present study was to gain insights into the functional and structural properties of infiltrated tissue. To this end, the mycobacterial load in lesions and dissemination to different tissue locations were investigated, as well as distribution, biological functions, and interactions of host immune cells. Analysis of early granuloma formation in formerly healthy lung tissue revealed a spatio-temporal sequence of cellular infiltration to sites of mycobacterial infection. A general structure of the developing granuloma was identified, comprising an inner cell layer with few CD8(+) cells surrounding the necrotic centre and an outer area of lymphocyte infiltration harbouring mycobacteria-containing antigen-presenting cells as well as CD4(+), CD8(+), and B cells in active follicle-like centres resembling secondary lymphoid organs. It is concluded that the follicular structures in the peripheral rim of granulomas serve as a morphological substrate for the orchestration of the enduring host response in pulmonary tuberculosis.

Although tuberculosis remains a substantial global threat, the mechanisms that enable mycobacterial persistence and replication within the human host are ill defined. This study represents the first genome-wide expression analysis of Mycobacterium tuberculosis from clinical lung samples, which has enabled the identification of M. tuberculosis genes actively expressed during pulmonary tuberculosis. To obtain optimal information from our DNA array analyses, we analyzed the differentially expressed genes within the context of computationally inferred protein networks. Protein networks were constructed using functional linkages established by the Rosetta stone, phylogenetic profile, conserved gene neighbor, and operon computational methods. This combined approach revealed that during pulmonary tuberculosis, M. tuberculosis actively transcribes a number of genes involved in active fortification and evasion from host defense systems. These genes may provide targets for novel intervention strategies.

Tuberculosis disease (TB) may progress at different rates and have different outcomes. Neutrophils have been implicated in TB progression; however, data on their role during TB are controversial. Here we show that in mice, TB progression is associated with the accumulation of cells that express neutrophilic markers Gr-1 and Ly-6G, but do not belong to conventional neutrophils. The cells exhibit unsegmented nuclei, have Gr-1dimLy-6GdimCD11b+ phenotype and express F4/80, CD49d, Ly-6C, CD117, CD135 markers characteristic not of neutrophils, but of immature myeloid cells. The cells accumulate in the lungs, bone marrow, spleen and blood at the advanced (pre-lethal) stage of M. tuberculosis infection and represent a heterogeneous population of myeloid cells at different stages of their differentiation. The accumulation of Gr-1dimCD11b+ cells is accompanied by the disappearance of conventional neutrophils (Gr-1hiLy-6Ghi-expressing cells). The Gr-1dimCD11b+ cells suppress T cell proliferation and IFN-γ production in vitro via NO-dependent mechanisms, i.e. they exhibit characteristics of myeloid-derived suppressor cells (MDSCs). These results document the generation of MDSCs during TB, suggesting their role in TB pathogenesis, and arguing that neutrophils do not contribute to TB pathology at the advanced disease stage.

Lung granulomas develop upon Mycobacterium tuberculosis (Mtb) infection as a hallmark of human tuberculosis (TB). They are structured aggregates consisting mainly of Mtb-infected and -uninfected macrophages and Mtb-specific T cells. The production of NO by granuloma macrophages expressing nitric oxide synthase-2 (NOS2) via L-arginine and oxygen is a key protective mechanism against mycobacteria. Despite this protection, TB granulomas are often hypoxic, and bacterial killing via NOS2 in these conditions is likely suboptimal. Arginase-1 (Arg1) also metabolizes L-arginine but does not require oxygen as a substrate and has been shown to regulate NOS2 via substrate competition. However, in other infectious diseases in which granulomas occur, such as leishmaniasis and schistosomiasis, Arg1 plays additional roles such as T-cell regulation and tissue repair that are independent of NOS2 suppression. To address whether Arg1 could perform similar functions in hypoxic regions of TB granulomas, we used a TB murine granuloma model in which NOS2 is absent. Abrogation of Arg1 expression in macrophages in this setting resulted in exacerbated lung granuloma pathology and bacterial burden. Arg1 expression in hypoxic granuloma regions correlated with decreased T-cell proliferation, suggesting that Arg1 regulation of T-cell immunity is involved in disease control. Our data argue that Arg1 plays a central role in the control of TB when NOS2 is rendered ineffective by hypoxia.

We conclude that differential regulation of the local immune response is crucial for the containment of M. tuberculosis and that a continuous antigen-specific cross talk between the host immune system and M. tuberculosis is ensured during latency. This activation requires sufficient supply of nutrients and well-coordinated structural organization, both of which are lost during reactivation of TB.

The diagnosis of mycobacterial infection depends on the Ziehl-Neelsen (ZN) stain, which detects mycobacteria because of their characteristic acid-fast cell wall composition and structure. The histological diagnosis of tuberculosis (TB) comprises various aspects: (1) sensitive detection of mycobacteria; (2) precise localization of mycobacteria in the context of granulomatous lesions; (3) 'staging' of disease according to mycobacterial spread and granulomatous tissue integrity. Thus, detection of minute numbers of acid-fast bacteria in tissue specimens is critical. The conventional ZN stain fails to identify mycobacteria in numbers less than 10(4) per ml. Hence many infections evade diagnosis. PCR is highly sensitive, but allows neither localization within tissues nor staging of mycobacterial disease, and positive findings frequently do not correlate with disease. In this study, an anti-Mycobacterium bovis bacille Calmette-Guérin polyclonal antiserum (pAbBCG) was used to improve immunostaining, which was compared to the ZN stain in histological samples. Screening of tissue samples including lungs, pleural lesions, lymph nodes, bone marrow, and skin for mycobacterial infection revealed that pAbBCG staining detects infected macrophages harbouring intracellular mycobacteria or mycobacterial material as well as free mycobacteria that are present at low abundance and not detected by the ZN stain. The positive pAbBCG staining results were confirmed either by PCR analysis of microdissected stained tissue or by culture from tissue. This immunostaining approach allows precise localization of the pathogen in infected tissue.

It is generally thought that Mycobacterium tuberculosis (Mtb)-specific CD4+ Th1 cells producing IFN-γ are essential for protection against tuberculosis (TB). In some studies, protection has recently been associated with polyfunctional subpopulation of Mtb-specific Th1 cells, i.e., with cells able to simultaneously secrete several type 1 cytokines. However, the role for Mtb-specific Th1 cells and their polyfunctional subpopulations during established TB disease is not fully defined. Pulmonary TB is characterized by a great variability of disease manifestations. To address the role for Mtb-specific Th1 responses during TB, we investigated how Th1 and other immune cells correlated with particular TB manifestations, such as the degree of pulmonary destruction, TB extent, the level of bacteria excretion, clinical disease severity, clinical TB forms, and “Timika X-ray score,” an integrative parameter of pulmonary TB pathology. In comparison with healthy Mtb-exposed controls, TB patients (TBP) did not exhibit deficiency in Mtb-specific cytokine-producing CD4+ cells circulating in the blood and differed by a polyfunctional profile of these cells, which was biased toward the accumulation of bifunctional TNF-α+IFN-γ+IL-2− lymphocytes. Importantly, however, severity of different TB manifestations was not associated with Mtb-specific cytokine-producing cells or their polyfunctional profile. In contrast, several TB manifestations were strongly correlated with leukocyte numbers, the percent or the absolute number of lymphocytes, segmented or band neutrophils. In multiple alternative statistical analyses, band neutrophils appeared as the strongest positive correlate of pulmonary destruction, bacteria excretion, and “Timika X-ray score.” In contrast, clinical TB severity was primarily and inversely correlated with the number of lymphocytes in the blood. The results suggest that: (i) different TB manifestations may be driven by distinct mechanisms; (ii) quantitative parameters and polyfunctional profile of circulating Mtb-specific CD4+ cells play a minor role in determining TB severity; and (iii) general shifts in production/removal of granulocytic and lymphocytic lineages represent an important factor of TB pathogenesis. Mechanisms leading to these shifts and their specific role during TB are yet to be determined but are likely to involve changes in human hematopoietic system.

Tuberculosis causes 2 million deaths per year, yet in most cases the immune response successfully contains the infection and prevents disease outbreak. Induced lymphoid structures associated with pulmonary granuloma are observed during tuberculosis in both humans and mice and could orchestrate host defense. To investigate whether granuloma perform lymphoid functions, mice lacking secondary lymphoid organs (SLO) were infected with Mycobacterium tuberculosis (MTB). As in WT mice, granuloma developed, exponential growth of MTB was controlled, and antigen-specific T-cell responses including memory T cells were generated in the absence of SLO. Moreover, adoptively transferred T cells were primed locally in lungs in a granuloma-dependent manner. T-cell activation was delayed in the absence of SLO, but resulted in a normal development program including protective subsets and functional recall responses that protected mice against secondary MTB infection. Our data demonstrate that protective immune responses can be generated independently of SLO during MTB infection and implicate local pulmonary T-cell priming as a mechanism contributing to host defense.Key words: Granuloma . Lymphoid neogenesis . T-cell development . Tuberculosis Supporting Information available online IntroductionMycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), currently persists in one-third of the world's population [1]. In most cases, the immune response generated upon exposure contains, but does not eradicate, the bacilli and an asymptomatic persistent infection develops, termed latent TB. Approximately one in ten latently infected individuals undergo reactivation to active TB disease during their lifetime. However, Ã These authors contributed equally to this work. ÃÃ These authors share equal senior authorship. [2,3]. Granuloma are considered central for control of MTB as loss of granuloma structure is associated with poor disease outcome in humans and animal models [3,4]. Yet, the precise mechanisms by which granuloma mediate control of MTB are poorly understood. Induction of an adaptive immune response is essential for host protection against MTB. Antigen-specific T-cell responses are generated within secondary lymphoid organs (SLO), such as lymph nodes (NO) and spleen. SLO maximize efficiency of encounters between APC and naïve T lymphocytes [5]. Therefore, SLO are critical for the generation of adaptive immunity. Ectopic accumulations of lymphoid cells, known as tertiary lymphoid organs, sometimes arise during chronic inflammation or infection. Tertiary lymphoid organs resemble SLO in structure and cell composition and evidence exists that some are capable of generating adaptive immunity [5,6]. However, whether immune responses generated outside of SLO are truly effective and thus beneficial in defense against pathogens is unclear.Recent reports have described lymphoid features associated with murine and human granuloma, such as B-cell follicles and the peripheral node addressin, which is expressed on high endothel...