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.