The influx of macrophages into the lungs is the major component of the granulomatous response to infection with Mycobacterium tuberculosis. In this investigation we used flow cytometric analysis to define macrophage populations entering the airways and lung tissues of infected mice. We demonstrate that by the judicious use of cell surface markers, especially CD11b and CD11c, several cell populations can be distinguished, allowing cell sorting and morphological definition. Primary populations of CD11b−/CD11c+/high were defined as alveolar macrophages, CD11bhigh/CD11c+/high as dendritic cells, and CD11b+/mid/CD11c+/mid as small macrophages or monocytes, and changes in the activation phenotype of these populations were followed over the early course of the infection. In further studies, these cell populations were compared with cells harvested during the chronic stage of the disease. During the chronic stage of infection, Ag-presenting class II molecules and activation markers were poorly expressed on dendritic, small macrophage, and monocyte cell populations, which may have important implications for the breakdown of the lesions during reactivation disease. This analytical approach may facilitate the further characterization of macrophage populations entering into the lung tissues and their relative contributions to host resistance to tuberculosis infection.

Mice lacking expression of granulocyte macrophage-colony stimulating factor (GM-CSF KO) are unable to contain Mycobacterium tuberculosis (M. tuberculosis) growth and succumb to infection by 35 days following pulmonary challenge. GM-CSF KO mice do not express normal levels of the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) nor the chemokines, regulated on activation, normal T expressed and secreted (RANTES), macrophage-inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and lymphotactin, which are required for recruitment of lymphocytes and expression of a T helper cell type 1 (TH1) response within the lungs. In contrast, transgenic mice overexpressing GM-CSF in the lungs but with a lack of GM-CSF in other organs (GM+) are able to recruit lymphocytes and to express a TH1 response with production of TNF-alpha and interferon-gamma in the lungs. However, GM+ mice succumb to infection between 60 and 90 days post-challenge, as they are unable to develop a normal granulomatous response. Although GM+ mice are able to express the chemokine RANTES, they lack the ability to express other inflammatory chemokines such as lymphotactin and MIP-1beta. We conclude that GM-CSF is essential to the recruitment of lymphocytes and expression of a TH1 response in the lung, to the generation of a normal mononuclear granuloma, and most importantly, to the containment of M. tuberculosis bacterial growth.

Both innate and adaptive immune systems contribute to host defense against infection with Mycobacterium tuberculosis. NK cells have been associated with early resistance against intracellular pathogens and are known to be potent producers of the cytokine IFN-γ. In C57BL/6 mice infected by aerosol exposure with M. tuberculosis, NK cells increased in the lungs over the first 21 days of infection. Expansion of the NK cell subset was associated with increased expression of activation and maturation markers. In addition, NK cells isolated from the infected lungs were capable of producing IFN-γ and became positive for perforin. In vivo depletion of NK cells using a lytic Ab had no influence on bacterial load within the lungs. These findings indicate that NK cells can become activated during the early response to pulmonary tuberculosis in the mouse model and are a source of IFN-γ, but their removal does not substantially alter the expression of host resistance.

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Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB.