The Sources of Inflammatory Mediators in the Lung after Silica Exposure

The expression of 10 genes implicated in regulation of the inflammatory processes in the lung was studied after exposure of alveolar macrophages (AMs) to silica in vitro or in vivo. Exposure of AMs to silica in vitro up-regulated the messenger RNA (mRNA) levels of three genes [interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2)] without a concomitant increase in the protein levels. AMs isolated after intratracheal instillation of silica up-regulated mRNA levels of four additional genes [granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-1β, IL-10, and inducible nitric oxide synthase]. IL-6, MCP-1, and MIP-2 protein levels were elevated in bronchoalveolar lavage fluid. Fibroblasts under basal culture conditions express much higher levels of IL-6 and GM-CSF compared with AMs. Coculture of AMs and alveolar type II cells, or coculture of AMs and lung fibroblasts, in contact cultures or Transwell chambers, revealed no synergistic effect. Therefore, such interaction does not explain the effects seen in vivo. Identification of the intercellular communication in vivo is still unresolved. However, fibroblasts appear to be an important source of inflammatory mediators in the lung

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective