Incubation of tissue with or isolated OMVs led to comparable damage scores, shown by a significant increase between 2 and 24 h compared to the control (Fig

Incubation of tissue with or isolated OMVs led to comparable damage scores, shown by a significant increase between 2 and 24 h compared to the control (Fig. collagenous structure (MARCO). Immunohistochemistry confirmed the downregulation of MARCO at sites of pathogen-induced tissue destruction. Neither host factor has ever been described in the context of infections. This work demonstrates that the tissue explant model reproduces realistic features of Legionnaires’ disease and reveals new functions for bacterial OMVs during infection. Our model allows us to characterize early steps of human infection which otherwise are not feasible for investigations. INTRODUCTION Histopathologically, Legionnaires’ disease, caused by the Gram-negative bacterium pneumonia Px-104 exhibit a massive infiltration of neutrophils and macrophages into the alveoli and destruction of alveolar septa. Moreover, the alveolar epithelium shows sloughs, and inflammatory cells exhibit intense necrosis. is present mainly in alveoli and tends to cluster inside macrophages. In late infection stages, bacteria disseminate to the patient’s spleen, kidneys, bone marrow, and lymph nodes (1,C4). Different models have been established to analyze specific aspects of infection. Besides human monocellular systems such as macrophages and epithelial cells, protozoa such as were used to study the cellular and molecular pathogenicity of (5,C9). These studies revealed that primarily enters phagocytes and resides within a unique membrane-bound compartment termed the vegetative cells also shed light on the cellular mechanisms of Legionnaires’ disease (13,C16). Moreover, proteomic approaches were shown to be powerful tools to characterize both sides of the host-pathogen interaction (17,C19). Mammalian models such as guinea pigs, mice, rhesus monkeys, and marmosets were used to address immunological, pathological, and pharmacological questions (20,C22). Despite providing enormous progress in the knowledge about mechanisms of infections, each of the current infection models has intrinsic limitations. Cell culture assays lack Rabbit Polyclonal to IRF-3 (phospho-Ser386) the complex interaction networks between the specialized cell types and extracellular components in the human lung. Guinea pig infections require intraperitoneal or intratracheal inoculation techniques, and owing to a different genetic and immunological background, the adequacy and transferability to humans can be questioned. Given the different model-immanent limitations, numerous intra- and extracellular interactions of factors with human lung tissue structures remain unknown. For example, early infection events appear to be underexplored, since histopathology studies were performed postmortem. Even conspicuous subcellular structures, such as the abundant outer membrane vesicles (OMVs) shed by wild-type and mutant strains in time course experiments with HLTEs. Moreover, we analyzed the contribution of OMVs to tissue destruction and demonstrated that the transcriptional response of Corby and a DotA-negative strain (25, 26) (kindly provided by Antje Flieger, Robert-Koch-Institut, Wernigerode, Germany) were cultivated in yeast extract broth (YEB) (with 20 g/ml kanamycin for the mutant) to the early stationary phase. For infection, the bacterial suspension was diluted to 107 bacteria/ml in RPMI 1640 (Gibco, Darmstadt, Germany) with 10% fetal calf serum (FCS), 20 mM HEPES, and 1 mM sodium pyruvate. OMVs were isolated from early-stationary-phase cultures as described previously (27) and diluted to 100 g/ml (total protein) in RPMI with supplements. Human lung tissue explants and assessment of bacterial replication. Tumor-free pulmonary tissue samples of approximately 1 cm3 were obtained from surgery patients as described previously (28). Samples were infected with the respective strain and incubated at 37C and 5% CO2 for up to 48 h. Microscopic inspection of untreated samples at different time points ensured tissue vitality (see below). For CFU determination, triplicate samples from eight donors were infected. At the indicated time points, samples were weighed and homogenized in phosphate-buffered saline (PBS). Dilutions were plated on buffered charcoal-yeast extract (BCYE) and incubated at 37C with 5% CO2 for Px-104 4 days. Extracellular replication of in HLTEs was excluded by control experiments which showed that acellular tissue homogenate or tissue supernatant does not support bacterial growth. The CFU/g of tissue were determined; means and standard deviations of results for samples were compared by using Student’s test. Px-104 Tissue processing and histology analysis. Tissue samples were fixed with the HEPES-glutamic acid organic solvent protection effect (HOPE) technique (DCS Diagnostics, Hamburg, Germany) (29). Briefly, samples were incubated in HOPE solution I at 4C for 18 h and dehydrated.