Transregional Collaborative Research Center SFB-TR 84 - “Innate Immunity of the Lung: Mechanisms of Pathogen Attack and Host Defence in Pneumonia“

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Z1a-Subproject

“High-end” microscopy in cellular microbiology of the lung (Hocke)

 

Microscopic procedures like wide-field, confocal, or life-cell-imaging (LCI), combined with digital image analysis, are of central importance in the field of cellular microbiology. In particular, these advanced microscopic techniques have become indispensable for uncovering innate immune mechanisms triggered by pathogen-host interaction in the pulmonary compartment. However, the elucidation of subcellular protein distribution, trafficking, or interaction with microbes requires a sophisticated methodological and technical basis. For example, the investigation of molecular interactions, trafficking, or measurement of pathophysiological parameters in single cells by fluorescence sensors is based on a broad expertise in molecular biology and cloning of fusion-vectors as well as “high-end” microscopy including spectral analysis combined with Förster-Resonance-Emission-Transfer (FRET) and Fluorescence-Recovery-After-Photobleaching (FRAP) applications. Next to this, insight and understanding of cellular and molecular interactions, especially in human lung tissue, is of utmost relevance for the field. Since strong autofluorescence of lung tissue conceals lots of specific fluorescence signals thereby hampering standard immunofluorescence, not to mention intravital imaging, antigen retrieval and multicolor labeling procedures have to be specifically designed and combined with advanced microscopy techniques like spectral imaging and linear unmixing.

This technical basis has been established in the first funding period and has already been applied within the SFB projects. For example, the cell-type specific and subcellular expression patterns of angiopoietin-2 (Ang-2), Chem23, or BLT-1 in human lung tissue were elucidated. Use of Z01a methodology brought inside into the cellular tropism of severe lung pathogens such as influenza A viruses (IAV), the Middle-East-respiratory-syndrome coronavirus (MERS-CoV), or Streptococcus pneumoniae (S.p.). Next to the known inflammatory role of alveolar macrophages (AM), the studies also highlighted alveolar type II epithelial cells (AEC II) as key players for the onset of infections, but also for the induction of alveolar damage in human lung tissue. Additionally, we tested and complemented new imaging methods such as super-resolution microscopy (SRM). By use of SRM unambiguous data about the morphological changes of the nuclear pore complex (NPC) after IAV infection have been obtained. Therefore, Z01a provides an advanced microscopic environment starting from sample preparation, protocol establishment, or cloning up to selection of adequate imaging procedures and microscopic education to fulfill the needs of the projects within the SFB-TR84. Moreover, Z01a combines and introduces innovative microscopic techniques and procedures to answer so far not addressable questions thereby significantly benefitting the field of lung cellular microbiology and innate immunity.