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Development and application of advanced fluorescence microscopy techniques for the investigation of inflammatory processes

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Neumann,  Jan
Multiphase Chemistry, Max Planck Institute for Chemistry, Max Planck Society;

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Citation

Neumann, J. (2018). Development and application of advanced fluorescence microscopy techniques for the investigation of inflammatory processes. PhD Thesis, Universität, Mainz.


Cite as: https://hdl.handle.net/21.11116/0000-0003-2F93-3
Abstract
The innate immune system is the first barrier against pathogens and induces fast immune responses. Hereby the Toll-like receptor 4 (TLR4) plays a key role by detecting damage- and pathogen-associated molecular patterns (DAMPs, PAMPs) like lipopolysaccharides (LPS), an outer cell wall component of gram-negative bacteria. Activation of TLR4 leads to a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) dependent signal transduction into the cell nucleus. TLR4 can be activated not only by LPS, but also by amylase trypsin inhibitors (ATI). ATI comprise a family of small proteins that are associated with gluten containing grains like wheat, barley or rice. Consumption of food containing ATI can therefore cause intestinal inflammation. Studies that microscopically investigate the effect of ATI on the molecules involved in the TLR4 signaling pathway are limited.
In this study, single molecule localization microscopy (SMLM) and high-content screening microscopy (HCS) were used to study both, the membrane distribution of TLR4 and the translocation of NF-κB into the cell nucleus in primary human macrophages after ATI or LPS stimulation. To investigate the membrane distribution of TLR4 below the optical diffraction limit, a SMLM microscope was constructed during this thesis and algorithms for quantitative evaluation of the measured distribution of TLR4 molecules were implemented.
SMLM microscopy revealed that more than 60 % of TLR4 molecules are part of a cluster, with diameters between 46 nm to 57 nm. Upon stimulation with ATI or LPS a tendency towards the formation of smaller and less dense clusters was observed. At the same time, the percentage of molecules that are part of a cluster increased. These results suggest that TLR4 molecules are recruited into specific domains consisting of monomers or lower order oligomers, which indicates the formation of signaling platforms. In addition, the use of HCS microscopy showed that ATI treatment induces the translocation of NF-κB into the cell nucleus. Quantitative differences in the response between the individual donors were observed.
Together, the results show a comparable influence of ATI on both the supramolecular distribution of TLR4 and the translocation of NF-κB into the cell nucleus, as observed for LPS. In addition, the algorithms implemented for evaluating the membrane distribution of TLR4 will be helpful in future experiments for quantifying SMLM data with high signal densities and a large number of samples.