Abstract
Toll-like receptor 4 (TLR4) plays a crucial role in the recognition of invading pathogens. Upon activation by lipopolysaccharides (LPS), TLR4 is recruited into specific membrane domains and dimerizes. In addition to LPS, TLR4 can be stimulated by wheat amylase-trypsin inhibitors (ATI). ATI are proteins associated with gluten containing grains, whose ingestion promotes intestinal and extraintestinal inflammation. However, the effect of ATI vs. LPS on the membrane distribution of TLR4 at the nanoscale has not been analyzed. In this study, we investigated the effect of LPS and ATI stimulation on the membrane distribution of TLR4 in primary human macrophages using single molecule localization microscopy (SMLM). We found that in unstimulated macrophages the majority of TLR4 molecules are located in clusters, but with donor-dependent variations from ∼51% to ∼75%. Depending on pre-clustering, we found pronounced variations in the fraction of clustered molecules and density of clusters on the membrane upon LPS and ATI stimulation. Although clustering differed greatly among the human donors, we found an almost constant cluster diameter of ∼44 nm for all donors, independent of treatment. Together, our results show donor-dependent but comparable effects between ATI and LPS stimulation on the membrane distribution of TLR4. This may indicate a general mechanism of TLR4 activation in primary human macrophages. Furthermore, our methodology visualizes TLR4 receptor clustering and underlines its functional role as a signaling platform.
Highlights
Toll-like receptors (TLR) represent a family of pattern recognition receptors (PRRs) that are part of the innate immune system.[1,2] TLRs detect damage and pathogen associated molecular patterns (DAMPs, PAMPs), which subsequently triggers the production and release of inflammatory mediators
Toll-like receptor 4 (TLR4) is pre-clustered in unstimulated primary human macrophages
To investigate the large and small-scale distribution of TLR4 on the cell membrane, unstimulated macrophages stained for TLR4 were imaged by conventional fluorescence and localization microscopy (Fig. 1A + B)
Summary
High-mobility group protein B1 (HMGB1) or heat-shock protein 60 (HSP60). TLR4 plays a central role in many acute and chronic inflammatory diseases such as chronic obstructive pulmonary disease (COPD), allergic asthma or non-celiac wheat sensitivity (NCWS).[5,6,7,8]. DBSCAN requires two user input parameters – the radius (ε) and the minimum number of neighboring molecule signals (minPts) within this radius Together, these values form a local density threshold that determines whether the point under investigation is part of a cluster. For every detected cluster the algorithm generated a table containing its area, diameter, center of mass, density, the number of molecules and the distance to the neighboring cluster as well as an assignment list for every point to which cluster it belongs This assignment list was used to calculate the distances of molecule signals outside of the clusters as shown in Fig. S3.† The maximum allowable cluster diameter was set to 1 μm.
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