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Papillon-Lefèvre syndrome (PLS) is characterized by nonfunctional neutrophil serine proteases (NSPs) and fulminant periodontal inflammation of unknown cause. Here we investigated neutrophil extracellular trap (NET)-associated aggregation and cytokine/chemokine-release/degradation by normal and NSP-deficient human and mouse granulocytes. Stimulated with solid or soluble NET inducers, normal neutrophils formed aggregates and both released and degraded cytokines/chemokines. With increasing cell density, proteolytic degradation outweighed release. Maximum output of cytokines/chemokines occurred mostly at densities between 2 × 10(7) and 4 × 10(7) neutrophils/cm(3). Assessment of neutrophil density in vivo showed that these concentrations are surpassed during inflammation. Association with aggregated NETs conferred protection of neutrophil elastase against α1-antitrypsin. In contrast, eosinophils did not influence cytokine/chemokine concentrations. The proteolytic degradation of inflammatory mediators seen in NETs was abrogated in Papillon–Lefèvre syndrome (PLS) neutrophils. In summary, neutrophil-driven proteolysis of inflammatory mediators works as a built-in safeguard for inflammation. The absence of this negative feedback mechanism might be responsible for the nonresolving periodontitis seen in PLS.—Hahn, J., Schauer, C., Czegley, C., Kling, L., Petru, L., Schmid, B., Weidner, D., Reinwald, C., Biermann, M. H. C., Blunder, S., Ernst, J., Lesner, A., Bäuerle, T., Palmisano, R., Christiansen, S., Herrmann, M., Bozec, A., Gruber, R., Schett, G., Hoffmann, M. H. Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.

Molecular checkpoints that trigger the onset of islet autoimmunity or progression to human type 1 diabetes (T1D) are incompletely understood. Using T cells from children at an early stage of islet autoimmunity without clinical T1D, we find that a microRNA181a (miRNA181a)-mediated increase in signal strength of stimulation and costimulation links nuclear factor of activated T cells 5 (NFAT5) with impaired tolerance induction and autoimmune activation. We show that enhancing miRNA181a activity increases NFAT5 expression while inhibiting FOXP3+ regulatory T cell (Treg) induction in vitro. Accordingly, Treg induction is improved using T cells from NFAT5 knockout (NFAT5ko) animals, whereas altering miRNA181a activity does not affect Treg induction in NFAT5ko T cells. Moreover, high costimulatory signals result in phosphoinositide 3-kinase (PI3K)-mediated NFAT5, which interferes with FoxP3+ Treg induction. Blocking miRNA181a or NFAT5 increases Treg induction in murine and humanized models and reduces murine islet autoimmunity in vivo. These findings suggest targeting miRNA181a and/or NFAT5 signaling for the development of innovative personalized medicines to limit islet autoimmunity.

β-site APP-cleaving enzyme 1 (BACE1)is a major player in the pathogenesis of Alzheimer’sdisease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques either require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduceSiR-BACE1, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photo- physical and pharmacologic properties, and evaluate its behavior in solution, in overexpression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion (STED), and dynamic single molecule microscopy. First functional studies withSiR-BACE1on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.

Intercellular junctions are crucial structural elements for the formation and maintenance of epithelial barrier functions to control homeostasis or protect against intruding pathogens in humans. Alterations in these complexes represent key events in the development and progression of numerous cancers as well as multiple infectious diseases. Many bacterial pathogens harbor type IV secretion systems (T4SSs), which translocate virulence factors into host cells to hijack cellular processes. The pathology of the gastric pathogen and type-I carcinogen Helicobacter pylori strongly depends on a T4SS encoded by the cag pathogenicity island (cagPAI). This T4SS forms a needle-like pilus and its activity is accomplished by the pilus-associated factors CagL, CagI and CagY which target the host integrin-β1 receptor followed by injection of the CagA oncoprotein into non-polarized AGS gastric epithelial cells. The finding of a T4SS receptor, however, suggested the presence of a sophisticated control mechanism for the injection of CagA. In fact, integrins constitute a group of basolateral receptors, which are normally absent at apical surfaces of the polarized epithelium in vivo. Our new results demonstrate that T4SS-pilus formation during H. pylori infection of polarized epithelial cells occurs preferentially at basolateral sites, and not at apical membranes (Tegtmeyer et al., 2017). We propose a stepwise process how H. pylori interacts with components of intercellular tight junctions (TJs) and adherens junctions (AJs), followed by contacting integrin-based focal adhesions to disrupt and transform the epithelial cell layer in the human stomach. The possible impact of this novel signaling cascade on pathogenesis during infection is reviewed.

Analysing cell migration and invasion is of interest to many investigators as they mimic a part of physiological or pathological events. In this chapter, methods to analyse MMP-dependent 2D and 3D cell migration are described in detail. To study 2D cell migration, the phagokinetic track motility assay can be used. To study 3D cell migration, Matrigel invasion assay or microcarrier beads invasion assay can be used to obtain quantitative results.

Upon specific interaction with APCs, T cells capture membrane fragments and surface molecules in a process termed trogocytosis. In this study, we demonstrate that human Ag-specific CD8+ T cells acquire the coinhibitory molecule programmed death ligand 1 (PD-L1) from mature dendritic cells (mDC) and tumor cells in an Ag-specific manner. Immature dendritic cells were less effective in transferring surface molecules onto CD8+ T cells than mDCs. Interestingly, trogocytosis of PD-L1 requires cell–cell contact and cannot be induced by uptake of soluble proteins obtained from mDC lysates. The transfer process is impaired by inhibition of vacuolar ATPases in T cells as well as by fixation of dendritic cells. Of importance, CD8+ T cells that acquired PD-L1 complexes were able to induce apoptosis of neighboring programmed death 1–expressing CD8+ T cells. In summary, our data demonstrate that human CD8+ T cells take up functionally active PD-L1 from APCs in an Ag-specific fashion, leading to fratricide of programmed death 1–expressing, neighboring T cells. The transfer of functionally active coinhibitory molecules from APCs onto human CD8+ T cells could have a regulatory role in immune responses.

Homodimerization of the membrane-bound collagenase MT1-MMP [membrane-type 1 MMP (matrix metalloproteinase)] is crucial for its collagenolytic activity. However, it is not clear whether this dimerization is regulated during cellular invasion into three-dimensional collagen matrices. To address this question, we established a fluorescence resonance energy transfer system to detect MT1-MMP dimerization and analysed the process in cells invading through three-dimensional collagen. Our data indicate that dimerization occurs dynamically and constantly at the leading edge of migrating cells, but not the trailing edge. We found that polarized dimerization was not due to ECM (extracellular matrix) attachment, but was rather controlled by reorganization of the actin cytoskeleton by the small GTPases, Cdc42 (cell division cycle 42) and Rac1. Our data indicate that cell-surface collagenolytic activity is regulated co-ordinately with cell migration events to enable penetration of the matrix physical barrier.

Obesity and type 2 diabetes are associated with metabolic defects and adipose tissue inflammation. Foxp3(+) regulatory Tcells (Tregs) control tissue homeostasis by counteracting local inflammation. However, if and how Tcells interlink environmental influences with adipocyte function remains unknown. Here, we report that enhancing sympathetic tone by cold exposure, beta3-adrenergic receptor (ADRB3) stimulation or a short-term high-calorie diet enhances Treg induction invitro and invivo. CD4(+) Tcell proteomes revealed higher expression of Foxp3 regulatory networks in response to cold or ADRB3 stimulation invivo reflecting Treg induction. Specifically, Ragulator-interacting protein C17orf59, which limits mTORC1 activity, was upregulated in CD4(+) Tcells by either ADRB3 stimulation or cold exposure, suggesting contribution to Treg induction. By loss- and gain-of-function studies, including Treg depletion and transfers invivo, we demonstrated that a Tcell-specific Stat6/Pten axis links cold exposure or ADRB3 stimulation with Foxp3(+) Treg induction and adipose tissue function. Our findings offer a new mechanistic model in which tissue-specific Tregs maintain adipose tissue function.

Human cytomegalovirus UL97-encoded protein kinase (pUL97) phosphorylates cellular and viral proteins and is critical for viral replication. To quantify the efficiency of nuclear translocation and to elucidate the role of putative nuclear localization signal (NLS) elements, immunofluorescence analysis of different pUL97 expression constructs was performed. Since manual quantitation of respective expression levels lacks objectivity and reproducibility, and is time-consuming as well, a computer-based model is established. This model enables objective quantitation of the degree of cytoplasmic localization λ. To determine the degree of cytoplasmic localization of different pUL97-GFP-β-gal fusion proteins automatically, a multi-channel segmentation of the nucleus and cytoplasm of transfected HeLa cells is performed in DAPI and GFP micrographs. A watershed transform-based segmentation scheme is used for the segmentation of the cell nuclei. Subsequently, the cytoplasm is segmented using a fast marching level set method. Based on the segmentation of cell nuclei and cytoplasm, λ can be determined for each HeLa cell by quantitation of the ratio of average signal intensity outside and inside the nucleus. The degree of cytoplasmic localization of an individual construct is then determined by evaluating the average and standard deviation of λ for the corresponding HeLa cells. Evaluation demonstrates that nuclear transport of pUL97 is a multilayered mechanism resulting in different efficiencies of nuclear translocation between a small and a large isoform and objective quantitation of the cytoplasmic localization is possible with a high accuracy (96.7% and 94.3%).

Sensing of infectious danger by toll-like receptors (TLRs) on macrophages causes not only a reprogramming of the transcriptome but also changes in the cytoskeleton important for cell spreading and motility. Since manual determination of cell contact areas from fluo-, rescence micrographs is very time-consuming and prone to bias, we have developed and tested algorithms for automated measurement of macrophage spreading. The two-step method combines identification of cells by nuclear staining with DAPI and cell surface staining of the integrin CD11b. Automated image analysis correlated very well with manual annotation in resting macrophages and early after stimulation, whereas at later time points the automated cell segmentation algorithm and manual annotation showed slightly larger variation. The method was applied to investigate the impact of genetic or pharmacological inhibition of known TLR signaling components. Deficiency in the adapter protein Myd88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of MEK1, the kinase activating the mitogen-activated protein kinases (MAPK) ERK1/2, indicating that ERK1/2 mediates Myd88-dependent macrophages spreading. In contrast, macrophages lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8-24 h after stimulation. The dichotomy of p38 and ERK1/2 MAPK effects on early and late macrophage spreading raises the question which of the respective substrate proteins mediate(s) cytoskeletal remodeling and spreading. The automated measurement of cell spreading described here increases the objectivity and greatly reduces the time required for such investigations and is therefore expected to facilitate larger throughput analysis of macrophage spreading, e.g., in siRNA knockdown screens.

Background The cause of sporadic amyotrophic lateral sclerosis (ALS) is largely unknown but hypotheses about disease mechanisms include oxidative stress, defective axonal transport, mitochondrial dysfunction and disrupted RNA processing. Whereas familial ALS is well represented by transgenic mutant SOD1 mouse models, the mouse mutant wobbler (WR) develops progressive motor neuron degeneration due to a point mutation in the Vps54 gene, and provides an animal model for sporadic ALS. VPS54 protein as a component of a protein complex is involved in vesicular Golgi trafficking; impaired vesicle trafficking might also be mechanistic in the pathogenesis of human ALS. Results In motor neurons of homozygous symptomatic WR mice, a massive number of endosomal vesicles significantly enlarged (up to 3 μm in diameter) were subjected to ultrastructural analysis and immunohistochemistry for the endosome-specific small GTPase protein Rab7 and for amyloid precursor protein (APP). Enlarged vesicles were neither detected in heterozygous WR nor in transgenic SOD1(G93A) mice; in WR motor neurons, numerous APP/Rab7-positive vesicles were observed which were mostly LC3-negative, suggesting they are not autophagosomes. Conclusions We conclude that endosomal APP/Rab7 staining reflects impaired vesicle trafficking in WR mouse motor neurons. Based on these findings human ALS tissues were analysed for APP in enlarged vesicles and were detected in spinal cord motor neurons in six out of fourteen sporadic ALS cases. These enlarged vesicles were not detected in any of the familial ALS cases. Thus our study provides the first evidence for wobbler-like aetiologies in human ALS and suggests that the genes encoding proteins involved in vesicle trafficking should be screened for pathogenic mutations.

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-alpha-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a K(i) of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.

We used multifocal two-photon laser scanning microscopy for local and selective protein activation and quantitative investigation of intracellular protein dynamics. The localized activation was realized with photo-activatable green-fluorescent-proteins (pa-GFP) and optical two-photon excitation in order to investigate the real-time intracellular dynamics in vivo. Such processes are of crucial importance for a deep understanding and modelling of regulatory and metabolic processes in living cells. Exemplarily, the intracellular dynamics of the Arabidopsis MYB transcription factor LHY/CCA1-like 1 (LCL1) that contains both a nuclear import and a nuclear export signal was quantitatively investigated. We used tobacco BY-2 protoplasts co-transfected with plasmids encoding photo-activatable green fluorescent protein (pa-GFP) fusion proteins and a red fluorescing transfection marker and measured the rapid nuclear export of pa-GFP-LCL1 I after its photo-activation in the nucleus. In contrast, an export-negative mutant of LCL1 remained trapped inside the nucleus. We detemined average time constants of 51s and 125s for the decrease of fluorescence in the nucleus due to active bi-directional nuclear transport of pa-GFP-LCL1 and diffusion of pa-GFP, respectively. (c) 2007 Elsevier Inc. All rights reserved.

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).