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Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Glucocorticoids (GCs) are widely prescribed therapeutics for the treatment of inflammatory diseases, and endogenous GCs play a key role in immune regulation. Toll-like receptors (TLRs) enable innate immune cells, such as macrophages, to recognize a wide variety of microbial ligands, thereby promoting inflammation. The interaction of GCs with macrophages in the immunosuppressive resolution phase upon prolonged TLR activation is widely unknown. Treatment of human alveolar macrophages (AMs) with the synthetic GC dexamethasone (Dex) did not alter the expression of TLRs -1, -4, and -6. In contrast, TLR2 was upregulated in a GC receptor-dependent manner, as shown by Western blot and qPCR. Furthermore, long-term lipopolysaccharide (LPS) exposure mimicking immunosuppression in the resolution phase of inflammation synergistically increased Dex-mediated TLR2 upregulation. Analyses of publicly available datasets suggested that TLR2 is induced during the resolution phase of inflammatory diseases, i.e., under conditions associated with high endogenous GC production. TLR2 induction did not enhance TLR2 signaling, as indicated by reduced cytokine production after treatment with TLR2 ligands in Dex- and/or LPS-primed AMs. Thus, we hypothesized that the upregulated membrane-bound TLR2 might serve as a precursor for soluble TLR2 (sTLR2), known to antagonize TLR2-dependent cell actions. Supernatants of LPS/Dex-primed macrophages contained sTLR2, as demonstrated by Western blot analysis. Activation of metalloproteinases resulted in enhanced sTLR2 shedding. Additionally, we detected full-length TLR2 and assumed that this might be due to the production of TLR2-containing extracellular vesicles (EVs). EVs from macrophage supernatants were isolated by sequential centrifugation. Both untreated and LPS/Dex-treated cells produced vesicles of various sizes and shapes, as shown by cryo-transmission electron microscopy. These vesicles were identified as the source of full-length TLR2 in macrophage supernatants by Western blot and mass spectrometry. Flow cytometric analysis indicated that TLR2-containing EVs were able to bind the TLR2 ligand Pam3CSK4. In addition, the presence of EVs reduced inflammatory responses in Pam3CSK4-treated endothelial cells and HEK Dual reporter cells, demonstrating that TLR2-EVs can act as decoy receptors. In summary, our data show that sTLR2 and full-length TLR2 are released by macrophages under anti-inflammatory conditions, which may contribute to GC-induced immunosuppression.
Natural variation in physiological responses of tunisian hedysarum carnosum under iron deficiency
Iron (Fe) is an essential element for plant growth and development. The cultivation of leguminous plants has generated strong interest because of their growth even on poor soils. Calcareous and saline soils with poor mineral availability are wide-spread in Tunisia. In an attempt to select better forage crops adapted to Tunisian soils, we characterized Fe deficiency responses of three different isolates of Hedysarum carnosum, an endemic Tunisian extremophile species growing in native stands in salt and calcareous soil conditions. H. carnosum is a non-model crop. The three isolates, named according to their habitats Karkar, Thelja, and Douiret, differed in the expression of Fe deficiency symptoms like morphology, leaf chlorosis with compromised leaf chlorophyll content and photosynthetic capacity and leaf metal contents. Across these parameters Thelja was found to be tolerant, while Karkar and Douiret were susceptible to Fe deficiency stress. The three physiological and molecular indicators of the iron deficiency response in roots, Fe reductase activity, growth medium acidification and induction of the IRON-REGULATED TRANSPORTER1 homolog, indicated that all lines responded to -Fe, however, varied in the strength of the different responses. We conclude that the individual lines have distinct adaptation capacities to react to iron deficiency, presumably involving mechanisms of whole-plant iron homeostasis and internal metal distribution. The Fe deficiency tolerance of Thelja might be linked with adaptation to its natural habitat on calcareous soil.
M2 polarization enhances silica nanoparticle uptake by macrophages
While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth. We employed different models of M1 and M2 polarization: granulocyte-macrophage colony-stimulating factor/lipopolysaccharide (LPS)/interferon (IFN)-γ was used to generate primary human M1 cells and macrophage colony-stimulating factor (M-CSF)/interleukin (IL)-10 to differentiate M2 monocyte-derived macrophages (MDM). PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-γ and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø26 and 41 nm) and microparticles (Ø1.75 μm) was quantified. At the concentration used (50 μg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human MDM compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue. In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but might also open up therapeutic perspectives allowing to specifically target M2 polarized macrophages.
Microenvironments designed to support growth and function of neuronal cells
Strategies for neural tissue repair heavily depend on our ability to temporally reconstruct the natural cellular microenvironment of neural cells. Biomaterials play a fundamental role in this context, as they provide the mechanical support for cells to attach and migrate to the injury site, as well as fundamental signals for differentiation. This review describes how different cellular processes (attachment, proliferation, and (directional) migration and differentiation) have been supported by different material parameters, in vitro and in vivo. Although incipient guidelines for biomaterial design become visible, literature in the field remains rather phenomenological. As in other fields of tissue regeneration, progress will depend on more systematic studies on cell-materials response, better understanding on how cells behave and understand signals in their natural milieu from neurobiology studies, and the translation of this knowledge into engineered microenvironments for clinical use.
Red blood cell passage of small capillaries is associated with transient Ca2+-mediated adaptations
When red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca2+-signaling by confocal microscopy of Fluo-4 loaded RBCs.We were able to confirm ourin vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca2+ when RBCs were passing through small capillaries in vivo. Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca2+-signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca2+ to enter the cell, leading to a transient activation of the Gardos-channel associated with K+, Cl−, and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constricti on.
Reducing the Effect of Spurious Phase Variations in Neural Oscillatory Signals
The phase-reset model of oscillatory EEG activity has received a lot of attention in the last decades for decoding different cognitive processes. Based on this model, the ERPs are assumed to be generated as a result of phase reorganization in ongoing EEG. Alignment of the phase of neuronal activities can be observed within or between different assemblies of neurons across the brain. Phase synchronization has been used to explore and understand perception, attentional binding and considering it in the domain of neuronal correlates of consciousness. The importance of the topic and its vast exploration in different domains of the neuroscience presses the need for appropriate tools and methods for measuring the level of phase synchronization of neuronal activities. Measuring the level of instantaneous phase (IP) synchronization has been used extensively in numerous studies of ERPs as well as oscillatory activity for a better understanding of the underlying cognitive binding with regard to different set of stimulations such as auditory and visual. However, the reliability of results can be challenged as a result of noise artifact in IP. Phase distortion due to environmental noise artifacts as well as different pre-processing steps on signals can lead to generation of artificial phase jumps. One of such effects presented recently is the effect of low envelope on the IP of signal. It has been shown that as the instantaneous envelope of the analytic signal approaches zero, the variations in the phase increase, effectively leading to abrupt transitions in the phase. These abrupt transitions can distort the phase synchronization results as they are not related to any neurophysiological effect. These transitions are called spurious phase variation. In this study, we present a model to remove generated artificial phase variations due to the effect of low envelope. The proposed method is based on a simplified form of a Kalman smoother, that is able to model the IP behavior in narrow-bandpassed oscillatory signals. In this work we first explain the details of the proposed Kalman smoother for modeling the dynamics of the phase variations in narrow-bandpassed signals and then evaluate it on a set of synthetic signals. Finally, we apply the model on ongoing-EEG signals to assess the removal of spurious phase variations.
Bayesian Modeling of the Dynamics of Phase Modulations and their Application to Auditory Event Related Potentials at Different Loudness Scales
We study the effect of long-term habituation signatures of auditory selective attention reflected in the instantaneous phase information of the auditory event-related potentials (ERPs) at four distinct stimuli levels of 60, 70, 80, and 90 dB SPL. The analysis is based on the single-trial level. The effect of habituation can be observed in terms of the changes (jitter) in the instantaneous phase information of ERPs. In particular, the absence of habituation is correlated with a consistently high phase synchronization over ERP trials. We estimate the changes in phase concentration over trials using a Bayesian approach, in which the phase is modeled as being drawn from a von Mises distribution with a concentration parameter which varies smoothly over trials. The smoothness assumption reflects the fact that habituation is a gradual process. We differentiate between different stimuli based on the relative changes and absolute values of the estimated concentration parameter using the proposed Bayesian model.
Vimentin intermediate filament rings deform the nucleus during the first steps of adhesion
During cell spreading, cells undergo many changes to their architecture and their mechanical properties. Vimentin, as an integral part of the cell architecture, and its mechanical stability must adapt to the new state of the cell. This study focuses on the structures formed by vimentin during the first steps of cell adhesion. Very early, ball-like structures, or “knots,” are seen and often vimentin filaments emerge in the shape of rings around the nucleus. Although intermediate filaments are not known to be associated to motor proteins to form contractile systems, these rings can nonetheless strongly deform the cell nucleus. In the first 6 to 12 h of adhesion, these vimentin knots and rings disappear, and the intermediate filament network returns to the state seen before detachment of the cells. As these vimentin structures are very transient in the early steps of cell spreading, they have rarely been described in the literature. However, they can also be seen during mitosis, which is an event that involves partial detachment and re-spreading of the cells. Interestingly, the turnover dynamics of vimentin are reduced in both the knots and rings, compared to vimentin in the lamellipodia. It remains to define how the force is transmitted from the ball-like structures to the rings, and to measure the impact of such strong nuclear deformation on gene expression during cell re-spreading and the rearrangement of the vimentin network. Copyright © 2019 Terriac, Schütz and Lautenschläger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.