Cell shape sensing licenses dendritic cells for homeostatic migration to lymph nodes.

Immune cells experience large cell shape changes during environmental patrolling because of the physical constraints that they encounter while migrating through tissues. These cells can adapt to such deformation events using dedicated shape-sensing pathways. However, how shape sensing affects immune cell function is mostly unknown. Here, we identify a shape-sensing mechanism that increases the expression of the chemokine receptor CCR7 and guides dendritic cell migration from peripheral tissues to lymph nodes at steady state. This mechanism relies on the lipid metabolism enzyme cPLA2, requires nuclear envelope tensioning and is finely tuned by the ARP2/3 actin nucleation complex. We also show that this shape-sensing axis reprograms dendritic cell transcription by activating an IKKß-NF-κB-dependent pathway known to control their tolerogenic potential. These results indicate that cell shape changes experienced by immune cells can define their migratory behavior and immunoregulatory properties and reveal a contribution of the physical properties of tissues to adaptive immunity.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

HIF2α is a direct regulator of neutrophil motility.

Orchestrated recruitment of neutrophils to inflamed tissue is essential during the initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it remains unclear how these factors influence the migration of neutrophils to and at the site of inflammation during their transmigration through the blood-endothelial cell barrier, as well as their motility in the interstitial space. Here, we reveal that activation of hypoxia-inducible factor 2 (HIF2α) as a result of a deficiency in HIF prolyl hydroxylase domain protein 2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNA sequencing analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation because it promotes neutrophil movement through highly confined tissue landscapes.

Critical role for TRIM28 and HP1ß/γ in the epigenetic control of T cell metabolic reprograming and effector differentiation.

Naive CD4+ T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4+ T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 ß and γ isoforms (HP1ß/γ, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K-AKT-mTOR axis and switch to glycolysis. While differentiation of naive TRIM28-/- T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28-/- regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1ß/γ control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.

Dynamics of the membrane-cytoskeleton interface in MHC class II-restricted antigen presentation.

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC-T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.

Connexin43 hemichannels mediate secondary cellular damage spread from the trauma zone to distal zones in astrocyte monolayers.

The mechanism of secondary damage spread after brain trauma remains unsolved. In this work, we redirected the attention to astrocytic communication pathways. Using an in vitro trauma model that consists of a scratch injury applied to an astrocyte monolayer, we found a significant and transient induction of connexin43 (Cx43) hemichannel activity in regions distal from the injury, which was maximal ∼1 h after scratch. Two connexin hemichannel blockers, La(3+) and the peptide Gap26, abolished the increased activity, which was also absent in Cx43 KO astrocytes. In addition, the scratch-induced increase of hemichannel activity was prevented by inhibition of P2 purinergic receptors. Changes in hemichannel activity took place with a particular spatial distribution, with cells located at ∼17 mm away from the scratch presenting the highest activity (dye uptake). In contrast, the functional state of gap junction channels (dye coupling) was not significantly affected. Cx43 hemichannel activity was also enhanced by the acute extracellular application of 60 mM K(+) . The increase in hemichannel activity was associated with an increment in apoptotic cells at 24 h after scratch that was totally prevented by Gap26 peptide. These findings suggest that Cx43 hemichannels could be a new approach to prevent or reduce the secondary cell damage of brain trauma.

Linoleic acid induces opening of connexin26 hemichannels through a PI3K/Akt/Ca(2+)-dependent pathway.

Connexin hemichannel (Cx HC) opening is involved in physiological and pathological processes, allowing the cellular release of autocrine/paracrine signaling molecules. Linoleic acid (LA) is known to modulate the functional state of connexin46 (Cx46) HCs. However, the molecular mechanism involved in this effect, or whether LA affects HCs constituted of other connexins, remains unknown. Here, we report the effects of LA on HCs in HeLa cells that express Cx26, one of the main Cxs in the cochlear sensory epithelium. Cx26 HC activity (dye uptake) was increased in a concentration-dependent manner by bath application of LA and inhibited by HC blockers. Moreover, intracellular BAPTA, a Ca(2+) chelator, and PI3K/AKT inhibitors were found to reduce the LA-induced Cx26 HC opening, suggesting that the LA effect is mediated by an increase of free intracellular Ca(2+) concentration and activation of the PI3K/Akt-dependent pathway. The LA-induced increase in free intracellular Ca(2+) concentration was mainly due to Ca(2+) influx through Cx26 HCs. In addition, the involvement of SH groups was ruled out, because dithiothreitol (DTT) did not block the LA-induced dye uptake. LA also increased the membrane current mediated by Cx26 HCs expressed in Xenopus oocytes and the dye uptake in HeLa cells expressing Cxs 32, 43 or 45. Since LA is an essential polyunsaturated fatty acid, its effect on HCs might be relevant to cell growth as well as to cellular functions of differentiated cells such as audition.

Regulation of hemichannels and gap junction channels by cytokines in antigen-presenting cells.

Autocrine and paracrine signals coordinate responses of several cell types of the immune system that provide efficient protection against different challenges. Antigen-presenting cells (APCs) coordinate activation of this system via homocellular and heterocellular interactions. Cytokines constitute chemical intercellular signals among immune cells and might promote pro- or anti-inflammatory effects. During the last two decades, two membrane pathways for intercellular communication have been demonstrated in cells of the immune system. They are called hemichannels (HCs) and gap junction channels (GJCs) and provide new insights into the mechanisms of the orchestrated response of immune cells. GJCs and HCs are permeable to ions and small molecules, including signaling molecules. The direct intercellular transfer between contacting cells can be mediated by GJCs, whereas the release to or uptake from the extracellular milieu can be mediated by HCs. GJCs and HCs can be constituted by two protein families: connexins (Cxs) or pannexins (Panxs), which are present in almost all APCs, being Cx43 and Panx1 the most ubiquitous members of each protein family. In this review, we focus on the effects of different cytokines on the intercellular communication mediated by HCs and GJCs in APCs and their impact on purinergic signaling.

Septins provide microenvironment sensing and cortical actomyosin partitioning in motile amoeboid T lymphocytes.

The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here, we report that septins participate in amoeboid motility of T cells, enabling the formation of F-actin and α-actinin-rich cortical rings at the sites of cell cortex-indenting collisions with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient "hourglass"-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results suggest that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots required for amoeboid motility of T cells in tissue-like microenvironments.

Human mesenchymal stem cells derived from adipose tissue reduce functional and tissue damage in a rat model of chronic renal failure.

Therapeutic approaches for CKD (chronic kidney disease) have been able to reduce proteinuria, but not diminish the disease progression. We have demonstrated beneficial effects by injection of BM (bone marrow)-derived MSCs (mesenchymal stem cells) from healthy donors in a rat model with CKD. However, it has recently been reported that BM-MSCs derived from uraemic patients failed to confer functional protection in a similar model. This suggests that autologous BM-MSCs are not suitable for the treatment of CKD. In the present study, we have explored the potential of MSCs derived from adipose tissue (AD-MSCs) as an alternative source of MSCs for the treatment of CKD. We have isolated AD-MSCs and evaluated their effect on the progression of CKD. Adult male SD (Sprague-Dawley) rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×10(6) AD-MSCs or MSC culture medium alone. The therapeutic effect was evaluated by plasma creatinine measurement, structural analysis and angiogenic/epitheliogenic protein expression. AD-MSCs were detected in kidney tissues from NPX animals. This group had a significant reduction in plasma creatinine levels and a lower expression of damage markers ED-1 and α-SMA (α-smooth muscle actin) (P<0.05). In addition, treated rats exhibited a higher level of epitheliogenic [Pax-2 and BMP-7 (bone morphogenetic protein 7)] and angiogenic [VEGF (vascular endothelial growth factor)] proteins. The expression of these biomarkers of regeneration was significantly related to the improvement in renal function. Although many aspects of the cell therapy for CKD remain to be investigated, we provide evidence that AD-MSCs, a less invasive and highly available source of MSCs, exert an important therapeutic effect in this pathology.

ATP is required and advances cytokine-induced gap junction formation in microglia in vitro.

Microglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs) and gap junction channels (GJCs), affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF-α/IFN-γ-induced dye coupling, probably through the induction of IL-1ß release. Moreover, TNF-α/IFN-γ, but not TNF-α plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-α/IFN-γ, but not TNF-α plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-α/IFN-γ in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.

Calcium channel ß3 subunit regulates ATP-dependent migration of dendritic cells.

Migratory dendritic cells (migDCs) continuously patrol tissues and are activated by injury and inflammation. Extracellular adenosine triphosphate (ATP) is released by damaged cells or actively secreted during inflammation and increases migDC motility. However, the underlying molecular mechanisms by which ATP accelerates migDC migration is not understood. Here, we show that migDCs can be distinguished from other DC subsets and immune cells by their expression of the voltage-gated calcium channel subunit ß3 (Cavß3; CACNB3), which exclusively facilitates ATP-dependent migration in vitro and during tissue damage in vivo. By contrast, CACNB3 does not regulate lipopolysaccharide-dependent migration. Mechanistically, CACNB3 regulates ATP-dependent inositol 1,4,5-trisphophate receptor-controlled calcium release from the endoplasmic reticulum. This, in turn, is required for ATP-mediated suppression of adhesion molecules, their detachment, and initiation of migDC migration. Thus, Cacnb3-deficient migDCs have an impaired migration after ATP exposure. In summary, we identified CACNB3 as a master regulator of ATP-dependent migDC migration that controls tissue-specific immunological responses during injury and inflammation.

Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens.

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.

Amyloid ß-induced death in neurons involves glial and neuronal hemichannels.

The mechanisms involved in Alzheimer's disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-ß peptide (Aß) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aß. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aß(25-35) increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aß. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aß(25-35)-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aß(25-35)-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aß leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aß.

Glucose increases intracellular free Ca(2+) in tanycytes via ATP released through connexin 43 hemichannels.

The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal-glial cells, termed tanycytes. The latter express glucose-sensing proteins, including glucose transporter 2, glucokinase, and ATP-sensitive K(+) (K(ATP) ) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose-induced rise of intracellular free Ca(2+) concentration ([Ca(2+) ](i) ) in cultured ß-tanycytes was examined. Fura-2AM time-lapse fluorescence images revealed that glucose increases the intracellular Ca(2+) signal in a concentration-dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a K(ATP) channel-dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y(1) receptors followed by inositol trisphosphate receptor activation and Ca(2+) release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca(2+) ](i) in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes.

c-Abl Activation Linked to Autophagy-Lysosomal Dysfunction Contributes to Neurological Impairment in Niemann-Pick Type A Disease.

Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.

Mesenchymal stem cell injection ameliorates chronic renal failure in a rat model.

CKD (chronic kidney disease) has become a public health problem. The therapeutic approaches have been able to reduce proteinuria, but have not been successful in limiting disease progression. In this setting, cell therapies associated with regenerative effects are attracting increasing interest. We evaluated the effect of MSC (mesenchymal stem cells) on the progression of CKD and the expression of molecular biomarkers associated with regenerative effects. Adult male Sprague-Dawley rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×106 MSC or culture medium. A sham group subjected to the same injection was used as the control. Rats were killed 5 weeks after MSC infusion. Dye tracking of MSC was followed by immunofluorescence analysis. Kidney function was evaluated using plasma creatinine. Structural damage was evaluated by H&E (haematoxylin and eosin) staining, ED-1 abundance (macrophages) and interstitial α-SMA (α-smooth muscle actin). Repairing processes were evaluated by functional and structural analyses and angiogenic/epitheliogenic protein expression. MSC could be detected in kidney tissues from NPX animals treated with intravenous cell infusion. This group presented a marked reduction in plasma creatinine levels and damage markers ED-1 and α-SMA (P<0.05). In addition, treated rats exhibited a significant induction in epitheliogenic [Pax-2, bFGF (basic fibroblast growth factor) and BMP-7 (bone morphogenetic protein-7)] and angiogenic [VEGF (vascular endothelial growth factor) and Tie-2] proteins. The expression of these biomarkers of regeneration was significantly related to the increase in renal function. Many aspects of the cell therapy in CKD remain to be investigated in more detail: for example, its safety, low cost and the possible need for repeated cell injections over time. Beyond the undeniable importance of these issues, what still needs to be clarified is whether MSC administration has a real effect on the treatment of this pathology. It is precisely to this point that the present study aims to contribute.

Pannexin Channel Regulation of Cell Migration: Focus on Immune Cells.

The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes (i.e., T cells, dendritic cells) exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells (including fibroblasts, keratinocytes, astrocytes, and cancer cells). Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration.

Pinching the cortex of live cells reveals thickness instabilities caused by myosin II motors.

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration, and division. Because its thickness is below optical resolution, there is a tendency to consider the cortex as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time-resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension because of actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape-changing capacity of highly contractile cells that use amoeboid-like migration.

The first junior European Calcium Society meeting: calcium research across scales, Kingdoms and countries.

The first junior European Calcium Society online meeting, held October 20-21, 2020, aimed to promote junior researchers in the Ca2+ community. The meeting included four scientific sessions, covering Ca2+ research from molecular detail to whole organisms. Each session featured one invited speaker and three speakers selected based on submitted abstracts, with the overall aim of actively involving early-career researchers. Consequently, the meeting underlined the diversity of Ca2+ physiology, by showcasing research across scales and Kingdoms, as presented by a correspondingly diverse speaker panel across career stages and countries. In this meeting report, we introduce the visions of the junior European Calcium Society board and summarize the meeting content.