Membrane curvature and PS localize coagulation proteins to filopodia and retraction fibers of endothelial cells.

Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)-containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α-stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation.

Performance assessment and economic analysis of a human Liver-Chip for predictive toxicology.

BACKGROUND: Conventional preclinical models often miss drug toxicities, meaning the harm these drugs pose to humans is only realized in clinical trials or when they make it to market. This has caused the pharmaceutical industry to waste considerable time and resources developing drugs destined to fail. Organ-on-a-Chip technology has the potential improve success in drug development pipelines, as it can recapitulate organ-level pathophysiology and clinical responses; however, systematic and quantitative evaluations of Organ-Chips' predictive value have not yet been reported. METHODS: 870 Liver-Chips were analyzed to determine their ability to predict drug-induced liver injury caused by small molecules identified as benchmarks by the Innovation and Quality consortium, who has published guidelines defining criteria for qualifying preclinical models. An economic analysis was also performed to measure the value Liver-Chips could offer if they were broadly adopted in supporting toxicity-related decisions as part of preclinical development workflows. RESULTS: Here, we show that the Liver-Chip met the qualification guidelines across a blinded set of 27 known hepatotoxic and non-toxic drugs with a sensitivity of 87% and a specificity of 100%. We also show that this level of performance could generate over $3 billion annually for the pharmaceutical industry through increased small-molecule R&D productivity. CONCLUSIONS: The results of this study show how incorporating predictive Organ-Chips into drug development workflows could substantially improve drug discovery and development, allowing manufacturers to bring safer, more effective medicines to market in less time and at lower costs.

Drug development is lengthy and costly, as it relies on laboratory models that fail to predict human reactions to potential drugs. Because of this, toxic drugs sometimes go on to harm humans when they reach clinical trials or once they are in the marketplace. Organ-on-a-Chip technology involves growing cells on small devices to mimic organs of the body, such as the liver. Organ-Chips could potentially help identify toxicities earlier, but there is limited research into how well they predict these effects compared to conventional models. In this study, we analyzed 870 Liver-Chips to determine how well they predict drug-induced liver injury, a common cause of drug failure, and found that Liver-Chips outperformed conventional models. These results suggest that widespread acceptance of Organ-Chips could decrease drug attrition, help minimize harm to patients, and generate billions in revenue for the pharmaceutical industry.

Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice.

Factor VIII (FVIII) replacement therapy for hemophilia A is complicated by development of inhibitory antibodies (inhibitors) in ∼30% of patients. Because endothelial cells (ECs) are the primary physiologic expression site, we probed the therapeutic potential of genetically restoring FVIII expression selectively in ECs in hemophilia A mice (FVIIInull). Expression of FVIII was driven by the Tie2 promoter in the context of lentivirus (LV)-mediated in situ transduction (T2F8LV) or embryonic stem cell-mediated transgenesis (T2F8Tg). Both endothelial expression approaches were associated with a strikingly robust immune response. Following in situ T2F8LV-mediated EC transduction, all FVIIInull mice developed inhibitors but had no detectable plasma FVIII. In the transgenic approach, the T2F8Tg mice had normalized plasma FVIII levels, but showed strong sensitivity to developing an FVIII immune response upon FVIII immunization. A single injection of FVIII with incomplete Freund adjuvant led to high titers of inhibitors and reduction of plasma FVIII to undetectable levels. Because ECs are putative major histocompatibility complex class II (MHCII)-expressing nonhematopoietic, "semiprofessional" antigen-presenting cells (APCs), we asked whether they might directly influence the FVIII immune responses. Imaging and flow cytometric studies confirmed that both murine and human ECs express MHCII and efficiently bind and take up FVIII protein in vitro. Moreover, microvascular ECs preconditioned ex vivo with inflammatory cytokines could functionally present exogenously taken-up FVIII to previously primed CD4+/CXCR5+ T follicular helper (Tfh) cells to drive FVIII-specific proliferation. Our results show an unanticipated immunogenicity of EC-expressed FVIII and suggest a context-dependent role for ECs in the regulation of inhibitors as auxiliary APCs for Tfh cells.

Tumor-Derived Extracellular Vesicles Breach the Intact Blood-Brain Barrier via Transcytosis.

The restrictive nature of the blood-brain barrier (BBB) creates a major challenge for brain drug delivery with current nanomedicines lacking the ability to cross the BBB. Extracellular vesicles (EVs) have been shown to contribute to the progression of a variety of brain diseases including metastatic brain cancer and have been suggested as promising therapeutics and drug delivery vehicles. However, the ability of native tumor-derived EVs to breach the BBB and the mechanism(s) involved in this process remain unknown. Here, we demonstrate that tumor-derived EVs can breach the intact BBB in vivo, and by using state-of-the-art in vitro and in vivo models of the BBB, we have identified transcytosis as the mechanism underlying this process. Moreover, high spatiotemporal resolution microscopy demonstrated that the endothelial recycling endocytic pathway is involved in this transcellular transport. We further identify and characterize the mechanism by which tumor-derived EVs circumvent the low physiologic rate of transcytosis in the BBB by decreasing the brain endothelial expression of rab7 and increasing the efficiency of their transport. These findings identify previously unknown mechanisms by which tumor-derived EVs breach an intact BBB during the course of brain metastasis and can be leveraged to guide and inform the development of drug delivery approaches to deliver therapeutic cargoes across the BBB for treatment of a variety of brain diseases including, but not limited to, brain malignancies.

Edible Mushrooms Reduce Atherosclerosis in Ldlr-/- Mice Fed a High-Fat Diet.

BACKGROUND: Commonly consumed mushrooms, portobello (PBM) and shiitake (SHM), are abundant in nutrients, soluble dietary fibers, and bioactive compounds that have been implicated as beneficial in reducing inflammation, improving lipid profiles, and ameliorating heart disease and atherosclerosis, an inflammatory disease of the arteries. OBJECTIVE: The aim of this study was to determine effects of PBM and SHM in preventing atherosclerosis and associated inflammation in an animal model. METHODS: Four-week-old Ldlr-/- male mice were divided into 5 dietary groups for 16 wk: a low-fat control (LF-C, 11 kcal% fat), high-fat control (HF-C, 18.9 kcal% fat), HF + 10% (wt:wt) PBM (HF-PBM, 19.5 kcal% fat) or SHM (HF-SHM, 19.7 kcal% fat) powder, and HF + mushroom control mix (MIX-C, 19.6 kcal% fat), a diet best matched to the average macronutrient content of both mushrooms. Body composition was measured using MRI. Aortic tricuspid valves and aortas were collected and stained to quantify plaque formation. Adhesion molecule expression was quantified by immunohistochemistry. Plasma lipid and cytokine concentrations were measured. RESULTS: We found that mice fed a HF-SHM diet had ∼86% smaller aortic lesion area than mice in both HF-C (P < 0.01) and MIX-C (P < 0.01) groups and also expressed 31-48% lower vascular cell adhesion molecule-1 levels (P < 0.05) than all other groups. Similarly, HF-PBM-fed mice displayed a 70% reduction in aortic lesion area in the tricuspid valve only (P < 0.05). Both mushroom-fed groups had lower weight gain and fat mass (P < 0.05) than the control groups. CONCLUSION: These results suggest that consumption of PBMs and particularly SHMs is effective in preventing development of high-fat diet-induced atherosclerosis in Ldlr-/- mice. Future studies will determine active components in mushrooms responsible for this beneficial effect.

The tumor suppressor p53 can promote collective cellular migration.

Loss of function of the tumor suppressor p53 is known to increase the rate of migration of cells transiting the narrow pores of the traditional Boyden chamber assay. Here by contrast we investigate how p53 impacts the rate of cellular migration within a 2D confluent cell layer and a 3D collagen-embedded multicellular spheroid. We use two human carcinoma cell lines, the bladder carcinoma EJ and the colorectal carcinoma HCT116. In the confluent 2-D cell layer, for both EJ and HCT cells the migratory speeds and effective diffusion coefficients for the p53 null cells were significantly smaller than in p53-expressing cells. Compared to p53 expressers, p53-null cells exhibited more organized cortical actin rings together with reduced front-rear cell polarity. Furthermore, loss of p53 caused cells to exert smaller traction forces upon their substrates, and reduced formation of cryptic lamellipodia. In the 3D multicellular spheroid, loss of p53 consistently reduced collective cellular migration into surrounding collagen matrix. As regards the role of p53 in cellular migration, extrapolation from the Boyden chamber assay to other cellular microenvironments is seen to be fraught even in terms of the sign of the effect. Together, these paradoxical results show that the effects of p53 on cellular migration are context-dependent.

T Lymphocyte-Endothelial Interactions: Emerging Understanding of Trafficking and Antigen-Specific Immunity.

Antigen-specific immunity requires regulated trafficking of T cells in and out of diverse tissues in order to orchestrate lymphocyte development, immune surveillance, responses, and memory. The endothelium serves as a unique barrier, as well as a sentinel, between the blood and the tissues, and as such it plays an essential locally tuned role in regulating T cell migration and information exchange. While it is well established that chemoattractants and adhesion molecules are major determinants of T cell trafficking, emerging studies have now enumerated a large number of molecular players as well as a range of discrete cellular remodeling activities (e.g., transmigratory cups and invadosome-like protrusions) that participate in directed migration and pathfinding by T cells. In addition to providing trafficking cues, intimate cell-cell interaction between lymphocytes and endothelial cells provide instruction to T cells that influence their activation and differentiation states. Perhaps the most intriguing and underappreciated of these "sentinel" roles is the ability of the endothelium to act as a non-hematopoietic "semiprofessional" antigen-presenting cell. Close contacts between circulating T cells and antigen-presenting endothelium may play unique non-redundant roles in shaping adaptive immune responses within the periphery. A better understanding of the mechanisms directing T cell trafficking and the antigen-presenting role of the endothelium may not only increase our knowledge of the adaptive immune response but also empower the utility of emerging immunomodulatory therapeutics.

Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway.

Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin 'foci'. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

Intravital imaging of mesenchymal stem cell trafficking and association with platelets and neutrophils.

Early events of mesenchymal stem/stromal cell (MSC) adhesion to and transmigration through the vascular wall following systemic infusion are important for MSC trafficking to inflamed sites, yet are poorly characterized in vivo. Here, we used intravital confocal imaging to determine the acute extravasation kinetics and distribution of culture-expanded MSC (2-6 hours postinfusion) in a murine model of dermal inflammation. By 2 hours postinfusion, among the MSC that arrested within the inflamed ear dermis, 47.8% ± 8.2% of MSC had either initiated or completed transmigration into the extravascular space. Arrested and transmigrating MSCs were equally distributed within both small capillaries and larger venules. This suggested existence of an active adhesion mechanism, since venule diameters were greater than those of the MSC. Heterotypic intravascular interactions between distinct blood cell types have been reported to facilitate the arrest and extravasation of leukocytes and circulating tumor cells. We found that 42.8% ± 24.8% of intravascular MSC were in contact with neutrophil-platelet clusters. A role for platelets in MSC trafficking was confirmed by platelet depletion, which significantly reduced the preferential homing of MSC to the inflamed ear, although the total percentage of MSC in contact with neutrophils was maintained. Interestingly, although platelet depletion increased vascular permeability in the inflamed ear, there was decreased MSC accumulation. This suggests that increased vascular permeability is unnecessary for MSC trafficking to inflamed sites. These findings represent the first glimpse into MSC extravasation kinetics and microvascular distribution in vivo, and further clarify the roles of active adhesion, the intravascular cellular environment, and vascular permeability in MSC trafficking.

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells ('APCs') referred to as 'immunological synapses'. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of 'semi-professional APCs'. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic 'planar cellular APC model' for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Role of BRAFV600E in the first preclinical model of multifocal infiltrating myopericytoma development and microenvironment.

Myopericytoma (MPC) is a rare tumor with perivascular proliferation of pluripotent stem-cell-like pericytes. Although indolent, MPC may be locally aggressive with recurrent disease. The pathogenesis and diagnostic biomarkers of MPC are poorly understood. We discovered that 15% of benign MPCs (thyroid, skin; 3 of 20 samples) harbored BRAF(WT/V600E); 33.3% (1 of 3 samples) of BRAF(WT/V600E)-MPCs were multifocal/infiltrative/recurrent. Patient-MPC and primary MPC cells harbored BRAF(WT/V600E), were clonal and expressed pericytic-differentiation biomarkers crucial for its microenvironment. BRAF(WT/V600E)-positive thyroid MPC primary cells triggered in vitro (8.8-fold increase) and in vivo (3.6-fold increase) angiogenesis. Anti-BRAF(V600E) therapy with vemurafenib disrupted angiogenic and metabolic properties (~3-fold decrease) with down-regulation (~2.2-fold decrease) of some extracellular-matrix (ECM) factors and ECM-associated long non-coding RNA (LincRNA) expression, with no effects in BRAF(WT)-pericytes. Vemurafenib also inhibited (~3-fold decrease) cell viability in vitro and in BRAF(WT/V600E)-positive thyroid MPC patient-derived xenograft (PDX) mice (n = 5 mice per group). We established the first BRAF(WT/V600E)-dependent thyroid MPC cell culture. Our findings identify BRAF(WT/V600E) as a novel genetic aberration in MPC pathogenesis and MPC-associated biomarkers and imply that anti-BRAF(V600E) agents may be useful adjuvant therapy in BRAF(WT/V600E)-MPC patients. Patients with BRAF(WT/V600E)-MPC should be closely followed because of the risk for multifocality/recurrence.

Probing the biomechanical contribution of the endothelium to lymphocyte migration: diapedesis by the path of least resistance.

Immune cell trafficking requires the frequent breaching of the endothelial barrier either directly through individual cells ('transcellular' route) or through the inter-endothelial junctions ('paracellular' route). What determines the loci or route of breaching events is an open question with important implications for overall barrier regulation. We hypothesized that basic biomechanical properties of the endothelium might serve as crucial determinants of this process. By altering junctional integrity, cytoskeletal morphology and, consequently, local endothelial cell stiffness of different vascular beds, we could modify the preferred route of diapedesis. In particular, high barrier function was associated with predominantly transcellular migration, whereas negative modulation of junctional integrity resulted in a switch to paracellular diapedesis. Furthermore, we showed that lymphocytes dynamically probe the underlying endothelium by extending invadosome-like protrusions (ILPs) into its surface that deform the nuclear lamina, distort actin filaments and ultimately breach the barrier. Fluorescence imaging and pharmacologic depletion of F-actin demonstrated that lymphocyte barrier breaching efficiency was inversely correlated with local endothelial F-actin density and stiffness. Taken together, these data support the hypothesis that lymphocytes are guided by the mechanical 'path of least resistance' as they transverse the endothelium, a process we term 'tenertaxis'.

Soluble adhesion molecules as markers for sepsis and the potential pathophysiological discrepancy in neonates, children and adults.

Sepsis is a severe and life-threatening systemic inflammatory response to infection that affects all populations and age groups. The pathophysiology of sepsis is associated with aberrant interaction between leukocytes and the vascular endothelium. As inflammation progresses, the adhesion molecules that mediate these interactions become shed from cell surfaces and accumulate in the blood as soluble isoforms that are being explored as potential prognostic disease biomarkers. We critically review the studies that have tested the predictive value of soluble adhesion molecules in sepsis pathophysiology with emphasis on age, as well as the underlying mechanisms and potential roles for inflammatory shedding. Five soluble adhesion molecules are associated with sepsis, specifically, E-selectin, L-selectin and P-selectin, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. While increased levels of these soluble adhesion molecules generally correlate well with the presence of sepsis, their degree of elevation is still poorly predictive of sepsis severity scores, outcome and mortality. Separate analyses of neonates, children and adults demonstrate significant age-dependent discrepancies in both basal and septic levels of circulating soluble adhesion molecules. Additionally, a range of both clinical and experimental studies suggests protective roles for adhesion molecule shedding that raise important questions about whether these should positively or negatively correlate with mortality. In conclusion, while predictive properties of soluble adhesion molecules have been researched intensively, their levels are still poorly predictive of sepsis outcome and mortality. We propose two novel directions for improving clinical utility of soluble adhesion molecules: the combined simultaneous analysis of levels of adhesion molecules and their sheddases; and taking age-related discrepancies into account. Further attention to these issues may provide better understanding of sepsis pathophysiology and increase the usefulness of soluble adhesion molecules as diagnostic and predictive biomarkers.

Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium.

ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins--including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein--was blocked by preincubation with PEG-catalase, which degrades H2O2. ADP treatment promoted the H2O2-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H2O2. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl-interacting protein CrkII are mediated by H2O2. We transfected endothelial cells with differentially targeted HyPer2 H2O2 biosensors and found that ADP promoted a marked increase in H2O2 levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H2O2 in control of ADP-mediated signaling responses in the vascular wall.

Novel role of CD47 in rat microvascular endothelium: signaling and regulation of T-cell transendothelial migration.

OBJECTIVE: Although endothelial CD47, a member of the immunoglobulin superfamily, has been implicated in leukocyte diapedesis, its capacity for intracellular signaling and physical localization during this process has not been addressed in detail. This study examined endothelial CD47 spatiotemporal behavior and signaling pathways involved in regulating T-cell transendothelial migration. APPROACH AND RESULTS: By biochemical methods, transmigration assays, and live-cell microscopy techniques, we show that endothelial CD47 engagement results in intracellular calcium mobilization, increased permeability, and activation of Src and AKT1/phosphoinositide 3-kinase in brain microvascular endothelial cells. These signaling pathways converge to induce cytoskeleton remodeling and vascular endothelial cadherin phosphorylation, which are necessary steps during T-cell transendothelial migration. In addition, during T-cell migration, transmigratory cups and podo-prints enriched in CD47 appear on the surface of the endothelium, indicating that the spatial distribution of CD47 changes after its engagement. Consistent with previous findings of intercellular adhesion molecule 1, blockade of CD47 results in decreased T-cell transmigration across microvascular endothelium. The overlapping effect of intercellular adhesion molecule 1 and CD47 suggests their involvement in different steps of the diapedesis process. CONCLUSIONS: These data reveal a novel role for CD47-mediated signaling in the control of the molecular network governing endothelial-dependent T-cell diapedesis.

Glassy dynamics, cell mechanics, and endothelial permeability.

A key feature of all inflammatory processes is disruption of the vascular endothelial barrier. Such disruption is initiated in part through active contraction of the cytoskeleton of the endothelial cell (EC). Because contractile forces are propagated from cell to cell across a great many cell-cell junctions, this contractile process is strongly cooperative and highly nonlocal. We show here that the characteristic length scale of propagation is modulated by agonists and antagonists that impact permeability of the endothelial barrier. In the presence of agonists including thrombin, histamine, and H2O2, force correlation length increases, whereas in the presence of antagonists including sphingosine-1-phosphate, hepatocyte growth factor, and the rho kinase inhibitor, Y27632, force correlation length decreases. Intercellular force chains and force clusters are also evident, both of which are reminiscent of soft glassy materials approaching a glass transition.

Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds.

Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.

The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood.

CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (T(FR) cells) inhibit humoral immunity mediated by CD4(+)CXCR5(+)Foxp3(-) follicular helper T cells (T(FH) cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of T(FR) cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of T(FR) cells in the lymph nodes and that those T(FR) cells had enhanced suppressive ability. We also found substantial populations of T(FR) cells in mouse blood and demonstrated that T(FR) cells in the blood homed to lymph nodes and potently inhibited T(FH) cells in vivo. T(FR) cells in the blood required signaling via the costimulatory receptors CD28 and ICOS but were inhibited by PD-1 and PD-L1. Our findings demonstrate mechanisms by which the PD-1 pathway regulates antibody production and help reconcile inconsistencies surrounding the role of this pathway in humoral immunity.