Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation.

The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.

Senescent endothelial cells promote pathogenic neutrophil trafficking in inflamed tissues.

Cellular senescence is a hallmark of advanced age and a major instigator of numerous inflammatory pathologies. While endothelial cell (EC) senescence is aligned with defective vascular functionality, its impact on fundamental inflammatory responses in vivo at single-cell level remain unclear. To directly investigate the role of EC senescence on dynamics of neutrophil-venular wall interactions, we applied high resolution confocal intravital microscopy to inflamed tissues of an EC-specific progeroid mouse model, characterized by profound indicators of EC senescence. Progerin-expressing ECs supported prolonged neutrophil adhesion and crawling in a cell autonomous manner that additionally mediated neutrophil-dependent microvascular leakage. Transcriptomic and immunofluorescence analysis of inflamed tissues identified elevated levels of EC CXCL1 on progerin-expressing ECs and functional blockade of CXCL1 suppressed the dysregulated neutrophil responses elicited by senescent ECs. Similarly, cultured progerin-expressing human ECs exhibited a senescent phenotype, were pro-inflammatory and prompted increased neutrophil attachment and activation. Collectively, our findings support the concept that senescent ECs drive excessive inflammation and provide new insights into the mode, dynamics, and mechanisms of this response at single-cell level.

Hepatocyte Growth Factor Receptor c-Met Instructs T Cell Cardiotropism and Promotes T Cell Migration to the Heart via Autocrine Chemokine Release.

Effector-T-cell-mediated immunity depends on the efficient localization of antigen-primed lymphocytes to antigen-rich non-lymphoid tissue, which is facilitated by the expression of a unique set of "homing" receptors acquired by memory T cells. We report that engagement of the hepatocyte growth factor (HGF) receptor c-Met by heart-produced HGF during priming in the lymph nodes instructs T cell cardiotropism, which was associated with a specialized homing "signature" (c-Met(+)CCR4(+)CXCR3(+)). c-Met signals facilitated T cell recruitment to the heart via the chemokine receptor CCR5 by inducing autocrine CCR5 ligand release. c-Met triggering was sufficient to support cardiotropic T cell recirculation, while CCR4 and CXCR3 sustained recruitment during heart inflammation. Transient pharmacological blockade of c-Met during T cell priming led to enhanced survival of heart, but not skin, allografts associated with impaired localization of alloreactive T cells to heart grafts. These findings suggest c-Met as a target for development of organ-selective immunosuppressive therapies.

Annexin A1 attenuates cardiac diastolic dysfunction in mice with inflammatory arthritis.

Rheumatoid arthritis (RA) carries a twofold increased incidence of heart failure with preserved ejection fraction, accompanied by diastolic dysfunction, which can lead to death. The causes of diastolic dysfunction are unknown, and there are currently no well-characterized animal models for studying these mechanisms. Current medications for RA do not have marked beneficial cardio-protective effects. K/BxN F1 progeny and KRN control mice were analyzed over time for arthritis development, monitoring left ventricular diastolic and systolic function using echocardiography. Excised hearts were analyzed by flow cytometry, qPCR, and histology. In pharmacological experiments, K/BxN F1 mice were treated with human recombinant AnxA1 (hrAnxA1, 1 µg/mouse) or vehicle daily. K/BxN F1 mice exhibited fully developed arthritis with normal cardiac function at 4 wk; however, by week 8, all mice displayed left ventricular diastolic dysfunction with preserved ejection fraction. This dysfunction was associated with cardiac hypertrophy, myocardial inflammation and fibrosis, and inflammatory markers. Daily treatment of K/BxN F1 mice with hrAnxA1 from weeks 4 to 8 halted progression of the diastolic dysfunction. The treatment reduced cardiac transcripts of proinflammatory cytokines and profibrotic markers. At the cellular level, hrAnxA1 decreased activated T cells and increased MHC IIlow macrophage infiltration in K/BxN F1 hearts. Similar effects were obtained when hrAnxA1 was administered from week 8 to week 15. We describe an animal model of inflammatory arthritis that recapitulates the cardiomyopathy of RA. Treatment with hrAnxA1 after disease onset corrected the diastolic dysfunction through modulation of both fibroblast and inflammatory cell phenotype within the heart.

ß1-Integrin-Mediated Uptake of Chondrocyte Extracellular Vesicles Regulates Chondrocyte Homeostasis.

Osteoarthritis (OA) is the most prevalent age-related degenerative disorder, which severely reduces the quality of life of those affected. Whilst management strategies exist, no cures are currently available. Virtually all joint resident cells generate extracellular vesicles (EVs), and alterations in chondrocyte EVs during OA have previously been reported. Herein, we investigated factors influencing chondrocyte EV release and the functional role that these EVs exhibit. Both 2D and 3D models of culturing C28I/2 chondrocytes were used for generating chondrocyte EVs. We assessed the effect of these EVs on chondrogenic gene expression as well as their uptake by chondrocytes. Collectively, the data demonstrated that chondrocyte EVs are sequestered within the cartilage ECM and that a bi-directional relationship exists between chondrocyte EV release and changes in chondrogenic differentiation. Finally, we demonstrated that the uptake of chondrocyte EVs is at least partially dependent on ß1-integrin. These results indicate that chondrocyte EVs have an autocrine homeostatic role that maintains chondrocyte phenotype. How this role is perturbed under OA conditions remains the subject of future work.

Galectin-9 mediates neutrophil capture and adhesion in a CD44 and ß2 integrin-dependent manner.

Neutrophil trafficking is a key component of the inflammatory response. Here, we have investigated the role of the immunomodulatory lectin Galectin-9 (Gal-9) on neutrophil recruitment. Our data indicate that Gal-9 is upregulated in the inflamed vasculature of RA synovial biopsies and report the release of Gal-9 into the extracellular environment following endothelial cell activation. siRNA knockdown of endothelial Gal-9 resulted in reduced neutrophil adhesion and neutrophil recruitment was significantly reduced in Gal-9 knockout mice in a model of zymosan-induced peritonitis. We also provide evidence for Gal-9 binding sites on human neutrophils; Gal-9 binding induced neutrophil activation (increased expression of ß2 integrins and reduced expression of CD62L). Intra-vital microscopy confirmed a pro-recruitment role for Gal-9, with increased numbers of transmigrated neutrophils following Gal-9 administration. We studied the role of both soluble and immobilized Gal-9 on human neutrophil recruitment. Soluble Gal-9 significantly strengthened the interaction between neutrophils and the endothelium and inhibited neutrophil crawling on ICAM-1. When immobilized, Gal-9 functioned as an adhesion molecule and captured neutrophils from the flow. Neutrophils adherent to Gal-9 exhibited a spread/activated phenotype that was inhibited by CD18 and CD44 neutralizing antibodies, suggesting a role for these molecules in the pro-adhesive effects of Gal-9. Our data indicate that Gal-9 is expressed and released by the activated endothelium and functions both in soluble form and when immobilized as a neutrophil adhesion molecule. This study paves the way for further investigation of the role of Gal-9 in leukocyte recruitment in different inflammatory settings.

Peripheral control of psychiatric disorders: Focus on OCD. Are we there yet?

"We are all in this together" - we often hear this phrase when we want to flag up a problem that is not for a single individual but concerns us all. A similar reflection has been recently made in the field of mental disorders where brain-centric scientists have started to zoom out their brain-focused graphical representations of the mechanisms regulating psychiatric diseases to include other organs or mediators that did not belong historically to the world of neuroscience. The brain itself - that has long been seen as a master in command secluded in its fortress (the blood brain barrier), has now become a collection of Airbnb(s) where all sorts of cells come in and out and sometimes even rearrange the furniture! Under this new framework of reference, mental disorders have become multisystem pathologies where different biological systems - not just the CNS -contribute 'all together' to the development and severity of the disease. In this narrative review article, we will focus on one of the most popular biological systems that has been shown to influence the functioning of the CNS: the immune system. We will specifically highlight the two main features of the immune system and the CNS that we think are important in the context of mental disorders: plasticity and memory.

The immunomodulatory effects of social isolation in mice are linked to temperature control.

Living in isolation is considered an emerging societal problem that negatively affects the physical wellbeing of its sufferers in ways that we are just starting to appreciate. This study investigates the immunomodulatory effects of social isolation in mice, utilising a two-week program of sole cage occupancy followed by the testing of immune-inflammatory resilience to bacterial sepsis. Our results revealed that mice housed in social isolation showed an increased ability to clear bacterial infection compared to control socially housed animals. These effects were associated with specific changes in whole blood gene expression profile and an increased production of classical pro-inflammatory cytokines. Interestingly, equipping socially isolated mice with artificial nests as a substitute for their natural huddling behaviour reversed the increased resistance to bacterial sepsis. Together these results suggest that the control of body temperature through social housing and huddling behaviour are important factors in the regulation of the host immune response to infection in mice and might provide another example of the many ways by which living conditions influence immunity.

Correction of Defective T-Regulatory Cells From Patients With Crohn's Disease by Ex Vivo Ligation of Retinoic Acid Receptor-α.

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4ß7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor-α (RARA), and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. The pathway that regulates the expression of integrin subunit α is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4ß7, as well as CD62L and FOXP3, compared with cells incubated with rapamycin or rapamycin and all-trans retinoic acid. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines, and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labeled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4ß7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4ß7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. ClinicalTrials.gov, Number: NCT03185000.

Galectin-3: A Positive Regulator of Leukocyte Recruitment in the Inflamed Microcirculation.

In vivo and ex vivo imaging were used to investigate the function of galectin-3 (Gal-3) during the process of leukocyte recruitment to the inflamed microcirculation. The cremasteric microcirculation of wild-type (C57BL/6), Gal-3-/-, and CX3CR1gfp/+ mice were assessed by intravital microscopy after PBS, IL-1ß, TNF-α, or recombinant Gal-3 treatment. These cellular responses were investigated further using flow-chamber assays, confocal microscopy, flow cytometry, PCR analysis, and proteome array. We show that mechanisms mediating leukocyte slow rolling and emigration are impaired in Gal-3-/- mice, which could be because of impaired expression of cell adhesion molecules and an altered cell surface glycoproteome. Local (intrascrotal) administration of recombinant Gal-3 to wild-type mice resulted in a dose-dependent reduction in rolling velocity associated with increased numbers of adherent and emigrated leukocytes, ∼50% of which were Ly6G+ neutrophils. Intrascrotal administration of Gal-3 to CX3CR1gfp/+ mice confirmed that approximately equal numbers of monocytes are also recruited in response to this lectin. Exogenous Gal-3 treatment was accompanied by increased proinflammatory cytokines and chemokines within the local tissue. In conclusion, this study unveils novel biology for both exogenous and endogenous Gal-3 in promoting leukocyte recruitment during acute inflammation.

Both MC1 and MC3 Receptors Provide Protection From Cerebral Ischemia-Reperfusion-Induced Neutrophil Recruitment.

OBJECTIVE: Neutrophil recruitment is a key process in the pathogenesis of stroke, and may provide a valuable therapeutic target. Targeting the melanocortin (MC) receptors has previously shown to inhibit leukocyte recruitment in peripheral inflammation, however, it is not known whether treatments are effective in the unique cerebral microvascular environment. Here, we provide novel research highlighting the effects of the MC peptides on cerebral neutrophil recruitment, demonstrating important yet discrete roles for both MC1 and MC3. APPROACH AND RESULTS: Using intravital microscopy, in 2 distinct murine models of cerebral ischemia-reperfusion (I/R) injury, we have investigated MC control for neutrophil recruitment. After global I/R, pharmacological treatments suppressed pathological neutrophil recruitment. MC1 selective treatment rapidly inhibited neutrophil recruitment while a nonselective MC agonist provided protection even when coadministered with an MC3/4 antagonist, suggesting the importance of early MC1 signaling. However, by 2-hour reperfusion, MC1-mediated effects were reduced, and MC3 anti-inflammatory circuits predominated. Mice bearing a nonfunctional MC1 displayed a transient exacerbation of neutrophil recruitment after global I/R, which diminished by 2 hours. However importantly, enhanced inflammatory responses in both MC1 mutant and MC3 (-/-) mice resulted in increased infarct size and poor functional outcome after focal I/R. Furthermore, we used an in vitro model of leukocyte recruitment to demonstrate these anti-inflammatory actions are also effective in human cells. CONCLUSIONS: These studies reveal for the first time MC control for neutrophil recruitment in the unique pathophysiological context of cerebral I/R, while also demonstrating the potential therapeutic value of targeting multiple MCs in developing effective therapeutics.

Endogenous galectin-1 exerts tonic inhibition on experimental arthritis.

Little is known about the role(s) of endogenous galectin-1 (Gal-1) in arthritis. In this study we queried whether antiarthritic functions for this effector of endogenous anti-inflammation could be unveiled by studying collagen-induced arthritis in Gal-1(-/-) mice. Gal-1(-/-) and C57BL/6J [wild-type (WT)] mice received an immunization of chicken type II collagen (CII) in CFA followed by a booster on day 21, which consisted of CII in IFA. Animals were monitored for signs of arthritis from day 14 onward. Clinical and histological signs of arthritis were recorded, and humoral and cellular immune responses against CII were analyzed. A distinct disease penetrance was apparent, with ~ 70% of Gal-1(-/-) mice developing arthritis compared with ~ 50% in WT animals. Gal-1(-/-) mice also exhibited an accelerated disease onset and more severe arthritis characterized by significantly elevated clinical scores. Postmortem analyses (day 42) revealed higher levels of IgG1 and IgG2b anti-CII Ig isotypes in the serum of Gal-1 null animals compared with WT. Finally, T cell responses following ex vivo stimulation with CII revealed a greater degree of proliferation in T cells of Gal-1(-/-) mice compared with WT, which was associated with increased production of IL-17 and IL-22. These data suggest the novel idea that endogenous Gal-1 is an inhibitory factor in the development of arthritis affecting disease severity. We have also highlighted the importance of endogenous Gal-1 in regulating T cell reactivity during experimental arthritis.

Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis.

A growing body of evidence indicates that resolution of acute inflammation is an active process. Resolvins are a new family of lipid mediators enzymatically generated within resolution networks that possess unique and specific functions to orchestrate catabasis, the phase in which disease declines. Resolvin D2 (RvD2) was originally identified in resolving exudates, yet its individual contribution in resolution remained to be elucidated. Here, we establish RvD2's potent stereoselective actions in reducing excessive neutrophil trafficking to inflammatory loci. RvD2 decreased leukocyte-endothelial interactions in vivo by endothelial-dependent nitric oxide production, and by direct modulation of leukocyte adhesion receptor expression. In mice with microbial sepsis initiated by caecal ligation and puncture, RvD2 sharply decreased both local and systemic bacterial burden, excessive cytokine production and neutrophil recruitment, while increasing peritoneal mononuclear cells and macrophage phagocytosis. These multi-level pro-resolving actions of RvD2 translate to increased survival from sepsis induced by caecal ligation and puncture and surgery. Together, these results identify RvD2 as a potent endogenous regulator of excessive inflammatory responses that acts via multiple cellular targets to stimulate resolution and preserve immune vigilance.

Glycobiology of leukocyte trafficking in inflammation.

To fulfill their potential, leukocytes must be able to exit the vasculature and reach the site of inflammation within the tissue. This process of leukocyte extravasation is a tightly regulated sequence of events that is governed by a host of cell adhesion molecules, cytokines, chemokines and lipid mediators. Of major importance to this process and the function of many of the proteins and lipids involved is the posttranslational modification of these moieties by glycosylation. The glycosylation process is coordinated by multiple enzymes that add and remove saccharides to/from glycan structures on proteins and lipids, resulting in a unique molecular signature that affords specificity to the molecules involved in leukocyte recruitment. This review will discuss how glycosylation impacts the function of these key molecules involved in the recruitment of leukocytes during inflammation and the function of specific lectins (carbohydrate-binding proteins) that have a role in leukocyte trafficking.

The peritoneal tumour microenvironment of high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSC) disseminates early and extensively throughout the peritoneal space, causing multiple lesions that are a major clinical problem. The aim of this study was to investigate the cellular composition of peritoneal tumour deposits in patient biopsies and their evolution in mouse models using immunohistochemistry, intravital microscopy, confocal microscopy, and 3D modelling. Tumour deposits from the omentum of HGSC patients contained a prominent leukocyte infiltrate of CD3(+) T cells and CD68(+) macrophages, with occasional neutrophils. Alpha-smooth muscle actin(+) (α-SMA(+) ) pericytes and/or fibroblasts surrounded these well-vascularized tumour deposits. Using the murine bowel mesentery as an accessible mouse peritoneal tissue that could be easily imaged, and two different transplantable models, we found multiple microscopic tumour deposits after i.p. injection of malignant cells. Attachment to the peritoneal surface was rapid (6-48 h) with an extensive CD45(+) leukocyte infiltrate visible by 48 h. This infiltrate persisted until end point and in the syngeneic murine ID8 model, it primarily consisted of CD3(+) T lymphocytes and CD68(+) macrophages with α-SMA(+) cells also involved from the earliest stages. A majority of tumour deposits developed above existing mesenteric blood vessels, but in avascular spaces new blood vessels tracked towards the tumour deposits by 2-3 weeks in the IGROV-1 xenografts and 6 weeks in the ID8 syngeneic model; a vigorous convoluted blood supply was established by end point. Inhibition of tumour cell cytokine production by stable expression of shRNA to CXCR4 in IGROV-1 cells did not influence the attachment of cells to the mesentery but delayed neovascularization and reduced tumour deposit size. We conclude that the multiple peritoneal tumour deposits found in HGSC patients can be modelled in the mouse. The techniques described here may be useful for assessing treatments that target the disseminated stage of this disease.

A possible role for G-quadruplexes formation and DNA methylation at IMOOD gene promoter in Obsessive Compulsive Disorder.

Obsessive Compulsive Disorder (OCD) is a mental health condition still classified and diagnosed with subjective interview-based assessments and which molecular clues have not completely been elucidated. We have recently identified a new regulator of anxiety and OCD-like behavior called Immuno-moodulin (IMOOD) and, here, we report that IMOOD gene promoter is differentially methylated in OCD subjects when compared to genomic material collected from healthy controls and this alteration is significantly correlated with the increased expression of the gene in OCD. We also demonstrated that IMOOD promoter can form G-quadruplexes and we suggest that, in homeostatic conditions, these structures could evoke DNA-methylation silencing the gene, whereas in pathological conditions, like OCD, could induce gene expression making the promoter more accessible to transcriptional factors. We here thus further suggest IMOOD as a new biomarker for OCD and also hypothesize new mechanisms of gene regulation.

The glucose transporter 2 regulates CD8+ T cell function via environment sensing.

T cell activation is associated with a profound and rapid metabolic response to meet increased energy demands for cell division, differentiation and development of effector function. Glucose uptake and engagement of the glycolytic pathway are major checkpoints for this event. Here we show that the low-affinity, concentration-dependent glucose transporter 2 (Glut2) regulates the development of CD8+ T cell effector responses in mice by promoting glucose uptake, glycolysis and glucose storage. Expression of Glut2 is modulated by environmental factors including glucose and oxygen availability and extracellular acidification. Glut2 is highly expressed by circulating, recently primed T cells, allowing efficient glucose uptake and storage. In glucose-deprived inflammatory environments, Glut2 becomes downregulated, thus preventing passive loss of intracellular glucose. Mechanistically, Glut2 expression is regulated by a combination of molecular interactions involving hypoxia-inducible factor-1 alpha, galectin-9 and stomatin. Finally, we show that human T cells also rely on this glucose transporter, thus providing a potential target for therapeutic immunomodulation.

Endogenous galectin-1 and acute inflammation: emerging notion of a galectin-9 pro-resolving effect.

The role of endogenous galectin-1 (Gal-1) in acute inflammation has been poorly investigated. We therefore performed the carrageenan-induced paw edema model in wild-type and Gal-1(-/-) mice. On subplantar injection of carrageenan, Gal-1(-/-) mice displayed a similar first phase of edema (≤24 hours) to wild-type mice; however, a much less pronounced second phase (48 to 96 hours) was evident in this genotype. This reduced inflammation was associated with lower paw expression of inflammatory genes and cell infiltrates. Analysis of galectin protein and mRNA expression revealed high expression of Gal-1 in wild-type paws during resolution (≥48 hours), with some expression of galectin-9 (Gal-9). Administration of stable Gal-1 to wild-type mice completely ablated the first phase of edema but was ineffective when administered therapeutically at the 24-hour time point. Conversely, Gal-9 administration did not alter the first phase of edema but significantly reduced the second phase when administered therapeutically. This suggests anti-inflammatory actions for both proteins in this model albeit at different phases of the inflammatory response. Collectively, these data indicate that the absence of endogenous Gal-1 results in an abrogated response during the second phase of the edema reaction.

Design and characterization of a cleavage-resistant Annexin A1 mutant to control inflammation in the microvasculature.

Human polymorphonuclear leukocytes adhesion to endothelial cells during the early stage of inflammation leads to cell surface externalization of Annexin A1 (AnxA1), an effector of endogenous anti-inflammation. The antiadhesive properties of AnxA1 become operative to finely tune polymorphonuclear leukocytes transmigration to the site of inflammation. Membrane bound proteinase 3 (PR3) plays a key role in this microenvironment by cleaving the N terminus bioactive domain of AnxA1. In the present study, we generated a PR3-resistant human recombinant AnxA1-named superAnxA1 (SAnxA1)-and tested its in vitro and in vivo properties in comparison to the parental protein. SAnxA1 bound and activated formyl peptide receptor 2 in a similar way as the parental protein, while showing a resistance to cleavage by recombinant PR3. SAnxA1 retained anti-inflammatory activities in the murine inflamed microcirculation (leukocyte adhesion being the readout) and in skin trafficking model. When longer-lasting models of inflammation were applied, SAnxA1 displayed stronger anti-inflammatory effect over time compared with the parental protein. Together these results indicate that AnxA1 cleavage is an important process during neutrophilic inflammation and that controlling the balance between AnxA1/PR3 activities might represent a promising avenue for the discovery of novel therapeutic approaches.

Activation of the annexin A1 pathway underlies the protective effects exerted by estrogen in polymorphonuclear leukocytes.

OBJECTIVE: The anti-inflammatory properties of the female sex hormone estrogen have been linked to a reduced incidence of cardiovascular disease. In the present study, we addressed whether estrogen could activate vasculoprotective mechanisms via annexin A1 (AnxA1) mobilization in human polymorphonuclear cells (PMNs). METHODS AND RESULTS: Using whole-blood flow cytometry, we demonstrated that premenopausal women expressed higher levels of surface AnxA1 on circulating PMNs compared with males. This correlated with high plasma estrogen during the menstrual cycle. The addition of estrogen in vitro to male PMNs induced rapid mobilization of AnxA1, optimal at 5 ng/mL and a 30-minute incubation period; this effect was abolished in the presence of the estrogen receptor antagonist ICI182780. Estrogen addition to human PMNs induced a distinct AnxA1(hi) CD62L(lo) CD11b(lo) phenotype, and this was associated with lower cell activation as measured by microparticle formation. Treatment of human PMNs with E(2) inhibited cell adhesion to an endothelial cell monolayer under shear, which was absent when endogenous AnxA1 was neutralized. Of interest, addition of estrogen to PMNs flowed over the endothelial monolayer amplified its upregulation of AnxA1 localization on the cell surface. Finally, in a model of intravital microscopy, estrogen inhibition of white blood cell adhesion to the postcapillary venule was absent in mice nullified for AnxA1. CONCLUSION: We unveil a novel AnxA1-dependent mechanism behind the inhibitory properties of estrogen on PMN activation, describing a novel phenotype with a conceivable impact on the vasculoprotective effects of this hormone.