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.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Leukocyte Trafficking: Time to Take Time Seriously.

Leukocyte trafficking is a key component of steady-state tissue homing and in mounting an inflammatory response. Two recent publications in Immunity by He et al. (2018) and Adrover et al. (2019) report on the diurnal regulation of these responses and the associated pathophysiological implications.

Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis.

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

Exacerbated atherosclerosis in progeria is prevented by progerin elimination in vascular smooth muscle cells but not endothelial cells.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here, we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice.

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.

Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo.

Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic.

The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo.

The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.

Leukocyte migration into inflamed tissues.

Leukocyte migration through activated venular walls is a fundamental immune response that is prerequisite to the entry of effector cells such as neutrophils, monocytes, and effector T cells to sites of infection, injury, and stress within the interstitium. Stimulation of leukocytes is instrumental in this process with enhanced temporally controlled leukocyte adhesiveness and shape-changes promoting leukocyte attachment to the inner wall of blood vessels under hydrodynamic forces. This initiates polarized motility of leukocytes within and through venular walls and transient barrier disruption facilitated sequentially by stimulated vascular cells, i.e., endothelial cells and their associated pericytes. Perivascular cells such as macrophages and mast cells that act as tissue inflammatory sentinels can also directly and indirectly regulate the exit of leukocytes from the vascular lumen. In this review, we discuss current knowledge and open questions regarding the mechanisms involved in the interactions of different effector leukocytes with peripheral vessels in extralymphoid organs.

Cardiovascular Progerin Suppression and Lamin A Restoration Rescue Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by premature aging and death mainly because of myocardial infarction, stroke, or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Patients look healthy at birth, and symptoms typically emerge in the first or second year of life. Assessing the reversibility of progerin-induced damage and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. METHODS: We used CRISPR-Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice engineered to ubiquitously express progerin while lacking lamin A and allowing progerin suppression and lamin A restoration in a time- and cell type-specific manner on Cre recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells and cardiomyocytes. RESULTS: Like patients with HGPS, HGPSrev mice appear healthy at birth and progressively develop HGPS symptoms, including failure to thrive, lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26 months in wild-type controls, P<0.0001). Ubiquitous progerin suppression and lamin A restoration significantly extended lifespan when induced in 6-month-old mildly symptomatic mice and even in severely ill animals aged 13 months, although the benefit was much more pronounced on early intervention (84.5% lifespan extension in mildly symptomatic mice, P<0.0001, and 6.7% in severely ill mice, P<0.01). It is remarkable that major vascular alterations were prevented and lifespan normalized in HGPSrev mice when progerin suppression and lamin A restoration were restricted to vascular smooth muscle cells and cardiomyocytes. CONCLUSIONS: HGPSrev mice constitute a new experimental model for advancing knowledge of HGPS. Our findings suggest that it is never too late to treat HGPS, although benefit is much more pronounced when progerin is targeted in mice with mild symptoms. Despite the broad expression pattern of progerin and its deleterious effects in many organs, restricting its suppression to vascular smooth muscle cells and cardiomyocytes is sufficient to prevent vascular disease and normalize lifespan.

Dynamic trafficking and turnover of JAM-C is essential for endothelial cell migration.

Junctional complexes between endothelial cells form a dynamic barrier that hinders passive diffusion of blood constituents into interstitial tissues. Remodelling of junctions is an essential process during leukocyte trafficking, vascular permeability, and angiogenesis. However, for many junctional proteins, the mechanisms of junctional remodelling have yet to be determined. Here, we used receptor mutagenesis, horseradish peroxidase (HRP), and ascorbate peroxidase 2 (APEX-2) proximity labelling, alongside light and electron microscopy (EM), to map the intracellular trafficking routes of junctional adhesion molecule-C (JAM-C). We found that JAM-C cotraffics with receptors associated with changes in permeability such as vascular endothelial cadherin (VE-Cadherin) and neuropilin (NRP)-1 and 2, but not with junctional proteins associated with the transmigration of leukocytes. Dynamic JAM-C trafficking and degradation are necessary for junctional remodelling during cell migration and angiogenesis. By identifying new potential trafficking machinery, we show that a key point of regulation is the ubiquitylation of JAM-C by the E3 ligase Casitas B-lineage lymphoma (CBL), which controls the rate of trafficking versus lysosomal degradation.

Breaching multiple barriers: leukocyte motility through venular walls and the interstitium.

The shuttling of leukocytes between the bloodstream and interstitial tissues involves different locomotion strategies that are governed by locally presented soluble and cell-bound signals. Recent studies have furthered our understanding of the rapidly advancing field of leukocyte migration, particularly regarding cellular and subcellular events at the level of the venular wall. Furthermore, emerging cellular models are now addressing the transition from an adherent mode to a non-adherent state, incorporating mechanisms that support an efficient migratory profile of leukocytes in the interstitial tissue beyond the venular wall.

Circulating BMP9 Protects the Pulmonary Endothelium during Inflammation-induced Lung Injury in Mice.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

Neutrophil trafficking to lymphoid tissues: physiological and pathological implications.

Recent advances have provided evidence for the involvement of neutrophils in both innate and adaptive immunity, robustly challenging the old dogma that neutrophils are short-lived prototypical innate immune cells solely involved in acute responses to microbes and exerting collateral tissue damage. There is now ample evidence showing that neutrophils can migrate into different compartments of the lymphoid system where they contribute to the orchestration of the activation and/or suppression of lymphocyte effector functions in homeostasis and during chronic inflammation, such as autoimmune disorders and cancer. In support of this notion, neutrophils can generate a wide range of cytokines and other mediators capable of regulating the survival, proliferation and functions of both T and B cells. In addition, neutrophils can directly engage with lymphocytes and promote antigen presentation. Furthermore, there is emerging evidence of the existence of distinct and diverse neutrophil phenotypes with immunomodulatory functions that characterise different pathological conditions, including chronic and autoimmune inflammatory conditions. The aim of this review is to discuss the mechanisms implicated in neutrophil trafficking into the lymphoid system and to provide an overview of the immuno-regulatory functions of neutrophils in health and disease in the context of adaptive immunity. Copyright © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Neutrophil elastase plays a non-redundant role in remodeling the venular basement membrane and neutrophil diapedesis post-ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury is a severe inflammatory insult associated with numerous pathologies, such as myocardial infarction, stroke and acute kidney injury. I/R injury is characterized by a rapid influx of activated neutrophils secreting toxic free radical species and degrading enzymes that can irreversibly damage the tissue, thus impairing organ functions. Significant efforts have been invested in identifying therapeutic targets to suppress neutrophil recruitment and activation post-I/R injury. In this context, pharmacological targeting of neutrophil elastase (NE) has shown promising anti-inflammatory efficacy in a number of experimental and clinical settings of I/R injury and is considered a plausible clinical strategy for organ care. However, the mechanisms of action of NE, and hence its inhibitors, in this process are not fully understood. Here we conducted a comprehensive analysis of the impact of NE genetic deletion on neutrophil infiltration in four murine models of I/R injury as induced in the heart, kidneys, intestine and cremaster muscle. In all models, neutrophil migration into ischemic regions was significantly suppressed in NE-/- mice as compared with wild-type controls. Analysis of inflamed cremaster muscle and mesenteric microvessels by intravital and confocal microscopy revealed a selective entrapment of neutrophils within venular walls, most notably at the level of the venular basement membrane (BM) following NE deletion/pharmacological blockade. This effect was associated with the suppression of NE-mediated remodeling of the low matrix protein expressing regions within the venular BM used by transmigrating neutrophils as exit portals. Furthermore, whilst NE deficiency led to reduced neutrophil activation and vascular leakage, levels of monocytes and prohealing M2 macrophages were reduced in tissues of NE-/- mice subjected to I/R. Collectively our results identify a vital and non-redundant role for NE in supporting neutrophil breaching of the venular BM post-I/R injury but also suggest a protective role for NE in promoting tissue repair. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Reverse Migration of Neutrophils: Where, When, How, and Why?

Neutrophil migration to injured and pathogen-infected tissues is a fundamental component of innate immunity. An array of cellular and molecular events mediate this response to collectively guide neutrophils out of the vasculature and towards the core of the ensuing inflammatory reaction where they exert effector functions. Advances in imaging modalities have revealed that neutrophils can also exhibit motility away from sites of inflammation and injury, although it is unclear under what circumstances this reverse migration is a physiological protective response, and when it has pathophysiological relevance. Here we review different types of neutrophil reverse migration and discuss the current understanding of the associated mechanisms. In this context we propose clarifications to the existing terminology used to describe the many facets of neutrophil reverse migration.

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.

Design of an Activity-Based Probe for Human Neutrophil Elastase: Implementation of the Lossen Rearrangement To Induce Förster Resonance Energy Transfers.

Human neutrophil elastase is an important regulator of the immune response and plays a role in host defense mechanisms and further physiological processes. The uncontrolled activity of this serine protease may cause severe tissue alterations and impair inflammatory states. The design of an activity-based probe for human neutrophil elastase reported herein relies on a sulfonyloxyphthalimide moiety as a new type of warhead that is linker-connected to a coumarin fluorophore. The inhibitory potency of the activity-based probe was assessed against several serine and cysteine proteases, and the selectivity for human neutrophil elastase (Ki = 6.85 nM) was determined. The adequate fluorescent tag of the probe allowed for the in-gel fluorescence detection of human neutrophil elastase in the low nanomolar range. The coumarin moiety and the anthranilic acid function of the probe, produced in the course of a Lossen rearrangement, were part of two different Förster resonance energy transfers.

Correction: Signatures of inflammation and impending multiple organ dysfunction in the hyperacute phase of trauma: A prospective cohort study.

[This corrects the article DOI: 10.1371/journal.pmed.1002352.].