The Lymphatic System: Integral Roles in Immunity.

The lymphatic vasculature is not considered a formal part of the immune system, but it is critical to immunity. One of its major roles is in the coordination of the trafficking of antigen and immune cells. However, other roles in immunity are emerging. Lymphatic endothelial cells, for example, directly present antigen or express factors that greatly influence the local environment. We cover these topics herein and discuss how other properties of the lymphatic vasculature, such as mechanisms of lymphatic contraction (which immunologists traditionally do not take into account), are nonetheless integral in the immune system. Much is yet unknown, and this nascent subject is ripe for exploration. We argue that to consider the impact of lymphatic biology in any given immunological interaction is a key step toward integrating immunology with organ physiology and ultimately many complex pathologies.

Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages.

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Non-classical monocytes scavenge the growth factor CSF1 from endothelial cells in the peripheral vascular tree to ensure survival and homeostasis.

Unlike sessile macrophages that occupy specialized tissue niches, non-classical monocytes (NCMs)-circulating phagocytes that patrol and cleanse the luminal surface of the vascular tree-are characterized by constant movement. Here, we examined the nature of the NCM's nurturing niche. Expression of the growth factor CSF1 on endothelial cells was required for survival of NCMs in the bloodstream. Lack of endothelial-derived CSF1 did not affect blood CSF1 concentration, suggesting that NCMs rely on scavenging CSF1 present on endothelial cells. Deletion of the transmembrane chemokine and adhesion factor CX3CL1 on endothelial cells impaired NCM survival. Mechanistically, endothelial-derived CX3CL1 and integrin subunit alpha L (ITGAL) facilitated the uptake of CSF1 by NCMs. CSF1 was produced by all tissular endothelial cells, and deletion of Csf1 in all endothelial cells except bone marrow sinusoids impaired NCM survival, arguing for a model where the full vascular tree acts as a niche for NCMs and where survival and patrolling function are connected.

Evidence for Pro-angiogenic Functions of VEGF-Ax.

The VEGF-A isoforms play a crucial role in vascular development, and the VEGF signaling pathway is a clinically validated therapeutic target for several pathological conditions. Alternative mRNA splicing leads to the generation of multiple VEGF-A isoforms, including VEGF165. A recent study reported the presence of another isoform, VEGF-Ax, arising from programmed readthrough translation. Compared to VEGF165, VEGF-Ax has a 22-amino-acid extension in the COOH terminus and has been reported to function as a negative regulator of VEGF signaling in endothelial cells, with potent anti-angiogenic effects. Here, we show that, contrary to the earlier report, VEGF-Ax stimulates endothelial cell mitogenesis, angiogenesis, as well as vascular permeability. Accordingly, VEGF-Ax induces phosphorylation of key tyrosine residues in VEGFR-2. Notably, VEGF-Ax was less potent than VEGF165, consistent with its impaired binding to the VEGF co-receptor neuropilin-1.

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.

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.

Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Hemodynamic Control of Endothelial Cell Fates in Development.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. From the onset of blood flow, the embryonic vasculature is continuously exposed to a variety of hemodynamic forces. These biomechanical stimuli are key determinants of vascular cell specification and remodeling and the establishment of vascular homeostasis. In recent years, major advances have been made in our understanding of mechano-activated signaling networks that control both spatiotemporal and structural aspects of vascular development. It has become apparent that a major site for mechanotransduction is situated at the interface of blood and the vessel wall and that this process is controlled by the vascular endothelium. In this review, we discuss the hemodynamic control of endothelial cell fates, focusing on arterial-venous specification, lymphatic development, and the endothelial-to-hematopoietic transition, and present some recent insights into the mechano-activated pathways driving these cell fate decisions in the developing embryo.

Endothelial cells give a boost to senescence surveillance.

Senescence is a specialized form of cell cycle arrest induced in response to damage and stress. In certain settings, senescent cells can promote their own removal by recruitment of the immune system, a process that is thought to decline in efficiency with age. In this issue of Genes & Development, Yin et al. (pp. 533-549) discover a surprising cross-talk where senescent cells instruct endothelial cells to help organize the clearance of the senescent population. This uncovers yet another layer of complexity in senescent cell biology, with implications for cancer treatment and aging.

Senescence-induced endothelial phenotypes underpin immune-mediated senescence surveillance.

Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.

Opening the window for endothelial-to-hematopoietic transition.

Definitive long-term hematopoietic stem cells (LT-HSCs) arise during embryogenesis in a process termed endothelial-to-hematopoietic transition (EHT), in which specialized hemogenic endothelial cells (HECs) transform into hematopoietic cells. The transcription factor RUNX1 marks HECs and is essential for EHT. Ectopic RUNX1 expression in non-HECs is sufficient to convert them into HECs. However, the conversion efficiency depends on the developmental timing of expression. In this issue of Genes & Development, Howell and colleagues (pp. 1475-1489) leverage this observation to further understand how RUNX1 mediates EHT. They engineered mice that ectopically express RUNX1 in endothelial cells at different developmental time points and doses. They then performed chromatin accessibility and other analyses and correlate this with hemogenic potential. They found that RUNX1 collaborates with TGFß signaling transcription factors to drive chromatin accessibility changes that specify HECs. They also highlight interesting parallels between EHT and endothelial-to-mesenchymal transition (EndoMT), which occurs during cardiac development. The results of Howell and colleagues provide new mechanistic insights into EHT and take us one step closer to generating patient-specific LT-HSCs from induced pluripotent stem cells.

Efficient hemogenic endothelial cell specification by RUNX1 is dependent on baseline chromatin accessibility of RUNX1-regulated TGFß target genes.

Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo from hemogenic endothelial cells (HECs) via an endothelial-to-hematopoietic transition (EHT) that requires the transcription factor RUNX1. Ectopic expression of RUNX1 alone can efficiently promote EHT and HSPC formation from embryonic endothelial cells (ECs), but less efficiently from fetal or adult ECs. Efficiency correlated with baseline accessibility of TGFß-related genes associated with endothelial-to-mesenchymal transition (EndoMT) and participation of AP-1 and SMAD2/3 to initiate further chromatin remodeling along with RUNX1 at these sites. Activation of TGFß signaling improved the efficiency with which RUNX1 specified fetal ECs as HECs. Thus, the ability of RUNX1 to promote EHT depends on its ability to recruit the TGFß signaling effectors AP-1 and SMAD2/3, which in turn is determined by the changing chromatin landscape in embryonic versus fetal ECs. This work provides insight into regulation of EndoMT and EHT that will guide reprogramming efforts for clinical applications.

Inflammation and thrombosis in COVID-19 pathophysiology: proteinase-activated and purinergic receptors as drivers and candidate therapeutic targets.

Evolving information has identified disease mechanisms and dysregulation of host biology that might be targeted therapeutically in coronavirus disease 2019 (COVID-19). Thrombosis and coagulopathy, associated with pulmonary injury and inflammation, are emerging clinical features of COVID-19. We present a framework for mechanisms of thrombosis in COVID-19 that initially derive from interaction of SARS-CoV-2 with ACE2, resulting in dysregulation of angiotensin signaling and subsequent inflammation and tissue injury. These responses result in increased signaling by thrombin (proteinase-activated) and purinergic receptors, which promote platelet activation and exert pathological effects on other cell types (e.g., endothelial cells, epithelial cells, and fibroblasts), further enhancing inflammation and injury. Inhibitors of thrombin and purinergic receptors may, thus, have therapeutic effects by blunting platelet-mediated thromboinflammation and dysfunction in other cell types. Such inhibitors include agents (e.g., anti-platelet drugs) approved for other indications, and that could be repurposed to treat, and potentially improve the outcome of, COVID-19 patients. COVID-19, caused by the SARS-CoV-2 virus, drives dysregulation of angiotensin signaling, which, in turn, increases thrombin-mediated and purinergic-mediated activation of platelets and increase in inflammation. This thromboinflammation impacts the lungs and can also have systemic effects. Inhibitors of receptors that drive platelet activation or inhibitors of the coagulation cascade provide opportunities to treat COVID-19 thromboinflammation.

Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions.

Migrating cells preferentially breach and integrate epithelial and endothelial monolayers at multicellular vertices. These sites are amenable to forces produced by the migrating cell and subsequent opening of the junctions. However, the cues that guide migrating cells to these entry portals, and eventually drive the transmigration process, are poorly understood. Here, we show that lymphatic endothelium multicellular junctions are the preferred sites of dendritic cell transmigration in both primary cell co-cultures and in mouse dermal explants. Dendritic cell guidance to multicellular junctions was dependent on the dendritic cell receptor CCR7, whose ligand, lymphatic endothelial chemokine CCL21, was exocytosed at multicellular junctions. Characterization of lymphatic endothelial secretory routes indicated Golgi-derived RAB6+ vesicles and RAB3+/27+ dense core secretory granules as intracellular CCL21 storage vesicles. Of these, RAB6+ vesicles trafficked CCL21 to the multicellular junctions, which were enriched with RAB6 docking factor ELKS (ERC1). Importantly, inhibition of RAB6 vesicle exocytosis attenuated dendritic cell transmigration. These data exemplify how spatially-restricted exocytosis of guidance cues helps to determine where dendritic cells transmigrate.

Transcellular Barriers to Glucose Delivery in the Body.

Glucose is the universal fuel of most mammalian cells, and it is largely replenished through dietary intake. Glucose availability to tissues is paramount for the maintenance of homeostatic energetics and, hence, supply should match demand by the consuming organs. In its journey through the body, glucose encounters cellular barriers for transit at the levels of the absorbing intestinal epithelial wall, the renal epithelium mediating glucose reabsorption, and the tight capillary endothelia (especially in the brain). Glucose transiting through these cellular barriers must escape degradation to ensure optimal glucose delivery to the bloodstream or tissues. The liver, which stores glycogen and generates glucose de novo, must similarly be able to release it intact to the circulation. We present the most up-to-date knowledge on glucose handling by the gut, liver, brain endothelium, and kidney, and discuss underlying molecular mechanisms and open questions. Diseases associated with defects in glucose delivery and homeostasis are also briefly addressed. We propose that the universal problem of sparing glucose from catabolism in favor of translocation across the barriers posed by epithelia and endothelia is resolved through common mechanisms involving glucose transfer to the endoplasmic reticulum, from where glucose exits the cells via unconventional cellular mechanisms.

A Razor's Edge: Vascular Responses to Acute Inflammatory Lung Injury/Acute Respiratory Distress Syndrome.

Historically considered a metabolically inert cellular layer separating the blood from the underlying tissue, the endothelium is now recognized as a highly dynamic, metabolically active tissue that is critical to organ homeostasis. Under homeostatic conditions, lung endothelial cells (ECs) in healthy subjects are quiescent, promoting vasodilation, platelet disaggregation, and anti-inflammatory mechanisms. In contrast, lung ECs are essential contributors to the pathobiology of acute respiratory distress syndrome (ARDS), as the quiescent endothelium is rapidly and radically altered upon exposure to environmental stressors, infectious pathogens, or endogenous danger signals into an effective and formidable regulator of innate and adaptive immunity. These dramatic perturbations, produced in a tsunami of inflammatory cascade activation, result in paracellular gap formation between lung ECs, sustained lung edema, and multi-organ dysfunction that drives ARDS mortality. The astonishing plasticity of the lung endothelium in negotiating this inflammatory environment and efforts to therapeutically target the aberrant ARDS endothelium are examined in further detail in this review.

Transcriptional regulatory network controlling the ontogeny of hematopoietic stem cells.

Hematopoietic stem cell (HSC) ontogeny is accompanied by dynamic changes in gene regulatory networks. We performed RNA-seq and histone mark ChIP-seq to define the transcriptomes and epigenomes of cells representing key developmental stages of HSC ontogeny in mice. The five populations analyzed were embryonic day 10.5 (E10.5) endothelium and hemogenic endothelium from the major arteries, an enriched population of prehematopoietic stem cells (pre-HSCs), fetal liver HSCs, and adult bone marrow HSCs. Using epigenetic signatures, we identified enhancers for each developmental stage. Only 12% of enhancers are primed, and 78% are active, suggesting the vast majority of enhancers are established de novo without prior priming in earlier stages. We constructed developmental stage-specific transcriptional regulatory networks by linking enhancers and predicted bound transcription factors to their target promoters using a novel computational algorithm, target inference via physical connection (TIPC). TIPC predicted known transcriptional regulators for the endothelial-to-hematopoietic transition, validating our overall approach, and identified putative novel transcription factors, including the broadly expressed transcription factors SP3 and MAZ. Finally, we validated a role for SP3 and MAZ in the formation of hemogenic endothelium. Our data and computational analyses provide a useful resource for uncovering regulators of HSC formation.

A ß-catenin chromobody-based probe highlights endothelial maturation during vascular morphogenesis in vivo.

Visualization of protein dynamics is a crucial step in understanding cellular processes. Chromobodies, fluorescently labelled single-domain antibodies, have emerged as versatile probes for live cell imaging of endogenous proteins. However, how these chromobodies behave in vivo and how accurately they monitor tissue changes remain poorly explored. Here, we generated an endothelial-specific ß-catenin chromobody-derived probe and analyzed its expression pattern during cardiovascular development in zebrafish. Using high-resolution confocal imaging, we show that the chromobody signal correlates with the localization of ß-catenin in the nucleus and at cell-cell junctions, and thereby can be used to assess endothelial maturation. Loss of Cadherin 5 strongly affects the localization of the chromobody at the cell membrane, confirming the cadherin-based adherens junction role of ß-catenin. Furthermore, using a genetic model to block blood flow, we observed that cell junctions are compromised in most endothelial cells but not in the endocardium, highlighting the heterogeneous response of the endothelium to the lack of blood flow. Overall, our data further expand the use of chromobodies for in vivo applications and illustrate their potential to monitor tissue morphogenesis at high resolution.

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.