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.

Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia.

Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.

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.

Targeting Extracellular Vesicles to the Arthritic Joint Using a Damaged Cartilage-Specific Antibody.

The targeted delivery of therapies to diseased tissues offers a safe opportunity to achieve optimal efficacy while limiting systemic exposure. These considerations apply to many disease indications but are especially relevant for rheumatoid arthritis (RA), as RA is a systemic autoimmune disease which affects multiple joints. We have identified an antibody that is specific to damaged arthritic cartilage (anti-ROS-CII) that can be used to deliver treatments specifically to arthritic joints, yielding augmented efficacy in experimental arthritis. In the current study, we demonstrate that scaffolds enriched with bioactive payloads can be delivered precisely to an inflamed joint and achieve superior efficacy outcomes consistent with the pharmacological properties of these payloads. As a scaffold, we have used extracellular vesicles (EVs) prepared from human neutrophils (PMNs), which possess intrinsic anti-inflammatory properties and the ability to penetrate inflamed arthritic cartilage. EV fortified with anti-ROS-CII (EV/anti-ROS-CII) retained anti-ROS-CII specificity and bound exclusively to the damaged cartilage. Following systemic administration, EV/anti-ROS-CII (a) exhibited the ability to localize specifically in the arthritic joint in vivo and (b) was able to specifically target single (viral IL-10 or anti-TNF) or combined (viral IL-10 and anti-TNF) anti-inflammatory treatments to the arthritic joint, which accelerated attenuation of clinical and synovial inflammation. Overall, this study demonstrates the attainability of targeting a pro-resolving biological scaffold to the arthritic joint. The potential of targeting scaffolds such as EV, nanoparticles, or a combination thereof alongside combined therapeutics is paramount for designing systemically administered broad-spectrum of anti-inflammatory treatments.

Heparanase-Dependent Remodeling of Initial Lymphatic Glycocalyx Regulates Tissue-Fluid Drainage During Acute Inflammation in vivo.

The glycocalyx is a dense layer of carbohydrate chains involved in numerous and fundamental biological processes, such as cellular and tissue homeostasis, inflammation and disease development. Composed of membrane-bound glycoproteins, sulfated proteoglycans and glycosaminoglycan side-chains, this structure is particularly essential for blood vascular barrier functions and leukocyte diapedesis. Interestingly, whilst the glycocalyx of blood vascular endothelium has been extensively studied, little is known about the composition and function of this glycan layer present on tissue-associated lymphatic vessels (LVs). Here, we applied confocal microscopy to characterize the composition of endothelial glycocalyx of initial lymphatic capillaries in murine cremaster muscles during homeostatic and inflamed conditions using an anti-heparan sulfate (HS) antibody and a panel of lectins recognizing different glycan moieties of the glycocalyx. Our data show the presence of HS, α-D-galactosyl moieties, α2,3-linked sialic acids and, to a lesser extent, N-Acetylglucosamine moieties. A similar expression profile was also observed for LVs of mouse and human skins. Interestingly, inflammation of mouse cremaster tissues or ear skin as induced by TNF-stimulation induced a rapid (within 16 h) remodeling of the LV glycocalyx, as observed by reduced expression of HS and galactosyl moieties, whilst levels of α2,3-linked sialic acids remains unchanged. Furthermore, whilst this response was associated with neutrophil recruitment from the blood circulation and their migration into tissue-associated LVs, specific neutrophil depletion did not impact LV glycocalyx remodeling. Mechanistically, treatment with a non-anticoagulant heparanase inhibitor suppressed LV HS degradation without impacting neutrophil migration into LVs. Interestingly however, inhibition of glycocalyx degradation reduced the capacity of initial LVs to drain interstitial fluid during acute inflammation. Collectively, our data suggest that rapid remodeling of endothelial glycocalyx of tissue-associated LVs supports drainage of fluid and macromolecules but has no role in regulating neutrophil trafficking out of inflamed tissues via initial LVs.

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.

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.

Endogenous TNFα orchestrates the trafficking of neutrophils into and within lymphatic vessels during acute inflammation.

Neutrophils are recognised to play a pivotal role at the interface between innate and acquired immunities following their recruitment to inflamed tissues and lymphoid organs. While neutrophil trafficking through blood vessels has been extensively studied, the molecular mechanisms regulating their migration into the lymphatic system are still poorly understood. Here, we have analysed neutrophil-lymphatic vessel interactions in real time and in vivo using intravital confocal microscopy applied to inflamed cremaster muscles. We show that antigen sensitisation of the tissues induces a rapid but transient entry of tissue-infiltrated neutrophils into lymphatic vessels and subsequent crawling along the luminal side of the lymphatic endothelium. Interestingly, using mice deficient in both TNF receptors p55 and p75, chimeric animals and anti-TNFα antibody blockade we demonstrate that tissue-release of TNFα governs both neutrophil migration through the lymphatic endothelium and luminal crawling. Mechanistically, we show that TNFα primes directly the neutrophils to enter the lymphatic vessels in a strictly CCR7-dependent manner; and induces ICAM-1 up-regulation on lymphatic vessels, allowing neutrophils to crawl along the lumen of the lymphatic endothelium in an ICAM-1/MAC-1-dependent manner. Collectively, our findings demonstrate a new role for TNFα as a key regulator of neutrophil trafficking into and within lymphatic system in vivo.

ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.

Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense.

Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF.

Microvascular plasma protein leakage is an essential component of the inflammatory response and serves an important function in local host defense and tissue repair. Mediators such as histamine and bradykinin act directly on venules to increase the permeability of endothelial cell (EC) junctions. Neutrophil chemoattractants also induce leakage, a response that is dependent on neutrophil adhesion to ECs, but the underlying mechanism has proved elusive. Through application of confocal intravital microscopy to the mouse cremaster muscle, we show that neutrophils responding to chemoattractants release TNF when in close proximity of EC junctions. In vitro, neutrophils adherent to ICAM-1 or ICAM-2 rapidly released TNF in response to LTB4, C5a, and KC. Further, in TNFR(-/-) mice, neutrophils accumulated normally in response to chemoattractants administered to the cremaster muscle or dorsal skin, but neutrophil-dependent plasma protein leakage was abolished. Similar results were obtained in chimeric mice deficient in leukocyte TNF. A locally injected TNF blocking antibody was also able to inhibit neutrophil-dependent plasma leakage, but had no effect on the response induced by bradykinin. The results suggest that TNF mediates neutrophil-dependent microvascular leakage. This mechanism may contribute to the effects of TNF inhibitors in inflammatory diseases and indicates possible applications in life-threatening acute edema.

Towards a KCC2 blocker pharmacophore model.

A multi-disciplinary approach was used to identify the first pharmacophore model for KCC2 blockers: several physico-chemical studies such as XRD and NMR were combined to molecular modelling techniques, SAR analysis and synthesis of constrained analogues in order to determine a minimal conformational space regrouping few potential bioactive conformations. These conformations were further compared to the conformational space of a different series of KCC2 blockers in order to identify the common pharmacophoric features. The synthesis of more potent analogues in this second series confirmed the usefulness of this KCC2 blocker pharmacophore model.

Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo.

Neutrophil transmigration through venular walls that are composed of endothelial cells (ECs), pericytes, and the venular basement membrane is a key component of innate immunity. Through direct analysis of leukocyte-pericyte interactions in inflamed tissues using confocal intravital microscopy, we show how pericytes facilitate transmigration in vivo. After EC migration, neutrophils crawl along pericyte processes to gaps between adjacent pericytes in an ICAM-1-, Mac-1-, and LFA-1-dependent manner. These gaps were enlarged in inflamed tissues through pericyte shape change and were used as exit points by neutrophils in breaching the venular wall. The findings identify previously unknown roles for pericytes in neutrophil transmigration in vivo and add additional steps to the leukocyte adhesion cascade that supports leukocyte trafficking into sites of inflammation.

Venular basement membranes ubiquitously express matrix protein low-expression regions: characterization in multiple tissues and remodeling during inflammation.

The venular basement membrane plays a critical role in maintaining the integrity of blood vessels and through its dense and highly organized network of matrix proteins also acts as a formidable barrier to macromolecules and emigrating leukocytes. Leukocytes can however penetrate the venular basement membrane at sites of inflammation, though the associated in vivo mechanisms are poorly understood. Using whole mount immunostained tissues and confocal microscopy, we demonstrate that the venular basement membrane of multiple organs expresses regions of low matrix protein (laminin-511 and type IV collagen) deposition that have been termed low-expression regions (LERs). In the multiple tissues analyzed (eg, cremaster muscle, skin, mesenteric tissue), LERs were directly aligned with gaps between adjacent pericytes and were more prevalent in small venules. As predicted by their permissive nature, LERs acted as "gates" for transmigrating neutrophils in all inflammatory reactions investigated (elicited by leukotriene B(4) [LTB(4)], CXCL1, tumor necrosis factor [TNF]alpha, endotoxin, and ischemia/reperfusion [I/R] injury), and this response was associated with an enhancement of the size of laminin-511 and type IV collagen LERs. Transmigrated neutrophils stained positively for laminins but not type IV collagen, suggesting that different mechanisms exist in remodeling of different basement membrane networks. Collectively the findings provide further insight into characteristics of specialized regions within venular basement membranes that are preferentially used and remodeled by transmigrating neutrophils.

Endothelial cell activation leads to neutrophil transmigration as supported by the sequential roles of ICAM-2, JAM-A, and PECAM-1.

Leukocyte transmigration is mediated by endothelial cell (EC) junctional molecules, but the associated mechanisms remain unclear. Here we investigate how intercellular adhesion molecule-2 (ICAM-2), junctional adhesion molecule-A (JAM-A), and platelet endothelial cell adhesion molecule (PECAM-1) mediate neutrophil transmigration in a stimulus-dependent manner (eg, as induced by interleukin-1beta [IL-1beta] but not tumor necrosis factor-alpha [TNF-alpha]), and demonstrate their ability to act in sequence. Using a cell-transfer technique, transmigration responses of wild-type and TNF-alpha p55/p75 receptor-deficient leukocytes (TNFR(-/-)) through mouse cremasteric venules were quantified by fluorescence intravital microscopy. Whereas wild-type leukocytes showed a normal transmigration response to TNF-alpha in ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) recipient mice, TNFR(-/-) leukocytes exhibited a reduced transmigration response. Hence, when the ability of TNF-alpha to directly stimulate neutrophils is blocked, TNF-alpha-induced neutrophil transmigration is rendered dependent on ICAM-2, JAM-A, and PECAM-1, suggesting that the stimulus-dependent role of these molecules is governed by the target cell being activated. Furthermore, analysis of the site of arrest of neutrophils in inflamed tissues from ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) mice demonstrated that these molecules act sequentially to mediate transmigration. Collectively, the findings provide novel insights into the mechanisms of action of key molecules implicated in leukocyte transmigration.

Separate functions for the two modules of the membrane-proximal cytokine binding domain of glycoprotein 190, the leukemia inhibitory factor low affinity receptor, in ligand binding and receptor activation.

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130. Both are members of the hematopoietic receptors family characterized by the cytokine receptor homology (CRH) domain, which consists of two barrel-like modules of around 100 amino acids each. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an immunoglobulin-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin-like repeats. A minimal D1IgD2 fragment is required for binding LIF. By using transmembrane forms of deletion mutants in gp190 ectodomain, we demonstrated that removal of D1 led to spontaneous activation of the receptor and that this property was devoted to a peptidic sequence localized within the last 42 amino acids of the carboxyl-terminal module of D2. By using soluble forms of deletion mutants made by progressive truncations from the end of the D1IgD2 fragment, we demonstrated that the carboxyl-terminal module of D2 was dispensable for LIF binding and that the correct conformation of the D1Ig fragment required a full amino-terminal module of D2. Therefore, the two constitutive modules of the membrane-proximal CRH domain D2 of gp190 fulfill two distinct roles in gp190 function, i.e. in stabilizing the conformation of gp190 allowing LIF binding and in activating the receptor.