Leukocyte Rolling Research Articles

L-Arginine Modulates Neonatal Leukocyte Recruitment in a Gestational Age-Dependent Manner.

(1) Background: L-arginine is a complex modulator of immune functions, and its levels are known to decrease under septic conditions. L-arginine may suppress leukocyte recruitment in vivo; however, little is known about the gestational age-specific effects of L-arginine on leukocyte recruitment in preterm infants. We now asked whether L-arginine alters leukocyte recruitment in preterm and term neonates. (2) Methods: Leukocytes were isolated from preterm (28 + 0 to 32 + 6 weeks of gestation) and term (>37 weeks of gestation) newborns as well as from healthy adults. After incubation with 10 µg/mL L-arginine, we assessed leukocyte rolling and adhesion in dynamic microflow chamber experiments and leukocyte transmigration in fluorescence assays. In addition, we measured the expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) in neutrophils by flow cytometry. (3) Results: Leukocyte rolling, adhesion, and transmigration increased with gestational age. Leukocyte rolling, adhesion, and transmigration were decreased by L-arginine in term-born infants and adults. Preterm leukocytes showed no change in recruitment upon L-arginine exposure. Leukocyte adhesion after L-arginine exposure reached similar levels among all groups. In line, the expression of iNOS and Arg-1 was similar in all three age groups. (4) Conclusion: L-arginine dampens the ex vivo recruitment capacity of leukocytes from term-born infants, whereas no effect was seen in premature infants. As levels of iNOS and Arg-1 in neutrophils remain ontogenetically unchanged, the anti-inflammatory effect of L-arginine on the leukocyte recruitment cascade needs further investigation. These results add to the controversial debate of L-arginine supplementation in premature infants in sepsis.

Mesenchymal stromal cells protect against vascular damage and depression-like behavior in mice surviving cerebral malaria

BackgroundMalaria is one of the most critical global infectious diseases. Severe systemic inflammatory diseases, such as cerebral malaria, lead to the development of cognitive and behavioral alterations, such as learning disabilities and loss of memory capacity, as well as increased anxiety and depression. The consequences are profound and usually contribute to reduce the patient’s quality of life. There are no therapies to treat the neurological sequelae of cerebral malaria. Mesenchymal stromal cells (MSCs) may be an alternative, since they have been used as therapy for neurodegenerative diseases and traumatic lesions of the central nervous system. So far, no study has investigated the effects of MSC therapy on the blood-brain barrier, leukocyte rolling and adherence in the brain, and depression like-behavior in experimental cerebral malaria.MethodsMale C57BL/6 mice were infected with Plasmodium berghei ANKA (PbA, 1 × 106 PbA-parasitized red blood cells, intraperitoneally). At day 6, PbA-infected animals received chloroquine (25 mg/kg orally for seven consecutive days) as the antimalarial treatment and were then randomized to receive MSCs (1 × 105 cells in 0.05 ml of saline/mouse) or saline (0.05 ml) intravenously. Parasitemia, clinical score, and survival rate were analyzed throughout the experiments. Evans blue assay was performed at 6, 7, and 15 days post-infection (dpi). Behavioral tests were performed at 5 and 15 dpi. Intravital microscopy experiments and brain-derived neurotrophic factor (BDNF) protein expression analyses were performed at 7 dpi, whereas inflammatory mediators were measured at 15 dpi. In vitro, endothelial cells were used to evaluate the effects of conditioned media derived from MSCs (CMMSC) on cell viability by lactate dehydrogenase (LDH) release.ResultsPbA-infected mice presented increased parasitemia, adherent leukocytes, blood-brain barrier permeability, and reduced BDNF protein levels, as well as depression-like behavior. MSCs mitigated behavioral alterations, restored BDNF and transforming growth factor (TGF)-β protein levels, and reduced blood-brain barrier dysfunction and leukocyte adhesion in the brain microvasculature. In a cultured endothelial cell line stimulated with heme, CMMSC reduced LDH release, suggesting a paracrine mechanism of action.ConclusionA single dose of MSCs as adjuvant therapy protected against vascular damage and improved depression-like behavior in mice that survived experimental cerebral malaria.

Endothelial BMP4 Promotes Leukocyte Rolling and Adhesion and Is Elevated in Patients After Survived Out-of-Hospital Cardiac Arrest.

Leukocyte recruitment is a fundamental step in the inflammatory response during ischemia/reperfusion injury (IRI). Rolling and adhesion of leukocytes to activated endothelium promote tissue inflammation after IRI and require presentation of adhesion molecules E-selectin and ICAM-1 on the endothelial surface. Bone morphogenetic protein (BMP) 4 is a prominent member of the BMP family expressed and secreted by endothelial cells. BMP4 derived from endothelial cells has important functions in vascular disease but its influence on the leukocyte adhesion cascade during inflammation is incompletely understood. In the present study, we challenged mice with an inducible endothelial-specific BMP4 deletion (referred to as EC-BMP4-/- mice) and their control littermates (EC-BMP4+/+) with thioglycollate i.p. and assessed extravasation of different leukocyte subsets during peritonitis. Peritoneal lavages were performed and peritoneal cells were counted. Total cell count in lavages of EC-BMP4-/- mice was markedly reduced compared with lavages of EC-BMP4+/+ mice. FACS analyses of thioglycollate-elicited peritoneal cells revealed that diverse leukocyte subsets were reduced in EC-BMP4-/- mice. Intravital microscopy of cremaster venules demonstrated that rolling and adhesion of leukocytes were significantly diminished in EC-BMP4-/- mice in comparison with control mice in response to TNFα. These observations indicate that endothelial BMP4 is essential for rolling, adhesion, and extravasation of leukocytes in vivo. To understand the underlying mechanisms, levels of endothelial adhesion molecules E-selectin and ICAM-1 were quantified in EC-BMP4-/- and EC-BMP4+/+ mice by quantitative PCR and Western blotting. Interestingly, ICAM-1 and E-selectin expressions were reduced in the hearts of EC-BMP4-/- mice. Next we confirmed pro-inflammatory properties of BMP4 in a gain of function experiments and found that administration of recombinant BMP4 in male C57BL/6 mice increased leukocyte rolling and adhesion in cremaster venules in vivo. To assess the regulation of BMP4 in inflammatory disease in humans, we collected plasma samples of patients from day 0 to day 7 after survived out-of-hospital cardiac arrest (OHCA, n = 42). Remarkably, plasma of OHCA patients contained significantly higher BMP4 protein levels compared with patients with coronary artery disease (CAD, n = 12) or healthy volunteers (n = 11). Subgroup analysis revealed that elevated plasma BMP4 levels after ROSC are associated with decreased survival and unfavorable neurological outcome. Collectively, endothelial BMP4 is a potent activator of inflammation in vivo that promotes rolling, adhesion, and extravasation of leukocyte subsets by induction of E-selectin and ICAM-1. Elevation of plasma BMP4 levels in the post-resuscitation period suggests that BMP4 contributes to pathophysiology and poor outcome of post-cardiac arrest syndrome.

Myeloperoxidase instigates proinflammatory responses in a cecal ligation and puncture rat model of sepsis.

Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.

Chronic ethanol consumption compromises neutrophil function in acute pulmonary Aspergillus fumigatus infection.

Chronic ethanol consumption is a leading cause of mortality worldwide, with higher risks to develop pulmonary infections, including Aspergillus infections. Mechanisms underlying increased susceptibility to infections are poorly understood. Chronic ethanol consumption induced increased mortality rates, higher Aspergillus fumigatus burden and reduced neutrophil recruitment into the airways. Intravital microscopy showed decrease in leukocyte adhesion and rolling after ethanol consumption. Moreover, downregulated neutrophil activation and increased levels of serum CXCL1 in ethanol-fed mice induced internalization of CXCR2 receptor in circulating neutrophils. Bone marrow-derived neutrophils from ethanol-fed mice showed lower fungal clearance and defective reactive oxygen species production. Taken together, results showed that ethanol affects activation, recruitment, phagocytosis and killing functions of neutrophils, causing susceptibility to pulmonary A. fumigatus infection. This study establishes a new paradigm in innate immune response in chronic ethanol consumers.

Sulforaphane Elicits Protective Effects in Intestinal Ischemia Reperfusion Injury.

Intestinal ischemia reperfusion injury (IRI) is an inherent, unavoidable event of intestinal transplantation, contributing to allograft failure and rejection. The inflammatory state elicited by intestinal IRI is characterized by heightened leukocyte recruitment to the gut, which is amplified by a cross-talk with platelets at the endothelial border. Sulforaphane (SFN), a naturally occurring isothiocyanate, exhibits anti-inflammatory characteristics and has been shown to reduce platelet activation and block leukocyte adhesion. Thus, the aim of this study was to investigate protective effects and mechanism of action of SFN in a murine model of intestinal IRI. Intestinal IRI was induced by superior mesenteric artery occlusion for 30 min, followed by reperfusion for 2 h, 8 h or 24 h. To investigate cellular interactions, leukocytes were in vivo stained with rhodamine and platelets were harvested from donor animals and ex vivo stained. Mice (C57BL/6J) were divided into three groups: (1) control, (2) SFN treatment 24 h prior to reperfusion and (3) SFN treatment 24 h prior to platelet donation. Leukocyte and platelet recruitment was analyzed via intravital microscopy. Tissue was analyzed for morphological alterations in intestinal mucosa, barrier permeability, and leukocyte infiltration. Leukocyte rolling and adhesion was significantly reduced 2 h and 8 h after reperfusion. Mice receiving SFN treated platelets exhibited significantly decreased leukocyte and platelet recruitment. SFN showed protection for intestinal tissue with less damage observed in histopathological and ultrastructural evaluation. In summary, the data presented provide evidence for SFN as a potential therapeutic strategy against intestinal IRI.

Leukocyte Tetraspanin CD53 Restrains α3 Integrin Mobilization and Facilitates Cytoskeletal Remodeling and Transmigration in Mice.

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of β2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.

Mitochondrial Alterations and Enhanced Human Leukocyte/Endothelial Cell Interactions in Type 1 Diabetes

Type 1 diabetes has been associated with oxidative stress. This study evaluates the rates of oxidative stress, mitochondrial function, leukocyte–endothelium interactions and adhesion molecules in type 1 diabetic patients. The study population consisted of 52 diabetic patients and 46 body-composition and age-matched controls. We assessed anthropometric and metabolic parameters, oxidative stress and mitochondrial function by evaluating reactive oxygen species (ROS) production, mitochondrial ROS production, mitochondrial membrane potential and superoxide dismutase (SOD) and catalase (CAT) expression in polymorphonuclear leukocytes from type 1 diabetic patients. In addition, we evaluated interactions between leukocytes and human umbilical vein endothelial cells (HUVEC), and serum expression of adhesion molecules (P-selectin, VCAM-1 and ICAM-1), proinflammatory cytokines (IL-6 and TNFα) and myeloperoxidase (MPO). HbA1C and glucose levels were higher in diabetic patients than in control subjects, as expected. Mitochondrial function was altered and leukocyte–endothelium interactions were enhanced in diabetic patients, which was evident in the increase in total and mitochondrial ROS production, higher mitochondrial membrane potential, enhanced leukocyte rolling and adhesion, and decreased rolling velocity. Furthermore, we observed an increase in levels of adhesion molecules P-selectin, VCAM-1, and ICAM-1 in these subjects. In addition, type 1 diabetic patients exhibited an increase in proinflammatory mediators TNFα and MPO, and a decreased expression of SOD. The enhancement of leukocyte–endothelium interactions and proinflammatory markers correlated with glucose and HbA1Clevels. Mitochondrial alteration, oxidative stress, and enhanced leukocyte–endothelium interactions are features of type 1 diabetes and may be related to cardiovascular implications.

Mn porphyrins as a novel treatment targeting sickle cell NOXs to reverse and prevent acute vaso-occlusion in vivo

AbstractIn sickle cell disease (SCD), adhesion of sickle red blood cells (SSRBCs) and activated leukocytes in inflamed venules affects blood rheology, causing vaso-occlusive manifestations and vital reduction in microvascular blood flow. Recently, we found that NADPH oxidases (NOXs) create a vicious feedback loop within SSRBCs. This positive feedback loop mediates SSRBC adhesion to the endothelium. We show for the first time the therapeutic effectiveness of the redox-active manganese (Mn) porphyrins MnTnBuOE-2-PyP5+ (MnBuOE; BMX-001) and MnTE-2-PyP5+ (MnE; BMX-010, AEOL10113) to treat established vaso-occlusion in a humanized sickle mouse model of an acute vaso-occlusive crisis using intravital microscopy. These Mn porphyrins can suppress SSRBC NOX activity. Subcutaneous administration of only 1 dose of MnBuOE or MnE at 0.1 to 2 mg/kg after the inflammatory trigger of vaso-occlusion, or simultaneously, reversed and reduced leukocyte and SSRBC adhesion, diminished leukocyte rolling, restored blood flow, and increased survival rate. Furthermore, MnBuOE and MnE administered to sickle mice subcutaneously at 0.1 to 1 mg/kg for 28 days (except on weekends) did not exacerbate anemia, which seemed to be due to downregulation of both SSRBC reactive oxygen species production and exposure of the eryptotic marker phosphatidylserine. In addition, Mn porphyrins ameliorated leukocytosis, venous blood gases, endothelial activation, and organ oxidative damage. Our data suggest that Mn porphyrins, likely by repressing NOX-mediated adhesive function of SSRBCs and activated leukocytes, could represent a novel, safe therapeutic intervention to treat or prevent the establishment of acute pain crises. These NOX-targeted antioxidants merit further assessment in SCD clinical trials.

Stabilization of Endothelial Receptor Arrays by a Polarized Spectrin Cytoskeleton Facilitates Rolling and Adhesion of Leukocytes

SUMMARYMultivalent complexes of endothelial adhesion receptors (e.g., selectins) engage leukocytes to orchestrate their migration to inflamed tissues. Proper anchorage and sufficient density (clustering) of endothelial receptors are required for efficient leukocyte capture and rolling. We demonstrate that a polarized spectrin network dictates the stability of the endothelial cytoskeleton, which is attached to the apical membrane, at least in part, by the abundant transmembrane protein CD44. Single-particle tracking revealed that CD44 undergoes prolonged periods of immobilization as it tethers to the cytoskeleton. The CD44-spectrin “picket fence” alters the behavior of bystander molecules—notably, selectins—curtailing their mobility, inducing their apical accumulation, and favoring their clustering within caveolae. Accordingly, depletion of either spectrin or CD44 virtually eliminated leukocyte rolling and adhesion to the endothelium. Our results indicate that a unique spectrin-based apical cytoskeleton tethered to transmembrane pickets—notably, CD44—is essential for proper extravasation of leukocytes in response to inflammation.

P0352P-SELECTIN BLOCKADE AMELIORATES RENAL HYPOXIA OF LUPUS NEPHRITIS IN MRI/LPR MICE

Abstract Background and Aims Lupus nephritis is one of the most common and severe complications of systemic lupus erythematosus, of which poor prognosis is indicated by aggravated renal hypoxia and tubulointerstitial fibrosis. Cell adhesion molecules play a key role in the progression of lupus nephritis tubulointerstitial lesion, including P-selectin, which mediates the rolling of leukocytes and subsequent adhesion and infiltration and then initiates the inflammatory immune response and ischemia and hypoxia injury. However, the effects and mechanisms of P-selectin in lupus nephritis remain to be investigated, and a noninvasive measurement of lupus nephritis tubulointerstitial hypoxia and fibrosis remains to be explored. Method Thirty-four MRL/lpr mice were randomly divided into the following three groups: MRL/lpr, saline, and anti–P-selectin, which consisted of no treatment, treatment with normal saline, and treatment with anti–P-selectin monoclonal antibody (mAb) from 12 to 16 weeks of age, respectively. Ten male C57BL/6 mice of the same age served as normal controls. Twenty-four-hour urinary protein, urinary albumin–creatinine ratio, and periodic acid–Schiff were used to assess kidney damage; Western blot or immunohistochemical staining of the hypoxia probe HypoxyprobeTM-1, hypoxia-inducible factor 1α (HIF-1α), and CD31 were used to evaluate hypoxia in renal tissue; and NADPH oxidase subunit gp91phox and p22phox were used to examine renal oxidative stress. The correlation between kidney injury and blood oxygen level–dependent magnetic resonance imaging (BOLD-MRI) was calculated to assess the clinical value of BOLD-MRI. Results P-selectin is upregulated in lupus nephritis. Blocking P-selectin with mAb alleviated renal tubulointerstitial fibrosis, renal hypoxia, and peritubular capillary loss, without alteration of the levels of lupus activity indicators, anti-dsDNA antibody, or complement C3. BOLD-MRI showed that the reduced R2* values in the renal cortex and medulla of lupus mice were increased when treated with anti–P-selectin mAb as compared with those treated with normal saline, which were negatively correlated with HypoxyprobeTM-1 hypoxia probe and the expression of HIF-1α. Conclusion Early intervention of lupus nephritis with anti–P-selectin mAb can significantly improve the hypoxic state of the kidney and reduce the severity of tubulointerstitial lesions. BOLD-MRI techniques are noninvasive and can dynamically evaluate the changes in renal lesions and intrarenal oxygenation levels before and after treatment in lupus nephritis.

P-selectin blockade in COVID-19-related ARDS

P-selectin blockade in COVID-19-related ARDS

Pretreatment with Warfarin Attenuates the Development of Ischemia/Reperfusion-Induced Acute Pancreatitis in Rats.

In acute pancreatitis (AP), pancreatic damage leads to local vascular injury, manifesting as endothelial damage and activation, increased vascular permeability, leukocyte rolling, sticking and transmigration to pancreatic tissue as well as activation of coagulation. Previous studies have shown that pretreatment with heparin or acenocoumarol inhibits the development of AP. The aim of the present study was to check the impact of pretreatment with warfarin, an oral vitamin K antagonist, on the development of ischemia/reperfusion-induced AP in rats. AP was induced by pancreatic ischemia followed by reperfusion of the gland. Warfarin (90, 180 or 270 µg/kg/dose) or vehicle were administered intragastrically once a day for 7 days before induction of AP. The effect of warfarin on the severity of AP was assessed 6 h after pancreatic reperfusion. The assessment included histological, functional, and biochemical analyses. Pretreatment with warfarin given at a dose of 90 or 180 µg/kg/dose increased the international normalized ratio and reduced morphological signs of pancreatic damage such as pancreatic edema, vacuolization of acinar cells, necrosis and the number of hemorrhages. These effects were accompanied by an improvement of pancreatic blood flow and a decrease in serum level amylase, lipase, pro-inflammatory interleukin-1β and plasma level of D-dimer. In contrast, pretreatment with warfarin given at a dose of 270 µg/kg/dose led to an increase in severity of pancreatic damage and biochemical indicators of AP. In addition, this dose of warfarin resulted in deaths in some animals. Pretreatment with low doses of warfarin inhibits the development of AP induced by pancreatic ischemia followed by reperfusion.

Leukocyte–Endothelium Interaction Is Associated with Fat Mass in Children

To study leukocyte-endothelium interaction, a measure of the initial phase of atheromatosis, in children with overweight or obesity. A prospective study was conducted in 77 children aged 7-16years; 47 were children with overweight/obesity and 30 were normal weight. Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells were isolated from venous blood samples and the interaction of leukocytes over a monolayer of human umbilical vein endothelial cells was analyzed using flow chamber microscopy. The variables studied included leukocyte rolling velocity, rolling flux, and adhesion to endothelial cells. These were compared between children with overweight/obesity and control children. Correlation between the measures of leukocyte-endothelium interaction and anthropometric and biochemical variables was evaluated. In comparison with normal weight children, the PMNs and peripheral blood mononuclear cells of the overweight/obesity group showed a reduction in rolling velocity (P=.000 and P=.001, respectively) and an increase in rolling flux (P=.001 and P=.004), and adhesion (P=.003 and P=.002). The homeostasis model of insulin resistance was correlated inversely with rolling velocity and positively with rolling flux in PMNs. C-reactive protein was correlated positively with rolling flux and adhesion in both types of leucocytes. Fat mass index was correlated with all measures of leukocyte-endothelial interaction and proved to be the main predictor of leukocyte adhesion in the multiple regression analysis (P=.001 for PMNs and P=.006 for peripheral blood mononuclear cells). Excess fat mass in children is related to the activation of the leukocyte-endothelium interaction, potentially contributing to the development of atherosclerosis.

Adaptive optics permits label-free intravital imaging of tissue resident and infiltrating immune cells in the mouse retina

Abstract The eye is ideally suited for unperturbed longitudinal intravital imaging of immune responses. No confounding inflammatory surgery, externalization or depressurization is required for access as it is uniquely transparent. Directly imaging immune cells in vivo would greatly advance our biological understanding but it is not currently possible in man, and only feasible in mice using exogenous fluorescent labeling or transgenic modification which can result in physiologically unrepresentative results. We discover both tissue resident and infiltrating immune cells can be visualized in the retina using a custom adaptive optics scanning light ophthalmoscope (AOSLO) employing phase contrast detection of reflected 796nm light. This is analogous to scatter characteristics observed in flow cytometry, with motile cells visible within the laminated structure of the retina. Anesthetized mice underwent intraocular injection of LPS to induce acute inflammation before repeated imaging at the same anatomical location across intervals from 6 hours to 2 months. Leukocyte rolling within post-capillary venules and tissue infiltration was visible without exogenous contrast. We confirmed 83% of this population were neutrophils by co-localization following tail vein injection of Alexa Fluor-647 anti-Ly6G antibody. Tracking the same location revealed reduced motility (24 vs 6 hours) despite cellular persistence preceding resolution of inflammation. Microglia could be imaged including their highly motile processes, confirmed using healthy eyes from Cx3cr1GFP/+ reporter mice. This is the first report of label-free immune cell imaging within the mouse retina and the approach could feasibly translate to existing human AOSLO platforms.

Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy.

Neutrophil and macrophage (Mϕ) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.

Physical exercise promotes astrocyte coverage of microvessels in a model of chronic cerebral hypoperfusion

BackgroundBrain circulation disorders such as chronic cerebral hypoperfusion have been associated with a decline in cognitive function during the development of dementia. Astrocytes together with microglia participate in the immune response in the CNS and make them potential sentinels in the brain parenchyma. In addition, astrocytes coverage integrity has been related to brain homeostasis. Currently, physical exercise has been proposed as an effective intervention to promote brain function improvement. However, the neuroprotective effects of early physical exercise on the astrocyte communication with the microcirculation and the microglial activation in a chronic cerebral hypoperfusion model are still unclear. The aim of this study was to investigate the impact of early intervention with physical exercise on cognition, brain microcirculatory, and inflammatory parameters in an experimental model of chronic cerebral hypoperfusion induced by permanent bilateral occlusion of the common carotid arteries (2VO).MethodsWistar rats aged 12 weeks were randomly divided into four groups: Sham-sedentary group (Sham-Sed), Sham-exercised group (Sham-Ex), 2VO-sedentary group (2VO-Sed), and 2VO-exercised group (2VO-Ex). The early intervention with physical exercise started 3 days after 2VO or Sham surgery during 12 weeks. Then, the brain functional capillary density and endothelial-leukocyte interactions were evaluated by intravital microscopy; cognitive function was evaluated by open-field test; hippocampus postsynaptic density protein 95 and synaptophysin were evaluated by western blotting; astrocytic coverage of the capillaries, microglial activation, and structural capillary density were evaluated by immunohistochemistry.ResultsEarly moderate physical exercise was able to normalize functional capillary density and reduce leukocyte rolling in the brain of animals with chronic cerebral hypoperfusion. These effects were accompanied by restore synaptic protein and the improvement of cognitive function. In addition, early moderate exercise improves astrocytes coverage in blood vessels of the cerebral cortex and hippocampus, decreases microglial activation in the hippocampus, and improves structural capillaries in the hippocampus.ConclusionsMicrocirculatory and inflammatory changes in the brain appear to be involved in triggering a cognitive decline in animals with chronic cerebral ischemia. Therefore, early intervention with physical exercise may represent a preventive approach to neurodegeneration caused by chronic cerebral hypoperfusion.

PSGL-1 restricts HIV-1 infectivity by blocking virus particle attachment to target cells

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-like, 120-kDa glycoprotein that binds to P-, E-, and L-selectins. PSGL-1 is expressed primarily on the surface of lymphoid and myeloid cells and is up-regulated during inflammation to mediate leukocyte tethering and rolling on the surface of endothelium for migration into inflamed tissues. Although it has been reported that PSGL-1 expression inhibits HIV-1 replication, the mechanism of PSGL-1-mediated anti-HIV activity remains to be elucidated. Here we report that PSGL-1 in virions blocks the infectivity of HIV-1 particles by preventing the binding of particles to target cells. This inhibitory activity is independent of the viral glycoprotein present on the virus particle; the binding of particles bearing the HIV-1 envelope glycoprotein or vesicular stomatitis virus G glycoprotein or even lacking a viral glycoprotein is impaired by PSGL-1. Mapping studies show that the extracellular N-terminal domain of PSGL-1 is necessary for its anti-HIV-1 activity, and that the PSGL-1 cytoplasmic tail contributes to inhibition. In addition, we demonstrate that the PSGL-1-related monomeric E-selectin-binding glycoprotein CD43 also effectively blocks HIV-1 infectivity. HIV-1 infection, or expression of either Vpu or Nef, down-regulates PSGL-1 from the cell surface; expression of Vpu appears to be primarily responsible for enabling the virus to partially escape PSGL-1-mediated restriction. Finally, we show that PSGL-1 inhibits the infectivity of other viruses, such as murine leukemia virus and influenza A virus. These findings demonstrate that PSGL-1 is a broad-spectrum antiviral host factor with a unique mechanism of action.

P2Y2 Receptor Modulates Leukocyte‐Endothelium Interaction

Nucleotide P2Y2 receptor (P2Y2R) plays important roles in inflammation. The leukocyte‐endothelium interaction is essential for leukocyte recruitment at the site of tissue damage or injury. Little is known about the role of P2Y2R in the regulation of in vivo leukocyte‐endothelium interaction. This study tested the hypothesis that P2Y2R mediates leukocyte‐endothelium interaction using the cremaster muscle. Leukocytes were labeled with Rhodamine 6G injected via tail vein. Leukocyte rolling and stable adhesion were analyzed using a semi‐automatic leukocyte tracking method. P2Y2R activation by UTP increased leukocyte rolling (EC50 = 4.7 ± 1.4 x 10−7 M) and adhesion (EC50= 4.3 ± 1.3 x 10−6 M) in a concentration‐dependent manner in C57BL/6 wild‐type (WT) mice (n=3). UTP (10−5 M) induced sustained increases in both leukocyte rolling and adhesion in WT mice (n=7–19, P<0.05) over 60 min. P2Y2R knockout (P2Y2R−/−) mice exhibited a 2‐fold increase in baseline leukocyte rolling (rolling fraction=37.8%, n=8, p < 0.001) compared to WT mice (rolling fraction=16.2%, n=12) and stayed at the high basal level without changes in rolling following stimulation of UTP (10−5 M) over 60 min. Baseline leukocyte adhesion in P2Y2R−/− mice was 1.5‐fold increase (15 ± 4 adherent cells.100 μm−1, n=12, p < 0.001) compared with WT mice (9 ± 4 adherent cells·100 μm−1; n=19). There was no change in leukocyte adhesion from the baseline in response to UPT (10−5 M) stimulation in P2Y2R−/− mice (n=5–12). We conclude that P2Y2R prevents in vivo leukocyte rolling and adhesion under resting condition while activation P2Y2R by UTP increases both leukocyte rolling and adhesion.Support or Funding InformationMissouri State University

Genesis of Antibiotic Resistance (AR) LVIII: Discordant Antibiotic Stewardship contort Early Goal Directed Fluid Resuscitation therapy (EGDT) with higher incidence of Mortality in severe Sepsis: A global melee

A retrospective analysis of EGDT trials from 2000 to 2018 registered in national clinical trials is presented here: (PRISM‐U) Study group attributed that the sepsis cohort with Mycobacterium tuberculosis (MTB) bacteremia for higher rate of in‐hospital mortality. FEAST (ISRCTN69856593) trial observed that fluid resuscitation in children with sepsis significantly increased mortality (48‐hour) possibly due to impaired perfusion. SSSP trail (NCT01449916) showed that though hypoxemic respiratory failure as contributing factor for mortality in addition to tissue hypoperfusion as inclusion criteria of patients with hypovolemia but excluding the patients with severe respiratory distress when ventilator support is not readily available could have potentially improved the survival rate. ARISE (NCT00975793) presented their report on the effectiveness of EGDT compare to that of usual sepsis care had similar (~18%) mortality rate concluding that EGDT did not reduce all‐cause mortality at 90 days. PRISM (NCT02030158) reported that, EGDT miss the mark to relieve mortality rate compare to that of usual care cohort. ProCESS (NCT00510835) reported that among 1341 patients, of whom 439 were randomly assigned to protocol‐based EGDT, 446 to protocol‐based standard therapy, and 456 to usual care in which protocol‐based resuscitation of patients in whom septic shock was diagnosed in the ER did not improve outcomes. ProMISe (ISRCTN36307479) concluded that septic shock cohort identified early and treated with intravenous antibiotics and adequate fluid resuscitation, hemodynamic management according to a strict EGDT protocol showed that no difference in mortality rate by 90 days. The IMPreSS study group found that compliance with “all of the evidence‐based bundle metrics” had a 40 % reduction in the odds of dying in hospital with the 3‐h bundle and 36 % for the 6‐h bundle. ProCESS Investigators (NCT00793442, NCT00510835 ) showed that increase in leucocyte rolling and adhering to dysfunctional endothelial surface in the cohort of septic shock compare to that of control, attributed to an increase in endothelial permeability‐hemostasis as cause for mortality. SSSP‐2 (NCT01663701) reported that cohort with sepsis and hypotension, most of whom were positive for HIV, in a resource‐limited setting, a protocol for early resuscitation with administration of intravenous fluids and vasopressors increased in‐hospital mortality compared with usual care. MOSAICS Study Group attributed the high mortality rate in severe sepsis treatment for the variation in adhering to the “all of the evidence‐based bundle metrics” for sepsis treatment. GENESIS project comparing 6‐hour resuscitation bundle (RB) for severe sepsis cohort showed decreased mortality rate compare to that of no RB bundle. Taken together it is suggested that lack coherent antibiotic stewardship practices across the globe warrant immediate review during EGDT and implementation of such guidelines would likely improve the survival rate in both low and high resource setting.Support or Funding InformationSupported by the professional development funds by SWTJC to Subburaj Kannan