Neutrophil Adhesion Research Articles

Primary adhered neutrophils increase JNK1-MARCKSL1-mediated filopodia to promote secondary neutrophil transmigration

During inflammation, leukocytes extravasate the vasculature to areas of inflammation in a process termed transendothelial migration. Previous research has shown that transendothelial migration hotspots exist, areas in the vasculature that are preferred by leukocytes to cross. Several factors that contribute to hotspot-mediated transmigration have been proposed already, but whether one leukocyte transmigration hotspot can be used subsequently by a second wave of leukocytes and thereby can increase the efficiency of leukocyte transmigration is not well understood. Here, we show that primary neutrophil adhesion to the endothelium triggers endothelial transmigration hotspots, allowing secondary neutrophils to cross the endothelium more efficiently. Mechanistically, we show that primary neutrophil adhesion increases the number of endothelial apical filopodia, resulting in an increase in the number of adherent secondary neutrophils. Using fluorescence resonance energy transfer (FRET)-based biosensors, we found that neutrophil adhesion did not trigger the activity of the small GTPase Cdc42. We used kinase translocation reporters to study the activity of mitogen-activated protein (MAP) kinases and Akt in endothelial cells on a single-cell level with a high temporal resolution during the process of leukocyte transmigration and found that c-Jun N-terminal kinase (JNK) is rapidly activated upon neutrophil adhesion, whereas extracellular regulated kinase (ERK), p38, and Akt are not. Additionally, we show that short-term chemical inhibition of endothelial JNK successfully prevents the adhesion of neutrophils to the endothelium. Furthermore, we show that neutrophil-induced endothelial JNK1 but not JNK2 increases the formation of filopodiaand thereby the adhesion of secondary neutrophils. JNK1 needs its downstream substrate MARCKSL1 to trigger additional apical filopodia and consequently neutrophil adhesion. Overall, our data show that primary neutrophils can trigger the endothelial transmigration hotspot by activating JNK1 and MARCKSL1 to induce filopodia that trigger more neutrophils to transmigrate at the endothelial hotspot area.

A key role for platelet GPVI in neutrophil recruitment, migration, and NETosis in the early stages of acute lung injury

AbstractAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. Here, we show that the immunoreceptor tyrosine-based activation motif–coupled immunoglobulin-like platelet receptor, glycoprotein VI (GPVI), plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In wild-type (WT) control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, GPVI-deficient mice as well as anti-GPVI–treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High-resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interactions with neutrophils and overall reduced platelet–neutrophil complex (PNC) formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells, resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, neutrophil activation was also diminished in anti-GPVI–treated animals, associated with strongly reduced formation of PNC clusters and neutrophil extracellular traps (NETs) compared with that in control mice. These results establish GPVI as a key mediator of neutrophil recruitment, PNC formation, and NET formation (ie, NETosis) in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation that causes ALI/ARDS.

Synthesis of Sialyl LewisX Mimetics with E- and P-Selectin Binding Properties and Immunosuppressive Activity.

E- and P-selectins are adhesion proteins implicated in immune cell recruitment at sites of infection, making them important drug targets for diseases involving excessive and uncontrolled inflammation. In this study, we developed an efficient strategy to synthesize bicyclic galactopyranosides through a key stereoselective equatorial C4-propiolate addition and TMSCN axial C-glycosidation. The nitrile group can then be converted to the carboxyl and different bioisosteres at a late stage in the synthesis, allowing for various derivatizations to potentially enhance biological activity. The sialyl LewisX glycomimetic featuring this rigidified bicyclic galactopyranoside moiety prevents neutrophil adhesion to endothelial cells in vitro by binding to both E- and P-selectins. We show here that the axial carboxyl analogue blocks immune cell recruitment in vivo, demonstrating its potential as an immunomodulator.

A myristoylated alanine-rich C-kinase substrate (MARCKS) inhibitor peptide attenuates neutrophil outside-in β2-integrin activation and signaling

ABSTRACT MARCKS is an actin and PIP2-binding protein that plays an essential role in neutrophil migration and adhesion; however, the molecular details regarding MARCKS function in these processes remains unclear. Neutrophil adhesion and migration also require the cell surface receptors β2-integrins. We hypothesized that MARCKS inhibition would alter neutrophil β2-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in β2-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out β2-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn2+-stimulated static adhesion, cell spreading and β2-integrin clustering, suggesting a role for MARCKS function in outside-in β2-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in β2-integrin activation and signaling.

Dissecting the Therapeutic Mechanisms of Sphingosine-1-Phosphate Receptor Agonism during Ischaemia and Reperfusion.

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PR) regulate many cellular processes, including lymphocyte migration and endothelial barrier function. As neutrophils are major mediators of inflammation, their transendothelial migration may be the target of therapeutic approaches to inflammatory conditions such as ischaemia-reperfusion injury (IRI). The aim of this project was to assess whether these therapeutic effects are mediated by S1P acting on neutrophils directly or indirectly through the endothelial cells. First, our murine model of peritoneum cell recruitment demonstrated the ability of S1P to reduce CXCL8-mediated neutrophil recruitment. Mechanistic in vitro studies revealed that S1P signals in neutrophils mainly through the S1PR1 and 4 receptors and induces phosphorylation of ERK1/2; however, this had no effect on neutrophil transmigration and adhesion. S1P treatment of endothelial cells significantly reduced TNF-α-induced neutrophil adhesion under flow (p < 0.01) and transendothelial migration towards CXCL8 during in vitro chemotaxis assays (p < 0.05). S1PR1 agonist CYM5442 treatment of endothelial cells also reduced neutrophil transmigration (p < 0.01) and endothelial permeability (p < 0.005), as shown using in vitro permeability assays. S1PR3 agonist had no effects on chemotaxis or permeability. In an in vivo mouse model of renal IRI, S1PR agonism with CYM5442 reduced endothelial permeability as shown by reduced Evan's Blue dye extravasation. Western blot was used to assess phosphorylation at different sites on vascular endothelial (VE)-cadherin and showed that CYM5442 reduced VEGF-mediated phosphorylation. Taken together, the results of this study suggest that reductions in neutrophil infiltration during IRI in response to S1P are mediated primarily by S1PR1 signalling on endothelial cells, possibly by altering phosphorylation of VE-cadherin. The results also demonstrate the therapeutic potential of S1PR1 agonist during IRI.

New-generation cytopharmaceuticals with powerfully boosted extravasation for enhanced cancer therapy.

Effective extravasation of therapeutic agents into solid tumors still faces huge challenges. Since the doubted effectiveness of enhanced penetration and retention effect, first-generation neutrophil cytopharmaceuticals with encapsulated drugs have been developed to improve the drug accumulation in tumors based on the active chemotaxis and extravasation of neutrophils. Herein, a new generation of neutrophil cytopharmaceuticals with enhanced tumor-specific extravasation is reported to satisfy more complex clinical demands. This neutrophil cytopharmaceutical is obtained by anchoring vascular endothelial growth factor receptor 2 (VEGFR2)-targeting peptide K237 on neutrophil membrane after endocytosis of chemotherapeutics by neutrophils. Leveraging the cytokine-mediated active migration of neutrophils, the specific-recognition of K237 peptide to tumor vascular endothelium expedites the migration and enhances tight adhesion of neutrophils to vascular endothelium, thus improving the extravasation of therapeutic agents to target sites. Moreover, anti-angiogenesis effect from VEGFR2-blocking by K237 peptide achieves a cooperative tumor destruction with cytotoxic effects from released chemotherapeutics. This study demonstrates the great potential of enhanced proactive extravasation of cytopharmaceuticals via a cell-anchoring technology, leading to expedited drug infiltration and boosted therapeutic effects, which can be applied in other cell therapies to improve efficacy.

Sfxn5 Regulation of Actin Polymerization for Neutrophil Spreading Depends on a Citrate-Cholesterol-PI(4,5)P2 Pathway.

Cell spreading is an initial and critical step in neutrophil adhesion and migration, leading to neutrophil recruitment to inflammatory tissues. Sideroflexin (Sfxn) family proteins are metabolite transporters located in the mitochondrial membrane. Recombinant SFXN5 protein is a citrate transporter invitro; however, whether Sfxn5 regulates any cellular behavior or function remains unknown. In this study, we found that small interfering RNA transfection or morpholino injection achieving Sfxn5 deficiency in neutrophils significantly decreased neutrophil recruitment in mice and zebrafish, respectively. Sfxn5 deficiency impaired neutrophil spreading and spreading-associated cellular phenotypes, such as cell adhesion, chemotaxis, and ROS production. Actin polymerization is critical for neutrophil spreading, and we found that actin polymerization in spreading neutrophils was partially inhibited by Sfxn5 deficiency. Mechanistically, we observed that the levels of cytosolic citrate and its downstream metabolic products, acetyl-CoA and cholesterol, were decreased in Sfxn5-deficient neutrophils. The levels of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a mediator for the regulation of actin polymerization by cholesterol, were reduced in the plasma membrane of Sfxn5-deficient neutrophils. Exogenous supplementation with citrate or cholesterol partially reversed the reduction in PI(4,5)P2 levels, defective neutrophil actin polymerization, and cell spreading. Altogether, we demonstrated that Sfxn5 maintains cytosolic citrate levels and ensures the synthesis of sufficient cholesterol to promote actin polymerization in a PI(4,5)P2-dependent manner during neutrophil spreading, which is essential for the eventual inflammatory recruitment of neutrophils. Our study revealed the importance of Sfxn5 in neutrophil spreading and migration, thus identifying, to our knowledge, for the first time, the physiological cellular functions of the Sfxn5 gene.

Intravital imaging of three different microvascular beds in SARS-CoV-2–infected mice

AbstractSevere acute respiratory syndrome coronavirus–2 (SARS-CoV-2) enters the respiratory tract, where it infects the alveoli epithelial lining. However, patients have sequelae that extend well beyond the alveoli into the pulmonary vasculature and, perhaps, beyond to the brain and other organs. Because of the dynamic events within blood vessels, histology does not report platelet and neutrophil behavior. Because of the rapid nontranscriptional response of these cells, neither single-cell RNA sequencing nor proteomics report robustly on their critical behaviors. We used intravital microscopy in level-3 containment to examine the pathogenesis of SARS-CoV-2 within 3 organs in mice expressing human angiotensin converting enzyme 2 (ACE-2) ubiquitously (CAG-AC-70) or on epithelium (K18-promoter). Using a neon-green SARS-CoV-2, we observed both the epithelium and endothelium infected in AC70 mice but only the epithelium in K18 mice. There were increased neutrophils in the microcirculation but not in the alveoli of the lungs of AC70 mice. Platelets formed large aggregates in the pulmonary capillaries. Despite only neurons being infected within the brain, profound neutrophil adhesion forming the nidus of large platelet aggregates were observed in the cerebral microcirculation, with many nonperfused microvessels. Neutrophils breached the brain endothelial layer associated with a significant disruption of the blood-brain-barrier. Despite ubiquitous ACE-2 expression, CAG-AC-70 mice had very small increases in blood cytokine, no increase in thrombin, no infected circulating cells, and no liver involvement suggesting limited systemic effects. In summary, our imaging of SARS-CoV-2–infected mice gave direct evidence that there is a significant perturbation locally in the lung and brain microcirculation induced by local viral infection leading to increased local inflammation and thrombosis in these organs.

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis.

Rac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens. Here, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control. All the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis. Collectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils.

INFLAMMATION AS A FUNDAMENTAL PATHOLOGICAL PROCESS: LECTURE 2 (CELLULAR COMPONENT)

The second part of the lecture describes cellular events occurring in the focus of inflammation. The first cells migrating to the focus of inflammation are neutrophils, the second are accumulate monocytes (macrophages), and the final cells of inflammation are fibroblasts. This sequence is determined by the spectrum of chemoattractants which are presented in the focus of inflammation. Neutrophil migration is initiated by tissue macrophages, which are activated upon tissue damage and begin to secrete cytokines, primarily IL-8 and IL-1. These cytokines ensure neutrophil adhesion to the vascular wall and their further chemotaxis along the gradient of these cytokines and other chemoattractants. There are several populations of neutrophils. Inflammation mainly involves high density neutrophils (mature cells) and low density neutrophils (immature cells, e.g. band forms). From a functional point of view, neutrophils are divided into pro-inflammatory (N1) and immunosuppressive (N2), the balance between these cells is very important for an adequate course of inflammation. Neutrophils begin the process of destroying the pathogen and tissue damage products. In addition to the long-known phagocytosis, this process is carried out with the help of neutrophil extracellular traps (NET), which consist of DNA, histones and lysosomal enzymes. Neutrophils in the focus of inflammation synthesize cytokines (IL-1, IL-6, TNFα, G-CSF and others), which stimulate the migration of monocytes and their differentiation into macrophages. Activated macrophages are divided into two groups: classic pro-inflammatory M1 and alternative anti-inflammatory M2. M1 are involved in the elimination of pathogen. They have powerful cytotoxic and antimicrobial activities. M2 macrophages produce anti-inflammatory cytokines (IL-10, TGFβ, etc.), growth factors, they stimulate angiogenesis, fibroblast proliferation processes and the formation of an extracellular matrix by fibroblasts. Fibroblasts, attracted by macrophage cytokines, complete the inflammatory process. They synthesize the basic substance of the connective tissue and provide the process of its organization. Dysregulation of cellular responses can underlie chronic inflammation.

A novel galactoxylan derived from Viola diffusa alleviates LPS-induced acute lung injury via antagonizing P-selectin-mediated adhesion function

Acute lung injury (ALI) greatly threatens human health worldwide. P-selectin is a potential target for the treatment of acute inflammatory diseases, and natural polysaccharides exhibit high-affinity for P-selectin. Viola diffusa, a traditional Chinese herbal, shows strong anti-inflammatory effects, but pharmacodynamic substances and underlying mechanisms are still unclear. In this study, a galactoxylan polysaccharide (VDPS) derived from Viola diffusa was isolated and characterized, evaluated the protective effect on LPS induced ALI and underlying mechanism. VDPS significantly alleviated LPS-induced pathological lung injury, and decreased the numbers of total cells and neutrophils as well as the total protein contents in the bronchoalveolar lavage fluid (BALF). Moreover, VDPS reduced proinflammatory cytokine production both in BALF and lung. Interestingly, VDPS significantly restrained the activation of NF-κB signaling in the lung of LPS-exposed mice, but it cannot inhibit LPS-induced inflammation in human pulmonary microvascular endothelial cells (HPMECs) in vitro. Additionally, VDPS disrupted neutrophil adhesion and rolling on the activated HPMECs. VDPS cannot impact the expression or cytomembrane translocation of endothelial P-selectin, but remarkably interrupt the binding of P-selectin and PSGL-1. Overall, this study demonstrated that VDPS can alleviate LPS-induced ALI via inhibiting P-selectin-dependent adhesion and recruitment of neutrophils on the activated endothelium, providing a potential treatment strategy for ALI.

Store-operated Ca2+ entry-sensitive glycolysis regulates neutrophil adhesion and phagocytosis in dairy cows with subclinical hypocalcemia

Hypocalcemia in dairy cows is associated with a decrease of neutrophil adhesion and phagocytosis, an effect driven partly by changes in the expression of store-operated Ca2+ entry (SOCE)-related molecules. It is well-established in non-ruminants that neutrophils obtain the energy required for immune function through glycolysis. Whether glycolysis plays a role in the acquisition of energy by neutrophils during hypocalcemia in dairy cows is unknown. To address this relationship, we performed a cohort study and then a clinical trial. Neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (average 2.83 ± 0.42 lactations, n = 6) diagnosed as clinically healthy (CON) or with plasma concentrations of Ca2+ < 2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (HYP, average 2.83 ± 0.42 lactations, n = 6). In the first experiment, neutrophils were isolated from blood of CON and HYP cows and used to analyze aspects of adhesion and phagocytosis function through QRT-PCR along with confocal laser scanning microscopy, mRNA expression of the glycolysis-related gene hexokinase 2 (HKII) and components of the SOCE moiety ORAI calcium release-activated calcium modulator 1 [ORAI1, ORAI2, ORAI3, stromal interaction molecule 1 (STIM1) and STIM2]. Results showed that adhesion and phagocytosis function were reduced in HYP cows. The mRNA expression of adhesion-related syndecan-4 (SDC4), integrin β9 (ITGB9) and integrin β3 (ITGB3) and phagocytosis-related molecules complement component 1 R subcomponent (C1R), CD36, tubulinß1 (TUBB1) were significantly decreased in the HYP group. In the second experiment, to address how glycolysis affects neutrophil adhesion and phagocytosis, neutrophils isolated from CON and HYP cows were treated with 2 μM of the HKII inhibitor Benserazide-d3 or 1 μM of the fructose-bisphosphatase 1 (FBP1) inhibitor MB05032 for 1 h. Results revealed that the HKII inhibitor Benserazide-d3 reduced phagocytosis and the mRNA abundance of ITGB9, and CD36 in the HYP group. The FBP1 inhibitor MB05032 increased adhesion and phagocytosis and increased mRNA abundance of HKII, ITGB9, and CD36 in the HYP group. Lastly, to investigate the mechanism whereby SOCE-sensitive glycolysis affects neutrophil adhesion and phagocytosis, isolated neutrophils were treated with 1 μM of the SOCE activator thapsigagin or 50 μM of the inhibitor 2-APB for 1 h. Results showed that thapsigargin increased mRNA abundance of HKII, ITGB9, and CD36, and increased adhesion and phagocytosis in the HYP group. In contrast, 2-APB decreased mRNA abundance of HKII and both adhesion and phagocytosis of neutrophils in the CON group. Overall, the data indicated that SOCE-sensitive intracellular Ca2+ levels affect glycolysis and help regulate adhesion and phagocytosis of neutrophils during hypocalcemia in dairy cows.

Modeling the Effects of IL-1β-mediated Inflammation During Ex Vivo Lung Perfusion Using a Split Human Donor Model.

The association between interleukin-1β (IL-1β) concentrations during ex vivo lung perfusion (EVLP) with donor organ quality and post-lung transplant outcome has been demonstrated in several studies. The mechanism underlying IL-1β-mediated donor lung injury was investigated using a paired single-lung EVLP model. Human lung pairs were dissected into individual lungs and perfused on identical separate EVLP circuits, with one lung from each pair receiving a bolus of IL-1β. Fluorescently labeled human neutrophils isolated from a healthy volunteer were infused into both circuits and quantified in perfusate at regular timepoints. Perfusates and tissues were subsequently analyzed, with perfusates also used in functional assays. Neutrophil numbers were significantly lower in perfusate samples collected from the IL-1β-stimulated lungs consistent with increased neutrophil adhesion ( P = 0.042). Stimulated lungs gained significantly more weight than controls ( P = 0.046), which correlated with soluble intercellular adhesion molecule-1 (R 2 = 0.71, P = 0.0043) and von-Willebrand factor (R 2 = 0.39, P = 0.040) in perfusate. RNA expression patterns for inflammatory genes were differentially regulated via IL-1β. Blockade of IL-1β significantly reduced neutrophil adhesion in vitro ( P = 0.025). These data illustrate the proinflammatory functions of IL-1β in the context of EVLP, suggesting this pathway may be susceptible to therapeutic modulation before transplantation.

Vascular traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk

During bloodstream infections, neutrophils home to the liver as part of an intravascular immune response to eradicate blood-borne pathogens, but the mechanisms regulating this crucial response are unknown. Using invivo imaging of neutrophil trafficking in germ-free and gnotobiotic mice, we demonstrate that the intestinal microbiota guides neutrophil homing to the liver in response to infection mediated by the microbial metabolite D-lactate. Commensal-derived D-lactate augments neutrophil adhesion in the liver independent of granulopoiesis in bone marrow or neutrophil maturation and activation in blood. Instead, gut-to-liver D-lactate signaling primes liver endothelial cells to upregulate adhesion molecule expression in response to infection and promote neutrophil adherence. Targeted correction of microbiota D-lactate production in a model of antibiotic-induced dysbiosis restores neutrophil homing to the liver and reduces bacteremia in a model of Staphylococcus aureus infection. These findings reveal long-distance traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk.

Severe Leukocyte Adhesion Deficiency-I (LAD-I) Lentiviral-Mediated Ex-Vivo Gene Therapy: Ongoing Phase 1/2 Study Results

BackgroundSevere leukocyte adhesion deficiency-I (LAD-I) is an autosomal recessive inborn error of immunity due to mutations in the ITGB2 gene that encodes the common CD18 subunit of β2-integrins, essential for neutrophil adhesion to the inflamed endothelium and transmigration into tissues. Severe LAD-I, defined as <2% of normal CD18 polymorphonucleocyte [PMN] expression, is characterized by frequent and often refractory bacterial and fungal infections, impaired wound healing, and significant pediatric mortality in the absence of allogeneic hematopoietic stem cell transplant (HSCT). HSCT is potentially curative but limited by donor availability, graft-versus-host disease, and graft failure. The gene therapy RP-L201-0318 (NCT03812263) employs autologous CD34+ cells transduced with a lentiviral vector carrying ITGB2 to restore CD18 expression. MethodsPatients ≥3 months old with severe LAD-I were enrolled. Hematopoietic stem cells (HSCs) were collected via apheresis after mobilization with granulocyte-colony stimulating factor and plerixafor and transduced ex-vivo with Chim-CD18-WPRE-LV. Myeloablative therapeutic drug monitoring (TDM) busulfan conditioning preceded RP-L201 infusion. Patients were followed for safety and efficacy measures, including survival to age two and ≥1-year post-infusion, peripheral blood [PB] PMN CD18 expression, PB vector copy number [VCN], neutrophilia improvement, decrease in infections/hospitalizations, and resolution of skin/periodontal abnormalities. ResultsNine patients (age five months to nine years) received RP-L201; follow-up was 3 to 24 months. RP-L201 cell doses ranged from 2.8 × 106 to 10 × 106 CD34+ cells/kg with a drug product VCN of 1.8 to 3.8. All nine patients demonstrated PMN CD18 restoration (median expression of 56.3%) with sustained, stable genetic markings (median PB mononuclear cell VCN of 1.53). At one year, the overall survival (OS) rate was 100% per Kaplan-Meier estimate. Pre-treatment leukocytosis improved uniformly. Hospitalizations and severe infections were significantly reduced following therapy. No RP-L201-related serious adverse events (SAEs) were reported. Insertion site analyses indicate highly polyclonal integration patterns across the entire cohort. ConclusionRP-L201 has a favorable safety profile and confers durable correction of the severe LAD-I phenotype with improved clinical course in all nine pediatric patients treated as demonstrated via all laboratory and clinical parameters.

ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis.

Peritoneal inflammation remains a major cause of treatment failure in patients with kidney failure who receive peritoneal dialysis. Peritoneal inflammation is characterized by an increase in neutrophil infiltration. However, the molecular mechanisms that control neutrophil recruitment in peritonitis are not fully understood. ELMO and DOCK proteins form complexes which function as guanine nucleotide exchange factors to activate the small GTPase Rac to regulate F-actin dynamics during chemotaxis. In the current study, we found that deletion of the Elmo1 gene causes defects in chemotaxis and the adhesion of neutrophils. ELMO1 plays a role in the fMLP-induced activation of Rac1 in parallel with the PI3K and mTORC2 signaling pathways. Importantly, we also reveal that peritoneal inflammation is alleviated in Elmo1 knockout mice in the mouse model of thioglycollate-induced peritonitis. Our results suggest that ELMO1 functions as an evolutionarily conserved regulator for the activation of Rac to control the chemotaxis of neutrophils both in vitro and in vivo. Our results suggest that the targeted inhibition of ELMO1 may pave the way for the design of novel anti-inflammatory therapies for peritonitis.

Seizure-induced neutrophil adhesion in brain capillaries leads to a decrease in postictal cerebral blood flow

Cerebral hypoperfusion has been proposed as a potential cause of postictal neurological dysfunction in epilepsy, but its underlying mechanism is still unclear. We show that a 30% reduction in postictal cerebral blood flow (CBF) has two contributing factors: the early hypoperfusion up to ∼30min post-seizure was mainly induced by arteriolar constriction, while the hypoperfusion that persisted for over an hour was due to increased capillary stalling induced by neutrophil adhesion to brain capillaries, decreased red blood cell (RBC) flow accompanied by constriction of capillaries and venules, and elevated intercellular adhesion molecule-1 (ICAM-1) expression. Administration of antibodies against the neutrophil marker Ly6G and against LFA-1, which mediates adhesive interactions with ICAM-1, prevented neutrophil adhesion and recovered the prolonged CBF reductions to control levels. Our findings provide evidence that seizure-induced neutrophil adhesion to cerebral microvessels via ICAM-1 leads to prolonged postictal hypoperfusion, which may underlie neurological dysfunction in epilepsy.

Mechano-Redox Control of Macrophage-1 Antigen De-Adhesion From ICAM-1 (Intercellular Adhesion Molecule 1) by Protein Disulfide Isomerase Promotes Directional Movement Under Flow.

Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMβ2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the βI domain of the β2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the βI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of β2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Livin upregulation in keratinocytes of psoriasis patients to promote adhesion molecule expression.

Activation of keratinocytes (KCs) is the main pathological feature of psoriasis. KCs recruit neutrophils by releasing various antimicrobial peptides and chemokines, which is also related to the expression of KC adhesion molecules. However, the regulatory mechanism governing their expression is still unclear. Livin, an inhibitor of theapoptosis protein family member involved in proliferation and metastasis of tumor cells, is significantly increased in psoriatic lesions. The aim of this study was to investigate the role of Livin in regulating adhesion molecule expression in KCs and release of chemokines that promote the activation and adhesion of neutrophils. The expression of Livin in psoriasis patients, imiquimod mouse model, and the combination of IL-17 alpha, IL-22, IL-1 alpha, OSM, and TNF-α (Mix M5)-treated HaCaT cells were detected by immunofluorescence staining, RT-qPCR, and ELISA. Livin-overexpression and knockdown in HaCaT cells transfected with HIV-1-based lentiviral vectors were used to study the function of Livin using RNA-seq. Moreover, differences in the expression of HaCaT cell adhesion molecules after regulation of Livin expression and activation of neutrophils in the co-culture model were verified. Livin was upregulated in the KCs of psoriasis patients, imiquimod mouse model and Mix M5-treated HaCaT cells compared with the control groups. Livin in HaCaT cells might regulate the expression of adhesion molecules in KCs. Thus, Livin may be a key effector molecule that regulates the expression of adhesion molecules in KCs and promotes the activation and adhesion of neutrophils.