Search

Abstract: Pulmonary defense mechanisms are critical for host integrity during pneumonia and sepsis. This defense is fundamentally dependent on the activation of neutrophils during the innate immune response. Recent work has shown that semaphorin 7A (Sema7A) holds significant impact on platelet function, yet its role on neutrophil function within the lung is not well understood. This study aimed to identify the role of Sema7A during pulmonary inflammation and sepsis. In patients with acute respiratory distress syndrome (ARDS), we were able to show a correlation between Sema7A and oxygenation levels. During subsequent workup, we found that Sema7A binds to the neutrophil PlexinC1 receptor, increasing integrins, and L-selectin on neutrophils. Sema7A prompted neutrophil chemotaxis in vitro and the formation of platelet-neutrophil complexes in vivo. We also observed altered adhesion and transmigration of neutrophils in Sema7A-/-animals in the lung during pulmonary inflammation. This effect resulted in increased number of neutrophils in the interstitial space of Sema7A-/- animals but reduced numbers of neutrophils in the alveolar space during pulmonary sepsis. This finding was associated with significantly worse outcome of Sema7A-/- animals in a model of pulmonary sepsis. Sema7A has an immunomodulatory effect in the lung, affecting pulmonary sepsis and ARDS. This effect influences the response of neutrophils to external aggression and might influence patient outcome. This trial was registered at www.ClinicalTrials.gov as #NCT02692118., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Ischemic events are associated with severe inflammation and are here referred to as ischemic inflammatory response (IIR). Recent studies identified the formation of platelet-neutrophil complexes (PNC) as key players in IIR. We investigated the role of extracellular platelet nucleotide signaling in the context of IIR and defined a cybernetic circle, including description of feedback loops. Cybernetic circles seek to integrate different levels of information to understand how biological systems function. Our study specifies the components of the cybernetic system of platelets in IIR and describes the theoretical progression of IIR passing the cybernetic cycle with positive and negative feedback loops based on nucleotide-dependent signaling and functional regulation. The cybernetic components and feedback loops were explored by cytometry, immunohistological staining, functional blocking antibodies, and ADP/ATP measurements. Using several ex vivo and in vivo approaches we confirmed cybernetic parameters, such as controller, sensor, and effector (VASP phosphorylation, P2Y 12 , ADORAs and GPIIb/IIIa activity), as well as set points (ADP, adenosine) and interfering control and disturbance variables (ischemia). We demonstrate the impact of the regulated platelet-neutrophil complex (PNC) formation in blood and the resulting damage to the affected inflamed tissue. Taken together, extracellular nucleotide signaling, PNC formation, and tissue damage in IIR can be integrated in a controlled cybernetic circle of platelet function, as introduced through this study.

Blood flow through left ventricular assist devices (LVAD) may induce activation and dysfunction of platelets. Dysfunctional platelets cause coagulation disturbances and form platelet-neutrophil conjugates (PNC), which contribute to inflammatory tissue damage. This prospective observational cohort study investigated patients, who underwent implantation of a LVAD (either HeartMate II (HM II) (n = 7) or HeartMate 3 (HM 3) (n = 6)) and as control patients undergoing coronary artery bypass grafting (CABG) and/or aortic valve replacement (AVR) (n = 10). We performed platelet and leukocyte flow cytometry, analysis of platelet activation markers, and platelet aggregometry. Platelet CD42b expression was reduced at baseline and perioperatively in HM II/3 compared to CABG/AVR patients. After surgery the platelet activation marker β-thromboglobulin and platelet microparticles increased in all groups while platelet aggregation decreased. Platelet aggregation was more significantly impaired in LVAD compared to CABG/AVR patients. PNC were higher in HM II compared to HM 3 patients. We conclude that LVAD implantation is associated with platelet dysfunction and proinflammatory platelet-leukocyte binding. These changes are less pronounced in patients treated with the newer generation LVAD HM 3. Future research should identify device-specific LVAD features, which are associated with the least amount of platelet activation to further improve LVAD therapy.

Ischemic preconditioning (IP) is a well-known strategy to protect organs against cell death following ischemia. The previous work has shown that vasodilator-stimulated phosphoprotein (VASP) is involved in cytoskeletal reorganization and that it holds significant importance for the extent of myocardial ischemia reperfusion injury. Yet, the role of VASP during myocardial IP is, to date, not known. We report here that VASP phosphorylation at serine 157 and serine 239 is induced during hypoxia in vitro and during IP in vivo. The preconditioning-induced VASP phosphorylation inactivates the GP IIb/IIIa integrin receptor on platelets, which results in the reduced formation of organ compromising platelet neutrophil complexes. Experiments in chimeric mice confirmed the importance of VASP phosphorylation during myocardial IP. When studying this in VASP -/- animals and in an isolated heart model, we were able to confirm the important role of VASP on myocardial IP. In conclusion, we were able to show that IP-induced VASP phosphorylation in platelets is a protective mechanism against the deleterious effects of ischemia.

Resolution of inflammation is now recognized as a biosynthetically active process involving pro-resolving mediators. Here, we show in zymosan-initiated peritoneal inflammation that the vagus nerve regulates local expression of netrin-1, an axonal guidance molecule that activates resolution, and that vagotomy reduced local pro-resolving mediators, thereby delaying resolution. In netrin-1(+/-) mice, resolvin D1 (RvD1) was less effective in reducing neutrophil influx promoting resolution of peritonitis compared with Ntn1(+/+). Netrin-1 shortened the resolution interval, decreasing exudate neutrophils, reducing proinflammatory mediators, and stimulating the production of resolvins, protectins, and lipoxins. Human monocytes incubated with netrin-1 produced proresolving mediators, including resolvins and lipoxins. Netrin-1 and RvD1 displayed bidirectional activation in that they stimulated each other's expression and enhanced efferocytosis. These results indicate that the vagus nerve regulates both netrin-1 and pro-resolving lipid mediators, which act in a bidirectional fashion to stimulate resolution, and provide evidence for a novel mechanism for local neuronal control of resolution., (© 2014 Mirakaj et al.)

Recent studies identified basic biological principles that are shared by the immune and the nervous system. One of these analogies applies to the orchestration of cellular migration where guidance proteins that serve as a stop signal for axonal migration can also serve as a stop signal for the migration of immune-competent cells. The control of leukocyte migration is of key interest during conditions associated with inflammatory tissue changes such as tissue hypoxia or hypoxic inflammation. Semaphorins are members of these axon guidance molecules. Previously unknown, we report here the expression and induction of semaphorin 7A (SEMA7A) on endothelium through hypoxia-inducible factor 1α during hypoxia. This induction of SEMA7A translates into increased transmigration of polymorphonuclear neutrophil granulocytes across endothelial cells. Extension of these findings demonstrated an attenuated extravasation of polymorphonuclear neutrophil granulocytes in Sema7a-deficient mice from the vasculature during hypoxia. Studies using chimeric animals identified the expression of Sema7A on nonhematopoietic tissue to be the underlying cause of the observed results. Taken together, our findings demonstrate that neuronal guidance proteins do not only serve as a stop signal for leukocyte migration but also can propagate the extravasation of leukocytes from the vascular space. Future anti-inflammatory strategies might be based on this finding.

Background: Recent work has suggested that the formation of platelet-neutrophil complexes (PNCs) aggravates the severity of inflammatory tissue injury. Given the importance of vasodilator-stimulated phosphoprotein (VASP) for platelet function, we pursued the role of VASP on the formation of PNCs and its impact on the extent of myocardial ischemia-reperfusion (IR) injury., Methods and Results: In initial in vitro studies we found that neutrophils facilitated the movement of platelets across endothelial monolayers. Phosphorylation of VASP reduced the formation of PNCs and transendothelial movement of PNCs. During myocardial IR injury, VASP(-/-) animals demonstrated reduced intravascular formation of PNCs and reduced presence of PNCs within the ischemic myocardial tissue. This was associated with reduced IR injury. Studies using platelet transfer and bone marrow chimeric animals showed that hematopoietic VASP expression was crucial for the intravascular formation of PNCs the presence of PNCs within ischemic myocardial tissue and the extent of myocardial IR injury. Furthermore, phosphorylation of VASP on Ser153 or Ser235 reduced intravascular PNC formation and presence of PNCs within ischemic myocardial tissue. This finding was associated with reduced myocardial IR injury., Conclusion: Previously unappreciated, the phosphorylation of VASP performs a key function for the formation of PNCs that is crucially important for the extent of myocardial IR injury.

Mucosal diseases are often characterized by an inflammatory infiltrate that includes polymorphonuclear leukocytes (PMNs), monocytes, lymphocytes, and platelets. A number of studies have suggested that the interaction of platelets with leukocytes has an essential proinflammatory role. Here, we examined whether platelets migrate across mucosal epithelium, as PMNs are known to do, and whether platelets influence epithelial cell function. Initial studies revealed that human platelets did not efficiently transmigrate across human epithelial cell monolayers. However, in the presence of human PMNs, platelet movement across the epithelium was proportional to the extent of PMN transmigration, and strategies that blocked PMN transmigration diminished platelet movement. Furthermore, platelet-PMN comigration was observed in intestinal tissue derived from human patients with inflammatory bowel disease (IBD). The translocated platelets were found to release large quantities of ATP, which was metabolized to adenosine via a 2-step enzymatic reaction mediated by ecto-nucleotidases, including CD73 and ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), expressed on the apical membrane of the intestinal epithelial cells. In vitro studies and a mouse model of intestinal inflammation were employed to define a mechanism involving adenosine-mediated induction of electrogenic chloride secretion, with concomitant water movement into the intestinal lumen. These studies demonstrate that ecto-NTPDases are expressed on the apical membrane of epithelial cells and are involved in what we believe to be a previously unappreciated function for platelets in the inflamed intestine, which might promote bacterial clearance under inflammatory conditions.

Objective: Several studies have suggested that intravenous hydroxyethyl starch treatment may dampen acute inflammatory responses. It is well documented that limited oxygen delivery to tissues (hypoxia) is common in acute inflammation, and numerous parallels exist between acute responses to hypoxia and to inflammation, including the observation that both are associated with increased vascular leakage and neutrophil infiltration of tissues. Therefore, we compared functional influences of hydroxyethyl starch on normoxic or posthypoxic endothelia., Design: Laboratory study., Setting: University hospital., Subjects: Cultured human microvascular endothelial cells and mice (C57BL/6/129 svj)., Interventions: We measured functional influences of hydroxyethyl starch on normoxic or posthypoxic endothelia., Measurements and Main Results: Studies to assess endothelial barrier function in vitro indicated that the addition of hydroxyethyl starch promotes endothelial barrier in a dose-dependent fashion and hydroxyethyl starch-barrier effects are increased following endothelial hypoxia exposure (human microvascular endothelial cells, 48 hrs, 2% oxygen). Treatment of human microvascular endothelial cells with hydroxyethyl starch resulted in a dose-dependent increase in 157-phosphorylated vasodilator-stimulated phosphoprotein, a protein responsible for controlling the geometry of actin-filaments. Neutrophil adhesion was decreased in the presence of physiologically relevant concentrations of hydroxyethyl starch in vitro, particularly after endothelial hypoxia exposure. Using a murine model of normobaric hypoxia, increases in vascular leakage and pulmonary edema associated with hypoxia exposure (4 hrs at 8% oxygen) were decreased in animals treated with intravenous hydroxyethyl starch. Increases of tissue neutrophil accumulation following hypoxia exposure were dampened in hydroxyethyl starch-treated mice., Conclusions: Taken together, these results indicate that hypoxia-induced increases in vascular leakage and acute inflammation are attenuated by hydroxyethyl starch treatment.

Myocardial ischemia is one of the leading health problems worldwide. Therapy consists of the restitution of coronary perfusion which is followed by myocardial inflammation. Platelet–neutrophil interaction is a crucial process during inflammation, yet its consequences are not fully understood. Here, we show that platelet–neutrophil complexes (PNCs) are increased in patients with acute myocardial infarction and that this is associated with increased levels of neuronal guidance protein semaphorin 7A (SEMA7A). To investigate this further, we injected WT animals with Sema7a and found increased infarct size with increased numbers of PNCs. Experiments in genetically modified animals identify Sema7a on red blood cells to be crucial for this condition. Further studies revealed that Sema7a interacts with the platelet receptor glycoprotein Ib (GPIb). Treatment with anti-Sema7a antibody protected from myocardial tissue injury. In summary, we show that Sema7a binds to platelet GPIb and enhances platelet thrombo-inflammatory activity, aggravating post-ischemic myocardial tissue injury. Reperfusion injury following myocardial ischemia is aggravated by inflammation and platelet–neutrophil complex formation. Here the authors show that semaphorin 7A binds to platelet GPIb, enhancing platelet–neutrophil interaction and increasing post-ischemic myocardial tissue injury, and that blockage of semaphorin 7A is protective. [ABSTRACT FROM AUTHOR]

The extent of pulmonary inflammation during lung injury ultimately determines patient outcome. Pulmonary inflammation is initiated by the migration of neutrophils into the alveolar space. Recent work has demonstrated that the guidance protein semaphorin 7A (SEMA7A) influences the migration of neutrophils into hypoxic tissue sites, yet, its role during lung injury is not well understood. Here, we report that the expression of SEMA7A is induced in vitro through pro-inflammatory cytokines. SEMA7A itself induces the production of pro-inflammatory cytokines in endothelial and epithelial cells, enhancing pulmonary inflammation. The induction of SEMA7A facilitates the transendothelial migration of neutrophils. In vivo, animals with deletion of SEMA7A expression showed reduced signs of pulmonary inflammatory changes following lipopolysaccharide challenge. We define here the role of SEMA7A in the development of lung injury and identify a potential pathway to interfere with these detrimental changes. Future anti-inflammatory strategies for the treatment of lung injury might be based on this finding. [ABSTRACT FROM AUTHOR]

Objectives: Sepsis is associated with a systemic inflammatory reaction, which can result in a life-endangering organ dysfunction. Pro-inflammatory responses during sepsis are characterized by increased activation of leukocytes and platelets, formation of platelet-neutrophil aggregates, and cytokine production. Sequestration of platelet-neutrophil aggregates in the microvasculature contributes to tissue damage during sepsis. At present no effective therapeutic strategy to ameliorate these events is available. In this preclinical pilot study, a novel anti-inflammatory approach was evaluated, which targets nucleoside triphosphate hydrolase activity toward activated platelets via a recombinant fusion protein combining a single-chain antibody against activated glycoprotein IIb/IIIa and the extracellular domain of CD39 (targ-CD39).Design: Experimental animal study and cell culture study.Setting: University-based experimental laboratory.Subjects: Human dermal microvascular endothelial cells 1, human platelets and neutrophils, and C57BL/6NCrl mice.Interventions: Platelet-leukocyte-endothelium interactions were evaluated under inflammatory conditions in vitro and in a murine lipopolysaccharide-induced sepsis model in vivo. The outcome of polymicrobial sepsis was evaluated in a murine cecal ligation and puncture model. To evaluate the anti-inflammatory potential of activated platelet targeted nucleoside triphosphate hydrolase activity, we employed a potato apyrase in vitro and in vivo, as well as targ-CD39 and as a control, nontarg-CD39 in vivo.Measurements and Main Results: Under conditions of sepsis, agents with nucleoside triphosphate hydrolase activity decreased platelet-leukocyte-endothelium interaction, transcription of pro-inflammatory cytokines, microvascular platelet-neutrophil aggregate sequestration, activation marker expression on platelets and neutrophils contained in these aggregates, leukocyte extravasation, and organ damage. Targ-CD39 had the strongest effect on these variables and retained hemostasis in contrast to nontarg-CD39 and potato apyrase. Most importantly, targ-CD39 improved survival in the cecal ligation and puncture model to a stronger extent then nontarg-CD39 and potato apyrase.Conclusions: Targeting nucleoside triphosphate hydrolase activity (CD39) toward activated platelets is a promising new treatment concept to decrease systemic inflammation and mortality of sepsis. This innovative therapeutic approach warrants further development toward clinical application. [ABSTRACT FROM AUTHOR]

Objectives: Hepatic ischemia-reperfusion injury is a disease pattern that is associated with an acute inflammatory reaction. It is well known that neutrophils play an essential role in the early phase of hepatic ischemia-reperfusion injury and determine the extent of tissue damage. Hepatic ischemia-reperfusion injury can result in organ failure, which is linked to high mortality. Recent data indicate that the neuronal guidance receptor Plexin C1 is involved in the control of the acute inflammatory response and, as such, modulates the transmigration of neutrophils. Hence, we investigated the functional role of Plexin C1 in a mouse model of early hepatic ischemia-reperfusion injury.Design: Animal study.Setting: University experimental laboratory.Subjects: Wild-type, PLXNC1 and chimeric mice.Interventions: Hepatic ischemia-reperfusion injury or sham operation.Measurements and Main Results: We found that the functional inhibition of Plexin C1 in wild-type mice treated with an anti-Plexin C1 antibody and a Semaphorin 7A peptide reduced hepatic ischemia-reperfusion injury, as measured by the levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. This reduction in ischemia-reperfusion injury was accompanied by reduced numbers of neutrophils in ischemic hepatic tissue and reduced serum levels of inflammatory cytokines. Experiments using Plexin C1 receptor-deficient (PLXNC1) mice also demonstrated decreased hepatic ischemia-reperfusion injury. Studies of chimeric mice revealed that the hematopoietic Plexin C1 knockout is crucial for reducing the extent of hepatic ischemia-reperfusion injury.Conclusions: These results describe a role for Plexin C1 during ischemia-reperfusion injury, highlight the role of hematopoietic Plexin C1 in the development of hepatic ischemia-reperfusion injury, and suggest that Plexin C1 is a potential drug target. [ABSTRACT FROM AUTHOR]

Abstract: Extracorporeal circulation (ECC) is an essential tool for the execution of cardiac operations. However, ECC is also associated with undesirable side effects. These include the induction of a systemic inflammatory response associated with leukocyte activation and cytokine release as well as potentially life-threatening complications. The volatile anesthetic sevoflurane has been reported to exert anti-ischemic and anti-inflammatory effects. We therefore investigated whether sevoflurane modulates the ECC-triggered inflammatory response. Heparinized human blood was circulated for 90min in a normothermic (37°C) ex vivo ECC circuit. An air–oxygen mixture was administered via an oxygenator in controls (n=5). Sevoflurane (2vol.%) was added to the gas mixture in a second group (n=5). At baseline and after 30, 60 and 90min of ECC, blood samples were taken. In each sample whole blood counts were determined. Expression of the activation-indicating Mac-1 receptor on granulocytes and monocytes as well as leukocyte–platelet aggregate formation was measured in flow cytometry. Levels of the granulocyte activation marker PMN-elastase and of the cytokines IL-1β, IL-8 and TNF-α were analyzed using ELISA. ECC induced significant increases in Mac-1 expression on granulocytes (p<0.001) and PMN-elastase release (p<0.001). Sevoflurane decreased granulocyte Mac-1 expression during ECC (p<0.05) and inhibited the ECC-induced PMN-elastase release (p<0.05). Sevoflurane had no effect on whole blood cell counts, leukocyte–platelet aggregate formation and cytokine release during ECC. Sevoflurane inhibits granulocyte activation during ex vivo ECC and therefore has the potential to decrease the ECC-triggered inflammatory response. This promising finding warrants further investigation under clinical conditions. [Copyright &y& Elsevier]