Neutrophil cerebrovascular transmigration triggers rapid neurotoxicity through release of proteases associated with decondensed DNA.
ABSTRACT: Cerebrovascular inflammation contributes to diverse CNS disorders through mechanisms that are incompletely understood. The recruitment of neutrophils to the brain can contribute to neurotoxicity, particularly during acute brain injuries, such as cerebral ischemia, trauma, and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. In this study, we show that mouse neutrophils are not inherently toxic to neurons but that transendothelial migration across IL-1-stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes the rapid death of cultured neurons. Neurotoxicity was induced by the addition of transmigrated neutrophils or conditioned medium, taken from transmigrated neutrophils, to neurons and was partially mediated by excitotoxic mechanisms and soluble proteins. Transmigrated neutrophils also released decondensed DNA associated with proteases, which are known as neutrophil extracellular traps. The blockade of histone-DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas the inhibition of key neutrophil proteases in the presence of transmigrated neutrophils rescued neuronal viability. We also show that neutrophil recruitment in the brain is IL-1 dependent, and release of proteases and decondensed DNA from recruited neutrophils in the brain occurs in several in vivo experimental models of neuroinflammation. These data reveal new regulatory and effector mechanisms of neutrophil-mediated neurotoxicity (i.e., the release of proteases and decondensed DNA triggered by phenotypic transformation during cerebrovascular transmigration). Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of antileukocyte therapies.
Project description:<b>Introduction:</b> The acute phase protein pentraxin 3 (PTX3) is known for its anti-inflammatory effects through downregulating neutrophil transmigration during peripheral inflammation. Furthermore, we have previously demonstrated a neuroprotective and neuroreparative effect of PTX3 after cerebral ischaemia. Here we investigated, to our knowledge for the first time, the role of PTX3 in neutrophil transmigration and neurotoxicity following lipopolysaccharide (LPS)-induced cerebral inflammation and cerebral ischaemia. <b>Methods:</b> Neutrophil transmigration through interleukin-1? (IL-1?) activated brain endothelium and neurotoxicity of neutrophils isolated from wild-type (WT) or PTX3 knock-out (KO) mice was assessed <i>in vitro</i>. Primary cortical neuronal death after treatment with transmigrated neutrophils was quantified by lactate dehydrogenase (LDH) assay. Cerebral inflammation or ischemia was induced in WT and PTX3 KO mice via intrastriatal LPS injection or by transient middle cerebral artery occlusion (MCAo) respectively. Subsequent neutrophil infiltration in the brain was assessed by immunohistochemistry and the expression of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1? by enzyme-linked immunosorbent assay (ELISA). <b>Results:</b> Neutrophils isolated from WT mice after intrastriatal LPS injection transmigrated significantly more through IL-1? activated brain endothelium compared to neutrophils from PTX3 KO mice. Transmigrated WT and PTX3 KO neutrophils were significantly more neurotoxic than corresponding non-transmigrated neutrophils; however, no significant differences in neurotoxicity between genotypes were observed. PTX3 reduced the number of transmigrated neutrophils to the brain after intrastriatal LPS injection. Furthermore, PTX3 KO mice showed significantly increased levels of neutrophils in the brain after LPS administration or in the ischaemic hemisphere after MCAo, compared to WT mice. <b>Conclusion:</b> Our study shows that PTX3 regulates neutrophil transmigration in the CNS during neuroinflammation, demonstrating the potential of PTX3 as an effective therapeutic target in neuroinflammatory conditions.
Project description:Neutrophils can form neutrophil extracellular traps (NETs) to capture microbes and facilitate their clearance. NETs consist of decondensed chromatin decorated with anti-microbial proteins. Here, we describe the effect of neutrophil proteases on the protein content of NETs. We show that the neutrophil serine proteases degrade several neutrophil proteins associated with NETs. Interestingly, the anti-bacterial proteins associated with NETs, such as myeloperoxidase, calgranulin B and neutrophil elastase (NE), seem to be less susceptible to proteolytic degradation than other NET proteins, such as actin and MNDA. NETs have been proposed to play a role in autoimmune reactions. Our data demonstrate that a large number of the autoepitopes of NET proteins that are recognized by autoantibodies produced by systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients are also removed by the proteases. In conclusion, neutrophil serine proteases have a major impact on the NET proteome and the proteolytic changes of NET-associated proteins may counteract autoimmune reactions to NET components.
Project description:Neutrophils are recruited to the airways of patients with acute respiratory distress syndrome (ARDS) where they acquire an activated pro-survival phenotype with an enhanced respiratory burst thought to contribute to ARDS pathophysiology. Our in vitro model enables blood neutrophil transepithelial migration into cell-free tracheal aspirate fluid from patients to recapitulate the primary airway neutrophil phenotype observed in vivo. Neutrophils transmigrated through our model toward airway fluid from children with lower respiratory viral infections coinfected with bacteria had elevated levels of neutrophil activation markers but paradoxically exhibited an inability to kill bacteria and a defective respiratory burst compared with children without bacterial coinfection. The airway fluid from children with bacterial coinfections had higher levels of neutrophil elastase activity, as well as myeloperoxidase levels compared to children without bacterial coinfection. Neutrophils transmigrated into the aspirate fluid from children with bacterial coinfection showed decreased respiratory burst and killing activity against H. influenzae and S. aureus compared to those transmigrated into the aspirate fluid from children without bacterial coinfection. Use of a novel transmigration model recapitulates this pathological phenotype in vitro that would otherwise be impossible in a patient, opening avenues for future mechanistic and therapeutic research.
Project description:Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production.
Project description:Neutrophils are potent effector cells and contain a battery of harmful substances and degrading enzymes. A silent neutrophil death, i.e., apoptosis, is therefore of importance to avoid damage to the surrounding tissue and to enable termination of the acute inflammatory process. There is a pile of evidence supporting the role for pro-inflammatory cytokines in extending the life-span of neutrophils, but relatively few studies have been devoted to mechanisms actively driving apoptosis induction in neutrophils. We have previously demonstrated that natural killer (NK) cells can promote apoptosis in healthy neutrophils. In this study, we set out to investigate how neutrophil sensitivity to NK cell-mediated cytotoxicity is regulated under inflammatory conditions. Using in vitro-activated neutrophils and a human skin chamber model that allowed collection of in vivo-transmigrated neutrophils, we performed a comprehensive characterization of neutrophil expression of ligands to NK cell receptors. These studies revealed a dramatic downregulation of HLA class I molecules in inflammatory neutrophils, which was associated with an enhanced susceptibility to NK cell cytotoxicity. Collectively, our data shed light on the complex regulation of interactions between NK cells and neutrophils during an inflammatory response and provide further support for a role of NK cells in the resolution phase of inflammation.
Project description:Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R) injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and ?-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent) transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen) mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and ?-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall.
Project description:Neutrophils, early mediators of the innate immune defense, are recruited to developing thrombi in different types of thrombosis. They amplify intravascular coagulation by stimulating the tissue factor-dependent extrinsic pathway via inactivation of endogenous anticoagulants, enhancing factor XII activation or decreasing plasmin generation. Neutrophil-dependent prothrombotic mechanisms are supported by the externalization of decondensed nucleosomes and granule proteins that together form neutrophil extracellular traps. These traps, either in intact or fragmented form, are causally involved in various forms of experimental thrombosis as first indicated by their role in the enhancement of both microvascular thrombosis during bacterial infection and carotid artery thrombosis. Neutrophil extracellular traps can be induced by interactions of neutrophils with activated platelets; vice versa, these traps enhance adhesion of platelets via von Willebrand factor. Neutrophil-induced microvascular thrombus formation can restrict the dissemination and survival of blood-borne bacteria and thereby sustain intravascular immunity. Dysregulation of this innate immune pathway may support sepsis-associated coagulopathies. Notably, neutrophils and extracellular nucleosomes, together with platelets, critically promote fibrin formation during flow restriction-induced deep vein thrombosis. Neutrophil extracellular traps/extracellular nucleosomes are increased in thrombi and in the blood of patients with different vaso-occlusive pathologies and could be therapeutically targeted for the prevention of thrombosis. Thus, during infections and in response to blood vessel damage, neutrophils and externalized nucleosomes are major promoters of intravascular blood coagulation and thrombosis.
Project description:Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.
Project description:Neutrophils have key roles in ischemic brain injury, thrombosis, and atherosclerosis. As such, neutrophils are of great interest as targets to treat and prevent ischemic stroke. After stroke, neutrophils respond rapidly promoting blood-brain barrier disruption, cerebral edema, and brain injury. A surge of neutrophil-derived reactive oxygen species, proteases, and cytokines are released as neutrophils interact with cerebral endothelium. Neutrophils also are linked to the major processes that cause ischemic stroke, thrombosis, and atherosclerosis. Thrombosis is promoted through interactions with platelets, clotting factors, and release of prothrombotic molecules. In atherosclerosis, neutrophils promote plaque formation and rupture by generating oxidized-low density lipoprotein, enhancing monocyte infiltration, and degrading the fibrous cap. In experimental studies targeting neutrophils can improve stroke. However, early human studies have been met with challenges, and suggest that selective targeting of neutrophils may be required. Several properties of neutrophil are beneficial and thus may important to preserve in patients with stroke including antimicrobial, antiinflammatory, and neuroprotective functions.
Project description:Neutrophils are important effector cells of antimicrobial immunity in an acute inflammatory response, with a primary role in the clearance of extracellular pathogens. However, in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD), there is excessive infiltration and activation of neutrophils, subsequent production of reactive oxygen species, and release of serine proteases, matrix metalloproteinases, and myeloperoxidase-resulting in collateral damage as the cells infiltrate into the tissue. Increased neutrophil survival through dysregulated apoptosis facilitates continued release of neutrophil-derived mediators to perpetuate airway inflammation and tissue injury. Several target mechanisms have been investigated to address pathologic neutrophil biology and thereby provide a novel therapy for respiratory disease. These include neutrophil influx through inhibition of chemokine receptors CXCR2, CXCR1, and PI3K? signaling and neutrophil weaponry by protease inhibitors, targeting matrix metalloproteinases and neutrophil serine proteases. In addition, neutrophil function can be modulated using selective PI3K? inhibitors. This review highlights the latest advances in targeting neutrophils and their function, discusses the opportunities and risks of neutrophil inhibition, and explores how we might better develop future strategies to regulate neutrophil influx and function for respiratory diseases in dire need of novel effective therapies.