Staphylococcus aureus and Neutrophil Extracellular Traps: The Master Manipulator Meets Its Match in Immunothrombosis

Over the past 10 years, neutrophil extracellular traps (NETs) have become widely accepted as an integral player in immunothrombosis, due to their complex interplay with both pathogens and components of the coagulation system. While the release of NETs is an attempt by neutrophils to trap pathogens and constrain infections, NETs can have bystander effects on the host by inducing uncontrolled thrombosis, inflammation, and tissue damage. From an evolutionary perspective, pathogens have adapted to bypass the host innate immune response. Staphylococcus aureus (S. aureus), in particular, proficiently overcomes NET formation using several virulence factors. Here we review mechanisms of NET formation and how these are intertwined with platelet activation, the release of endothelial von Willebrand factor, and the activation of the coagulation system. We discuss the unique ability of S. aureus to modulate NET formation and alter released NETs, which helps S. aureus to escape from the host’s defense mechanisms. We then discuss how platelets and the coagulation system could play a role in NET formation in S. aureus-induced infective endocarditis, and we explain how targeting these complex cellular interactions could reveal novel therapies to treat this disease and other immunothrombotic disorders

Involvement of peptidylarginine deiminase 4 in eosinophil extracellular trap formation and contribution to citrullinated histone signal in thrombi

Background: Extracellular traps formed by neutrophils (NETs) and eosinophils (EETs) have been described in coronary thrombi, contributing to thrombus stability. A key mechanism during NET formation is histone modification by the enzyme PAD4. Citrullinated histones, the product of PAD4 activity, are often attributed to neutrophils. Eosinophils also express high levels of PAD4. Objectives: We aimed to explore the contribution of PAD4 to EET formation. Methods: We performed immunohistological analyses on thrombi, including a large, intact, and eosinophil-containing thrombus retrieved from the right coronary artery using an aspiration catheter and stroke thrombi from thrombectomy retrieval. We studied eosinophils for their capability to form PAD4-dependent EETs in response to strong ET-inducing agonists as well as activated platelets and bacteria. Results: Histopathology and immunofluorescence microscopy identified a coronary thrombus rich in platelets and neutrophils, with distinct areas containing von Willebrand factor and citrullinated histone H3 (H3Cit). Eosinophils were also identified in leukocyte-rich areas. The majority of the H3Cit+ signal colocalized with myeloperoxidase, but some colocalized with eosinophil peroxidase, indicating EETs. Eosinophils isolated from healthy volunteers produced H3Cit+ EETs, indicating an involvement of PAD4 activity. The selective PAD4 inhibitor GSK484 blocked this process, supporting PAD4 dependence of H3Cit+ EET release. Citrullinated histones were also present in EETs produced in response to live Staphylococci. However, limited evidence for EETs was found in mouse models of venous thrombosis or infective endocarditis. Conclusion: As in NETosis, PAD4 can catalyze the formation of EETs. Inhibition of PAD4 decreases EET formation, supporting the future utility of PAD4 inhibitors as possible antithrombotic agents

Neutrophils Protect Against Staphylococcus aureus Endocarditis Progression Independent of Extracellular Trap Release

Background: Infective endocarditis (IE) is characterized by an infected thrombus at the heart valves. How bacteria bypass the immune system and cause these thrombi remains unclear. Neutrophils releasing NETs (neutrophil extracellular traps) lie at this interface between host defense and coagulation. We aimed to determine the role of NETs in IE immunothrombosis. Methods: We used a murine model of Staphylococcus aureus endocarditis in which IE is provoked on inflamed heart valves and characterized IE thrombus content by immunostaining identifying NETs. Antibody-mediated neutrophil depletion and neutrophil-selective PAD4 (peptidylarginine deiminase 4)-knockout mice were used to clarify the role of neutrophils and NETs, respectively. S. aureus mutants deficient in key virulence factors related to immunothrombosis (nucleases or staphylocoagulases) were investigated. Results: Neutrophils releasing NETs were present in infected thrombi and within cellular infiltrates in the surrounding vasculature. Neutrophil depletion increased occurrence of IE, whereas neutrophil-selective impairment of NET formation did not alter IE occurrence. Absence of S. aureus nuclease, which degrades NETs, did not affect endocarditis outcome. In contrast, absence of staphylocoagulases (coagulase and von Willebrand factor binding protein) led to improved survival, decreased bacteremia, smaller infiltrates, and decreased tissue destruction. Significantly more NETs were present in these vegetations, which correlated with decreased bacteria and cell death in the adjacent vascular wall. Conclusions: Neutrophils protect against IE independent of NET release. Absence of S. aureus coagulases, but not nucleases, reduced IE severity and increased NET levels. Staphylocoagulase-induced fibrin likely hampers NETs from constraining infection and the resultant tissue damage, a hallmark of valve destruction in IE

Maresins: novel macrophage mediators with potent antiinflammatory and proresolving actions

The endogenous cellular and molecular mechanisms that control acute inflammation and its resolution are of wide interest. Using self-resolving inflammatory exudates and lipidomics, we have identified a new pathway involving biosynthesis of potent antiinflammatory and proresolving mediators from the essential fatty acid docosahexaenoic acid (DHA) by macrophages (MΦs). During the resolution of mouse peritonitis, exudates accumulated both 17-hydroxydocosahexaenoic acid, a known marker of 17S-D series resolvin (Rv) and protectin biosynthesis, and 14S-hydroxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid from endogenous DHA. Addition of either DHA or 14S-hydroperoxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid to activated MΦs converted these substrates to novel dihydroxy-containing products that possessed potent antiinflammatory and proresolving activity with a potency similar to resolvin E1, 5S,12R,18R-trihydroxyeicosa-6Z,8E,10E,14Z,16E-pentaenoic acid, and protectin D1, 10R,17S-dihydroxydocosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. Stable isotope incorporation, intermediate trapping, and characterization of physical and biological properties of the products demonstrated a novel 14-lipoxygenase pathway, generating bioactive 7,14-dihydroxydocosa-4Z,8,10,12,16Z,19Z-hexaenoic acid, coined MΦ mediator in resolving inflammation (maresin), which enhances resolution. These findings suggest that maresins and this new metabolome may be involved in some of the beneficial actions of DHA and MΦs in tissue homeostasis, inflammation resolution, wound healing, and host defense

Resolving Thrombosis Resting and Polarized Macrophages Differentially Degrade Neutrophil Extracellular Traps

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Immunothrombosis and thromboinflammation in host defense and disease

Platelets are increasingly being recognized for playing roles beyond thrombosis and hemostasis. Today we know that they mediate inflammation by direct interactions with innate immune cells or secretion of cytokines/chemokines. Here we review their interactions with neutrophils and monocytes/macrophages in infection and sepsis, stroke, myocardial infarction and venous thromboembolism. We discuss new roles for platelet surface receptors like GPVI or GPIb and also look at platelet contributions to the formation of neutrophil extracellular traps (NETs) as well as to deep vein thrombosis during infection, e.g. in COVID-19 patients.status: publishe

Neutrophil Extracellular Traps in Arterial and Venous Thrombosis

Thrombotic complications are still a major health risk worldwide. Our view on the pathophysiology of thrombosis has significantly changed since the discovery of neutrophil extracellular traps (NETs) and their prothrombotic characteristics. Generated by neutrophils that release their decondensed chromatin as a network of extracellular fibers, NETs promote thrombus formation by serving as a scaffold that activates platelets and coagulation. The thrombogenic involvement of NETs has been described in various settings of thrombosis, including stroke, myocardial infarction, and deep vein thrombosis. The aim of this review is to summarize existing evidence showing the presence of NETs in human thrombus material. Following an introduction on NETs and their role in thrombus formation, the authors address studies showing the presence of NETs in arterial or venous thrombi. In addition, they focus on potential novel therapeutic opportunities to resolve or prevent thrombosis by targeting NETs.status: publishe

Extracellular DNA NET-Works With Dire Consequences for Health

Neutrophils play a central role in innate immune defense. Advances in neutrophil biology have brought to light the capacity of neutrophils to release their decondensed chromatin and form large extracellular DNA networks called neutrophil extracellular traps (NETs). NETs are produced in response to many infectious and noninfectious stimuli and, together with fibrin, block the invasion of pathogens. However, their formation in inflamed blood vessels produces a scaffold that supports thrombosis, generates neo-antigens favoring autoimmunity, and aggravates damage in ischemia/reperfusion injury. NET formation can also be induced by cancer and promotes tumor progression. Formation of NETs within organs can be immediately detrimental, such as in lung alveoli, where they affect respiration, or they can be harmful over longer periods of time. For example, NETs initiate excessive deposition of collagen, resulting in fibrosis, thus likely contributing to heart failure. Here, we summarize the latest knowledge on NET generation and discuss how excessive NET formation mediates propagation of thrombosis and inflammation and, thereby, contributes to various diseases. There are many ways in which NET formation could be averted or NETs neutralized to prevent their detrimental consequences, and we will provide an overview of these possibilities.status: publishe

Recombinant Human ADAMTS13 Treatment Improves Myocardial Remodeling and Functionality After Pressure Overload Injury in Mice

Background: A disintegrin‐like metalloproteinase with thrombospondin motif type 1 member 13 (ADAMTS13), the von Willebrand factor–cleaving enzyme, decreases leukocyte and platelet recruitment and, thus, reduces thrombosis and inflammation. Recombinant human ADAMTS13 (rhADAMTS13) is a novel drug candidate for ischemia/reperfusion injury and has shown short‐term benefits in mouse models of myocardial injury, but long‐term outcome has not been investigated. Methods and Results: We evaluated the impact of rhADAMTS13 on cardiac remodeling, scarring, and contractile function, under chronic left ventricular pressure overload. The role of von Willebrand factor and the effect of rhADAMTS13 treatment were studied. This model of heart failure, based on ascending aortic constriction, produces a coronary inflammatory response and microvascular dysfunction, resulting in fibrotic remodeling and cardiac failure. Mice were treated with either rhADAMTS13 or vehicle and assessed for coronary vascular inflammation and ventricular function at several postsurgical time points, as well as for cardiac fibrosis after 4 weeks. Early upon induction of pressure overload under rhADAMTS13 treatment, we detected less endothelial‐lumen–associated von Willebrand factor, fewer platelet aggregates, and decreased activated transforming growth factor‐β1 levels than in vehicle‐treated mice. We observed significant preservation of cardiac function and decrease in fibrotic remodeling as a result of rhADAMTS13 administration. Conclusions: Herein, we show that rhADAMTS13 decreases coronary vascular dysfunction and improves cardiac remodeling after left ventricular pressure overload in mice. We propose that this effect may, at least in part, be the result of decreased von Willebrand factor–mediated recruitment of platelets, a major source of the activated profibrotic cytokine transforming growth factor‐β1. Our study further supports the therapeutic potential of rhADAMTS13 for conditions characterized by inflammatory cardiac damage that results in fibrosis

Solid peripheral tumor leads to systemic inflammation, astrocyte activation and signs of behavioral despair in mice.

Prevalence of depression is higher in patients with cancer than in the general population. Sustained systemic inflammation has been associated with depressive behavior and it has been reported that depressed patients commonly display alterations in their immune system. We previously showed that cancer in mice induces a systemic environment that promotes neutrophil activation and leukocytosis. We thus hypothesized that the peripheral systemic response to a solid tumor leads to endothelial activation, which may promote inflammatory changes in the brain with behavioral consequences. Using the Lewis lung carcinoma (LLC) model, we show that tumor growth induces a progressive increase in peripheral inflammation as observed by elevated interleukin-6 (IL-6). In behavioral studies, tumor-bearing mice showed no sign of motor, coordination or short term working memory deficits as assessed by rotarod, balance-beam, and novel object recognition tests. However, there was an impairment in the grip strength test and interestingly, an anxious and despair-like phenotype in the elevated plus-maze, and tail suspension tests, respectively. Immunostaining of perfused brains revealed fibrin accumulation in the vasculature with some leakage into the parenchyma, a process known to activate endothelial cells. Taken together, our results suggest that the inflamed and prothrombotic systemic environment created by the growth of a peripherally-located solid tumor induces endothelial activation, accumulation of fibrin in the brain and astrocyte activation, perhaps leading to depressive-like behavior