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Mycobacterium tuberculosis (Mtb)-infected neutrophils are often found in the airways of patients with active tuberculosis (TB), and excessive recruitment of neutrophils to the lung is linked to increased bacterial burden and aggravated pathology in TB. The basis for the permissiveness of neutrophils for Mtb and the ability to be pathogenic in TB has been elusive. Here, we identified metabolic and functional features of neutrophils that contribute to their permissiveness in Mtb infection. Using single-cell metabolic and transcriptional analyses, we found that neutrophils in the Mtb-infected lung displayed elevated mitochondrial metabolism, which was largely attributed to the induction of activated neutrophils with enhanced metabolic activities. The activated neutrophil subpopulation was also identified in the lung granulomas from Mtb-infected non-human primates. Functionally, activated neutrophils harbored more viable bacteria and displayed enhanced lipid uptake and accumulation. Surprisingly, we found that interferon-γ promoted the activation of lung neutrophils during Mtb infection. Lastly, perturbation of lipid uptake pathways selectively compromised Mtb survival in activated neutrophils. These findings suggest that neutrophil heterogeneity and metabolic diversity are key to their permissiveness for Mtb and that metabolic pathways in neutrophils represent potential host-directed therapeutics in TB., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Streptococcus pneumoniae colonization in the upper respiratory tract is linked to pneumococcal disease development, predominantly affecting young children and older adults. As the global population ages and comorbidities increase, there is a heightened concern about this infection. We investigated the immunological responses of older adults to pneumococcal-controlled human infection by analyzing the cellular composition and gene expression in the nasal mucosa. Our comparative analysis with data from a concurrent study in younger adults revealed distinct gene expression patterns in older individuals susceptible to colonization, highlighted by neutrophil activation and elevated levels of CXCL9 and CXCL10. Unlike younger adults challenged with pneumococcus, older adults did not show recruitment of monocytes into the nasal mucosa following nasal colonization. However, older adults who were protected from colonization showed increased degranulation of cluster of differentiation 8+ T cells, both before and after pneumococcal challenge. These findings suggest age-associated cellular changes, in particular enhanced mucosal inflammation, that may predispose older adults to pneumococcal colonization., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease., Objectives: Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis., Methods: Here we describe a novel, high-throughput ex vivo whole blood-induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model., Results: We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α., Conclusion: These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment., Competing Interests: Declaration of competing interests K.Z., F.S., B.A., J.C., and T.K.K. are paid employees of Volition. M.E. and A.R. are paid consultants of Volition., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Plasmalogens are a class of phospholipids containing vinyl ether linked aliphatic groups at the sn-1 position. Plasmalogens are known to contain 16- and 18-carbon aliphatic groups at the sn-1 position. Here, we reveal that the human neutrophil plasmenylethanolamine pool uniquely includes molecular species with very long carbon chain (VLC) aliphatic groups, including 20-, 22- and 24-carbon vinyl ether linked aliphatic groups at the sn-1 position. We identified these novel VLC plasmalogen species by electrospray ionization mass spectrometry methods. VLC plasmalogens were only found in the neutrophil plasmenylethanolamine pool. During neutrophil activation, VLC plasmenylethanolamines undergo myeloperoxidase-dependent oxidation to produce VLC 2-chlorofatty aldehyde and its oxidation product, 2-chlorofatty acid (2-ClFA). Furthermore, plasma concentrations of VLC 2-ClFA are elevated in human sepsis. These studies demonstrate for the first time VLC plasmenylethanolamine molecular species, their myeloperoxidase-mediated chlorolipid products and the presence of these chlorolipids in human sepsis.

Background: Venous thromboembolism (VTE), particularly unprovoked VTE, is associated with occult cancer. The optimal screening regimen remains controversial. Neutrophil extracellular traps (NETs) are implicated in cancer-associated thrombosis, and elevated biomarkers of NET formation are associated with poor prognosis., Objectives: To investigate the association between NET formation and occult cancer in patients with VTE., Methods: Blood biomarkers associated with NETs and neutrophil activation (nucleosomal citrullinated histone H3 [H3Cit-DNA], cell-free DNA, and neutrophil elastase) were quantified in patients with VTE. The primary outcome was cancer diagnosed during a one-year follow-up., Results: This study included 460 patients with VTE, of which 221 (48%) had isolated deep vein thrombosis. Forty-three patients had active cancer at inclusion and were excluded from the primary analysis Cancer during follow-up was diagnosed in 29 of 417 (7.0%) patients. After adjustment for age and unprovoked VTE, the hazard ratio of cancer during follow-up per 500 ng/mL increase of H3Cit-DNA was 1.79 (95% CI, 1.03-3.10). Furthermore, patients with cancer-associated VTE (known active cancer or cancer diagnosed during follow-up) had higher levels of H3Cit-DNA than cancer-free patients with VTE after adjustment for age, hemoglobin, gender, chronic obstructive pulmonary disease, previous cancer, and start of anticoagulant treatment (odds ratio 2.06 per 500 ng/mL increase of H3Cit-DNA [95% CI, 1.35-3.13])., Conclusions: H3Cit-DNA is an independent predictor for occult cancer in patients with VTE and elevated in cancer-associated VTE, suggesting that H3Cit-DNA is potentially a useful diagnostic marker for cancer in patients with VTE and that elevated NET formation is a hallmark of cancer-associated VTE., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transfusion-related acute lung injury (TRALI) can occur during or after a transfusion, and remains a leading cause of transfusion-associated morbidity and mortality. TRALI is caused by the transfusion of either anti-leukocyte antibodies or biological response modifiers (BRMs). Experimental evidence suggests at least six different pathways that antibody-mediated TRALI might follow: (i) two hit neutrophil activation; (ii) monocyte and neutrophil dependent; (iii) endothelial cell, neutrophil Fc receptor, platelet and neutrophil extracellular trap dependent; (iv) direct monocyte activation; (v) direct endothelial cell activation; and (vi) endothelial cell, complement and monocyte dependent. Two of these pathways (i and v) also apply to BRM-mediated TRALI. Different antibodies or BRMs might initiate different pathways. Even though six pathways are described, these might not be distinct, and might instead be interlinked or proceed concurrently. The different pathways converge upon reactive oxygen species release which damages pulmonary endothelium, precipitating fluid leakage and the clinical symptoms of TRALI. Additional pathways to the six described are likely to also contribute to TRALI pathogenesis, and this requires further investigation. This review also discusses evidence of protective mechanisms and their implications for clinical TRALI treatment. Finally, it suggests directions for future research to support the translation of these findings into strategies to prevent and treat clinical TRALI., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Modulation of neutrophil recruitment and function is crucial for targeting inflammatory cells to sites of infection to combat invading pathogens while, at the same time, limiting host tissue injury or autoimmunity. The underlying mechanisms regulating recruitment of neutrophils, 1 of the most abundant inflammatory cells, have gained increasing interest over the years. The previously described classical recruitment cascade of leukocytes has been extended to include capturing, rolling, adhesion, crawling, and transmigration, as well as a reverse-transmigration step that is crucial for balancing immune defense and control of remote organ endothelial leakage. Current developments in the field emphasize the importance of cellular interplay, tissue environmental cues, circadian rhythmicity, detection of neutrophil phenotypes, differential chemokine sensing, and contribution of distinct signaling components to receptor activation and integrin conformations. The use of therapeutics modulating neutrophil activation responses, as well as mutations causing dysfunctional neutrophil receptors and impaired signaling cascades, have been defined in translational animal models. Human correlates of such mutations result in increased susceptibility to infections or organ damage. This review focuses on current advances in the understanding of the regulation of neutrophil recruitment and functionality and translational implications of current discoveries in the field with a focus on acute inflammation and sepsis., (© 2022 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.)

There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell-specific PKM2-/- mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe-/-). Controls were littermate PKM2fl/flLysMCre- or PKM2fl/flLysMCre-Apoe-/- mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion., (© 2022 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.)

Severe coronavirus disease 2019 (COVID-19) increases the risk of myocardial injury that contributes to mortality. This study used multiparameter immunofluorescence to extensively examine heart autopsy tissue of 7 patients who died of COVID-19 compared to 12 control specimens, with or without cardiovascular disease. Consistent with prior reports, no evidence of viral infection or lymphocytic infiltration indicative of myocarditis was found. However, frequent and extensive thrombosis was observed in large and small vessels in the hearts of the COVID-19 cohort, findings that were infrequent in controls. The endothelial lining of thrombosed vessels typically lacked evidence of cytokine-mediated endothelial activation, assessed as nuclear expression of transcription factors p65 (RelA), pSTAT1, or pSTAT3, or evidence of inflammatory activation assessed by expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tissue factor, or von Willebrand factor (VWF). Intimal EC lining was also generally preserved with little evidence of cell death or desquamation. In contrast, there were frequent markers of neutrophil activation within myocardial thrombi in patients with COVID-19, including neutrophil-platelet aggregates, neutrophil-rich clusters within macrothrombi, and evidence of neutrophil extracellular trap (NET) formation. These findings point to alterations in circulating neutrophils rather than in the endothelium as contributors to the increased thrombotic diathesis in the hearts of COVID-19 patients., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Activation of neutrophils is necessary for the protection of the host against microbial infection. This property can be used as mode of therapy for cancer treatment. Neutrophils have conflicting dual functions in cancer as either a tumor promoter or inhibitor. Neutrophil-based drug delivery has achieved increased attention in pre-clinical models. This review addresses in detail the different neutrophil constituents, the conflicting function of neutrophils and activation of the neutrophil as an important target of therapy for cancer treatment, and use of neutrophils or neutrophil membrane-derived vesicles as vehicles for drug delivery and targeting., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Arteriovenous fistula (AVF) stenosis leading to its failure is a major cause of morbidity in hemodialysis patients; however, detailed pathogenesis of AVF stenosis is still under investigation. To date, monocytes/macrophages have been considered pivotal players in chronic inflammation of vascular disease including atherosclerosis and AVF stenosis. However, recent evidence strongly suggests that neutrophils and neutrophil granule proteins are important contributors to vascular disease. The aim of the present study was to evaluate the relationship between AVF stenosis and neutrophil activation by measuring circulating levels of neutrophil elastase (NE) and lactoferrin, enzymes released on neutrophil activation, as well as other inflammation markers including neutrophil counts., Methods: This was a single-center, prospective observational study conducted on 83 prevalent hemodialysis patients with AVF. Blood levels of biomarkers and sonography (US) measurement were assessed at baseline and 1 year after enrollment. Clinical follow-up continued for one more year (a total of 2 years for each patient) to observe any AVF events., Results: Circulating levels of both NE and lactoferrin positively correlated with the degree of AVF stenosis. Patients with significant AVF stenosis had older AVFs, higher neutrophil-to-lymphocyte ratio (NLR), and higher circulating levels of NE and lactoferrin. On multivariate logistic regression analysis, both circulating levels of NE and NLR remained independent predictors of significant AVF stenosis., Conclusions: Circulating levels of NE and the NLR were identified as independent predictors of at-risk AVF with significant stenosis. Our data suggest the potential role of neutrophil and innate immunity activation on the development of AVF stenosis., (Copyright © 2021. Published by Elsevier Inc.)

Recent studies suggest an essential role of the innate immune effector cells neutrophils and monocytes in protection or disease progression in the early course of Leishmania infection. In areas endemic for cutaneous leishmaniasis in Ethiopia most individuals are exposed to bites of infected sandflies. Still only a minor ratio of the inhabitants develops symptomatic disease. Neutrophils, followed by monocytes, are the first cells to be recruited to the site of Leishmania infection, the initial response of neutrophils to parasites appears to be crucial for the protective response and disease outcome. Our working hypothesis is that neutrophils and/or monocytes in localized cutaneous leishmaniasis (LCL) patients may have defects in function of innate immune cell that contribute to failure to parasite clearance that lead to establishment of infection. The response of cells in Ethiopian LCL patients and healthy controls to Leishmania aethiopica and to the Toll like receptor (TLR) agonists lipopolysaccharide (LPS) and macrophage activating lipopeptide-2 (MALP-2) was investigated by assessing the cell surface expression of CD62L (on neutrophil and monocyte) and CD66b (only on neutrophil), as well as reactive oxygen species (ROS) production by using whole blood-based assays in vitro. No impaired response of neutrophils and monocytes to the microbial constituents LPS and MALP-2 was observed. Neutrophils and monocytes from LCL patients responded stronger to Leishmania aethiopica in the applied whole blood assays than cells from healthy individuals. These experimental findings do not support the hypothesis regarding a possible dysfunction of neutrophils and monocytes in cutaneous leishmaniasis. On the contrary, these cells react stronger in LCL patients as compared to healthy controls. The differential response to L. aethiopica observed between LCL patients and healthy controls have the potential to serve as biomarker to develop FACS based diagnostic/ prognostic techniques for LCL., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Peptidyl arginine deiminase 4 (PAD4) is an enzyme that converts arginine into citrulline. PAD4 is expressed in neutrophils that, when activated, can drive the formation of neutrophil extracellular traps (NETs). Uncontrolled activation of PAD4 and subsequent citrullination of proteins is increasingly recognized as a driver of (auto)immune diseases. Currently, our understanding of PAD4 structure-function relationships and activity control in vivo is incomplete., Aims: To provide the current state-of-the-art on PAD4 structure-activity relationships and involvement of PAD4 in autoimmune disorders as well as in thrombo-inflammatory disease., Materials & Methods: Literature review and molecular modelling Results: In this review, we used molecular modelling to generate a three-dimensional structure of the complete PAD4 molecule. Using our model, we discuss the catalytic conversion of the arginine substrate to citrulline. Besides mechanistic insight into PAD4 function, we give an overview of biological functions of PAD4 and mechanisms that influence its activation. In addition, we discuss the crucial role of PAD4-mediated citrullination of histones during the formation of NETs. Subsequently, we focus on the role of PAD4-mediated NET formation and its role in pathogenesis of rheumatoid arthritis, sepsis and (immune-)thrombosis. Finally, we summarize current efforts to design different classes of PAD4 inhibitors that are being developed for improved treatment of autoimmune disorders as well as thrombo-inflammatory disease., Discussion: Advances in PAD4 structure-function are still necessary to gain a complete insight in mechanisms that control PAD4 activity in vivo. The involvement of PAD4 in several diseases signifies the need for a PAD4 inhibitor. Although progress has been made to produce an isotype specific and potent PAD4 inhibitor, currently no PAD4 inhibitor is ready for clinical use., Conclusion: More research into PAD4 structure and function and into the regulation of its activity is required for the development of PAD4 specific inhibitors that may prove vital to combat and prevent autoimmune disorders and (thrombo)inflammatory disease., (© 2021 School for Cardiovascular Diseases Maastricht University. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Bacterial pneumonia is a significant cause of morbidity, mortality, and health care expenditures. Optimum neutrophil recruitment and their function are critical defense mechanisms against respiratory pathogens. The nucleotide-binding oligomerization domain-like receptor (NLRP) 6 controls gut microbiota and immune response to systemic and enteric infections. However, the importance of NLRP6 in neutrophil homeostasis following lung infection remains elusive. To investigate the role of NLRs in neutrophil homeostasis, we used Nlrp6 gene-deficient (Nlrp6 -/- ) mice in a model of Klebsiella pneumoniae-induced pneumonia-derived sepsis. We demonstrated that NLRP6 is critical for host survival, bacterial clearance, neutrophil influx, and CXC-chemokine production. Kp-infected Nlrp6 -/- mice have reduced numbers of hematopoietic stem cells and granulocyte-monocyte progenitors but increased retention of matured neutrophils in bone marrow. Neutrophil extracellular trap (NET) formation and NET-mediated bacterial killing were also impaired in Nlrp6 -/- neutrophils in vitro. Furthermore, recombinant CXCL1 rescued the impaired host defense, granulopoietic response, and NETosis in Kp-infected Nlrp6 -/- mice. Using A/J background mice and co-housing experiments, our findings revealed that the susceptible phenotype of Nlrp6 -/- mice is not strain-specific and gut microbiota-dependent. Taken together, these data unveil NLRP6 as a central regulator of neutrophil recruitment, generation, and function during bacterial pneumonia followed by sepsis.

Objectives: Critical illness in COVID-19 is attributed to an exaggerated host immune response. Since neutrophils are the major component of innate immunity, we hypothesize that the quantum of activated neutrophils in the blood may predict an adverse outcome., Design: In a retrospective study of 300 adult patients with confirmed COVID-19, we analyzed the impact of neutrophil activation (NEUT-RI), interleukin-6 (IL-6) and the established clinical risk factors of age, diabetes, obesity and hypertension on the clinical outcome., Results: Significant predictors of the need for mechanical ventilation were NEUT-RI (Odds Ratio (OR) = 1.22, P < 0.001), diabetes (OR = 2.56, P = 0.00846) and obesity (OR = 6.55, P < 0.001). For death, the significant predictors were NEUT-RI (OR = 1.14, P = 0.00432), diabetes (OR = 4.11, P = 0.00185) and age (OR = 1.04, P = 0.00896). The optimal cut-off value for NEUT-RI to predict mechanical ventilation and death was 52 fluorescence intensity units (sensitivity 44%, specificity 88%, area under the curve 0.67 and 44%, 86%, 0.64, respectively)., Conclusion: This finding supports an aberrant neutrophil response in COVID-19, likely due to uncontained viral replication, tissue hypoxia and exacerbated inflammation, introduces a novel biomarker for rapid monitoring and opens new avenues for therapeutic strategies., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dysregulated neutrophil (PMN) transmigration across epithelial surfaces (TEpM) significantly contributes to chronic inflammatory diseases, yet mechanisms defining this process remain poorly understood. In the intestine, uncontrolled PMN TEpM is a hallmark of disease flares in ulcerative colitis. Previous in vitro studies directed at identifying molecular determinants that mediate TEpM have shown that plasma membrane proteins including CD47 and CD11b/CD18 play key roles in regulating PMN TEpM across monolayers of intestinal epithelial cells. Here, we show that CD47 modulates PMN TEpM in vivo using an ileal loop assay. Importantly, using novel tissue-specific CD47 knockout mice and in vitro approaches, we report that PMN-expressed, but not epithelial-expressed CD47 plays a major role in regulating PMN TEpM. We show that CD47 associates with CD11b/CD18 in the plasma membrane of PMN, and that loss of CD47 results in impaired CD11b/CD18 activation. In addition, in vitro and in vivo studies using function blocking antibodies support a role of CD47 in regulating CD11b-dependent PMN TEpM and chemotaxis. Taken together, these findings provide new insights for developing approaches to target dysregulated PMN infiltration in the intestine. Moreover, tissue-specific CD47 knockout mice constitute an important new tool to study contributions of cells expressing CD47 to inflammation in vivo.

In addition to their well characterized role in mediating IgE-dependent allergic diseases, aberrant accumulation and activation of mast cells (MCs) is associated with many non-allergic inflammatory diseases, whereby their activation is likely triggered by non-IgE stimuli (e.g., IL-33). Siglec-8 is an inhibitory receptor expressed on MCs and eosinophils that has been shown to inhibit IgE-mediated MC responses and reduce allergic inflammation upon ligation with a monoclonal antibody (mAb). Herein, we evaluated the effects of an anti-Siglec-8 mAb (anti-S8) in non-allergic disease models of experimental cigarette-smoke-induced chronic obstructive pulmonary disease and bleomycin-induced lung injury in Siglec-8 transgenic mice. Therapeutic treatment with anti-S8 inhibited MC activation and reduced recruitment of immune cells, airway inflammation, and lung fibrosis. Similarly, using a model of MC-dependent, IL-33-induced inflammation, anti-S8 treatment suppressed neutrophil influx, and cytokine production through MC inhibition. Transcriptomic profiling of MCs further demonstrated anti-S8-mediated downregulation of MC signaling pathways induced by IL-33, including TNF signaling via NF-κB. Collectively, these findings demonstrate that ligating Siglec-8 with an antibody reduces non-allergic inflammation and inhibits IgE-independent MC activation, supporting the evaluation of an anti-Siglec-8 mAb as a therapeutic approach in both allergic and non-allergic inflammatory diseases in which MCs play a role.

Background: Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening blood disorder, primarily resulting from autoantibodies against ADAMTS13. Infection or inflammation often precedes acute iTTP. However, the association of inflammation and inflammatory mediators with disease severity and outcome of acute iTTP is not fully assessed., Objectives: Here, we determined plasma levels of S100A8/A9, histone/DNA complexes, citrullinated histone H3 (CitH3), and cell-free DNA (cfDNA) in a cohort of 108 acute episodes from 94 unique iTTP patients and healthy controls, and assessed the association of each of these biomarkers with the disease severity and mortality., Results: All acute iTTP patients had significantly increased plasma levels of S100A8/A9 (median 84.8, interquartile range [IQR] 31.2-157.4 µg/mL), histone/DNA complexes (median 55.7, IQR 35.8-130.8 U/mL), CitH3 (median 3.8, IQR 2.2-6.4 ng/mL), and cfDNA (median 937.7, IQR 781.3-1420.0 ng/mL) on the admission blood samples when compared with healthy controls. An increased plasma level of S100A8/A9, histone/DNA complex and cfDNA was associated with organ damage, coagulopathy, and mortality in iTTP. After being adjusted for age and history of hypertension, Cox proportional hazard regression analysis demonstrated that a hazard ratio (95% confidence interval) for an elevated plasma level of S100A8/A9, histone/DNA complexes, and cfDNA was 11.5 (1.4-90.9) (P = .021), 10.3 (2.7-38.5) (P = .001), and 12.8 (3.9-42.0) (P = .014), respectively., Conclusion: These results indicate that inflammation or plasma inflammatory mediators such as S100A8/A9 or NETosis markers such as histone/DNA complexes and cfDNA may play a role in pathogenesis of iTTP, which may help stratify patients with a high risk of death during acute iTTP episodes., (© 2020 International Society on Thrombosis and Haemostasis.)

Activated polymorphonuclear neutrophils play an important role in host defense system against invading pathogens via releasing reactive oxygen species through respiratory bursts. Nowadays, neutrophil activation process has been found to be modeled as a two-stages continuum from dormant to primed, and to fully activated. Although the differences of salivary and circulatory neutrophils have been investigated in various experimental designs, priming state of salivary polymorphonuclear neutrophils with respiratory burst has not been comprehensively studied. Here, for the first time so far, a programmable flow injection analysis based on Stop in Flow Cell operation mode has been applied to batch analyzing comparative study of neutrophil activation. The high-sensitivity luminol-dependent chemiluminescence assay has been employed with just 330 μL of cell suspension consumption per sample, and the process has been conducted rapidly and efficiently within 15 min to keep the neutrophils viability. ROS from either unstimulated or substimulated or stimulated salivary polymorphonuclear neutrophils, in comparison with those from autologous circulatory polymorphonuclear neutrophils, have been determined, together with further monitoring the impact of extracellular and intracellular calcium ion (Ca 2+ ) on neutrophils ROS generation. Conclusively, the priming state of salivary polymorphonuclear neutrophils during activation has been testified from multiple aspects., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this paper, a model is proposed of the pathophysiological processes of COVID-19 starting from the infection of human type II alveolar epithelial cells (pneumocytes) by SARS-CoV-2 and culminating in the development of ARDS. The innate immune response to infection of type II alveolar epithelial cells leads both to their death by apoptosis and pyroptosis and to alveolar macrophage activation. Activated macrophages secrete proinflammatory cytokines and chemokines and tend to polarise into the inflammatory M1 phenotype. These changes are associated with activation of vascular endothelial cells and thence the recruitment of highly toxic neutrophils and inflammatory activated platelets into the alveolar space. Activated vascular endothelial cells become a source of proinflammatory cytokines and reactive oxygen species (ROS) and contribute to the development of coagulopathy, systemic sepsis, a cytokine storm and ARDS. Pulmonary activated platelets are also an important source of proinflammatory cytokines and ROS, as well as exacerbating pulmonary neutrophil-mediated inflammatory responses and contributing to systemic sepsis by binding to neutrophils to form platelet-neutrophil complexes (PNCs). PNC formation increases neutrophil recruitment, activation priming and extraversion of these immune cells into inflamed pulmonary tissue, thereby contributing to ARDS. Sequestered PNCs cause the development of a procoagulant and proinflammatory environment. The contribution to ARDS of increased extracellular histone levels, circulating mitochondrial DNA, the chromatin protein HMGB1, decreased neutrophil apoptosis, impaired macrophage efferocytosis, the cytokine storm, the toll-like receptor radical cycle, pyroptosis, necroinflammation, lymphopenia and a high Th17 to regulatory T lymphocyte ratio are detailed., Competing Interests: Declaration of competing interest MB is supported by a NHMRC Senior Principal Research Fellowship (1059660 and 1156072). MB has received Grant/Research Support from the NIH, Cooperative Research Centre, Simons Autism Foundation, Cancer Council of Victoria, Stanley Medical Research Foundation, Medical Benefits Fund, National Health and Medical Research Council, Medical Research Futures Fund, Beyond Blue, Rotary Health, A2 milk company, Meat and Livestock Board, Woolworths, Avant and the Harry Windsor Foundation, has been a speaker for Astra Zeneca, Lundbeck, Merck, Pfizer, and served as a consultant to Allergan, Astra Zeneca, Bioadvantex, Bionomics, Collaborative Medicinal Development, Lundbeck Merck, Pfizer and Servier – all unrelated to this work. LO is supported by a NHMRC Early Career Fellowship (1158487). WM is currently funded by an Alfred Deakin Postdoctoral Research Fellowship and a Multiple Sclerosis Research Australia early-career fellowship. WM has previously received funding from the Cancer Council Queensland and university grants/fellowships from La Trobe University, Deakin University, University of Queensland, and Bond University. WM has received industry funding and has attended events funded by Cobram Estate Pty. Ltd. WM has received travel funding from Nutrition Society of Australia. WM has received consultancy funding from Nutrition Research Australia. WM has received speaker honoraria from The Cancer Council Queensland and the Princess Alexandra Research Foundation. The Food & Mood Centre has received Grant/Research support from Fernwood Foundation, Wilson Foundation, the A2 Milk Company, and Be Fit Foods. AO is supported by a Future Leader Fellowship (#101160) from the Heart Foundation Australia and Wilson Foundation. She has received research funding from National Health & Medical Research Council, Australian Research Council, University of Melbourne, Deakin University, Sanofi, Meat and Livestock Australia and Woolworths Limited and Honoraria from Novartis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

ANCA vasculitis is an autoimmune disease with increased expression of the autoantigen genes, myeloperoxidase (MPO) and proteinase 3 (PRTN3), but the origin and significance of expression is less distinct. To clarify this, we measured MPO and PRTN3 messenger RNA in monocytes, normal-density neutrophils, and in enriched leukocytes from peripheral blood mononuclear cells. Increased autoantigen gene expression was detected in normal-density neutrophils and enriched leukocytes from patients during active disease compared to healthy individuals, with the largest difference in enriched leukocytes. RNA-seq of enriched leukocytes comparing active-remission pairs identified a gene signature for low-density neutrophils. Cell sorting revealed low-density neutrophils contained mature and immature neutrophils depending on the presence or absence of CD10. Both populations contributed to autoantigen expression but the frequency of immature cells in low-density neutrophils did not correlate with low-density neutrophil MPO or PRTN3 expression. Low-density neutrophils were refractory to MPO-ANCA induced oxidative burst, suggesting an alternative role for low-density neutrophils in ANCA vasculitis pathogenesis. In contrast, normal-density neutrophils were activated by MPO-ANCA and monoclonal anti-PR3 antibody. Normal-density neutrophil activation correlated with MPO and PRTN3 mRNA. Increased autoantigen gene expression originating from the mature low-density and normal-density neutrophils suggests transcriptional dysregulation is a hallmark of ANCA vasculitis. Thus, the correlation between autoantigen gene expression and antibody-mediated normal-density neutrophil activation connects autoantigen gene expression with disease pathogenesis., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Neutrophils are important phagocytic cells for host defense against pathogens. They are rapidly recruited to the site of infection, release antimicrobial peptides and cytokines, and engulf and kill microbes. Neutrophils also accumulate in allergic inflammatory sites. Here we characterized neutrophil accumulation in the nasal mucosa using a mouse model of allergic rhinitis, in which mice were sensitized by intraperitoneal injection of ovalbumin (OVA) and then challenged by intranasal administration of OVA or PBS. In the nasal mucosa of both PBS- and OVA-challenged mice, we found a cell subset expressing the eosinophil marker Siglec-F in the Ly-6G + neutrophil population. Morphological analysis of the sorted Ly-6G + Siglec-F + cells revealed that they were devoid of eosinophilic granules in the cytosol and were apparently neutrophils, but compared to conventional Ly-6G + Siglec-F - neutrophils, they had a more lobulated, "botryoid" nucleus. Siglec-F + neutrophils were barely found in the nasopharynx-associated lymphoid tissue, cervical lymph nodes, the spleen, or blood. Both Siglec-F + neutrophils and conventional neutrophils showed increased numbers in the nasal mucosa of OVA-challenged mice. Compared to conventional Siglec-F - neutrophils, Siglec-F + neutrophils exhibited an activated phenotype and enhanced effector functions. Taken together, our findings identify Siglec-F + neutrophils as a novel neutrophil subset with an activated phenotype that resides specifically in the nasal mucosa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The aim of this study was to evaluate neutrophil extracellular traps (NETs) in kidney transplant recipients (KTR) and their potential involvement in acute antibody-mediated rejection (AAMR)., Methods: We studied 3 groups: KTR with AAMR (KTR-Cases, n = 14); KTR without any immunologic event (KTR-Controls, n = 14) and donors (n = 12). Spontaneous and lipopolysaccharide-induced NETosis were evaluated by immunofluorescence indirect (IFI) (NET/cells ratio). Plasmatic cH3-DNA complexes were evaluated by ELISA, (Optic Density Index - ODI). The expression of MPO and citrullinated histone 4 (cH4) was evaluated in renal biopsies., Results: We found an enhanced spontaneous NETosis in KTR regardless of whether they had rejection. The Nets/cells ratio in spontaneous NETosis was 0.203 (IQR 0.12-0.34) in Total-KTR and 0.094 (IQR 0.01-0.17) in donors, p = .011. Likewise, the ODI of cH3-DNA was 1.41 (IQR 0.94-1.72) in Total-KTR, and 0.95 (IQR 0.83-1.27) in donors, p = .019. KTR-Cases had the higher amount of NETs 1.70 (IQR 1.19-1.91). In two KTR-Cases, expression of MPO and cH4 was found in biopsies., Conclusions: KTR show enhanced NETosis. This may indicate a permanent activation of neutrophils. Although more studies are needed, the higher amount of NETs and netting neutrophils in biopsies of KTR-Cases suggest a role of NETosis in AAMR., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2Y 2 R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFAR2 when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free calcium ions (Ca 2+ ). Furthermore, following AZ1729/Cmp58 activation, the signaling by the desensitized FFAR2s was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca 2+ . The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFAR2 modulators represent a new regulatory mechanism that controls receptor signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Acute intestinal inflammation includes the early accumulation of neutrophils (PMN). Based on recent evidence that PMN infiltration "imprints" changes in the local tissue environment through local oxygen depletion and the release of adenine nucleotides, we hypothesized that the interaction between transmigrating PMN and intestinal epithelial cells (IECs) results in inflammatory acidification of the tissue. Using newly developed tools, we revealed that active PMN transepithelial migration (TEM) significantly acidifies the local microenvironment, a decrease of nearly 2 pH units. Using unbiased approaches, we sought to define acid-adaptive pathways elicited by PMN TEM. Given the significant amount of adenosine (Ado) generated during PMN TEM, we profiled the influence of Ado on IECs gene expression by microarray and identified the induction of SLC26A3, the major apical Cl - /HCO 3 - exchanger in IECs. Utilizing loss- and gain-of-function approaches, as well as murine and human colonoids, we demonstrate that Ado-induced SLC26A3 promotes an adaptive IECs phenotype that buffers local pH during active inflammation. Extending these studies, chronic murine colitis models were used to demonstrate that SLC26A3 expression rebounds during chronic DSS-induced inflammation. In conclusion, Ado signaling during PMN TEM induces an adaptive tissue response to inflammatory acidification through the induction of SLC26A3 expression, thereby promoting pH homeostasis.

Mucosal barriers segregate commensal microbes from the intestinal epithelia to maintain gut homeostasis. Ly6/Plaur domain-containing 8 (Lypd8), a highly glycosylated glycosylphosphatidylinositol-anchored protein selectively expressed on colonic enterocytes, promotes this segregation by inhibiting bacterial invasion of the inner mucus layer and colonic epithelia. However, it remains unclear whether Lypd8 prevents infection with enteric bacterial pathogens. Here, we demonstrate that Lypd8 strongly contributes to early-phase defense against Citrobacter rodentium, which causes colitis by inducing attachment and effacement (A/E) lesions on colonic epithelia. Lypd8 inhibits C. rodentium attachment to intestinal epithelial cells by binding to intimin, thereby suppressing the interaction between intimin and translocated intimin receptor. Lypd8 deficiency leads to rapid C. rodentium colonization in the colon, resulting in severe colitis with Th17-cell and neutrophil expansion in the lamina propria. This study identifies a novel function for Lypd8 against A/E bacteria and highlights the role of enterocytes as crucial players in innate immunity for protection against enteric bacterial pathogens.

Introduction: Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated "load & go" flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting., Methods: Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65 years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® "load & go" flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20 min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation., Results: The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at T = 0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1 h (T = 1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) p = .028, CD11b: 2.5× (1.7×-3.1×) p = .023, CD10: 2.5× (2.1×-2.7×) p = .009) and a further increase in activation markers was observed after 2 and 3 h. Monocytes also showed a increase in activation markers in 1 h (mean (95%Cl) CD35: 1.8× (1.3×-2.2×) p = .058, CD11b: 2.13× (1.6×-2.4×) p = .025) and also a further significant increase in 2 and 3 h was observed., Conclusion: This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2 h to only 15-20 min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. FUND: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Neutrophil extracellular traps (NETs) are web-like extrusions of genetic material, which are released upon neutrophil activation. NETs consist of a chromatin substructure, onto which a vast array of proteins with various properties is dispersed. NETs production was initially described as an unrecognized defense mechanism of neutrophils, due to their ability to entrap and possibly eliminate a wide range of pathogens. Nevertheless, growing evidence suggests that NETs are implicated in a multitude of pathophysiological conditions, such as autoimmunity, cancer, diabetes mellitus and Alzheimer's disease. Importantly, NETs may also play a decisive role in atherosclerosis and thrombosis. In this context, it has been demonstrated that NETs are present in atherosclerotic lesions of both humans and animal models and are implicated in various mechanisms leading to atherogenesis. Among others, NETs induce oxidative stress and oxidize high-density lipoprotein particles, thus reducing their beneficial cholesterol efflux capacity. NETs also induce endothelial cell dysfunction and apoptosis and promote the generation of anti-double-stranded-DNA autoantibodies. NETs may also play a prothrombotic role, since they form a fibrin-like base for platelet adhesion, activation and aggregation. Furthermore, NETs promote the accumulation of prothrombotic molecules, like von Willebrand factor and fibrinogen, thus significantly contributing to thrombus formation. Notably, there is vast data linking NETs to arterial and venous thrombosis in animal models, as well as in humans. Future large-scale studies should incorporate NETs and their individual components as disease markers, as well as potential therapeutic targets, to reduce atherosclerosis and to prevent thrombosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Collagen derived materials offer distinct advantages over synthetic polymers, considering their natural inherited biocompatibility and mechanical properties. However, because of the extraction procedure, the latter frequently need to be enhanced through the use of different crosslinking methods. Aldehydes are often used for the stabilization of biomaterials but the introduction of crosslinkers slightly alters the protein's surface reactivity hence calling for new biocompatibility studies. At the same time, silicate modification of natural polymers has gained interest within the biomaterials field for their strengthening potential and ease of manipulation, giving rise to different surfaces and bulk materials. In the present work, collagen gels modified with glutaraldehyde (ColGA) or glutaraldehyde and an aminosilane (ColGASi) were evaluated in vitro and in vivo with the aim to obtain biomaterials for wound dressings. The results obtained were compared to those derived from unmodified collagen matrices (Col). In vitro assays focused on the interaction of the materials with elements present in the human blood whereas in vivo assays evaluated their ability to support cell proliferation and angiogenesis for a period of 30 days in a rodent model. Col gels induced an increase in platelet aggregation while ColGA gels decreased it. On the other hand, ColGASi had no effect on platelet aggregation but induced IL-1β and nitric oxide platelet secretion. All gels induced lower IL-6 levels in PMN cells cultures when compared to controls. Col and ColGA gels decreased IL-1β concentration whereas ColGASi induced high expression of TGF-β in PMN cells. All gels decreased nitric oxide secretion but Col and ColGA gels increased IL-1β production by monocytes. Definitely, all gels induced an anti and pro-inflammatory profile depending on the cell type with which they interact. In vivo, an increased cellular infiltration was observed along with new blood vessel formation in those matrices containing silicified collagen, while glutaraldehyde fixed collagen induced a foreign body reaction and appeared surrounded by a fibrous capsule after 30 days of subcutaneous implantation. Overall, the results obtained show that the silicification of collagen has advantages not only through the enhancement of its mechanical properties but also through the stimulation of the integration of the material with the surrounding tissue., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tumor progression locus 2 (TPL2) is a serine/threonine kinase that belongs to the MAP3K family. The activated TPL2 regulates the innate immune-relevant signaling pathways, such as ERK, JNK, and NF-κB, and the differentiation of immune cells, for example, CD4+ T and NK cells. Therefore, TPL2 plays a critical role in regulating the innate immune response. The present review summarizes the recent advancements in the TPL2-regulated innate immune response., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Human T cell lymphotropic Virus type-1 (HTLV-1) induces lymphocyte activation and proliferation, but little is known about the innate immune response due to HTLV-1 infection. We evaluated the percentage of neutrophils that metabolize Nitroblue tetrazolium (NBT) to formazan in HTLV-1 infected subjects and the association between neutrophil activation and IFN-gamma and TNF-alpha levels. Blood was collected from 35 HTLV-1 carriers, from 8 patients with HAM/TSP (HTLV-1- associated myelopathy); 22 healthy individuals were evaluated for spontaneous and lipopolysaccharide (LPS)-stimulated neutrophil activity (reduction of NBT to formazan). The production of IFN-gamma and TNF-alpha by unstimulated mononuclear cells was determined by ELISA. Spontaneous NBT levels, as well as spontaneous IFN-gamma and TNF-alpha production, were significantly higher (p

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def ++ ) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def ++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def ++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def ++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis., (© 2019 by The American Society of Hematology.)

Background: Recent evidence suggests that adaptive immunity develops during abdominal aortic aneurysm evolution. Uncertainties remain about the antigens implicated and their role in inducing rupture. Because antigens from the extracellular matrix (ECM) have been suspected, the aim of this experimental study was to characterize the role of adaptive immunity directed against antigens from the aortic ECM., Methods: In a first step, an experimental model of abdominal aortic aneurysm rupture based on adaptive immunity against the ECM was developed and characterized. Forty 4-week-old male Lewis rats were divided into two groups. In the ECM group (n = 20), rats were presensitized against the guinea pig aortic ECM before implantation of a decellularized aortic xenograft (DAX). In the control group (n = 20), rats were not presensitized before DAX implantation. In each group, half the rats were sacrificed at day 3 to analyze early mechanisms involved after DAX implantation. In a second step, we aimed to assess which ECM component was most efficient in inducing rupture. For this purpose, the nonfibrillar and fibrillar ECM components were sequentially extracted from the guinea pig aortic wall. Forty Lewis rats were then divided into four groups. Each group was presensitized against one ECM component (structural glycoproteins and proteoglycans, collagen, elastin alone, and elastin-associated glycoproteins) before DAX implantation. Apart from those that experienced rupture, rats were sacrificed at day 21. Xenografts were harvested for histologic, immunofluorescence, and conditioned medium analyses., Results: In total, early aortic rupture occurred in 80% of the ECM group vs 0% of the control group (P < .001). In the ECM group, major circumferential immunoglobulin deposits were observed in combination with the C3 complement fraction, without cell infiltration. Conditioned medium analysis revealed that matrix metalloproteinase 9 and myeloperoxidase levels and elastase activities were significantly increased in this group. Immunofluorescence analysis demonstrated that myeloperoxidase co-localized with tissue-free DNA and histone H4, highlighting local neutrophil activation and formation of neutrophil extracellular traps. Following differential presensitization, it appeared that rats presensitized against structural glycoproteins and proteoglycans were significantly more susceptible to rupture after DAX implantation., Conclusions: Stimulating adaptive immunity against the aortic ECM, especially structural glycoproteins and proteoglycans, triggers rupture after DAX implantation. Further studies are needed to assess the precise proteins involved., (Copyright © 2018 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Neutrophils are recruited to infection sites and kill bacteria by phagocytosis and reactive oxygen species (ROS) production. It has been reported that vacuoles are present in neutrophils that produce ROS and are present in large numbers in blood smears of patients with bacterial infections. The leukocyte differentiation function on the Sysmex automated hematology analyzer classifies leukocytes by flow cytometry. Particularly, side-scattered light is known to reflect the quantity of organelles. This study investigated the possibility of detecting vacuoles or invagination of cell membrane in neutrophils producing ROS using a hematology analyzer. Whole blood and polymorphonuclear (PMN) cell fractions were activated with phorbol myristate acetate (PMA) or formylmethionylleucylphenylalanine (fMLP) and analyzed using the Sysmex XE-2100 automated hematology analyzer. PMN fractions were morphologically analyzed with a confocal laser scanning microscope (CLSM), electron microscope (EM), and general-purpose conventional flow cytometer. In the white blood cell differentiation scattergram obtained in this analysis, a new cluster separate from the original neutrophil cluster appeared in the eosinophil area in an area of higher side-scattering (SSC) intensity. Flow cytometry analysis of the PMN fractions revealed that the cells in this new cluster were CD16b- and APF-positive, indicating that the cells were activated neutrophils that produced ROS. CLSM and EM findings revealed that ROS production occurred in the cytoplasm and that the activated neutrophils contained some vacuole-like structures of vacuoles or invagination of cell membrane. Vacuole-like Sstructures were found within the cytoplasm of neutrophils producing ROS. These neutrophils were detected as an independent cluster in the eosinophil area with higher SSC intensity than that shown by neutrophils in the traditional cluster on the white blood cell differentiation scattergram, likely because the vacuole-like structures increased the SSC intensity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

The prospects for complement-targeted therapy in ANCA-associated vasculitis have been enhanced by a recent clinical trial in which C5a receptor 1 (C5aR1) inhibition safely replaced glucocorticoids in induction treatment. C5aR1 primes neutrophils for activation by anti-neutrophil cytoplasmic antibody (ANCA) and is therefore required in models of glomerulonephritis induced by anti-myeloperoxidase antibody. Although humoral and cellular autoimmunity play essential roles in ANCA-associated vasculitis, a role for C5aR1 in these responses has not been described. Here, we use murine models to dissect the role of C5aR1 in the generation of anti-myeloperoxidase autoimmunity and the effector responses resulting in renal injury. The genetic absence or pharmacological inhibition of C5aR1 results in reduced autoimmunity to myeloperoxidase with an attenuated Th1 response, increased Foxp3 + regulatory T cells and reduction in generation of myeloperoxidase-ANCA. These changes are mediated by C5aR1 on dendritic cells, which promotes activation, and thus myeloperoxidase autoimmunity and glomerulonephritis. We also use renal intravital microscopy to determine the effect of C5aR1 inhibition on ANCA induced neutrophil dysfunction. We found that myeloperoxidase-ANCA induce neutrophil retention and reactive oxygen species burst within glomerular capillaries. These pathological behaviors are abrogated by C5aR1 inhibition. Thus, C5aR1 inhibition ameliorates both autoimmunity and intra-renal neutrophil activation in ANCA-associated vasculitis., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Myeloperoxidase produces the two-electron oxidant HOCl, which targets plasmalogen phospholipids liberating 2-chlorofatty aldehyde. 2-Chlorofatty aldehyde has four known fates: 1) oxidation to 2-chlorofatty acid; 2) reduction to 2-chlorofatty alcohol; 3) Schiff base adduct formation with proteins and amines; and 4) reactivity with glutathione through nucleophilic attack of the α-chlorinated carbon. 2-Chlorofatty acid does not undergo conventional fatty acid β-oxidation due to the presence of the α-chlorinated carbon; however, 2-chlorofatty acid does undergo sequential ω-oxidation and β-oxidation from the ω-end, ultimately resulting in 2-chloroadipic acid urinary excretion. Recent studies have demonstrated that 2-chlorofatty acid clearance is increased by treatment with the PPAR-α agonist WY14643, which increases the enzymatic machinery responsible for hepatic ω-oxidation. Furthermore, 2-chlorofatty acid has been shown to be a PPAR-α agonist, and thus accelerates its own clearance. The roles of 2-chlorofatty aldehyde and 2-chlorofatty acid on leukocyte and endothelial function have been explored by several groups, suggesting that chlorinated lipids induce endothelial cell dysfunction, neutrophil chemotaxis, monocyte apoptosis, and alterations in vascular tone. Thus, the chlorinated lipidome, produced in response to leukocyte activation, is a potential biomarker and therapeutic target to modulate host response in inflammatory diseases., (Copyright © 2018. Published by Elsevier Inc.)