Aberrant Neutrophil Activation Research Articles

Blockade of the CCR3 receptor reduces neutrophil recruitment to the lung during acute inflammation.

Neutrophils represent one of the host's first lines of defense against invading pathogens. However, an aberrant activation can cause damage to the host. In the case of respiratory infections with viral or bacterial pathogens, one of the most common complications is the development of acute respiratory distress syndrome, in which neutrophil infiltration into the lung is a hallmark. Neutrophils gain expression of chemokine receptors under inflammatory conditions, and their activation can amplify the neutrophil responses. Earlier studies showed that neutrophils recruited to the lung mucosa during bacterial infection upregulate expression of CCR3 and ex vivo stimulation of CCR3 results in an increased neutrophil activation. Therefore, the modulation of effector functions or migration of neutrophils to target sites through chemokine receptors constitutes an opportunity for pharmacological intervention. We aimed to determine whether the blockade of the CCR3 using the specific antagonist SB-328437 reduces neutrophil recruitment and inflammation in the lung in the lipopolysaccharide (LPS)-induced lung injury model and influenza infection in mice. We found that neutrophils acquire CCR3 expression in the lung alveolar space. The intraperitoneal administration of SB-328437 reduced neutrophil recruitment to the lung alveolar space and reduced tissue damage in both the LPS-induced lung injury model and influenza infection. Moreover, treatment with SB-328437 reduced the percentage of neutrophils producing TNFα and neutrophil activation in the alveolar space. Together, these data suggest that CCR3 blockade might be a pharmacological strategy to prevent the aberrant neutrophil activation that results detrimental for the host but preserves sufficient effector response to control the pathogen.

Circulating Neutrophil Profiles Undergo a Dynamic Shift during Metabolic Dysfunction-Associated Steatohepatitis (MASH) Progression.

Neutrophils play a crucial role in host defense against infection. Aberrant neutrophil activation may induce tissue damage via sterile inflammation. Neutrophil accumulation has been identified as a feature of the inflammatory response observed in metabolic dysfunction-associated steatohepatitis (MASH) and has been associated with liver fibrosis and cirrhosis. Here, we performed the transcriptomic analysis of circulating neutrophils from mild and advanced MASH patients to identify the potential mechanism behind neutrophil contribution to MASH progression. Our findings demonstrated that circulating neutrophils from mild and advanced MASH display an increased activated transcriptional program, with the expression of pro-inflammatory factors and an amplified lifespan compared to cells from non-diseased controls. Our results also suggest that MASH progression is associated with a dynamic shift in the profile of circulating neutrophils. In the early stages of MASH, mature neutrophils predominate in the bloodstream. As hepatic inflammation and fibrosis progress, the premature release of immature neutrophils into the circulation occurs. These immature neutrophils exhibit a pro-inflammatory profile that may exacerbate inflammation and promote fibrosis in MASH.

Single-Cell Meta-Analysis of Neutrophil Activation in Kawasaki Disease and Multisystem Inflammatory Syndrome in Children Reveals Potential Shared Immunological Drivers.

Kawasaki disease (KD) and multisystem inflammatory syndrome in children (MIS-C) share similar clinical manifestations, including cardiovascular complications, suggesting similar underlying immunopathogenic processes. Aberrant neutrophil activation may play a crucial role in the shared pathologies of KD and MIS-C; however, the associated pathogenic mechanisms and molecular drivers remain unknown. We performed a single-cell meta-analysis of neutrophil activation with 103 pediatric single-cell transcriptomic peripheral blood mononuclear cell data across 9 cohorts, including healthy controls, KD, MIS-C, compared with dengue virus infection, juvenile idiopathic arthritis, and pediatric celiac disease. We used a series of computational analyses to investigate the shared neutrophil transcriptional programs of KD and MIS-C that are linked to systemic damage and cardiac pathologies, and suggested Food and Drug Administration-approved drugs to consider as KD and MIS-C treatment. We meta-analyzed 521 950 high-quality cells. We found that blood signatures associated with risks of cardiovascular events are enriched in neutrophils of KD and MIS-C. We revealed the expansion of CD177+ neutrophils harboring hyperactivated effector functions in both KD and MIS-C, but not in healthy controls or in other viral-, inflammatory-, or immune-related pediatric diseases. KD and MIS-C CD177+ neutrophils had highly similar transcriptomes, marked by conserved signatures and pathways related to molecular damage. We found the induction of a shared neutrophil expression program, potentially regulated by SPI1 (Spi-1 proto-oncogene), which confers enhanced effector functions, especially neutrophil degranulation. CD177 and shared neutrophil expression program expressions were associated with acute stages and attenuated during KD intravenous immunoglobulin treatment and MIS-C recovery. Network analysis identified hub genes that correlated with the high activation of CD177+ neutrophils. Disease-gene association analysis revealed that the KD and MIS-C CD177+ neutrophils' shared expression program was associated with the development of coronary and myocardial disorders. Last, we identified and validated TSPO (translocator protein) and S100A12 (S100 calcium-binding protein A12) as main molecular targets, for which the Food and Drug Administration-approved drugs methotrexate, zaleplon, metronidazole, lorazepam, clonazepam, temazepam, and zolpidem, among others, are primary candidates for drug repurposing. Our findings indicate that CD177+ neutrophils may exert systemic pathological damage contributing to the shared morbidities in KD and MIS-C. We uncovered potential regulatory drivers of CD177+ neutrophil hyperactivation and pathogenicity that may be targeted as a single therapeutic strategy for either KD or MIS-C.

Neutrophils' Contribution to Periodontitis and Periodontitis-Associated Cardiovascular Diseases.

Neutrophils represent the primary defense against microbial threats playing a pivotal role in maintaining tissue homeostasis. This review examines the multifaceted involvement of neutrophils in periodontitis, a chronic inflammatory condition affecting the supporting structures of teeth summarizing the contribution of neutrophil dysfunction in periodontitis and periodontal-related comorbidities. Periodontitis, a pathological condition promoted by dysbiosis of the oral microbiota, is characterized by the chronic inflammation of the gingiva and subsequent tissue destruction. Neutrophils are among the first immune cells recruited to the site of infection, releasing antimicrobial peptides, enzymes, and reactive oxygen species to eliminate pathogens. The persistent inflammatory state in periodontitis can lead to aberrant neutrophil activation and a sustained release of proinflammatory mediators, finally resulting in tissue damage, bone resorption, and disease progression. Growing evidence now points to the correlation between periodontitis and systemic comorbidities. Indeed, the release of inflammatory mediators, immune complexes, and oxidative stress by neutrophils, bridge the gap between local and systemic immunity, thus highlighting neutrophils as key players in linking periodontal inflammation to chronic conditions, including cardiovascular diseases, diabetes mellitus, and rheumatoid arthritis. This review underscores the crucial role of neutrophils in the pathogenesis of periodontitis and the complex link between neutrophil dysfunction, local inflammation, and systemic comorbidities. A comprehensive understanding of neutrophil contribution to periodontitis development and their impact on periodontal comorbidities holds significant implications for the management of oral health. Furthermore, it highlights the need for the development of novel approaches aimed at limiting the persistent recruitment and activation of neutrophils, also reducing the impact of periodontal inflammation on broader health contexts, offering promising avenues for improved disease management and patient care.

The involvement of NETs in ANCA-associated vasculitis.

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a serious autoimmune disease that is characterized by vascular necrosis. The pathogenesis of AAV includes ANCA-mediated neutrophil activation, subsequent release of inflammatory cytokines and reactive oxygen species (ROS), and formation of neutrophil extracellular traps (NETs). Excessive NETs could participate not only in ANCA-mediated vascular injury but also in the production of ANCAs per se as autoantigens. Thus, a vicious cycle of NET formation and ANCA production is critical for AAV pathogenesis. Elucidating the molecular signaling pathways in aberrant neutrophil activation and NETs clearance systems will allow specific therapeutics to regulate these pathways. Currently, standard therapy with high doses of glucocorticoids and immunosuppressants has improved outcomes in patients with AAV. However, AAV frequently develops in elderly people, and adverse effects such as severe infections in the standard regimens might contribute to the mortality. Mechanistically, cytokines or complement factors activate and prime neutrophils for ANCA-binding; thus, C5a receptor blocker has garnered attention as potential replacement for glucocorticoids in clinical settings. Recent studies have demonstrated that receptor-interacting protein kinases (RIPK3) and cyclophilin D (CypD), which regulate cell necrosis, may be involved in ANCA-induced NETs formation. Meanwhile, targeting NETs clearance, including the addition of deoxyribonuclease I (DNase I) and macrophage engulfment, may improve vasculitis. In this review, we focus on the pathogenesis of NETs and discuss potential targeted therapies for AAV based on recent experimental evidence.

Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

Neutrophils mediate Th17 promotion in COVID-19 patients.

From the beginning of 2020, an urgent need to understand the pathophysiology of SARS‐CoV‐2 disease (COVID‐19), much of which is due to dysbalanced immune responses, resonates across the world. COVID‐19‐associated neutrophilia, increased neutrophil‐to‐lymphocyte ratio, aberrant neutrophil activation, and infiltration of neutrophils into lungs suggest that neutrophils are important players in the disease immunopathology. The main objective of this study was to assess the phenotypic and functional characteristics of neutrophils in COVID‐19 patients, with particular focus on the interaction between neutrophils and T cells. We hypothesize that the altered functional characteristics of COVID‐19 patient‐derived neutrophils result in skewed Th1/Th17 adaptive immune response, thus contributing to disease pathology. The expansion of G‐MDSC and immature forms of neutrophils was shown in the COVID‐19 patients. In the COVID‐19 neutrophil/T cell cocultures, neutrophils caused a strong polarity shift toward Th17, and, conversely, a reduction of IFNγ‐producing Th1 cells. The Th17 promotion was NOS dependent. Neutrophils, the known modulators of adaptive immunity, skew the polarization of T cells toward the Th17 promotion and Th1 suppression in COVID‐19 patients, contributing to the discoordinated orchestration of immune response against SARS‐CoV‐2. As IL‐17 and other Th17‐related cytokines have previously been shown to correlate with the disease severity, we suggest that targeting neutrophils and/or Th17 represents a potentially beneficial therapeutic strategy for severe COVID‐19 patients.

Nanoparticle-Directed Targeting of Clustered PSGL-1 Mitigates Neutrophil-Derived Thrombosis in Antiphospholipid Antibody Syndrome

Multiorgan thromboses and subsequent organ failure are among the more devastating consequences of antiphospholipid antibody syndrome (APS). APS poses a relatively unique risk to patients by virtue of its propensity to cause thrombi of both the arterial and venous vascular systems. While recent studies have explored mechanisms of thrombosis in singular vascular beds, there remains a dearth of targetable effectors of arteriovenous thrombosis in APS. Here, we examine the potential of neutrophils as key players of both arterial and venous thrombosis in APS. From this and previous studies, we have also identified a critical transcriptional regulator in neutrophils whose loss mimics arterial and venous thrombosis susceptibility seen in APS. This factor, Krüppel-like factor 2 (KLF2), is down regulated in APS patients as well as in cells treated with antiphospholipid antibody (APLA) ex vivo. Loss of KLF2 in neutrophils contributes to classic neutrophil activation phenotypes including NETosis, increased tissue factor release, and heightened chemotaxis; remarkably, blocking each of these functions greatly abrogates the thrombotic phenotype. Utilizing the myeloid KLF2 knockout (K2KO) system as a model for aberrant neutrophil activation seen in APS, we have identified clustering of P-selectin glycoprotein ligand 1 (PSGL-1) secondary to actin rearrangement to be operative in neutrophil-mediated thrombosis in arterial and venous systems. While neutralizing antibodies against PSGL-1 have been explored as therapeutic options in APS, there remains a challenge in establishing local specificity to activated immune cells in order to increase efficacy. To circumvent this, we have designed a nanoparticle (NP) based approach that utilizes NPs decorated with anti-PSGL-1 immunoglobulins to better target clustered PSGL-1 on activated neutrophils. PSGL-1 NPs are more effective at inhibiting neutrophil rolling and adhesion in in vitro assays while, incubating activated neutrophils in PSGL-1 NPs prior to infusion attenuates their ability to contribute to both arterial and venous thrombosis. Further, injection of PSGL-1 NPs directly into K2KO mice reduces their extensive thrombotic phenotype. Importantly, this effect occurs at a substantially lower dose of PSGL-1 NPs than that which would be needed of PSGL-1 antibody alone. Finally, PSGL-1 NPs greatly reduce thrombotic burden in mice injected with APLA, diminishing clotting in both arteries and veins. Together, this work identifies key molecular and cellular players in APLA-induced arteriovenous thrombosis. Furthermore, it provides early pre-clinical evidence at specifically targeting clustered PSGL-1 on neutrophils as a viable option in combatting thrombosis in this particularly vulnerable patient population. Disclosures No relevant conflicts of interest to declare.

Identification of a Novel HIF-1α-αMβ2 Integrin-NET Axis in Fibrotic Interstitial Lung Disease.

Neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ILD) particularly in the most severe form, idiopathic pulmonary fibrosis (IPF), although the underlying mechanisms remain unclear. Neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. Hypoxia has an important role in inflammation and may also contribute to pulmonary disease. We aimed to determine how neutrophil activation occurs in ILD and the relative importance of hypoxia. Using lung biopsies and bronchoalveolar lavage (BAL) fluid from ILD patients we investigated the extent of hypoxia and neutrophil activation in ILD lungs. Then we used ex vivo neutrophils isolated from healthy volunteers and BAL from patients with ILD and non-ILD controls to further investigate aberrant neutrophil activation in hypoxia and ILD. We demonstrate for the first time using intracellular staining, HIF-1α stabilization in neutrophils and endothelial cells in ILD lung biopsies. Hypoxia enhanced both spontaneous (+1.31-fold, p < 0.05) and phorbol 12-myristate 13-acetate (PMA)-induced (+1.65-fold, p < 0.001) neutrophil extracellular trap (NET) release, neutrophil adhesion (+8.8-fold, <0.05), and trans-endothelial migration (+1.9-fold, p < 0.05). Hypoxia also increased neutrophil expression of the αM (+3.1-fold, p < 0.001) and αX (+1.6-fold, p < 0.01) integrin subunits. Interestingly, NET formation was induced by αMβ2 integrin activation and prevented by cation chelation. Finally, we observed NET-like structures in IPF lung sections and in the BAL from ILD patients, and quantification showed increased cell-free DNA content (+5.5-fold, p < 0.01) and MPO-citrullinated histone H3 complexes (+21.9-fold, p < 0.01) in BAL from ILD patients compared to non-ILD controls. In conclusion, HIF-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β2 integrins. Our results identify a novel HIF-1α- αMβ2 integrin axis in NET formation for future exploration in therapeutic approaches to fibrotic ILD.

Neutrophils are key mediators in crescentic glomerulonephritis and targets for new therapeutic approaches.

Crescentic glomerulonephritis (CGN) results from a diverse set of diseases associated with immune dysregulation and the breakdown of self-tolerance to a wide range of autoantigens, some known and some that remain unknown. Experimental data demonstrate that neutrophils have an important role in the pathogenesis of CGN. Upon activation, neutrophils generate reactive oxygen species, release serine proteases and form neutrophil extracellular traps (NETs), all of which can induce direct tissue damage. In addition, serine proteases such as myeloperoxidase and proteinase 3, presented on NETs, can be processed and recognized as autoantigens, leading to the generation and maintenance of autoimmune responses in susceptible individuals. The basis of the specificity of autoimmune responses in different patients to NET proteins is unclear, but relates at least in part to differences in human leucocyte antigen expression. Conditions associated with CGN are often characterized by aberrant neutrophil activation and NETosis and, in some, impaired NET degradation. Targeting neutrophil degranulation and NETosis is now possible using a variety of novel compounds and may provide a promising therapeutic alternative to glucocorticoid use, which has been a mainstay of management in CGN for decades and is associated with significant adverse effects. In this review, we discuss the evidence supporting the role of neutrophils in the development of CGN and the pathways identified in neutrophil degranulation and NETosis that may translate to novel therapeutic applications.

Monocyte-Driven Atypical Cytokine Storm and Aberrant Neutrophil Activation as Key Mediators of COVID19 Disease Severity

The host immune response in symptomatic COVID-19 patients seems ineffective in clearing SARS-CoV-2, yet excessive in causing local tissue damage and hypercytokinemia. To elucidate the immunopathology underlying COVID-19 severity, cytokine profiling was performed in mild-moderate and critically-ill COVID-19 patients. Hypercytokinemia in COVID-19 differed from the IFN-γ-driven cytokine storm in macrophage activation syndrome, but was increased in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels followed by deep-immune profiling showed that classical monocytes drive this hyper-inflammatory phenotype and that both a quantitative and qualitative reduction in T-lymphocytes correlate with disease severity, with CD8+ cells being disproportionately affected. Expression of antigen presentation and co-stimulatory molecules was reduced in critical disease, while also neutrophils contributed to disease severity and local tissue damage by amplifying hypercytokinemia and neutrophil extracellular trap formation. We suggest a monocyte-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.Funding: This work was funded by KU Leuven (internal fund and grant C14/17/084 and C16/17/010), VIB (Grand Challenge project), UZ Leuven (KOOR project), FWO grant I007418N, the Rega Foundation (research expert fellowship to G.M.) and ‘het Leuvens Kankerinstituut’. E.D. is a postdoctoral research fellow of the Research Foundation – Flanders (FWO), Belgium (grant number 12X9420N). J.G. holds a postdoctoral research fellowship granted by the clinical research and education council of the University Hospitals Leuven. Y.S. received funding from the Flemish Government (AI Research Program). L.V. is supported by an FWO PhD fellowship (grant number 11E9819N). P.V.M. is supported by an FWO PhD fellowship (grant number 1S66020N). S.V.G. is an ISAC Marylou Ingram Scholar and supported by an FWO postdoctoral research grant (Research Foundation – Flanders). E.W. is supported by Stichting tegen Kanker (Mandate for basic & clinical oncology research). J.W. is supported by an FWO Fundamental Clinical Mandate (1833317N). Conflict of Interest: The authors declare no competing interests.Ethical Approval: All study procedures were approved by the Ethics Committee of the University Hospitals Leuven. Informed consent was obtained from all individuals or their legal guardians.

O Papel da Imunidade Inata na COVID-19

O sistema imune apresenta papel importante na pandemia da COVID-19, pois está envolvido na patogênese e agravamento da doença. O objetivo desta revisão é abordar os aspectos da imunidade inata na COVID-19, especialmente o papel dos neutrófilos. O sistema imune inato corresponde à primeira defesa do organismo, porém é necessário um equilíbrio para obter efetividade contra o invasor sem lesar excessivamente o hospedeiro. O desequilíbrio imune está relacionado com quadros mais graves e à ativação aberrante de neutrófilos, sendo a linfopenia e a neutrofilia preditores de pior prognóstico em pacientes com COVID-19. Especula-se que a neutrofilia seja uma importante fonte para formação excessiva de NET (Neutrophil Extracellular Traps), levando ao aumento da resposta inflamatória e evolução desfavorável da doença. As NET também estão associadas à tempestade de citocinas, outro mecanismo relacionado a gravidade da COVID-19. Portanto, estratégias que envolvam a imunomodulação podem ter um papel importante no controle da doença.

Progress in Model Systems of Cystic Fibrosis Mucosal Inflammation to Understand Aberrant Neutrophil Activity.

In response to recurrent infection in cystic fibrosis (CF), powerful innate immune signals trigger polymorphonuclear neutrophil recruitment into the airway lumen. Exaggerated neutrophil proteolytic activity results in sustained inflammation and scarring of the airways. Consequently, neutrophils and their secretions are reliable clinical biomarkers of lung disease progression. As neutrophils are required to clear infection and yet a direct cause of airway damage, modulating adverse neutrophil activity while preserving their pathogen fighting function remains a key area of CF research. The factors that drive their pathological behavior are still under investigation, especially in early disease when aberrant neutrophil behavior first becomes evident. Here we examine the latest findings of neutrophils in pediatric CF lung disease and proposed mechanisms of their pathogenicity. Highlighted in this review are current and emerging experimental methods for assessing CF mucosal immunity and human neutrophil function in the laboratory.

Tumor stem-like cell-derived exosomal RNAs prime neutrophils for facilitating tumorigenesis of colon cancer

BackgroundCell-cell interactions maintain tissue homeostasis and contribute to dynamic alteration of the tumor microenvironment (TME). Communication between cancer and host cells not only promotes advanced disease aggression but also determines therapeutic response in cancer patients. Despite accumulating evidence supporting the role of tumor-infiltrating immunocytes in modulating tumor immunity, the interplay between heterogeneous tumor subpopulations and immunocytes is elusive.MethodsWe expanded colorectal cancer stem cells (CRCSCs) as cancer spheroids from the murine colorectal cancer (CRC) cell line CT26 to interrogate tumor-host interactions using a syngeneic tumor model. RNA-sequencing analysis of host cells and tumor exosomes was performed to identify molecular determinants that mediate the crosstalk between CRCSCs and immunocytes. The Cancer Genome Atlas (TCGA) database was used to validate the clinical significance in CRC patients.ResultsThe expanded CT26 cancer spheroids showed increased stemness gene expression, enhanced spheroid and clonogenicity potential, and an elevated tumor-initiating ability, characteristic of CRCSCs. By examining immune cell composition in syngeneic tumor-bearing mice, a systemic increase in CD11b+/Ly6GHigh/Ly6CLow neutrophils was observed in mice bearing CRCSC-derived tumors. An increased secretion of CRCSC exosomes was observed in vitro, and through in vivo tracking, CRCSC exosomes were found to be transported to the bone marrow. Moreover, CRCSC exosomes prolonged the survival of bone marrow-derived neutrophils and engendered a protumoral phenotype in neutrophils. Mechanistically, tumor exosomal tri-phosphate RNAs induced the expression of interleukin-1β (IL-1β) through a pattern recognition-NF-κB signaling axis to sustain neutrophil survival. CRCSC-secreted CXCL1 and CXCL2 then attracted CRCSC-primed neutrophils to promote tumorigenesis of CRC cells via IL-1β. Moreover, neutrophil depletion using a Ly6G-specific antibody (clone 1A8) attenuated the tumorigenicity of CRCSCs. In human specimens, CRC patients exhibiting an active CRCSC signal (Snail+IL8+) showed elevated tumor infiltration of MPO+ neutrophils, and high (in the top 10%) MPO expression predicted poor survival of CRC patients.ConclusionsThis study elucidates a multistep CRCSC-neutrophil interaction during advanced cancer progression. Strategies targeting aberrant neutrophil activation may be developed for combating CSC-related malignancy.

Impact of anti-glomerular basement membrane antibodies and glomerular neutrophil activation on glomerulonephritis in experimental myeloperoxidase-antineutrophil cytoplasmic antibody vasculitis

Antineutrophil cytoplasmic antibody (ANCA) and neutrophil interactions play important roles in ANCA-associated vasculitis (AAV) pathogenesis. However, mechanisms underlying the pathogenesis of crescent formation in ANCA-associated vasculitis have not been completely elucidated. To ascertain the involvement of these interactions in necrotizing crescentic glomerulonephritis (NCGN), we used an AAV rat model and investigated the effects of the anti-myeloperoxidase (MPO) antibody (Ab) titer, tumor necrosis factor α (TNF-α), granulocyte colony-stimulating factor (G-CSF) and subnephritogenic anti-glomerular basement membrane (GBM) Abs, as proinflammatory stimuli. NCGN was induced in Wistar Kyoto rats by human MPO (hMPO) immunization. Renal function, pathology, and glomerular cytokine and chemokine expression were evaluated in hMPO-immunized rats with/without several co-treatments (TNF-α, G-CSF or subnephritogenic anti-GBM Abs). Rat neutrophils activation by IgG purified from rat serum in each group was examined in vitro. The hMPO-immunized rats had significantly higher level of anti-hMPO Ab production. The induced anti-hMPO Abs cross-reacted with TNF-α- or G-CSF-primed rat neutrophils secreting TNF-α and interleukin-1β in vitro. The reactivity of anti-MPO Abs against rat MPO, crescent formation with neutrophil extracellular traps and glomerular-activated neutrophil infiltration in the rat model were significantly enhanced by subnephritogenic anti-GBM Ab but not by TNF-α or G-CSF administration. The model rats injected with the subnephritogenic anti-GBM Abs showed increased urinary albumin excretion and serum TNF-α, chemokine (C-X-C) ligand 1 (CXCL1) and CXCL2 levels. TNF-α, CXCL1, CXCL2 and CXCL8 increased in the glomeruli with significant amounts of crescent formation. In addition, in vitro activated neutrophils decreased CXC chemokine receptor 1 (CXCR1) and CXCR2 expressions. The coexistence of subnephritogenic anti-GBM Abs leads to the inflammatory environment in glomeruli that is amplified by the interaction of ANCA and neutrophils. Development of NCGN in MPO-AAV may be necessary for not only the accumulation of neutrophils in glomeruli, but also the aberrant neutrophil activation on glomerulonephritis.

Elevated Levels of Circulating DNA and Chromatin Are Independently Associated With Severe Coronary Atherosclerosis and a Prothrombotic State

Aberrant neutrophil activation occurs during the advanced stages of atherosclerosis. Once primed, neutrophils can undergo apoptosis or release neutrophil extracellular traps. This extracellular DNA exerts potent proinflammatory, prothrombotic, and cytotoxic properties. The goal of this study was to examine the relationships among extracellular DNA formation, coronary atherosclerosis, and the presence of a prothrombotic state. In a prospective, observational, cross-sectional cohort of 282 individuals with suspected coronary artery disease, we examined the severity, extent, and phenotype of coronary atherosclerosis using coronary computed tomographic angiography. Double-stranded DNA, nucleosomes, citrullinated histone H4, and myeloperoxidase-DNA complexes, considered in vivo markers of cell death and NETosis, respectively, were established. We further measured various plasma markers of coagulation activation and inflammation. Plasma double-stranded DNA, nucleosomes, and myeloperoxidase-DNA complexes were positively associated with thrombin generation and significantly elevated in patients with severe coronary atherosclerosis or extremely calcified coronary arteries. Multinomial regression analysis, adjusted for confounding factors, identified high plasma nucleosome levels as an independent risk factor of severe coronary stenosis (odds ratio, 2.14; 95% confidence interval, 1.26-3.63; P=0.005). Markers of neutrophil extracellular traps, such as myeloperoxidase-DNA complexes, predicted the number of atherosclerotic coronary vessels and the occurrence of major adverse cardiac events. Our report provides evidence demonstrating that markers of cell death and neutrophil extracellular trap formation are independently associated with coronary artery disease, prothrombotic state, and occurrence of adverse cardiac events. These biomarkers could potentially aid in the prediction of cardiovascular risk in patients with chest discomfort.

Special issue on neutrophils

For nearly a century, ever since their discovery byMetchnikoff, neutrophils have been viewed as mundane, plodding footmen of the immune system, not much unlike the archetypal London Bobby: first to arrive at the scene of a crime and to secure the situation by a cursory neutralisation of the intruding culprits. The true detective work, in this case the recognition and processing of foreign pathogenic antigens, would be assumed by the superintendent T and B lymphocytes. This paradigm of the dull neutrophil and the immunologically exciting lymphocyte has seen challenges by a series of recent findings. Of these the most sensational was the discovery that neutrophils can extrude their genomic DNA into the extracellular environment as Neutrophil Extracellular Traps (NETs). While it was initially thought that NETs mainly served to ensnare and debilitate pathogenic microorganisms, they have truly opened up a new window to investigate neutrophil physiology. As is evident from several reviews in this special issue on Neutrophils (Hahn et al., Mantovani et al., Branzk and Papayannopoulos), these specialized structures not only interact with bacteria or yeast, but also with multicellular eukaryotic parasites (P. vivax and P. falciparum in Malaria) and with viruses (HIV in AIDS). Not unexpectedly, in a scenario reminiscent of a cold-war spy vs. spy thriller, both neutrophils and pathogens have developed an array of strategies and counter-strategies to secure or prevent entrapment and destruction. One of the most intriguing is the ability of HIV virions to induce the production of the immune modulating cytokine IL-10 by dendritic cells, which effectively hinders NETosis and normal neutrophil activity, thereby paving the way for frequently lethal secondary infections such as with Mycobacterium tuberculosis. On the other hand, in a process dubbed immune-thrombosis, the immune system may use components of the clotting cascade that interact with NETs to form tight complexes around invading pathogens. It has also become evident that the host micro-environment plays a significant role in modulating neutrophil activity, including the respiratory system or systemic influences present in pregnancy (refer to Hahn et al.). The extent of neutrophil interaction is also be associatedwith the seeding of cancer stem cells, the process of metastasis and cancer progression (refer to Mantovani et al., and Geffner et al.). In addition, a number of recent findings implicating aberrant neutrophil activity in a diverse array of human pathologies have rung the death knell of the arcane perception of the mundane, crude granulocyte. These include observations that neutrophil NETs may be implicated in autoimmune or auto-inflammatory conditions such as systemic lupus erythromatosus (SLE), anti-neutrophil cytoplasmic antibody (ANCA)-associated small vessel vasculitis and gout, or contribute to the pathology of cystic fibrosis (refer to reviews by Carmon-Rivera and Kaplan, Marion and Radic, Ritis et al.). Indeed, it appears that neutrophils may exist in discrete forms or distinct sub-populations, such as low-density granulocytes, and that the latter may play a crucial role in the pathology of SLE (refer to CarmonRivera and Kaplan). The potential involvement of cytokines such as Activin-A in promoting neutrophil mediated inflammation is discussed by Sideras and colleagues. These findings concerning overt or aberrant activity have fuelled the search for pharmacological agents to effectively tailor or modulate neutrophil behaviour; strategies targeting the cAMP or calcium signalling pathways or the inhibition of granular components are discussed in the reports by Anderson et al., and Kormacs et al., while the underlying signal transducing pathways involved in NETosis are This article is a contribution to the special issue on Neutrophils–Guest Editors: Paul Hasler and Sinuhe Hahn

Extracorporeal immune therapy with immobilized agonistic anti-Fas antibodies leads to transient reduction of circulating neutrophil numbers and limits tissue damage after hemorrhagic shock/resuscitation in a porcine model

BackgroundHemorrhagic shock/resuscitation is associated with aberrant neutrophil activation and organ failure. This experimental porcine study was done to evaluate the effects of Fas-directed extracorporeal immune therapy with a leukocyte inhibition module (LIM) on hemodynamics, neutrophil tissue infiltration, and tissue damage after hemorrhagic shock/resuscitation.MethodsIn a prospective controlled double-armed animal trial 24 Munich Mini Pigs (30.3 ± 3.3 kg) were rapidly haemorrhaged to reach a mean arterial pressure (MAP) of 35 ± 5 mmHg, maintained hypotensive for 45 minutes, and then were resuscitated with Ringer' solution to baseline MAP. With beginning of resuscitation 12 pigs underwent extracorporeal immune therapy for 3 hours (LIM group) and 12 pigs were resuscitated according to standard medical care (SMC). Haemodynamics, haematologic, metabolic, and organ specific damage parameters were monitored. Neutrophil infiltration was analyzed histologically after 48 and 72 hours. Lipid peroxidation and apoptosis were specifically determined in lung, bowel, and liver.ResultsIn the LIM group, neutrophil counts were reduced versus SMC during extracorporeal immune therapy. After 72 hours, the haemodynamic parameters MAP and cardiac output (CO) were significantly better in the LIM group. Histological analyses showed reduction of shock-related neutrophil tissue infiltration in the LIM group, especially in the lungs. Lower amounts of apoptotic cells and lipid peroxidation were found in organs after LIM treatment.ConclusionsTransient Fas-directed extracorporeal immune therapy may protect from posthemorrhagic neutrophil tissue infiltration and tissue damage.

Clinical imatinib mesylate treatment induces early normalisation of aberrant neutrophil leukotriene C4 synthase expression and activity in chronic myeloid leukaemia

Clinical imatinib mesylate treatment induces early normalisation of aberrant neutrophil leukotriene C4 synthase expression and activity in chronic myeloid leukaemia

In vivo inhibition of neutrophil activity by a FAS (CD95) stimulating module: arterial in-line application in a porcine cardiac surgery model

Objective Cardiac surgery with cardiopulmonary bypass is associated with aberrant neutrophil activation and potentially severe pathogenic sequelae. This experimental study was done to evaluate a leukocyte inhibition module that rapidly inactivates neutrophils through CD95 stimulation. Methods German landrace pigs (4 groups, each n = 5) underwent cardiac surgery without cardiopulmonary bypass (group I), with cardiopulmonary bypass (group II), with cardiopulmonary bypass plus a leukocyte filter (group III), and with cardiopulmonary bypass plus a leukocyte inhibition module (group IV). The leukocyte filter or leukocyte inhibition module was introduced into the arterial line of the heart-lung machine. Results Leukocyte counts were decreased by up to 43% in group IV compared with values in group II ( P = .023). In group IV, but not in groups I to III, no delay in spontaneous neutrophil apoptosis was observed after annexin V–propidium iodide staining. Late apoptotic (11.7%) or necrotic neutrophils (9.3%) were detected in 2 animals (group IV). Tumor necrosis factor α serum levels increased over time in groups I to III (>2-fold) but remained at baseline levels in group IV ( P < .05). Interleukin 8–mediated chemotactic neutrophil transmigration activity increased over time in groups I to III but was totally abrogated in group IV at any time point. The perioperative increase of creatine kinase and creatine kinase MB levels was lower in groups III (1.5-fold and 1.3-fold, respectively) and IV (1.2-fold and 1.5-fold, respectively) compared with values in group II (both 1.9-fold). Conclusions The leukocyte inhibition module downregulated cardiopulmonary bypass–related neutrophil activity and thus might be beneficial in cardiac surgery and other clinical settings with unappreciated neutrophil activation.