Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA.

Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly release various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator in eosinophil function against pathogens and in eosinophil-associated diseases. While MBP-1 targets both microorganisms and host cells, its precise mechanism remains elusive. We demonstrate that formation of small pores by MBP-1 in lipid bilayers induces membrane permeabilization and disrupts potassium balance. Additionally, we reveal that mitochondrial DNA (mtDNA) present in eosinophil extracellular traps (EETs) amplifies MBP-1 toxic effects, underscoring the pivotal role of mtDNA in EETs. Furthermore, we present evidence indicating that absence of CpG methylation in mtDNA contributes to the regulation of MBP-1-mediated toxicity. Taken together, our data suggest that the mtDNA scaffold within extracellular traps promotes MBP-1 toxicity.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

The BK Channel Limits the Pro-Inflammatory Activity of Macrophages.

Macrophages play a crucial role in the innate immune response, serving as key effector cells in the defense against pathogens. Although the role of the large-conductance voltage and calcium-activated potassium channel, also known as the KCa1.1 or BK channel, in regulating neurotransmitter release and smooth muscle contraction is well known, its potential involvement in immune regulation remains unclear. We employed BK-knockout macrophages and noted that the absence of a BK channel promotes the polarization of macrophages towards a pro-inflammatory phenotype known as M1 macrophages. Specifically, the absence of the BK channel resulted in a significant increase in the secretion of the pro-inflammatory cytokine IL-6 and enhanced the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2 kinases), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the transcription factor ATF-1 within M1 macrophages. Additionally, the lack of the BK channel promoted the activation of the AIM2 inflammasome without affecting the activation of the NLRC4 and NLRP3 inflammasomes. To further investigate the role of the BK channel in regulating AIM2 inflammasome activation, we utilized BK channel inhibitors, such as paxilline and iberiotoxin, along with the BK channel activator NS-11021. Pharmacological inactivation of the BK channel increased, and its stimulation inhibited IL-1ß production following AIM2 inflammasome activation in wild-type macrophages. Moreover, wild-type macrophages displayed increased calcium influx when activated with the AIM2 inflammasome, whereas BK-knockout macrophages did not due to the impaired extracellular calcium influx upon activation. Furthermore, under conditions of a calcium-free medium, IL-1ß production following AIM2 inflammasome activation was increased in both wild-type and BK-knockout macrophages. This suggests that the BK channel is required for the influx of extracellular calcium in macrophages, thus limiting AIM2 inflammasome activation. In summary, our study reveals a regulatory role of the BK channel in macrophages under inflammatory conditions.

Dupilumab-associated ocular surface disease is characterized by a shift from Th2/Th17 toward Th1/Th17 inflammation.

BACKGROUND: Dupilumab is used for the treatment of atopic dermatitis (AD). Approximately one third of AD patients develop a dupilumab-associated ocular surface disease (DAOSD), of which the pathomechanism is poorly understood. This study aimed at investigating inflammatory markers in tear fluids of patients on dupilumab therapy. METHODS: Tear fluids were collected from AD patients with DAOSD (ADwDAOSD), AD patients without DAOSD (ADw/oDAOSD), and non-AD patients before and during dupilumab therapy, and analyzed using a specialized proteomic approach quantifying inflammatory markers. The ocular surface microbiome was determined by next generation sequencing technology. RESULTS: Upon dupilumab therapy, an upregulation of 31 inflammatory markers was observed in DAOSD tear fluids compared to baseline in AD patients. While IL-12B was upregulated in both ADwDAOSD and ADw/oDAOSD groups, the pattern of inflammatory markers significantly differed between groups and over time. In the ADwDAOSD group, a shift from a mixed Th2/Th17 pattern at baseline toward a Th1/Th17 profile under dupilumab was observed. Furthermore, an upregulation of remodeling and fibrosis markers was seen in DAOSD. Semantic map and hierarchical cluster analyses of baseline marker expression revealed four clusters distinguishing between AD and non-AD as well as ADwDAOSD and ADw/oDAOSD patient groups. In a pilot study, dupilumab therapy was associated with a decrease in richness of the ocular surface microbiome. CONCLUSIONS: DAOSD is characterized by a Th1/Th17 cytokine profile and an upregulation of markers known to promote remodeling and fibrosis. The expression pattern of inflammatory markers in tear fluids at baseline might serve as a prognostic factor for DAOSD.

A Multicenter Long-Term Cohort Study of Eosinophilic Esophagitis Variants and Their Progression to Eosinophilic Esophagitis Over Time.

INTRODUCTION: Eosinophilic esophagitis (EoE) variants have been recently characterized as conditions with symptoms of esophageal dysfunction resembling EoE, but absence of significant esophageal eosinophilia. Their disease course and severity have yet to be determined. METHODS: Patients from 6 EoE centers with symptoms of esophageal dysfunction, but peak eosinophil counts of <15/hpf in esophageal biopsies and absence of gastroesophageal reflux disease with at least one follow-up visit were included. Clinical, (immuno)histological, and molecular features were determined and compared with EoE and healthy controls. RESULTS: We included 54 patients with EoE variants (EoE-like esophagitis 53.7%; lymphocytic esophagitis 13.0%; and nonspecific esophagitis 33.3%). In 8 EoE-like esophagitis patients, EoE developed after a median of 14 months (interquartile range 3.6-37.6). Such progression increased over time (17.6% year 1, 32.0% year 3, and 62.2% year 6). Sequential RNA sequencing analyses revealed only 7 genes associated with this progression (with TSG6 and ALOX15 among the top 3 upregulated genes) with upregulation of a previously attenuated Th2 pathway. Immunostaining confirmed the involvement of eosinophil-associated proteins (TSG6 and ALOX15) and revealed a significantly increased number of GATA3-positive cells during progression, indicating a Th1/Th2 switch. Transition from one EoE variant (baseline) to another variant (during follow-up) was seen in 35.2% (median observation time of 17.3 months). DISCUSSION: Transition of EoE variants to EoE suggests the presence of a disease spectrum. Few genes seem to be associated with the progression to EoE with upregulation of a previously attenuated Th2 signal. These genes, including GATA3 as a Th1/Th2 switch regulator, may represent potential therapeutic targets in early disease pathogenesis.

Histological Phenotyping in Eosinophilic Esophagitis: Localized Proximal Disease Is Infrequent but Associated with Less Severe Disease and Better Disease Outcome.

INTRODUCTION: It is still unknown whether eosinophilic esophagitis (EoE) patients with localized disease are different from those with extended disease. METHODS: We evaluated prospectively included patients in the Swiss EoE cohort. Data on all patients with active disease at baseline, no concomitant gastroesophageal reflux disease, no strictures at baseline, and at least one follow-up visit were analyzed. We compared patients with histologically localized proximal versus distal versus extended (=proximal and distal) disease with regard to patient, disease characteristics, disease presentation, and development of complications. RESULTS: We included 124 patients with a median of 2.5 years of follow-up (73.4% males, median age 35.0 years). Ten patients had proximal (8.1%), 46 patients had distal (37.1%), and 68 patients had extended disease (54.8%). Patients with proximal disease were significantly more often females (80%) compared with patients with distal (26.1%, p = 0.002) or extended disease (19.1%, p < 0.001) and reported less severe symptoms (VAS 0 vs. VAS 1, p = 0.001). Endoscopic and histological disease was less pronounced in the proximal esophagus of proximal EoE compared to extended disease (EREFS 1.0 vs. 3.0, p = 0.001; 27.0 eos/hpf vs. 52.5 eos/hpf, p = 0.008). Patients with proximal disease were less likely to undergo dilation compared to patients with distal disease in the follow-up (3.3% vs. 23.3%, p = 0.010). In a multivariate Cox regression model, proximal eosinophilia was less likely to be associated with treatment failure compared to distal eosinophilia. CONCLUSION: Although isolated proximal EoE is infrequent, it is associated with less severe disease and better disease outcome. Proximal disease appears to present a unique EoE phenotype.

Phytochemicals Target Multiple Metabolic Pathways in Cancer.

Cancer metabolic reprogramming is a complex process that provides malignant cells with selective advantages to grow and propagate in the hostile environment created by the immune surveillance of the human organism. This process underpins cancer proliferation, invasion, antioxidant defense, and resistance to anticancer immunity and therapeutics. Perhaps not surprisingly, metabolic rewiring is considered to be one of the "Hallmarks of cancer". Notably, this process often comprises various complementary and overlapping pathways. Today, it is well known that highly selective inhibition of only one of the pathways in a tumor cell often leads to a limited response and, subsequently, to the emergence of resistance. Therefore, to increase the overall effectiveness of antitumor drugs, it is advisable to use multitarget agents that can simultaneously suppress several key processes in the tumor cell. This review is focused on a group of plant-derived natural compounds that simultaneously target different pathways of cancer-associated metabolism, including aerobic glycolysis, respiration, glutaminolysis, one-carbon metabolism, de novo lipogenesis, and ß-oxidation of fatty acids. We discuss only those compounds that display inhibitory activity against several metabolic pathways as well as a number of important signaling pathways in cancer. Information about their pharmacokinetics in animals and humans is also presented. Taken together, a number of known plant-derived compounds may target multiple metabolic and signaling pathways in various malignancies, something that bears great potential for the further improvement of antineoplastic therapy.

Syntaxin-4 and SNAP23 are involved in neutrophil degranulation, but not in the release of mitochondrial DNA during NET formation.

Neutrophils are a specialized subset of white blood cells, which have the ability to store pre-formed mediators in their cytoplasmic granules. Neutrophils are well-known effector cells involved in host protection against pathogens through diverse mechanisms such as phagocytosis, degranulation, extracellular traps, and oxidative burst. In this study, we provide evidence highlighting the significance of the SNARE proteins syntaxin-4 and synaptosomal-associated protein (SNAP) 23 in the release of azurophilic granules, specific granules, and the production of reactive oxygen species in human neutrophils. In contrast, the specific blockade of either syntaxin-4 or SNAP23 did not prevent the release of mitochondrial dsDNA in the process of neutrophil extracellular trap (NET) formation. These findings imply that degranulation and the release of mitochondrial dsDNA involve at least partially distinct molecular pathways in neutrophils.

Eosinophils-from cradle to grave: An EAACI task force paper on new molecular insights and clinical functions of eosinophils and the clinical effects of targeted eosinophil depletion.

Over the past years, eosinophils have become a focus of scientific interest, especially in the context of their recently uncovered functions (e.g. antiviral, anti-inflammatory, regulatory). These versatile cells display both beneficial and detrimental activities under various physiological and pathological conditions. Eosinophils are involved in the pathogenesis of many diseases which can be classified into primary (clonal) and secondary (reactive) disorders and idiopathic (hyper)eosinophilic syndromes. Depending on the biological specimen, the eosinophil count in different body compartments may serve as a biomarker reflecting the underlying pathophysiology and/or activity of distinct diseases and as a therapy-driving (predictive) and monitoring tool. Personalized selection of an appropriate therapeutic strategy directly or indirectly targeting the increased number and/or activity of eosinophils should be based on the understanding of eosinophil homeostasis including their interactions with other immune and non-immune cells within different body compartments. Hence, restoring as well as maintaining homeostasis within an individual's eosinophil pool is a goal of both specific and non-specific eosinophil-targeting therapies. Despite the overall favourable safety profile of the currently available anti-eosinophil biologics, the effect of eosinophil depletion should be monitored from the perspective of possible unwanted consequences.

Methyltransferase Set7/9 as a Multifaceted Regulator of ROS Response.

Reactive oxygen species (ROS) induce multiple signaling cascades in the cell and hence play an important role in the regulation of the cell's fate. ROS can cause irreversible damage to DNA and proteins resulting in cell death. Therefore, finely tuned regulatory mechanisms exist in evolutionarily diverse organisms that are aimed at the neutralization of ROS and its consequences with respect to cellular damage. The SET domain-containing lysine methyltransferase Set7/9 (KMT7, SETD7, SET7, SET9) post-translationally modifies several histones and non-histone proteins via monomethylation of the target lysines in a sequence-specific manner. In cellulo, the Set7/9-directed covalent modification of its substrates affects gene expression, cell cycle, energy metabolism, apoptosis, ROS, and DNA damage response. However, the in vivo role of Set7/9 remains enigmatic. In this review, we summarize the currently available information regarding the role of methyltransferase Set7/9 in the regulation of ROS-inducible molecular cascades in response to oxidative stress. We also highlight the in vivo importance of Set7/9 in ROS-related diseases.

Eosinophils from A to Z.

Eosinophils are bone marrow-derived granulocytes and are found in low numbers in the peripheral blood of healthy subjects. In type 2 inflammatory diseases, eosinopoiesis in the bone marrow is increased, resulting in a rise in the number of mature eosinophils released in the circulation. From the blood, eosinophils can migrate in multiple tissues and organs under both physiological and pathological conditions. Eosinophils exert their various functions through the synthesis and release of a variety of granule proteins and pro-inflammatory mediators. Despite being present in all species of vertebrates, the functional role of eosinophils is still a matter of debate. Eosinophils may play a role in host defense against various pathogens. In addition, eosinophils have been reported to be involved in tissue homeostasis and exhibit immunomodulatory activities. In this review, we aim to provide a broad overview of eosinophil biology and eosinophilic diseases in a lexicon-style format using keywords starting from A until Z with cross-references to other chapters indicated in italics in the text or specified in parentheses.

Transcriptional Insights of Oxidative Stress and Extracellular Traps in Lung Tissues of Fatal COVID-19 Cases.

Neutrophil extracellular traps (NETs) and oxidative stress are considered to be beneficial in the innate immune defense against pathogens. However, defective clearance of NETs in the lung of acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients could lead to severe respiratory syndrome infection, the so-called coronavirus disease 2019 (COVID-19). To elucidate the pathways that are related to NETs within the pathophysiology of COVID-19, we utilized RNA sequencing (RNA-seq) as well as immunofluorescence and immunohistochemistry methods. RNA-seq analysis provided evidence for increased oxidative stress and the activation of viral-related signaling pathways in post-mortem lungs of COVID-19 patients compared to control donors. Moreover, an excess of neutrophil infiltration and NET formation were detected in the patients' lungs, where the extracellular DNA was oxidized and co-localized with neutrophil granule protein myeloperoxidase (MPO). Interestingly, staining of the lipid peroxidation marker 4-hydroxynonenal (4-HNE) depicted high colocalization with NETs and was correlated with the neutrophil infiltration of the lung tissues, suggesting that it could serve as a suitable marker for the identification of NETs and the severity of the disease. Moreover, local inhalation therapy to reduce the excess lipid oxidation and NETs in the lungs of severely infected patients might be useful to ameliorate their clinical conditions.

NET formation is independent of gasdermin D and pyroptotic cell death.

Neutrophil extracellular traps (NETs) are DNA scaffolds coated with granule proteins that are released by neutrophils to ensnare and kill bacteria. NET formation occurs in response to many stimuli through independent molecular pathways. Although NET release has been equated to a form of lytic cell death, live neutrophils can rapidly release antimicrobial NETs. Gasdermin D (GSDMD), which causes pyroptotic death in macrophages, is thought to be required for NET formation by neutrophils. Through experiments with known physiological activators of NET formation and ligands that activate canonical and noncanonical inflammasome signaling pathways, we demonstrated that Gsdmd-deficient mouse neutrophils were as competent as wild-type mouse neutrophils in producing NETs. Furthermore, GSDMD was not cleaved in wild-type neutrophils during NET release in response to inflammatory mediators. We found that activation of both canonical and noncanonical inflammasome signaling pathways resulted in GSDMD cleavage in wild-type neutrophils but was not associated with cell death. Moreover, NET formation as a result of either pathway of inflammasome activation did not require GSDMD. Together, these data suggest that NETs can be formed by viable neutrophils after inflammasome activation and that this function does not require GSDMD.

Established and emerging roles for mitochondria in neutrophils.

Neutrophils are the most abundant innate immune cells in human blood, emerging as important players in a variety of diseases. Mitochondria are bioenergetic, biosynthetic, and signaling organelles critical for cell fate and function. Mitochondria have been overlooked in neutrophil research owing to the conventional view that neutrophils contain few, if any, competent mitochondria and do not rely on these organelles for adenosine triphosphate production. A growing body of evidence suggests that mitochondria participate in neutrophil biology at many levels, ranging from neutrophil development to chemotaxis, effector function, and cell death. Moreover, mitochondria and mitochondrial components, such as mitochondrial deoxyribonucleic acid, can be released by neutrophils to eliminate infection and/or shape immune response, depending on the specific context. In this review, we provide an update on the functional role of mitochondria in neutrophils, highlight mitochondria as key players in modulating the neutrophil phenotype and function during infection and inflammation, and discuss the possibilities and challenges to exploit the unique aspects of mitochondria in neutrophils for disease treatment.

Pharmacological Induction of Granulocyte Cell Death as Therapeutic Strategy.

Apoptosis is central for the maintenance of health. In the immune system, apoptosis guarantees proper development of immune cells and shutdown of immune reactions by the coordinated elimination of activated immune cells. Limitation of the life span of granulocytes is important, as overactivation of these cells is associated with chronic inflammation and collateral tissue damage. Consequently, targeted induction of granulocyte apoptosis may be beneficial in the course of respective immune disorders. Anti-inflammatory drugs such as glucocorticoids and monoclonal antibodies against IL-5Rα exert their function in part by triggering eosinophil apoptosis. Agonistic antibodies targeting Siglec-8 or death receptors are tested (pre)clinically. Moreover, a new class of inhibitors targeting antiapoptotic BCL-2 proteins shows great promise for anticancer treatments. Because of their specificity and tolerable side effects, these so-called BH3 mimetics may be worthwhile to evaluate in inflammatory disorders. Here, we review past and recent data on pharmacological apoptosis induction of granulocytes and highlight respective therapeutic potential.

Proposed refined diagnostic criteria and classification of eosinophil disorders and related syndromes.

Eosinophilia and eosinophil activation are recurrent features in various reactive states and certain hematologic malignancies. In patients with hypereosinophilia (HE), HE-induced organ damage is often encountered and may lead to the diagnosis of a hypereosinophilic syndrome (HES). A number of known mechanisms and etiologies contribute to the development of HE and HES. Based on these etiologies and the origin of eosinophils, HE and HES are divided into primary forms where eosinophils are clonal cells, reactive forms where an underlying reactive or neoplastic condition is detected and eosinophils are considered to be "non-clonal" cells, and idiopathic HE and HES in which neither a clonal nor a reactive underlying pathology is detected. Since 2012, this classification and the related criteria have been widely accepted and regarded as standard. However, during the past few years, new developments in the field and an increasing number of markers and targets have created a need to update these criteria and the classification of HE and HES. To address this challenge, a Working Conference on eosinophil disorders was organized in 2021. In this conference, a panel of experts representing the relevant fields, including allergy, dermatology, hematology, immunology, laboratory medicine, and pathology, met and discussed new markers and concepts as well as refinements in definitions, criteria and classifications of HE and HES. The outcomes of this conference are presented in this article and should assist in the diagnosis and management of patients with HE and HES in daily practice and in the preparation and conduct of clinical trials.

The regulatory role of eosinophils in adipose tissue depends on autophagy.

Introduction: Obesity is a metabolic condition that elevates the risk of all-cause mortality. Brown and beige adipose tissues, known for their thermogenic properties, offer potential therapeutic targets for combating obesity. Recent reports highlight the role of immune cells, including eosinophils, in adipose tissue homeostasis, while the underlying mechanisms are poorly understood. Methods: To study the role of autophagy in eosinophils in this process, we used a genetic mouse model lacking autophagy-associated protein 5 (Atg5), specifically within the eosinophil lineage (Atg5 eoΔ). Results: The absence of Atg5 in eosinophils led to increased body weight, impaired glucose metabolism, and alterations in the cellular architecture of adipose tissue. Our findings indicate that Atg5 modulates the functional activity of eosinophils within adipose tissue rather than their abundance. Moreover, RNA-seq analysis revealed upregulation of arginase 2 (Arg2) in Atg5-knockout eosinophils. Increased Arg2 activity was shown to suppress adipocyte beiging. Furthermore, we observed enrichment of the purine pathway in the absence of Atg5 in eosinophils, leading to a pro-inflammatory shift in macrophages and a further reduction in beiging. Discussion: The data shed light on the importance of autophagy in eosinophils and its impact on adipose tissue homeostasis by suppressing Arg2 expression and limiting inflammation in adipose tissue.