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.

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.

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.

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 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.

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.

Nascent RHOH acts as a molecular brake on actomyosin-mediated effector functions of inflammatory neutrophils.

In contrast to molecular changes associated with increased inflammatory responses, little is known about intracellular counter-regulatory mechanisms that control signaling cascades associated with functional responses of neutrophils. Active RHO GTPases are typically considered as effector proteins that elicit cellular responses. Strikingly, we show here that RHOH, although being constitutively GTP-bound, limits neutrophil degranulation and the formation of neutrophil extracellular traps (NETs). Mechanistically, RHOH is induced under inflammatory conditions and binds to non-muscle myosin heavy chain IIA (NMHC IIA) in activated neutrophils in order to inhibit the transport of mitochondria and granules along actin filaments, which is partially reverted upon disruption of the interaction with NMHC IIA by introducing a mutation in RhoH at lysine 34 (RhoHK34A). In parallel, RHOH inhibits actin polymerization presumably by modulating RAC1 activity. In vivo studies using Rhoh-/- mice, demonstrate an increased antibacterial defense capability against Escherichia coli (E. coli). Collectively, our data reveal a previously undefined role of RHOH as a molecular brake for actomyosin-mediated neutrophil effector functions, which represents an intracellular regulatory axis involved in controlling the strength of an antibacterial inflammatory response.

Autophagy and Skin Diseases.

Autophagy is a highly conserved lysosomal degradation system that involves the creation of autophagosomes, which eventually fuse with lysosomes and breakdown misfolded proteins and damaged organelles with their enzymes. Autophagy is widely known for its function in cellular homeostasis under physiological and pathological settings. Defects in autophagy have been implicated in the pathophysiology of a variety of human diseases. The new line of evidence suggests that autophagy is inextricably linked to skin disorders. This review summarizes the principles behind autophagy and highlights current findings of autophagy's role in skin disorders and strategies for therapeutic modulation.

Physiological and Pathophysiological Roles of Metabolic Pathways for NET Formation and Other Neutrophil Functions.

Neutrophils are the most numerous cells in the leukocyte population and essential for innate immunity. To limit their effector functions, neutrophils are able to modulate glycolysis and other cellular metabolic pathways. These metabolic pathways are essential not only for energy usage, but also for specialized effector actions, such as the production of reactive oxygen species (ROS), chemotaxis, phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). It has been demonstrated that activated viable neutrophils can produce NETs, which consists of a DNA scaffold able to bind granule proteins and microorganisms. The formation of NETs requires the availability of increased amounts of adenosine triphosphate (ATP) as it is an active cellular and therefore energy-dependent process. In this article, we discuss the glycolytic and other metabolic routes in association with neutrophil functions focusing on their role for building up NETs in the extracellular space. A better understanding of the requirements of metabolic pathways for neutrophil functions may lead to the discovery of molecular targets suitable to develop novel anti-infectious and/or anti-inflammatory drugs.

Mycobacterial infection aggravates Helicobacter pylori-induced gastric preneoplastic pathology by redirection of de novo induced Treg cells.

The two human pathogens Helicobacter pylori and Mycobacterium tuberculosis (Mtb) co-exist in many geographical areas of the world. Here, using a co-infection model of H. pylori and the Mtb relative M. bovis bacillus Calmette-Guérin (BCG), we show that both bacteria affect the colonization and immune control of the respective other pathogen. Co-occurring M. bovis boosts gastric Th1 responses and H. pylori control and aggravates gastric immunopathology. H. pylori in the stomach compromises immune control of M. bovis in the liver and spleen. Prior antibiotic H. pylori eradication or M. bovis-specific immunization reverses the effects of H. pylori. Mechanistically, the mutual effects can be attributed to the redirection of regulatory T cells (Treg cells) to sites of M. bovis infection. Reversal of Treg cell redirection by CXCR3 blockade restores M. bovis control. In conclusion, the simultaneous presence of both pathogens exacerbates the problems associated with each individual infection alone and should possibly be factored into treatment decisions.

Characterization of eosinophilic esophagitis variants by clinical, histological, and molecular analyses: A cross-sectional multi-center study.

OBJECTIVE: Physicians are increasingly confronted with patients presenting with symptoms of esophageal dysfunction resembling eosinophilic esophagitis (EoE), but absence of significant esophageal eosinophilia. The purpose of this study was to characterize and classify this group of EoE variants. DESIGN: Patients from six EoE-centers with symptoms of esophageal dysfunction, but peak eosinophil counts of <60/mm2 (<15/hpf) in esophageal biopsies and absence of gastro-esophageal reflux disease (GERD) were included. Clinical, endoscopic, (immuno)-histological, and molecular features were determined and compared with EoE, GERD, and healthy controls. RESULTS: We included 69 patients with EoE variants. Endoscopic abnormalities were found in 53.6%. We identified three histological subtypes: EoE-like esophagitis (36/69, 52.2%), lymphocytic esophagitis (14/69, 20.3%), and non-specific esophagitis (19/69, 27.5%). Immunohistochemistry revealed-in contrast to EoE-no significant increase in inflammatory cell infiltrates compared with GERD and healthy controls, except for lymphocytes in lymphocytic esophagitis. EoE-typical Th2-response was absent in all EoE variants. However, considerable structural changes were detected based on histology and protein expression. Using next generation mRNA sequencing, we found the three EoE variants to have distinct molecular fingerprints partially sharing pronounced traits of EoE. Hierarchical sample clustering of RNA sequencing data confirmed the presence of an EoE-like (characterized by eotaxin-3 expression), non-specific, and lymphocytic variant cluster (characterized by CD3 cells and TSLP expression). CONCLUSION: All EoE variants are clinically and histologically active conditions despite the absence of esophageal eosinophilia. EoE variants appear to be part of a disease spectrum, where classical EoE represents the most common and apparent phenotype.

ATG5 and ATG7 Expression Levels Are Reduced in Cutaneous Melanoma and Regulated by NRF1.

Autophagy is a highly conserved cellular process in which intracellular proteins and organelles are sequestered and degraded after the fusion of double-membrane vesicles known as autophagosomes with lysosomes. The process of autophagy is dependent on autophagy-related (ATG) proteins. The role of autophagy in cancer is very complex and still elusive. We investigated the expression of ATG proteins in benign nevi, primary and metastatic melanoma tissues using customized tissue microarrays (TMA). Results from immunohistochemistry show that the expression of ATG5 and ATG7 is significantly reduced in melanoma tissues compared to benign nevi. This reduction correlated with changes in the expression of autophagic activity markers, suggesting decreased basal levels of autophagy in primary and metastatic melanomas. Furthermore, the analysis of survival data of melanoma patients revealed an association between reduced ATG5 and ATG7 levels with an unfavourable clinical outcome. Currently, the mechanisms regulating ATG expression levels in human melanoma remains unknown. Using bioinformatic predictions of transcription factor (TF) binding motifs in accessible chromatin of primary melanocytes, we identified new TFs involved in the regulation of core ATGs. We then show that nuclear respiratory factor 1 (NRF1) stimulates the production of mRNA and protein as well as the promoter activity of ATG5 and ATG7. Moreover, NRF1 deficiency increased in vitro migration of melanoma cells. Our results support the concept that reduced autophagic activity contributes to melanoma development and progression, and identifies NRF1 as a novel TF involved in the regulation of both ATG5 and ATG7 genes.

The Enigma of Eosinophil Degranulation.

Eosinophils are specialized white blood cells, which are involved in the pathology of diverse allergic and nonallergic inflammatory diseases. Eosinophils are traditionally known as cytotoxic effector cells but have been suggested to additionally play a role in immunomodulation and maintenance of homeostasis. The exact role of these granule-containing leukocytes in health and diseases is still a matter of debate. Degranulation is one of the key effector functions of eosinophils in response to diverse stimuli. The different degranulation patterns occurring in eosinophils (piecemeal degranulation, exocytosis and cytolysis) have been extensively studied in the last few years. However, the exact mechanism of the diverse degranulation types remains unknown and is still under investigation. In this review, we focus on recent findings and highlight the diversity of stimulation and methods used to evaluate eosinophil degranulation.

Evidence for Lysosomal Dysfunction within the Epidermis in Psoriasis and Atopic Dermatitis.

Atopic dermatitis and psoriasis are frequent chronic inflammatory skin diseases. Autophagy plays a substantial role in the homeostasis of an organism. Loss or impairment of autophagy is associated with multiple diseases. To investigate the possibility that autophagy plays a role in atopic dermatitis and psoriasis, we investigated the levels of key ATG proteins in human skin specimens as well as in primary human epidermal keratinocytes exposed to inflammatory stimuli in vitro. Although TNF-α facilitated the induction of autophagy in an initial phase, it reduced the levels and enzymatic activities of lysosomal cathepsins in later time periods, resulting in autophagy inhibition. Therefore, TNF-α appears to play a dual role in the regulation of autophagy. The relevance of these in vitro findings was supported by the observation that the protein levels of cathepsins D and L are decreased in both psoriasis and atopic dermatitis skin specimens. Taken together, this study suggests that TNF-α blocks autophagy in keratinocytes after long-term exposure, a mechanism that may contribute to the chronicity of inflammatory diseases of the skin and, perhaps, of other organs.

Patients with COVID-19: in the dark-NETs of neutrophils.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19.