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

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

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

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

The regulatory role of eosinophils in adipose tissue depends on autophagy

IntroductionObesity 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.MethodsTo 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 (Atg5eoΔ).ResultsThe 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.DiscussionThe 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

Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production

Optic atrophy 1 (OPA1) is a mitochondrial inner membrane protein that has an important role in mitochondrial fusion and structural integrity. Dysfunctional OPA1 mutations cause atrophy of the optic nerve leading to blindness. Here, we show that OPA1 has an important role in the innate immune system. Using conditional knockout mice lacking Opa1 in neutrophils (Opa1(N Delta)), we report that lack of OPA1 reduces the activity of mitochondrial electron transport complex I in neutrophils. This then causes a decline in adenosine-triphosphate (ATP) production through glycolysis due to lowered NAD(+) availability. Additionally, we show that OPA1-dependent ATP production in these cells is required for microtubule network assembly and for the formation of neutrophil extracellular traps. Finally, we show that Opa1(N Delta) mice exhibit a reduced antibacterial defense capability against Pseudomonas aeruginosa.Peer reviewe

Actin polymerization and its glutathionylation are required for NET formation

Neutrophils are the most abundant cells in blood and their antimicrobial defense capabilities are defined, at least partially, by their formation of neutrophil extracellular traps (NETs). For the past decade, efforts have been made to elucidate the molecular mechanisms of NET formation. In this study, we demonstrate that disruption of the cellular cytoskeleton in neutrophils using pharmacological inhibitors or in knockout mice having defects in genes regulating the actin network prevents the DNA release and degranulation required for NET formation. For instance, Wiskott–Aldrich syndrome protein (WASP)deficient mouse (Was-/-) neutrophils, unable to polymerize actin, were consequently unable to release DNA or to degranulate. Furthermore, activation of mouse and human neutrophils exhibiting a genetic defect of the NADPH oxidase also failed to cause actin polymerization and subsequent NET formation. Finally, activated glutaredoxin 1 (Grx1) – deficient mouse (Grx1-/-) neutrophils accumulated high levels of glutathionylated actin that prevented actin polymerization, DNA release and degranulation. Taken together, we conclude that a functional actin network is achieved by a balance between ROS-mediated polymerization and glutathionylation and Grx1-mediated de-glutathionylation that is required for NET formation. Thus, these findings enlighten us about the molecular mechanisms involved in NET formation and provide new strategies for increasing the anti-microbial activity of neutrophils in patients with defects in the innate immune syste

REDUCTION OF NON-TECHNICAL LOSSES BASED ON TIME DOMAIN REFLECTOMETER (TDR) PRINCIPLES AND FUNCTION

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