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[This corrects the article DOI: 10.3389/fimmu.2021.715766.].[This corrects the article DOI: 10.3389/fimmu.2021.715766.].

PAD4-mediated hypercitrullination of histone H4 arginine 3 (H4R3) has been previously found to promote the formation of Neutrophil Extracellular Traps in inflamed tissues and the resulting histone H4 citrulline 3 (H4Cit3) modification is thought to play a key role in extracellular trap (ET) formation by promoting chromatin decondensation. In addition to neutrophils, macrophages have also recently been found to generate functional extracellular traps (METs). However, a role for PADs in ET formation in macrophages has not been previously described. Transcripts for PAD2 and PAD4 are found in mature macrophages and these cells can be induced to citrullinate proteins, thus raising the possibility that PADs may play a direct role in ET formation in macrophages via histone hypercitrullination. In breast and visceral white adipose tissue from obese patients, infiltrating macrophages are often seen to surround dead adipocytes forming characteristic \"crown-like structures\" (CLS) and the presence of these lesions is associated with increased levels of inflammatory mediators. In light of these observations, we have initiated studies to test whether PADs are expressed in CLS macrophages and whether these macrophages might form METs. Our preliminary findings show that PAD2 (and to a lesser extent, PAD4) is expressed in both in the macrophage cell line (RAW 264.7) and in CLS lesions. Additionally, we provide evidence that macrophage-derived extracellular histones are seen around presumptive macrophages within CLS lesions and that these histones contain the H4Cit3 modification. These initial findings support our hypothesis that obesity-induced adipose tissue inflammation promotes the formation of METs within CLS lesions via PAD-mediated histone hypercitrullination. Subsequent studies are underway to further validate these findings and to investigate the role in PAD-mediated MET formation in CLS function in the mammary gland.

Eosinophils are an enigmatic white blood cell, whose immune functions are still under intense investigation. Classically, the eosinophil was considered to fulfill a protective role against parasitic infections, primarily large multicellular helminths. Although eosinophils are predominantly associated with parasite infections, evidence of a role for eosinophils in mediating immunity against bacterial, viral, and fungal infections has been recently reported. Among the mechanisms by which eosinophils are proposed to exert their protective effects is the production of DNA-based extracellular traps (ETs). Remarkably, DNA serves a role that extends beyond its biochemical function in encoding RNA and protein sequences; it is also a highly effective substance for entrapment of bacteria and other extracellular pathogens, and serves as valuable scaffolding for antimicrobial mediators such as granule proteins from immune cells. Extracellular trap formation from eosinophils appears to fulfill an important immune response against extracellular pathogens, although overproduction of traps is evident in pathologies. Here, we discuss the discovery and characterization of eosinophil extracellular traps (EETs) in response to a variety of stimuli, and suggest a role for these structures in the pathogenesis of disease as well as the establishment of autoimmunity in chronic, unresolved inflammation.

Abstract Cytolytic ETosis is a type of programmed cell death that is distinct from apoptosis and necrosis and plays a major role in the innate immune system and disease progression. Through the process of ETosis, cells release their chromatin with diverse antimicrobial proteins into the extracellular milieu, forming extracellular traps (ETs). Although ETosis has been reported in several leukocyte types, few studies have compared ETosis and the component proteins of ETs in leukocytes. The aim of this study was to better understand the characteristics of eosinophil ETosis (EETosis) compared with other leukocytes. We isolated human blood eosinophils, neutrophils, basophils, monocytes, and lymphocytes and stimulated them with known ETosis inducers: the protein kinase C activator PMA and calcium ionophore A23187. Both stimuli induced eosinophil cell death and ET release after 180 minutes of stimulation in a nicotinamide adenine dinucleotide phosphate oxidase–dependent manner. PMA also induced nicotinamide adenine dinucleotide phosphate oxidase–dependent ETosis in neutrophils, whereas little or no significant ETosis was observed in basophils, monocytes, or lymphocytes at 180 minutes. Mass spectrometry–based proteomic analysis of eosinophil- and neutrophil-derived ETs identified 997 and 1,415 proteins, respectively. Among the physiological stimuli tested, immobilized IgA and IgG induced EETosis. CCL11 (C-C motif chemokine ligand 11) and IL-5 were weak inducers of EETosis, but costimulation significantly induced rapid EETosis. Under high serum or albumin conditions, costimulation with CCL11 and IL-5 paradoxically prolonged cell survival by preventing spontaneous apoptosis. This study provides an in-depth characterization of EETosis and highlights the precise regulation of eosinophil survival and cell death pathways.

Freshwater crayfish immunity has received great attention due to the need for urgent conservation. This concern has increased the understanding of the cellular and humoral defense systems, although the regulatory mechanisms involved in these processes need updating. There are, however, aspects of the immune response that require clarification and integration. The particular issues addressed in this review include an overall description of the oomycete Aphanomyces astaci , the causative agent of the pandemic plague disease, which affects freshwater crayfish, and an overview of crustaceans’ immunity with a focus on freshwater crayfish. It includes a classification system of hemocyte sub-types, the molecular factors involved in hematopoiesis and the differential role of the hemocyte subpopulations in cell-mediated responses, including hemocyte infiltration, inflammation, encapsulation and the link with the extracellular trap cell death pathway (ETosis). In addition, other topics discussed include the identity and functions of hyaline cells, the generation of neoplasia, and the emerging topic of the role of sessile hemocytes in peripheral immunity. Finally, attention is paid to the molecular execution of the immune response, from recognition by the pattern recognition receptors (PRRs), the role of the signaling network in propagating and maintaining the immune signals, to the effector elements such as the putative function of the Down syndrome adhesion molecules (Dscam) in innate immune memory.

Filariae are parasitic roundworms, which can cause debilitating diseases such as lymphatic filariasis and onchocerciasis. Lymphatic filariasis, also known as elephantiasis, and onchocerciasis, commonly referred to as river blindness, can lead to stigmatizing pathologies and present a socio-economic burden for affected people and their endemic countries. Filariae typically induce a type 2 immune response, which is characterized by cytokines, i.e., IL-4, IL-5 and IL-13 as well as type 2 immune cells including alternatively activated macrophages, innate lymphoid cells and Th2 cells. However, the hallmark characteristic of filarial infections is a profound eosinophilia. Eosinophils are innate immune cells and pivotal in controlling helminth infections in general and filarial infections in particular. By modulating the function of other leukocytes, eosinophils support and drive type 2 immune responses. Moreover, as primary effector cells, eosinophils can directly attack filariae through the release of granules containing toxic cationic proteins with or without extracellular DNA traps. At the same time, eosinophils can be a driving force for filarial pathology as observed during tropical pulmonary eosinophilia in lymphatic filariasis, in dermatitis in onchocerciasis patients as well as adverse events after treatment of onchocerciasis patients with diethylcarbamazine. This review summarizes the latest findings of the importance of eosinophil effector functions including the role of eosinophil-derived proteins in controlling filarial infections and their impact on filarial pathology analyzing both human and experimental animal studies.

The release of extracellular traps (ETs) is a recently described mechanism of innate immune response to infection. Although ETs have been intensely investigated in the context of neutrophil antimicrobial effector mechanisms, other immune cells such as mast cells, eosinophils, and macrophages can also release these structures. The different ETs have several features in common, regardless of the type of cells from which they originated, including a DNA backbone with embedded antimicrobial peptides, proteases, and histones. However, they also exhibit remarkable individual differences such as the type of sub-cellular compartments from where the DNA backbone originates (e.g., nucleus or mitochondria), the proportion of responding cells within the pool, and/or the molecular mechanism/s underlying the ETs formation. This review summarizes the knowledge accumulated in recent years regarding the complex and expanding world of ETs and their role in immune function with particular emphasis on the role of other immune cells rather than on neutrophils exclusively.

Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates μ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express μ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca2+]i) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca2+]i. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of μ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between μ-opioid and TLR4 receptors.

Allergic bronchopulmonary aspergillosis (ABPA) is characterized by an early allergic response and late-phase lung injury in response to repeated exposure to antigens, as a consequence of persistent fungal colonization of the airways. Here, we summarize the clinical and pathological features of ABPA, focusing on thick mucus plugging, a key observation in ABPA. Recent findings have indicated that luminal eosinophils undergo cytolytic extracellular trap cell death (ETosis) and release filamentous chromatin fibers (extracellular traps, ETs) by direct interaction with . Production of ETs is considered to be an innate immune response against non-phagocytable pathogens using a \"trap and kill\" mechanism, although eosinophil ETs do not promote damage or killing. Compared with neutrophils, eosinophil ETs are composed of stable and condensed chromatin fibers and thus might contribute to the higher viscosity of eosinophilic mucus. The major fate of massively accumulated eosinophils in the airways is ETosis, which potentially induces the release of toxic granule proteins and damage-associated molecular patterns, epithelial damage, and further decreases mucus clearance. This new perspective on ABPA as a luminal hypereosinophilic disease with ETosis/ETs could provide a better understanding of airway mucus plugging and contribute to future therapeutic strategies for this challenging disease.

Over the past nearly two decades, increasing evidence has uncovered how immune cells can actively extrude genetic material to entrap invading pathogens or convey sterile inflammatory signals that contribute to shaping immune responses. Originally identified in neutrophils, the release of decondensed chromatin fibers decorated with antimicrobial proteins, called extracellular traps (ETs), has been recognized as a specific form of programmed inflammatory cell death, which is now known to occur in several other leukocytes. Subsequent reports have shown that self-DNA can be extruded from immune cells even in the absence of cell death phenomena. More recent data suggest that ETs formation could exacerbate neuroinflammation in several disorders of the central nervous system (CNS). This review article provides an overview of the varied types, sources, and potential functions of extracellular DNA released by immune cells. Key evidence suggesting the involvement of ETs in neurodegenerative, traumatic, autoimmune, and oncological disorders of the CNS will be discussed, outlining ongoing challenges and drawing potentially novel lines of investigation.