Process Of NETosis Research Articles

How Do ROS Induce NETosis? Oxidative DNA Damage, DNA Repair, and Chromatin Decondensation.

Neutrophil extracellular traps (NETs) are intricate, DNA-based, web-like structures adorned with cytotoxic proteins. They play a crucial role in antimicrobial defense but are also implicated in autoimmune diseases and tissue injury. The process of NET formation, known as NETosis, is a regulated cell death mechanism that involves the release of these structures and is unique to neutrophils. NETosis is heavily dependent on the production of reactive oxygen species (ROS), which can be generated either through NADPH oxidase (NOX) or mitochondrial pathways, leading to NOX-dependent or NOX-independent NETosis, respectively. Recent research has revealed an intricate interplay between ROS production, DNA repair, and NET formation in different contexts. UV radiation can trigger a combined process of NETosis and apoptosis, known as apoNETosis, driven by mitochondrial ROS and DNA repair. Similarly, in calcium ionophore-induced NETosis, both ROS and DNA repair are key components, but only play a partial role. In the case of bacterial infections, the early stages of DNA repair are pivotal. Interestingly, in serum-free conditions, spontaneous NETosis occurs through NOX-derived ROS, with early-stage DNA repair inhibition halting the process, while late-stage inhibition increases it. The intricate balance between DNA repair processes and ROS production appears to be a critical factor in regulating NET formation, with different pathways being activated depending on the nature of the stimulus. These findings not only deepen our understanding of the mechanisms behind NETosis but also suggest potential therapeutic targets for conditions where NETs contribute to disease pathology.

Unique Interplay Between Antinuclear Antibodies and Nuclear Molecules in the Pathogenesis of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that primarily affects young women and causes a wide range of inflammatory manifestations. The hallmark of SLE is the production of antibodies to components of the cell nucleus (antinuclear antibodies [ANAs]). These antibodies can bind to DNA, RNA, and protein complexes with nucleic acids. Among ANAs, antibodies to DNA (anti-DNA) are markers for classification and disease activity, waxing and waning disease activity in many patients. In the blood, anti-DNA antibodies can bind to DNA to form immune complexes with two distinct roles in pathogenesis: (1) renal deposition to provoke nephritis and (2) stimulation of cytokine production following uptake into innate immune cells and interaction with internal nucleic acid sensors. These sensors are part of an internal host defense system in the cell cytoplasm that can respond to DNA from infecting organisms; during cell stress, DNA from nuclear and mitochondrial sources can also trigger these sensors. The formation of immune complexes requires a source of extracellular DNA in an immunologically accessible form. As shown in in vivo and in vitro systems, extracellular DNA can emerge from dead and dying cells in both a free and a particulate form. Neutrophils undergoing the process of NETosis can release DNA in mesh-like structures called neutrophil extracellular traps. In SLE, therefore, the combination of ANAs and immunologically active DNA can create new structures that can promote inflammation throughout the body as well as drive organ inflammation and damage.

Regulation of miRNA-155-5p ameliorates NETosis in pulmonary fibrosis rat model via inhibiting its target cytokines IL-1β, TNF-α and TGF-β1

Idiopathic pulmonary fibrosis (IPF) is an age-related inflammatory disease with no cure up till now.It is accompanied by neutrophils infiltration as the main responders to inflammation and fibrosis. Importantly, neutrophils release neutrophil extracellular traps (NETs) through NETosis process. The function of microRNAs during inflammation became of great biological attention. Owing to microRNAs’ central role in immune system, microRNA-155-5p (miR-155-5p) is intensely involved in the inflammatory response. Capsaicin (Cap) is a bioactive compound that exhibits antioxidative and anti-inflammatory functions. Recent studies have shown its role in regulation of certain microRNAs’ expressions. Accordingly, the present study aims to investigate the effect of miR-155-5p regulation in suppressing NETs production via ameliorating its target inflammatory cytokines, IL-1ß, TNF-α and TGF-ß1, in bleomycin (BLM)-induced pulmonary fibrosis rat model treated by Cap. The obtained results demonstrated that miR-155-5p downregulation was associated with significant decrease in IL-1ß, TNF-α, TGF-β1, which consequently, reduced hydroxyproline (HYP), NETs activity markers as NE and PAD-4, and alleviated CTGF levels in lung tissues of animals treated by Cap. Furthermore, NETosis ultrastructure examination by transmission electron microscope (TEM), MPO immunohistochemical staining and histopathological studies confirmed an abolishment in NETs formation and an improvement in lung tissue architecture in Cap-treated rats. This study concluded that Cap quenched the inflammatory response through interrupting IL-1β, TNF-α and TGF-β1 pathway via modulating miR-155-5p expression. In addition, Cap was able to alleviate pulmonary NETosis markers by restraining NETs activity markers. These findings provide novel insight into the application of Cap-based treatment in ameliorating pulmonary damage in IPF.

THU493 The Role Of Liver Receptor Homolog1 (LRH-1) In Regulating Breast Cancer Progression By Modulating The Immune Response

Abstract Disclosure: Y. Wang: None. N. Krawczynska: None. S. Bendre: None. H. Vidana Gamage: None. A. Nelczyk: None. A. Das Gupta: None. E.R. Nelson: None. Breast cancer is the most common type of cancer and the second leading cause of death among American women. While immunotherapy holds much promise, the current FDA-approved immunotherapies are effective for only a subset of patients lacking expression of estrogen receptor, progesterone receptor and HER2, but positive for PD-L1. Thus, there is strong impetus to develop novel ways to engage the immune system. We have demonstrated that cholesterol metabolism and homeostasis within myeloid cells can either promote or suppress anti-cancer immunity. One of the regulators of cholesterol homeostasis is Liver Receptor Homolog 1 (LRH-1/NR5A2). We have found that elevated LRH-1 expression in breast tumors is associated with an increased survival. LRH-1 is highly expressed in myeloid cells, particularly neutrophils, a major immune population of the tumor microenvironment. Therefore, we hypothesized that LRH-1 plays modulatory roles in myeloid cells, that subsequently impacts breast cancer progression. Specifically, we evaluated the impact of LRH-1 on myeloid cell biology, including NETosis and phagocytosis. Treatment of neutrophils with an agonist of LRH-1, DLPC, resulted in decreased NETosis, a function of neutrophils previously implicated in recurrence and metastasis. Treatment with an antagonist resulted in increased NETosis. Both the agonist and antagonist exhibited dose-depended effects, strongly suggesting that LRH-1 regulates the process of NETosis. Macrophage phagocytosis is an important initial step towards antigen presentation and activation of T cells. Bone marrow derived macrophages treated with the LRH-1 antagonist exhibited decreased phagocytosis activity, in a dose dependent manner. Treatment with the agonist (DLPC) did not alter phagocytosis, but this may be because basal phagocytosis was already very high. Collectively, our data indicate that LRH-1 plays important roles in the regulation of myeloid cell function, including NETosis in neutrophils and phagocytosis in macrophages. Overall, these findings suggest that LRH-1 activity would be beneficial for an anti-cancer immune response, and therefore position LRH-1 as a therapeutic target. Our ongoing studies will assess the role of LRH-1 in other myeloid cell-related functions including efferocytosis, T-cell expansion and differentiation. Presentation: Thursday, June 15, 2023

Fluorescence imaging deformability cytometry: integrating nuclear structure with mechanical phenotyping

Mechanical measurements of cells can provide unique insights into cell state and disease processes. The overall mechanical properties of cells can be heavily affected by the stiffest organelle, the nucleus. However, it is challenging to fully characterize internal nuclear structures in most cell mechanical measurement platforms. Here, we demonstrate single-cell deformability measurements of whole cells and stained nuclei in a fluorescence imaging flow cytometry platform. We also introduce bending energy derived metrics as a way to normalize measurements of cytoskeletal cortex and nuclear shape changes of cells and demonstrate the utility of relative deformability distributions to characterize populations of cells. We apply the platform to measure changes in cell biophysical properties during the process of NETosis, whereby neutrophils undergo drastic nuclear restructuring. We characterize cell size, deformability, and nuclear structure changes and their correlations in thousands of neutrophils undergoing NETosis, a process implicated in development of critical disease states, such as sepsis. This platform can aid in understanding heterogeneity in deformability in cell populations and how this may be influenced by nuclear or internal structure changes.Graphical

Abstract P182: Neutrophil Extracellular Traps Are Formed In Response To Hypertensive Stretch And Contribute To Vascular Dysfunction In Hypertension

Background: Neutrophils play a crucial role in the innate immune response, in part by the release of neutrophil extracellular traps (NETs), which consist of decondensed chromatin and proteins including citrullinated histone 3 (H3-Cit). The process of NETosis involves the disassociation of DNA from the nucleosome catalyzed by the enzyme peptidyl arginine deiminase 4 (PAD4). TRPV4 is a mechanosensitive calcium channel expressed in neutrophils. Studies of human subjects and rodent models have described the accumulation of NETs in renal and vascular tissue in hypertension (HTN), however the causative role of NETs has not been described. We hypothesized that NETs form in response to hypertensive stretch and contribute to endothelial cell (EC) dysfunction and HTN. Methods: Mice deficient for Padi4 lack the PAD4 enzyme and cannot efficiently form NETs. Padi4 -/- and C57Bl/6J mice were treated with 490 ng/kg/min angiotensin (Ang) II for 4 weeks. Blood pressure was measured by radiotelemetry. Vascular reactivity studies were performed on mesenteric arteries. Vascular infiltration was measured by flow cytometry. Murine neutrophils and aortic ECs were co-cultured and subjected to uniaxial stretch under normotensive (5%) and hypertensive (10%) conditions. Neutrophils were treated with a TRPV4 agonist (GSK1016790A) and NETs were quantified by immunofluorescence. Results: Padi4 -/- mice exhibit attenuated HTN and improved vascular reactivity following Ang II treatment (systolic BP 159.4 ± 1.4 mmHg in C57Bl/6J mice vs 147.9 ± 2.6 mmHg in Padi4 -/- mice, n= 7-8, p= 0.007). Flow cytometry revealed a marked reduction in aortic infiltration of immune cells in Padi4 -/- mice compared to C57Bl/6J mice. Co-culture of murine neutrophils with ECs improves neutrophil survival and augments NETosis. Hypertensive stretch of co-cultured neutrophils and ECs augmented NETosis compared to normotensive stretch. Immunoblot revealed increased H3-Cit in neutrophils exposed to hypertensive stretch. Neutrophils treated with GSK1016790A exhibit augmented NETosis. Conclusions: These findings suggest a vital role of stretch-induced NETosis as a mediator of EC dysfunction and HTN. Mechanistically, both agonism of neutrophil TRPV4 and hypertensive stretch of ECs augment NETosis.

Immunoaging - the effect of age on serum levels of NET biomarkers in men: a pilot study.

The study aimed to evaluate the impact of aging on the formation of neutrophil extracellular traps (NETs). The impaired formation of NETs is the cause of an abnormal innate immune response. The study included a total of 45 healthy male subjects of different age groups. Whole blood was collected from the subjects, and the concentration of myeloperoxidase (MPO), the main biocidal protein in NETs, was determined in serum using ELISA. The serum levels of circulating free DNA (cfDNA), which are the structural basis of NETs, were also measured by fluorescence. In addition, the white blood cell count was determined, whole blood smear was evaluated, and the neutrophillymphocyte ratio was calculated. The variations in the levels of NET biomarkers were analyzed in different age groups. The low levels of MPO (243.70 ng/ml) and cfDNA (6.24 ng/100 μl) in boys indicated neutrophil insufficiency for NETosis in children. A progressive increase in the levels of MPO and cfDNA with age was observed among adolescents (420.91, p = 0.04; 13.55, p = 0.03, respectively), with the highest level noted in the healthy adult group (466.58, p = 0.01; 14.07, p = 0.01, respectively). The levels of the studied parameters were comparable in adolescents and young adults, which proved that the NETosis process was appropriate and suggested the attainment of neutrophil maturity for the release of NETs in adolescence. The levels of MPO and cfDNA were low in older men (225.46, p < 0.01; 5.19, p < 0.01, respectively) indicating impaired NET formation. Data on the generation of NETs in different age groups obtained in this study can allow a better understanding of the ontogenesis of the immune system in terms of the course of NETosis, and also indicate the need to support nonspecific responses in children and adults. Further research should be performed to determine the possibility of regulating the NETosis process. Int J Occup Med Environ Health. 2023;36(3):333-48.

A Synthetic Signaling Network Imitating the Action ofImmune Cells in Response to Bacterial Metabolism.

State-of-the-art bottom-up synthetic biology allows to replicate many basic biological functions in artificial-cell-like devices. To mimic more complex behaviors, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here, a sophisticated biological response is considered, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. A consortium consisting of two synthetic agents is designed-responsive DNA-based particles and antibiotic-loaded lipid vesicles-whose coordinated action mimics the sought immune-like response when triggered by bacterial metabolism. The artificial netosis-like response emerges from a series of interlinked sensing and communication pathways between the live and synthetic agents, and translates into both physical and chemical antimicrobial actions, namely bacteria immobilization and exposure to antibiotics. The results demonstrate how advanced life-like responses can be prescribed with a relatively small number of synthetic molecular components, and outlines a new strategy for artificial-cell-based antimicrobial solutions.

NET-(works) in arterial and venous thrombo-occlusive diseases.

Formation of Neutrophil Extracellular Traps (NETosis), accompanied by the release of extracellular decondensed chromatin and pro-inflammatory as well as pro-thrombotic factors, is a pivotal element in the development and progression of thrombo-occlusive diseases. While the process of NETosis is based on complex intracellular signalling mechanisms, it impacts a wide variety of cells including platelets, leukocytes and endothelial cells. Consequently, although initially mainly associated with venous thromboembolism, NETs also affect and mediate atherothrombosis and its acute complications in the coronary, cerebral and peripheral arterial vasculature. In this context, besides deep vein thrombosis and pulmonary embolism, NETs in atherosclerosis and especially its acute complications such as myocardial infarction and ischemic stroke gained a lot of attention in the cardiovascular research field in the last decade. Thus, since the effect of NETosis on platelets and thrombosis in general is extensively discussed in other review articles, this review focusses on the translational and clinical relevance of NETosis research in cardiovascular thrombo-occlusive diseases. Consequently, after a brief summary of the neutrophil physiology and the cellular and molecular mechanisms underlying NETosis are presented, the role of NETosis in atherosclerotic and venous thrombo-occlusive diseases in chronic and acute settings are discussed. Finally, potential prevention and treatment strategies of NET-associated thrombo-occlusive diseases are considered.

Neutrophil Extracellular Traps and Cancer: Trapping Our Attention with Their Involvement in Ovarian Cancer.

Neutrophils, the most abundant circulating leukocytes, play a well-known role in defense against pathogens through phagocytosis and degranulation. However, a new mechanism involving the release of neutrophil extracellular traps (NETs) composed of DNA, histones, calprotectin, myeloperoxidase, and elastase, among others, has been described. The so-called NETosis process can occur through three different mechanisms: suicidal, vital, and mitochondrial NETosis. Apart from their role in immune defense, neutrophils and NETs have been involved in physiopathological conditions, highlighting immunothrombosis and cancer. Notably, neutrophils can either promote or inhibit tumor growth in the tumor microenvironment depending on cytokine signaling and epigenetic modifications. Several neutrophils' pro-tumor strategies involving NETs have been documented, including pre-metastatic niche formation, increased survival, inhibition of the immune response, and resistance to oncologic therapies. In this review, we focus on ovarian cancer (OC), which remains the second most incidental but the most lethal gynecologic malignancy, partly due to the presence of metastasis, often omental, at diagnosis and the resistance to treatment. We deepen the state-of-the-art on the participation of NETs in OC metastasis establishment and progression and their involvement in resistance to chemo-, immuno-, and radiotherapies. Finally, we review the current literature on NETs in OC as diagnostic and/or prognostic markers, and their contribution to disease progression at early and advanced stages. The panoramic view provided in this article might pave the way for enhanced diagnostic and therapeutic strategies to improve the prognosis of cancer patients and, specifically, OC patients.

Molecular and genetic aspects of the pathogenesis of COVID-associated thrombosis

Annotation. COVID-19 is a viral infectious disease that reached pandemic proportions in 2020. The SARS-CoV-2 virus, which is the etiological factor of the coronavirus disease, has a complex mechanism of impact on the human body, causing various manifestations and causing complications of the disease. As you know, a severe form of coronavirus infection is associated with dire consequences, among which the main step is a violation of the coagulation system, which has the appearance of CVD syndrome, but its main symptom is thrombosis of arterial and venous vessels. The frequency of occurrence of thrombocytopenia forces us to investigate the pathogenesis of the development of this process to reduce the number of cases. However, there is still no clear opinion about the pathogenesis of such thrombosis. Therefore, the purpose of this review was to analyse the most probable mechanisms of development of venous and/or arterial thrombosis associated with coronavirus disease. In the course of the study, 64 information sources were analysed, extracted from PubMed, Web of Science, Google Scholar, and Elsevier databases. At the beginning of a thorough analysis of information, the following main links of COVID-associated thrombosis were identified: direct hyperactivation of blood platelets, which leads, through the participation of their surface molecules (P-selectin, CD40L, etc.), to the activation of aggregation and adhesion of platelets; ACE2 - mediated cell activation and endothelial dysfunction, which together have the property of stimulating thrombus formation; activation of the NETosis process, the MAPK pathway, Toll-like receptors and the Nox2 enzyme system, which also through a cascade of various reactions, which are described below, cause thrombosis. We understood that these several pathogenetic chains can work relatively separately, but the difficulty in describing the development of thrombotic disease caused by the SARS-CoV-2 virus is that these aspects of pathogenesis are closely related and intertwined at different links, forming both direct and feedback loops, and vicious circles. The obtained structured data can serve as a basis for further original research, which will allow the development of targeted therapy for the treatment and prevention of post-covid thromboses, directed at the described molecular genetic aspects.

Release of neutrophil extracellular traps in response to Candida albicans yeast, as a secondary defense mechanism activated by phagocytosis.

Candida albicans is one of the main pathogens responsible for the development of difficult-to-fight fungal infections called candidiasis. Neutrophils are the major effector cells involved in the eradication of fungal pathogens. This group of immune cells uses several mechanisms that enable the rapid neutralization of pathogens. The most frequently identified mechanisms are phagocytosis and the release of neutrophil extracellular traps (NETs). The mechanism for selecting the type of neutrophil immune response is still unknown. In our study, we analyzed the relationship between the activation of phagocytosis and netosis. We detected the presence of two neutrophil populations characterized by different response patterns to contact with C. albicans blastospores. The first neutrophil population showed an increased ability to rapidly release NETs without prior internalization of the pathogen. In the second population, the netosis process was inherently associated with phagocytosis. Differences between populations also referred to the production of reactive oxygen species. Our results suggest that neutrophils use different strategies to fight C. albicans and, contrary to previous reports, these mechanisms are not mutually exclusive.

Increased levels of NETosis biomarkers in high-grade serous ovarian cancer patients' biofluids: Potential role in disease diagnosis and management.

High-grade serous ovarian cancer (HGSOC) is the second most frequent gynecological malignancy but the most lethal, partially due to the spread of the disease through the peritoneal cavity. Recent evidence has shown that, apart from their role in immune defense through phagocytosis and degranulation, neutrophils are able to participate in cancer progression through the release of neutrophil extracellular traps (NETs) in a process called NETosis. NETs are composed of DNA, histones, calprotectin, myeloperoxidase (MPO) and elastase and the NETosis process has been proposed as a pre-requisite for the establishment of omental metastases in early stages of HGSOC. Nevertheless, its role in advanced stages remains to be elucidated. Therefore, our principal aim is to characterize a NETosis biomarker profile in biofluids from patients with advanced HGSOC and control women. Specifically, five biomarkers of NETosis (cell-free DNA (cfDNA), nucleosomes, citrullinated histone 3 (citH3), calprotectin and MPO) were quantified in plasma and peritoneal fluid (PF) samples from patients (n=45) and control women (n=40). Our results showed that HGSOC patients presented a higher concentration of cfDNA, citH3 and calprotectin in plasma and of all five NETosis biomarkers in PF than control women. Moreover, these biomarkers showed a strong ability to differentiate the two clinical groups. Interestingly, neoadjuvant treatment (NT) seemed to reduce NETosis biomarkers mainly systemically (plasma) compared to the tumor environment (PF). In conclusion, NETosis biomarkers are present in the tumor environment of patients with advanced HGSOC, which might contribute to the progression of the disease. Besides, plasma cfDNA and calprotectin could represent minimally invasive surrogate biomarkers for HGSOC. Finally, NT modifies NETosis biomarkers levels mainly at the systemic level.

Unobvious Neutrophil Extracellular Traps Signification in the Course of Oral Squamous Cell Carcinoma: Current Understanding and Future Perspectives.

Background: The current standards of treatment for oral squamous cell carcinoma (OSCC) include surgery, radiotherapy, and chemotherapy. In recent years, research on the effectiveness of immunotherapy in the treatment of OSCC has also been conducted.Purpose: Studies indicate that nonspecific immune mechanisms involved in the course of the anticancer response also need to be taken into account.Research Design: This review summarizes the results of our research on the active participation of neutrophils, which are previously underestimated, in the antitumor response in the course of OSCC, taking into account the ability of these cells to generate neutrophil extracellular traps (NETs).Results: We proved that the formation of NETs accompanies not only inflammatory changes but also the neoplastic process and that lipopolysaccharide (LPS) or interleukin 17 (IL-17) plays a critical role in inducing the formation of NETs during the OSCC. The greatest achievement of our published findings was the demonstration of the formation and release of NETs from neutrophils cocultured with tumor cells, as well as after stimulation with supernatant from the SCC culture with a PI3K-independent Akt kinase activation mechanism. Moreover, the pioneering achievement of our studies was the localization of NET structures in the tumor tissue, as well as the observation of high concentrations of NET markers in the serum of OSCC patients with low concentrations in the saliva, indicating the differences in the course of immune response between the periphery and the local reactions.Conclusions: The data presented here provide surprising but important information on the role of NETs in the course of OSCC, thus pointing to a promising new direction in the development of management strategies for early noninvasive diagnosis and monitoring of the disease course, and perhaps immunotherapy. Furthermore, this review raises further questions and elaborates on the process of NETosis in cancer.

Direct blood fluorescence signal intensity of neutrophils (NEU-SFL): A predictive marker of death in hospitalized COVID-19 patients?

Coronavirus disease 2019 (COVID-19) is a respiratory disease triggered by immunopathological mechanisms that cause excessive inflammation and leukocyte dysfunction. Neutrophils play a critical role in the innate immunity and are able to produce neutrophil extracellular traps (NETs: NETosis process) to combat infections. Some NETs markers are increased in patients who died from COVID-19. Recently, the neutrophil fluorescence variable (NEU-SFL), available on certain automated complete blood count (CBC) analyzers, has been correlated with NET formation and may reflect NETosis in patients. Here we evaluate whether NEU-SFL measured after admission of COVID-19 patients is associated with in-hospital survival or death. 1,852 patients admitted for severe COVID-19 at Nîmes University Hospital in 2021 were retrospectively included in the study: 1,564 who survived the hospital stay and 288 who did not. The NEU-SFL was obtained on the Sysmex™ XN-10® analyzer and values for survivors and non-survivors were compared. The intra-patient NEU-SFL variations between the hospital entry and the last day of hospitalization were also analyzed (IRB 22.06.01, NCT05413824). Non-survivors presented higher NEU-SFL values. NEU-SFL values above the 4th quartile were independently associated with a 2.88-fold risk of death. Furthermore, the difference of NEU-SFL values between the first and the last available data during hospitalization revealed that a decrease in NEU-SFL was associated to survivors and vice versa. Our study reinforces the role of neutrophils and NETosis in the pathophysiology and prognosis of COVID-19. Further studies combining NEU-SFL with other NETosis markers could improve the management of COVID-19 patients.

Therapeutic effect of Shaoyao-Gancao Decoction on TLR9-mediated NETosis in MRL/lpr mice.

Systemic lupus erythematosus (SLE) is a chronic, devastating autoimmune disorder associated with severe organ damage. The roles of Toll-like receptor 9 (TLR9) and NETosis in SLE have been described, suggesting the involvement of NETosis signaling in the development of SLE. Shaoyao-Gancao Decoction (SGT) is a potential medication for the treatment of SLE; however, its potential therapeutic mechanism remains unexplored. To determine the function of SGT in SLE, we treated MRL/lpr female mice with SGT, the main components of which were paeoniflorin (56.949 μg·mL-1) and glycyrrhizin (459.393 μg·mL-1). We found that SGT treatment relieved lymphadenectasis and splenomegaly, reduced urine protein and anti-dsDNA antibody concentrations, and relieved kidney pathology in MRL/lpr mice. SGT could also effectively regulate the oxidation/antioxidant balance, significantly reduce malondialdehyde (MDA) and nitric oxide (NO) contents and significantly increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in MRL/lpr mice. The neutrophil extracellular trap (NET) content of MRL/lpr mice also decreased to a certain extent after SGT treatment. All these results suggested that SGT might improve the inflammatory damage to tissues caused by oxygen free radicals, thereby regulating the NETosis process mediated by TLR9 and exerting a good therapeutic effect on SLE.

Histone Citrullination Mediates a Protective Role in Endothelium and Modulates Inflammation.

NETosis is a key host immune process against a pathogenic infection during innate immune activation, consisting of a neutrophil "explosion" and, consequently, NET formation, containing mainly DNA, histones, and other nuclear proteins. During sepsis, an exacerbated immune host response to an infection occurs, activating the innate immunity and NETosis events, which requires histone H3 citrullination. Our group compared the circulating histone levels with those citrullinated H3 levels in plasma samples of septic patients. In addition, we demonstrated that citrullinated histones were less cytotoxic for endothelial cells than histones without this post-translational modification. Citrullinated histones did not affect cell viability and did not activate oxidative stress. Nevertheless, citrullinated histones induced an inflammatory response, as well as regulatory endothelial mechanisms. Furthermore, septic patients showed elevated levels of circulating citrullinated histone H3, indicating that the histone citrullination is produced during the first stages of sepsis, probably due to the NETosis process.

Abstract 130: Isolevuglandins Drive Neutrophil Migration In Hypertension And Are Essential For The Formation Of Neutrophil Extracellular Traps

Introduction: Neutrophil extracellular traps (NETs) consist of decondensed chromatin and proteins including citrullinated histone 3, myeloperoxidase, and neutrophil elastase . Previous studies describe a role of neutrophils and NETs in hypertension and associated end-organ damage. Isolevuglandins (IsoLGs) are products of lipid peroxidation that play a role in immune activation in hypertension. We hypothesized that isoLGs are necessary for the process of NETosis and neutrophil migration in hypertension. Methods: C57BL/6 mice were treated with 490 ng/kg/min angiotensin (Ang) II and vehicle or the isoLG scavenger, 2-hydroxybenzylamine (2HOBA). Single cell sequencing was performed on spleens. NETs were quantified by flow cytometry and immunofluorescence. Human neutrophils were isolated and treated with ionomycin and vehicle or ethyl-2HOBA (Et2HOBA). Neutrophil chromatin expansion was determined by live cell confocal microscopy. Results: Single cell sequencing revealed increased splenic neutrophil accumulation following Ang II treatment. This was attenuated with 2HOBA co-treatment (Ang II 7.78%, Ang II + 2HOBA 2.46%, n=3). Flow cytometry revealed a marked increase in aortic NET accumulation that was reduced with 2HOBA (Sham 304 ± 110.6, Ang II 3,522 ± 928.6, Ang II + 2HOBA 1,007 ± 340.9 NETs, n=6-7). Immunofluorescence staining of kidneys revealed an increase in NET area in Ang II treated mice and a reduction with 2HOBA co-treatment (Sham 75.64 ± 27.2, Ang II 636.9 ± 105.0, Ang II + 2HOBA 14.98 ± 7.5 μm 2 , n=4-6). Ionomycin induced NETosis of isolated human neutrophils was associated with isoLG adduct accumulation that was attenuated with Et2HOBA co-treatment (54.24 ± 11.77% vs 1.78 ± 0.41%, n=5). Live cell confocal microscopy revealed NET area was increased 9.2-fold with ionomycin treatment compared to control. Pretreatment with Et2HOBA attenuated this response. Finally, isoLGs directly disrupt nucleosome assembly in vitro . Conclusion: These findings demonstrate an essential role of isoLGs in NETosis and neutrophil migration in hypertension. Moreover, they suggest a role of isoLG scavengers in the treatment of hypertension and additional NET-associated diseases including atherosclerosis and lupus.

Increased histone citrullination in juvenile idiopathic arthritis.

ObjectivePosttranslational modifications (PTMs) of proteins are crucial for regulating various biological processes. However, protein alteration via PTMs, and consequently, the creation of new epitopes, can induce abnormal autoimmune responses in predisposed individuals. Immunopathogenesis of several rheumatic diseases, including the most common childhood form, juvenile idiopathic arthritis (JIA), is associated with the generation of autoantibodies against such modified proteins. Dysregulated generation of neutrophil extracellular traps (NETs) can be a source of post-translationally altered proteins. Thus, we investigated the role of PTMs and the presence of NET-associated markers in JIA patients.MethodsWe recruited 30 pediatric patients with JIA (20 with active disease and 10 in remission) and 30 healthy donors. The serum concentrations of citrullinated histone H3 (citH3), peptidyl arginine deiminases (PADs), and NET-related products were detected using ELISA, and the number of citH3+ neutrophils was assessed using flow cytometry.ResultsThe serum levels of citH3 and PADs were higher in active as well as in remission JIA patients than in healthy donors. Similarly, the number of citH3+ neutrophils was higher in the peripheral blood of patients with JIA, implying an enhanced process of NETosis. This was effectively reflected by elevated serum levels of NET-associated products, such as neutrophil elastase, LL37, and cell-free DNA-histone complexes. Additionally, 16.7% of active JIA patients were seropositive for carbamylated autoantibodies, the levels of which declined sharply after initiation of anti-TNFα therapy.ConclusionCollectively, our data suggest that the accelerated process of NETosis and PTMs in JIA may result in the generation of anti-citrullinated/carbamylated autoantibodies against various epitopes later in life, which could be prevented by effectively regulating inflammation using immune therapy.

Understanding patient‐level immune response to COVID‐19 can inform treatment options

COVID‐19 is caused by an evolving pathogen that results in a disease that exhibits variability in progression and treatment response, highlighting the need for developing new and more precise therapies. Over the past two years, we have built a collection of tools to understand variability and mechanisms of COVID‐19 immune responses, including the SARS‐CoV‐2 evolutionary/structural dynamicome, curated lists of SARS‐CoV‐2 protein‐protein interactions (PPI), NCBI SRA data integrations for SARS‐CoV‐2 exposed cells, data from thousands of blood‐based RNA‐Seq analyzed, and created a clinical cohort of blood‐based RNA‐Seq of COVID‐19 and other critical illness pathologies. The dynamicome and PPI list elucidated several viral proteins that modulate the host immune response, including the nucleocapsid (N) role in suppressing nonsense‐mediated decay leading to rare disease phenotypes, ORF8 interaction with innate immune components, and the Papain‐like protease (NSP3) regulation of intracellular ISG15 interferon signaling. This stimulation in hospitalized COVID‐19 patients exerts three diverse systemic blood RNA‐Seq changes noted on five independent cohorts processed with a precision transcriptomics multi‐data analysis pipeline. The first was patients that are immunosuppressed, which showed blunted interferon response and often secondary viral activation, suggesting antiviral treatments and immune stimulation. The second patient group is those who go into cytokine storm, often seen with overly active ISG15 levels in peripheral blood, who benefit from immunosuppressive agents. The third group often has normal interferon activation but a triggered systemic process of NETosis in blood, activating neutrophils through controlled death to build DNA/histone nets to contain the virus. The third group likely requires specialized treatment strategies to prevent the NETosis from driving vascular dysfunction and COVID long hauler syndromes. These three groups resulting from varied pathologic mechanisms, showcase the need for different therapeutic approaches, and may explain the many failed clinical trials for COVID‐19. Thus, it would be beneficial to develop a deeper understanding of each patient’s immune response moving forward to optimize treatment and outcomes.