A therapeutic strategy of parthenolide in improving imiquimod-induced psoriasis-like skin inflammation targeting IL-36/NETs through skin transdermal therapeutic system.

BACKGROUND: Psoriasis is an inflammatory skin disease that occurs repeatedly over time. The natural product of sesquiterpene lactones, Parthenolide (Par), is isolated from Tanacetum parthenium L. (feverfew) which has significant effects on anti-inflammatory. The therapeutic effect of the medication itself is crucial, but different routes of administration of the same drug can also produce different effects. PURPOSE: The aim of our research sought to investigate the ameliorating effects of Par in psoriasis-like skin inflammation and its related mechanism of action. RESULTS: In the IMQ-induced model, intragastric administration of Par reduced the Psoriasis Area and Severity Index (PASI) score, improved skin erythema, scaling, and other symptoms. And Par decreased the expression of Ki67, keratin14, keratin16 and keratin17, and increased the expression of keratin1. Par could reduce IL-36 protein expressions, meanwhile the expression of Il1b, Cxcl1 and Cxcl2 mRNA were also decreased. Par regulated the expression levels of F4/80, MPO and NE. However, skin transdermal administration of Par was more effective. Similarly, Par attenuated IL-36γ, IL-1ß and caspase-1 activated by Poly(I:C) in in vitro and ex vivo. In addition, Par also reduced NE, PR3, and Cathepsin G levels in explant skin tissues. CONCLUSION: Par ameliorated psoriasis-like skin inflammation in both in vivo and in vitro, especially after treatment with transdermal drug delivery, possibly by inhibiting neutrophil extracellular traps and thus by interfering IL-36 signaling pathway. It indicated that Par provides a new research strategy for the treatment of psoriasis-like skin inflammation and is expected to be a promising drug.

Neutrophil Extracellular Traps in Tumors and Potential Use of Traditional Herbal Medicine Formulations for Its Regulation.

Neutrophil extracellular traps (NETs) are extracellular fibers composed of deoxyribonucleic acid (DNA) and decorated proteins produced by neutrophils. Recently, NETs have been associated with the development of many diseases, including tumors. Herein, we reviewed the correlation between NETs and tumors. In addition, we detailed active compounds from traditional herbal medicine formulations that inhibit NETs, related nanodrug delivery systems, and antibodies that serve as "guiding moieties" to ensure targeted delivery to NETs. Furthermore, we discussed the strategies used by pathogenic microorganisms to evade NETs.

[Traditional Chinese medicines target neutrophil extracellular traps to treat diseases: a review].

Neutrophil extracellular traps(NETs) are fibrous networks formed by neutrophils after a procedure called NETosis, with the function of capturing and killing pathogens. NETs are widely involved in the pathological processes of major diseases such as immune system diseases, respiratory diseases, metabolic diseases, cancers, and reperfusion injury. Therefore, regulating NETs has become one of the important ways to prevent and treat the above diseases. As an excellent traditional culture in China, traditional Chinese medicine has made outstanding contributions to the treatment of diseases. In recent years, studies have discovered that a variety of active components in traditional Chinese medicines, Chinese medicine compound prescriptions, and single traditional Chinese medicines can alleviate the symptoms by regulating NETs in the pathological process of major diseases. This article reviews the research progress in the regulation of NETs by the active components of traditional Chinese medicines, Chinese medicine compound prescriptions, and single traditional Chinese medicines in the last five years, aiming to serve as a reference for related research.

Mercury-containing preparations attenuate neutrophil extracellular trap formation in mice and humans through inhibiting the ERK1/2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Neutrophil extracellular trap (NET) formation plays a crucial role in wound healing disorders, including chronic skin ulcers and diabetic foot ulcers (DFUs). Over the years, traditional Chinese topical medications, such as Cinnabar (composed of HgS and soluble mercury salt) and hydrargyria oxydum rubrum (containing HgO and soluble mercury salt), have been utilized for treating these ailments. Nevertheless, the fundamental processes remain mostly ambiguous. AIM OF THE STUDY: This study sought to investigate the potential effects of topical mercury-containing preparations on the process of NET formation. MATERIALS AND METHODS: Neutrophils isolated from healthy individuals and mouse models of type 1 and type 2 diabetes were cultured with phorbol 12-myristate 13-acetate (PMA), both with and without the mercury-containing preparations (MCP). The formation of NETs was monitored using confocal and scanning electron microscopes. Immunofluorescence and fluorescent probes were employed to assess the levels of citrulline histone H3 (Cit-H3) and intracellular reactive oxygen species (ROS), respectively. The impact of MCP extracts on cytokine expression, peptidylarginine deiminase 4 (PAD4), and myeloperoxidase (MPO) was measured through Luminex and ELISA assays. Phagocytosis of human neutrophils was analyzed using Flow Cytometry. Finally, the phosphorylation levels of ERK were detected by western blotting. RESULTS: Treatment with MCP led to a reduction in PAD4, Cit-H3, and MPO expressions in neutrophils, consequently inhibiting PMA-induced NET formation. MCP treatment also dampened ERK1/2 activation in neutrophils. Furthermore, MCP exhibited inhibitory effects on the secretion of the cytokine IL-8 and ROS production while enhancing neutrophil phagocytosis. CONCLUSION: Our findings suggest that MCP can mitigate the release of NETs, likely by suppressing the ERK1/2 signaling pathway.

Rosavin inhibits neutrophil extracellular traps formation to ameliorate sepsis-induced lung injury by regulating the MAPK pathway.

BACKGROUND: Sepsis is a systemic organ dysfunction caused by infection, and the most affected organ is the lungs. Rosavin, a traditional Tibetan medicine, exerts an impressive anti--inflammatory effect. However, its effects on sepsis-related lung damage have not been investigated. PURPOSE: This study aimed to investigate the effects of Rosavin on cecal ligation and puncture (CLP)-induced lung injury. METHODS: The sepsis mouse model was established by CLP, and the mice were pretreated with Rosavin to explore whether it contributed to the alleviation of lung injury. Hematoxylin-eosin (H&E) stain and lung injury score were used to assess the severity of lung injury. The bronchoalveolar lavage fluid (BALF) inflammatory mediators (tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6], IL-1ß, and IL-17A) were detected by ELISA. The number of neutrophils in BALF was detected using flow cytometry. The immunofluorescence assay was used to detect histone and myeloperoxidase (MPO) in lung tissues. Then, the western blot was performed to detect the expression of mitogen-activated protein kinase (MAPK) pathways (extracellular regulated kinase [ERK], p-ERK, p38, p-p38, Jun N-terminal kinase 1/2 [JNK1/2], and p-JNK1/2) in lung tissues. RESULTS: We found that Rosavin significantly attenuated sepsis-induced lung injury. Specifically, Rosavin significantly inhibited inflammation response by decreasing the secretion of inflammatory mediators. The level of neutrophil extracellular traps (NETs) and MPO activity in CLP were decreased after administration with Rosavin. Moreover, the western blot showed that Rosavin could suppress NETs formation by inhibiting the MAPK/ERK/p38/JNK signaling pathway. CONCLUSION: These findings demonstrated that Rosavin inhibited NETs formation to attenuate sepsis-induced lung injury, and the inhibitory effect may be exerted via deregulation of the MAPK pathways.

Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.

The association between neutrophil extracellular traps (NETs) and response to inhaled corticosteroids (ICS) in asthma is unclear. To better understand this relationship, we analyzed the blood transcriptomes from children with controlled and uncontrolled asthma in the Taiwanese Consortium of Childhood Asthma Study using weighted gene coexpression network analysis and pathway enrichment methods. We identified 298 uncontrolled asthma-specific differentially expressed genes and one gene module associated with neutrophil-mediated immunity, highlighting a potential role for neutrophils in uncontrolled asthma. We also found that NET abundance was associated with nonresponse to ICS in patients. In a neutrophilic airway inflammation murine model, steroid treatment could not suppress neutrophilic inflammation and airway hyperreactivity. However, NET disruption with deoxyribonuclease I (DNase I) efficiently inhibited airway hyperreactivity and inflammation. Using neutrophil-specific transcriptomic profiles, we found that CCL4L2 was associated with ICS nonresponse in asthma, which was validated in human and murine lung tissue. CCL4L2 expression was also negatively correlated with pulmonary function change after ICS treatment. In summary, steroids fail to suppress neutrophilic airway inflammation, highlighting the potential need to use alternative therapies such as leukotriene receptor antagonists or DNase I that target the neutrophil-associated phenotype. Furthermore, these results highlight CCL4L2 as a potential therapeutic target for individuals with asthma refractory to ICS.

Selenium-rich yeast counteracts the inhibitory effect of nanoaluminum on the formation of porcine neutrophil extracellular traps.

Aluminum is widely used in daily life due to its excellent properties. However, aluminum exposure to the environment severely threatens animal and human health. Conversely, selenium (Se) contributes to maintaining the balance of the immune system. Neutrophils exert immune actions in several ways, including neutrophil extracellular traps (NETs) that localize and capture exogenous substances. Despite the recent investigations on the toxic effects of aluminum and its molecular mechanisms, the immunotoxicity of aluminum nanoparticles on pigs and the antagonistic effect of selenium on aluminum toxicity are poorly understood. Here, we treated porcine peripheral blood neutrophils with zymosan for 3 h to induce NETs formation. Then, we investigated the effect of nanoaluminum on NETs formation in pigs and its possible molecular mechanisms. Microscopy observations revealed that NETs formation was inhibited by nanoaluminum. Using a multifunctional microplate reader, the production of extracellular DNA and the burst of reactive oxygen species (ROS) in porcine neutrophils were inhibited by nanoaluminum. Western blot analyses showed that nanoaluminum caused changes in amounts of cellular selenoproteins. After Se supplementation, the production of porcine NETs, the burst of ROS, and selenoprotein levels were restored. This study indicated that nanoaluminum inhibited the zymosan-induced burst of ROS and release of NETs from porcine neutrophils, possibly through the selenoprotein signaling pathway. In contrast, Se supplementation reduced the toxic effects of nanoaluminum and restored NETs formation.

Curdione ameliorates sepsis-induced lung injury by inhibiting platelet-mediated neutrophil extracellular trap formation.

Sepsis-associated acute lung injury remains to be a major cause of morbidity and mortality worldwide, and there is a lack of effective therapeutic drugs. Curdione, an activeingredient of Curcuma zedoary, a traditional Chinese medicine (TCM), possesses a variety of pharmacological actions, such as anti-inflammatory, antioxidant and inhibition of platelet aggregation. However, whether curdione protects against sepsis-induced lung injury is still undetermined. In this study, we investigated the effects of curdione on sepsis-induced lung injury. Cecal ligation and puncture (CLP) surgery was performed in mice to establish a model of sepsis. Twenty-four hours after CLP, bronchoalveolar lavage fluid (BALF) and lung tissue samples were harvested for investigation. The protective effects of curdione on acute lung injury and potential mechanisms were explored by detecting pathological sections, exudative proteins, oxidative responses, inflammatory factors, platelet activation, neutrophil infiltration, and neutrophil extracellular trap (NET) formation in the lung and were further verified in vitro. We showed that treatment with curdione clearly relieved histopathological changes, reduced inflammatory cytokine elevation and total protein concentrations in BALF, and decreased oxidative stress responses in lung tissues. In addition, curdione inhibited platelet activation, further blocking the interaction between platelets and neutrophils. Finally, neutrophil infiltration and NET formation was also reduced in mice treated with curdione. In conclusion, curdione alleviates sepsis-induced lung injury by inhibiting platelet-mediated neutrophil recruitment, infiltration, and NET formation as well as its anti-inflammatory and antioxidant properties. Curdione has great therapeutic potential in sepsis.

Epigallocatechin-3-gallate inhibits the formation of neutrophil extracellular traps and suppresses the migration and invasion of colon cancer cells by regulating STAT3/CXCL8 pathway.

Colon cancer is a common malignant tumor of the digestive tract. Tea catechin exerts anti-tumor effects in colon cancer. This work aimed to determine the functions of epigallocatechin-3-gallate (EGCG), one of the main active components of Tea catechins, in the progression of colon cancer. In this work, enzyme-linked immune-sorbent assay, quantitative real-time PCR and western blotting was utilized to examine the levels of IL-1ß, TNF-α, STAT3, p-STAT3 and CXCL8 in colon cancer patients and healthy controls. Compared with healthy controls, the levels of IL-1ß and TNF-α were significantly increased in the peripheral blood of colon cancer patients, and the expression of STAT3, p-STAT3 and CXCL8 was elevated in the neutrophils derived from colon cancer patients. Moreover, neutrophils were treated with phorbol ester (PMA) or DNase I to induce or impede the formation of neutrophil extracellular traps (NETs). Both STAT3 overexpression and PMA treatment promoted the expression of CXCL8, myeloperoxidase (MPO) and citrullinated histone H3 (H3Cit) in the colon cancer-derived neutrophils, indicating that STAT3 overexpression facilitated the formation of NETs. STAT3 deficiency suppressed the formation of NETs, which consistent with the results of DNase I treatment. Transwell assay was utilized to detect the migration and invasion of colon cancer cell line SW480. EGCG treatment suppressed the formation of NETs and the expression of STAT3 and CXCL8 in the colon cancer-derived neutrophils, and then inhibited the migration and invasion of SW480 cells. In conclusion, this work demonstrated that EGCG inhibited the formation of NETs and subsequent suppressed the migration and invasion of colon cancer cells by regulating STAT3/CXCL8 signalling pathway. Thus, this study suggests that EGCG may become a potential drug for colon cancer therapy.

Huang Qin Decoction inhibits the initiation of experimental colitis associated carcinogenesis by controlling the PAD4 dependent NETs.

BACKGROUND: Colorectal cancer is associated with ulcerative colitis (UC). The infiltration of neutrophils is the main cause of DNA damage produced by inflammation in the intestinal epithelium. Under the action of peptidyl arginine deaminase 4 (PAD4), neutrophils dissociate chromatin and form neutrophil extracellular traps (NETs), which can aggravate tissue inflammation and encourage tumor development. Although Huang Qin Decoction (HQD) was found to be useful in treating UC and was used to gradually prevent and treat digestive tract cancers, the underlying reasons were unclear. METHODS: To demonstrate HQD could inhibits the initiation of colitis associated carcinogenesis by controlling NETs related inflammation, we first performed an AOM/DSS-generated colitis-associated carcinogenesis model to assess the efficacy of HQD in reducing neutrophil infiltration and anti-tumor activity. Then, using network pharmacology research, we investigated the potential mechanisms underlying those medicinal effects, as demonstrated by the detection of NETs aggregation and PAD4 expression changes in the colon. RESULTS: HQD substantially reduced the number of colon cancers and the expression of Ki67, restored the level of intestinal tight junction protein occludin and ZO-1, and relieved the intestinal inflammation caused by TNF-α, IL-1ß. At the same time, it inhibited neutrophil infiltration in the colon and improved the immunosurveillance of CD8+T cells. The potential mechanisms of HQD intervention against UC and UC with neoplasia (UCN) were studied using network pharmacology, and 156 conjunct genes as well as numerous inflammation-related pathways were identified. Protein-protein interaction (PPI) analysis indicated that HQD inhibition of intestinal tumors might be related to the deactivation of PAD4, which was verified by the down-regulation of NETs, MPO-DNA complex levels, and PAD4 expression after HQD treatment. CONCLUSION: Huang Qin Decoction inhibits the initiation of colitis associated carcinogenesis by controlling PAD4-dependent neutrophil extracellular traps.

An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation.

BACKGROUND: Owing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PURPOSE: To obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. METHODS: Diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. RESULTS: From HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of →4,6)-α-Glcp-(1→, →3,4)-α-Glcp-(1→, →4)-α-Glcp-(1→, →4,6)-α-Galp-(1→, →5)-α-Araf-(1→, α-Rhap-(1→, α-Araf-(1→, α-GlcpA-(1→, →4)-ß-Manp-(1→, ß-Manp-(1→ and →3)-ß-Manp-(1→. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 µg/ml and 307 ± 11 µg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. CONCLUSION: An anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.

Salvianolic Acid A Protects against Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting Neutrophil NETosis.

Salvianolic acid A (SAA) is one of bioactive polyphenol extracted from a Salvia miltiorrhiza (Danshen), which was widely used to treat cardiovascular disease in traditional Chinese medicine. SAA has been reported to be protective in cardiovascular disease and ischemia injury, with anti-inflammatory and antioxidative effect, but its role in acute lung injury (ALI) is still unknown. In this study, we sought to investigate the therapeutic effects of SAA in a murine model of lipopolysaccharide- (LPS-) induced ALI. The optimal dose of SAA was determined by comparing the attenuation of lung injury score after administration of SAA at three different doses (low, 5 mg/kg; medium, 10 mg/kg; and, high 15 mg/kg). Dexamethasone (DEX) was used as a positive control for SAA. Here, we showed that the therapeutic effect of SAA (10 mg/kg) against LPS-induced pathologic injury in the lungs was comparable to DEX. SAA and DEX attenuated the increased W/D ratio and the protein level, counts of total cells and neutrophils, and cytokine levels in the BALF of ALI mice similarly. The oxidative stress was also relieved by SAA and DEX according to the superoxide dismutase and malondialdehyde. NET level in the lungs was elevated in the injured lung while SAA and DEX reduced it significantly. LPS induced phosphorylation of Src, Raf, MEK, and ERK in the lungs, which was inhibited by SAA and DEX. NET level and phosphorylation level of Src/Raf/MEK/ERK pathway in the neutrophils from acute respiratory distress syndrome (ARDS) patients were also inhibited by SAA and DEX in vitro, but the YEEI peptide reversed the protective effect of SAA completely. The inhibition of NET release by SAA was also reversed by YEEI peptide in LPS-challenged neutrophils from healthy volunteers. Our data demonstrated that SAA ameliorated ALI via attenuating inflammation, oxidative stress, and neutrophil NETosis. The mechanism of such protective effect might involve the inhibition of Src activation.

Glycyrrhizin alleviates sepsis-induced acute respiratory distress syndrome via suppressing of HMGB1/TLR9 pathways and neutrophils extracellular traps formation.

BACKGROUND: Neutrophil extracellular traps (NETs) are involved in the development of sepsis-induced acute respiratory distress syndrome (ARDS). Glycyrrhizin (GL), the main active ingredient of the traditional Chinese medicine Glycyrrhiza glabra, has anti-inflammatory, anti-viral, and immunomodulatory effects. OBJECTIVE: The study aims to explore the efficacy and potential mechanism of GL on sepsis-induced ARDS in mice. MATERIALS AND METHODS: Mice were randomly divided into 3 groups: Control, CLP, and GL + CLP. Mice sepsis ARDS model was induced by cecal ligation and puncture (CLP) followed by intraperitoneal GL treatment. Then, the 7-day survival rate of mice was recorded. The lung function of mice was determined by whole-body plethysmography. Lung pathology and scores were observed by hematoxylin-eosin staining. The wet/dry ratio (W/D) of the lung was measured by weighing method. The protein concentration in bronchoalveolar lavage fluid (BALF) was measured by the BCA method. NETs formation in lung tissue was detected by immunofluorescence. Furthermore, HMGB1、TLR9、MyD88 and IL6 expression in lung tissue were detected by western blot and by quantitative real-time PCR, respectively. RESULTS: The results showed that GL improved the survival rate, attenuated lung tissue injury and reduced the expression of inflammatory factors in mice with CLP-induced sepsis. Meanwhile, we confirmed that GL could inhibit TLR9 / MyD88 activation from reducing NETs formation by decreasing HMGB1 expression. The formation of NETs is regulated by HMGB1 / TLR9 / MyD88. In addition, GL improved lung function in mice with sepsis-induced ARDS. Lung function suggested that GL increased alveolar ventilation, alleviated ventilator fatigue and reduced airway resistance in mice with ARDS induced by sepsis. CONCLUSIONS: GL ameliorated sepsis-induced ARDS and reduced the NETs formation in lung tissues, which may be associated with the inhibition of the HMGB1 / TLR9 pathway.

Alumina nanoparticles-induced heterophil extracellular traps exacerbate liver injury by regulating oxidative stress and inflammation in chickens.

Widely used alumina nanoparticles (Al2O3 NPs) exposed to the environment pose a serious threat to human and animal health. The formation of heterophil extracellular traps (HETs) is a mechanism of innate immune defense against infection, but excessive HETs cause pathological damage. Here, we aim to explore the influence and mechanism of Al2O3 NPs on the formation of HETs in vitro, and further investigate the role of HETs release in histopathological damage after Al2O3 NPs treatment. Immunofluorescence analysis showed that Al2O3 NPs induced the formation of HETs, which was characterized by modified histones and elastase in the DNA backbone. Fluorescence microplate analysis showed that HETs formation was dependent on NADPH oxidase, P38, extracellular regulated protein kinases (ERK1/2) pathways and glycolysis. In vivo investigation showed that Al2O3 NPs significantly caused HETs release and liver damage. Biochemical analysis showed that Al2O3 NPs inhibited the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). Real-time fluorescence quantification results showed that Al2O3 NPs caused the overexpression of inflammation-related molecules interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), caspase-1 and caspase-11. All these changes were significantly changed by DNase I (Degradation reagent for HETs). Together, these suggest that Al2O3 NPs-induced HETs exacerbate liver injury by regulating oxidative stress and inflammatory responses, which provide a new perspective and potential prophylaxis and treatment targets for Al2O3 NPs toxicological research.

Effect of Vitamin D3 in combination with Omega-3 Polyunsaturated Fatty Acids on NETosis in Type 2 Diabetes Mellitus Patients.

An understanding of the consequences of oxidative/halogenative stress triggered by neutrophil activation is impossible without considering NETosis. NETosis, formation of neutrophil extracellular traps (NETs), is known to promote microthrombus formation and impair wound healing in type 2 diabetes mellitus (T2DM) patients. Therefore, there is a need to search for drugs and treatment approaches that could prevent excessive NET formation. We aimed to evaluate the effect of vitamin D3 in combination with omega-3 polyunsaturated fatty acids (vitamin D3/omega-3 PUFAs) on NETosis in T2DM patients with purulent necrotizing lesions of the lower extremities. Patients and healthy subjects had vitamin D3 deficiency. Patients received, beyond standard treatment, 6000 IU of vitamin D3 and 480 mg of omega-3 PUFAs, and healthy subjects 1000 IU of vitamin D3 and 240 mg of omega-3 PUFAs daily for seven days. Neutrophil activation in ex vivo blood by phorbol-12-myristate-13-acetate (PMA) was used as a NETosis model. The percentage of blood NETs relative to leukocytes (NETbackground) before vitamin D3/omega-3 PUFA supplementation was 3.2%-4.9% in healthy subjects and 1.7%-10.8% in patients. These values rose, respectively, to 7.7%-9.1% and 4.0%-17.9% upon PMA-induced NETosis. In addition, the leukocyte count decreased by 700-1300 per 1 µL in healthy subjects and 700-4000 per 1 µL in patients. For both patients and healthy subjects, taking vitamin D3/omega-3 PUFAs had no effect on NETbackground but completely inhibited PMA-induced NET formation, though neutrophils exhibited morphological features of activation. Also, leukocyte loss was reduced (to 500 per 1 µL). For patients on standard treatment alone, changes occurred neither in background NETs and leukocytes nor in their amount after PMA stimulation. The decreased ability of neutrophils to generate NETs, which can be achieved by vitamin D3/omega-3 PUFA supplementation, could have a positive effect on wound healing in T2DM patients and reduce the incidence and severity of complications.

Do free radical NETwork and oxidative stress disparities in African Americans enhance their vulnerability to SARS-CoV-2 infection and COVID-19 severity?

This review focuses on the hypothetical mechanisms for enhanced vulnerability of African Americans to SARS-CoV-2 infection, COVID-19 severity, and increased deaths. A disproportionately higher number of African Americans are afflicted with autoimmune and inflammatory diseases (e.g., diabetes, hypertension, obesity), and SARS-CoV-2 has helped expose these health disparities. Several factors including socioeconomic status, inferior health care, and work circumstances contribute to these disparities. Identifying potential inflammatory biomarkers and decreasing basal levels in high-risk individuals with comorbidities through preventive measures is critical. Immune cells, particularly neutrophils, protect us against pathogens (bacteria, fungi, and viruses) through increased generation of free radicals or oxidants and neutrophil extracellular traps (NETs) that ensnare pathogens, killing them extracellularly. However, continued generation of NETs coupled with the lack of prompt removal pose danger to host cells. NET levels are increased during pro-inflammatory diseases. COVID-19 patients exhibit elevated NET levels, depending upon disease severity. Conceivably, high-risk individuals with elevated basal NET levels would exhibit hyper-inflammation when infected with SARS-CoV-2, amplifying disease severity and deaths. Drugs inhibiting oxidant formation and vitamin supplements decreased NET formation in mice models of inflammation. Thus, it is conceivable that preventive treatments lowering NET levels and inflammation in high-risk individuals could mitigate SARS-CoV-2-induced complications and decrease mortality.

Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusion.

BACKGROUND: The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis. METHODS: The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays. FINDINGS: The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively. INTERPRETATION: NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions. FUNDING: This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD).

Resolution of inflammation in arthritis.

Rheumatoid arthritis is among the most frequent and severe chronic inflammatory diseases. The disease is characterized by ongoing synovial inflammation, which leads to the destruction of cartilage and bone. In RA, the mechanisms of resolution of inflammation, which are normally intact in the joints, are either suppressed or overruled. Little efforts have been undertaken to understand the mechanisms of resolution of arthritis until recently, when several molecular mechanisms have been identified that determine the chronicity and resolution of inflammation in the joints, respectively. This review describes the key concepts of resolution of arthritis mentioning the key mechanisms involved, such as regulatory macrophages, pro-resolving lipid, fatty acid and cytokine mediators, aggregated neutrophil extracellular trap formation, antibody glycosylation changes, and stromal cell alterations that are involved in determining the decision between chronicity and resolution of arthritis. Each of these mechanisms represents a potential therapeutic approach that allows skewing the balance of the inflammatory processes towards resolution.

Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs).

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.

Enhanced Neutrophil Extracellular Trap Formation in Acute Pancreatitis Contributes to Disease Severity and Is Reduced by Chloroquine.

Background: Neutrophil extracellular traps (NETs) are generated when activated neutrophils, driven by PAD4, release their DNA, histones, HMGB1, and other intracellular granule components. NETs play a role in acute pancreatitis, worsening pancreatic inflammation, and promoting pancreatic duct obstruction. The autophagy inhibitor chloroquine (CQ) inhibits NET formation; therefore, we investigated the impact of CQ mediated NET inhibition in murine models of pancreatitis and human correlative studies. Methods: L-arginine and choline deficient ethionine supplemented (CDE) diet models of acute pancreatitis were studied in wild type and PAD4-/- mice, incapable of forming NETs. Isolated neutrophils were stimulated to induce NET formation and visualized with fluorescence microscopy. CQ treatment (0.5 mg/ml PO) was initiated after induction of pancreatitis. Biomarkers of NET formation, including cell-free DNA, citrullinated histone H3 (CitH3), and MPO-DNA conjugates were measured in murine serum and correlative human patient serum samples. Results: We first confirmed the role of NETs in the pathophysiology of acute pancreatitis by demonstrating that PAD4-/- mice had decreased pancreatitis severity and improved survival compared to wild-type controls. Furthermore, patients with severe acute pancreatitis had elevated levels of cell-free DNA and MPO-DNA conjugates, consistent with NET formation. Neutrophils from mice with pancreatitis were more prone to NET formation and CQ decreased this propensity to form NETs. CQ significantly reduced serum cell-free DNA and citrullinated histone H3 in murine models of pancreatitis, increasing survival in both models. Conclusions: Inhibition of NETs with CQ decreases the severity of acute pancreatitis and improves survival. Translating these findings into clinical trials of acute pancreatitis is warranted.