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Although the role of low-density granulocytes (LDGs), neutrophils in the peripheral blood mononuclear cell (PBMC) fraction, and neutrophil extracellular traps (NETs) in assessing lupus disease severity is acknowledged, data specific to childhood-onset lupus remains scarce. This study analyzed 46 patients with childhood-onset systemic lupus erythematosus (82.6% females, mean age 14.5 ± 0.3 years), including 26 cases with normal complement levels and 20 with low complement levels, along with 20 healthy adult volunteers. Key parameters that distinguished healthy volunteers from lupus patients and differentiated between lupus patients with low and normal complement were serum interferon (IFN)-α, serum citrullinated histone 3 (CitH3), and extracellular traps (ETs) in LDGs. However, NETs (assessed by nuclear staining morphology), LDG abundance, and other parameters (such as endotoxemia, cytokines, and double-stranded (ds) DNA) did not show such differentiation. When lipopolysaccharide (LPS) was administered to LDGs in the PBMC fraction, it induced ETs in both low and normal complement groups, indicating the inducible nature of ETs. In adult healthy volunteers, activation by recombinant IFN-α or dsDNA in isolated neutrophils induced LDGs and NETs (identified using immunofluorescent staining for CitH3, myeloperoxidase, and neutrophil elastase) at 45 min and 3 h post-stimulation, respectively. Additionally, approximately half of the LDGs underwent late apoptosis at 3 h post-stimulation, as determined by flow cytometry analysis. Activation by IFN-α or dsDNA in LDGs also led to a more pronounced expression of CD66b, an adhesion molecule, compared to regular-density neutrophils, suggesting higher activity in LDGs. In conclusion, IFN-α and/or dsDNA in serum may transform regular-density neutrophils into LDGs before progressing to NETosis and apoptosis, potentially exacerbating lupus severity through cell death-induced self-antigens. Therefore, LDGs and ETs in LDGs could provide deeper insights into the pathophysiology of childhood-onset lupus., (© 2024. The Author(s).)

Background: Vitamin C is a potent scavenger of reactive oxygen species, which induce neutrophil extracellular trap (NET) formation. NETs are a major source of autoantigens and are involved in systemic lupus erythematosus (SLE) pathogenesis. We determined vitamin C status and evaluated NET formation and inflammatory cytokines in children with lupus nephritis., Methods: Serum vitamin C was measured in 46 patients (82.6% females, mean age 14.5 ± 0.3 years). Vitamin C levels < 0.3 mg/dL indicated vitamin C deficiency. Patients were divided into two groups according to serum vitamin C levels: normal and low (< 0.3 mg/dL). We compared NET formation and levels of SLE-related cytokines, including interleukin (IL)-8, IL-10, and tumor necrosis factor-α (TNF-α), between groups. NET formation was determined through measurement of serum citrullinated histone 3 levels and mRNA expression of peptidyl arginine deiminase-4 and assessment of the percentage of neutrophils with NETs by immunofluorescence., Results: Nine patients (19.6%) had vitamin C deficiency. Kidney pathology assessment at disease onset revealed that histological activity index and number of kidney biopsies containing crescentic glomeruli were higher in vitamin C-deficient patients, but chronicity index was not. NET formation and serum IL-8 were more prominent in vitamin C-deficient patients. Serum IL-8 levels were 12.9 ± 5.2 pg/mL in low vitamin C group and 5.2 ± 0.9 pg/mL in normal vitamin C group (p = 0.03). Serum IL-10 and TNF-α were similar between groups., Conclusions: Our study demonstrated correlation among vitamin C deficiency, increased NET formation, and IL-8 upregulation in children with lupus nephritis. A prospective study is required to evaluate cause‒effect relationships of vitamin C status, NET formation and IL-8 expression., (© 2023. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

Neutrophil Extracellular Traps (NETs) are a contributing factor of vascular thrombosis and alveolar damage in COVID-19 patients. As enoxaparin is currently used to inhibit vascular thrombosis, this study aimed to investigate whether enoxaparin also reduced inflammation and NETs in COVID-19 patients. Patients with COVID-19 infection were classified into three groups: mild, moderate, and severe (n = 10 for all groups). Plasma was collected from patients and healthy donors (n = 10). Neutrophils isolated from healthy controls were incubated with COVID-19 or healthy plasma, and with or without enoxaparin pretreatment in vitro. Neutrophils and plasma isolated from patients treated with enoxaparin were also investigated. The levels of inflammatory cytokines and NET products such as dsDNA, NE, MPO−DNA and Histone−DNA complexes in plasma and supernatants were measured using immunofluorescence staining and ELISA kits. The expression of inflammatory signaling genes by neutrophils (RELA, SYK, ERK and PKC) was measured using real-time qPCR. The levels of NET products were elevated in the plasma of COVID-19 patients, particularly in the severe group (p < 0.01). Moreover, plasma from the severe group enhanced NET formation (p < 0.01) from neutrophils in vitro. Enoxaparin pretreatment in vitro decreased plasma-induced NETs in a dose-dependent manner and down-regulated the expression of inflammatory genes (p < 0.05). Patients treated with prophylactic enoxaparin showed lower inflammatory cytokine levels and expression of inflammatory genes (p < 0.05). Increased NETs were associated with the severity of COVID-19 infection, particularly in patients with severe pneumonia, and could be used as biomarkers to assess disease severity. Enoxaparin pretreatment inhibited NETs and reduced the expression of inflammatory cytokines, and these effects mostly persisted in patients treated with prophylactic enoxaparin.

Due to limited data on the link between gut barrier defects (leaky gut) and neutrophil extracellular traps (NETs) in coronavirus disease 2019 (COVID-19), blood samples of COVID-19 cases-mild (upper respiratory tract symptoms without pneumonia; n = 27), moderate (pneumonia without hypoxia; n = 28), and severe (pneumonia with hypoxia; n = 20)-versus healthy control ( n = 15) were evaluated, together with in vitro experiments. Accordingly, neutrophil counts, serum cytokines (IL-6 and IL-8), lipopolysaccharide (LPS), bacteria-free DNA, and NETs parameters (fluorescent-stained nuclear morphology, dsDNA, neutrophil elastase, histone-DNA complex, and myeloperoxidase-DNA complex) were found to differentiate COVID-19 severity, whereas serum (1→3)-β-D-glucan (BG) was different between the control and COVID-19 cases. Despite non-detectable bacteria-free DNA in the blood of healthy volunteers, using blood bacteriome analysis, proteobacterial DNA was similarly predominant in both control and COVID-19 cases (all severities). In parallel, only COVID-19 samples from moderate and severe cases, but not mild cases, were activated in vitro NETs, as determined by supernatant dsDNA, Peptidyl Arginine Deiminase 4 , and nuclear morphology. With neutrophil experiments, LPS plus BG (LPS + BG) more prominently induced NETs, cytokines, NFκB , and reactive oxygen species, when compared with the activation by each molecule alone. In conclusion, pathogen molecules (LPS and BG) from gut translocation along with neutrophilia and cytokinemia in COVID-19-activated, NETs-induced hyperinflammation.

Because of gut-barrier defect (gut-leakage) after acute kidney injury (AKI) and higher abundance of Candida albicans in human intestines compared with mouse guts, Candida administration in renal ischemia reperfusion injury (I/R) mice possibly more closely resemble patients with AKI than non-Candida model. Fungi in feces were detectable only in mice with Candida administration. Candida renal-I/R mice, when compared with non-Candida I/R, demonstrated more profound injuries, including (i) gut-leakage; FITC-dextran assay and serum (1→3)-β-D-glucan (BG), (ii) systemic inflammation (serum cytokines), and (iii) neutrophil extracellular traps (NETs); gene expression of peptidyl arginase 4 (PAD4) and IL-1β, nuclear morphology staining by 4',6-diamidino-2-phenylindole (DAPI) and co-staining of myeloperoxidase (MPO) with neutrophil elastase (NE) in peripheral blood neutrophils. Although renal excretory function (serum creatinine) and renal histology score were nondifferent between renal-I/R mice with and without Candida, prominent renal NETs (PAD4 and IL-1β expression with MPO and NE co-staining) was demonstrated in Candida renal-I/R mice. Additionally, neutrophil activation by lipopolysaccharide (LPS) plus BG (LPS + BG), when compared with LPS alone, caused (i) NETs formation; dsDNA, DAPI-stained nuclear morphology and MPO with NE co-staining, (ii) inflammatory responses; Spleen tyrosine kinase (Syk) and NFκB expression, and (iii) reduced cell energy status (maximal respiratory capacity using extracellular flux analysis). Also, LPS + BG-activated NETs formation was inhibited by a dectin-1 inhibitor, supporting an impact of BG signaling. In conclusion, Candida-renal I/R demonstrated more prominent serum BG and LPS from gut translocation that increased systemic inflammation and NETs through TLR-4 and dectin-1 activation. The influence of gut fungi in AKI should be concerned., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Renal ischemia is the most common cause of acute kidney injury (AKI) that might be exacerbate lupus activity through neutrophil extracellular traps (NETs) and apoptosis. Here, the renal ischemia reperfusion injury (I/R) was performed in Fc gamma receptor 2b deficient (Fcgr2b-/-) lupus mice and the in vitro experiments. At 24 h post-renal I/R injury, NETs in peripheral blood neutrophils and in kidneys were detected using myeloperoxidase (MPO), neutrophil elastase (NE) and citrullinated histone H3 (CitH3), as well as kidney apoptosis (activating caspase-3), which were prominent in Fcgr2b-/- mice more compared to wild-type (WT). After 120 h renal-I/R injury, renal NETs (using MPO and NE) were non-detectable, whereas glomerular immunoglobulin (Ig) deposition and serum anti-dsDNA were increased in Fcgr2b-/- mice. These results imply that renal NETs at 24 h post-renal I/R exacerbated the lupus nephritis at 120 h post-renal I/R injury in Fcgr2b-/- lupus mice. Furthermore, a Syk inhibitor attenuated NETs, that activated by phorbol myristate acetate (PMA; a NETs activator) or lipopolysaccharide (LPS; a potent inflammatory stimulator), more prominently in Fcgr2b-/- neutrophils than the WT cells as determined by dsDNA, PAD4 and MPO. In addition, the inhibitors against Syk and PAD4 attenuated lupus characteristics (serum creatinine, proteinuria, and anti-dsDNA) in Fcgr2b-/- mice at 120 h post-renal I/R injury. In conclusion, renal I/R in Fcgr2b-/- mice induced lupus exacerbation at 120 h post-I/R injury partly because Syk-enhanced renal NETs led to apoptosis-induced anti-dsDNA, which was attenuated by a Syk inhibitor., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Saisorn, Saithong, Phuengmaung, Udompornpitak, Bhunyakarnjanarat, Visitchanakun, Chareonsappakit, Pisitkun, Chiewchengchol and Leelahavanichkul.)