Sandwich ELISA for Circulating Myeloperoxidase- and Neutrophil Elastase-DNA Complexes Released from Neutrophil Extracellular Traps

Kano, Hideki; Huq, Muhammad Aminul; Tsuda, Masanobu; Noguchi, Hiroshi; Takeyama, Naoshi

doi:10.4172/2379-1764.1000196

Cited by 29 publications

(33 citation statements)

References 9 publications

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“…Our binding assay allows detection of only histone H3cit-bound cell-free DNA and therefore, it is specific for evaluation of NETosis. Various sandwich ELISAs have also been developed to detect extracellular DNA complexed with NET-specific proteins such as MPO or NE [ 22 , 23 , 26 , 33 , 34 ]. However, it has been shown that the release of NE and MPO is not strictly specific to NETosis.…”

Section: Discussionmentioning

confidence: 99%

Quantification of Citrullinated Histone H3 Bound DNA for Detection of Neutrophil Extracellular Traps

Lin

Leso

et al. 2020

Cancers

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Formation of neutrophil extracellular traps (NETs) has been associated with multiple pathologies including cancer. While the visualization of NETs by microscopy is a routine technique, their quantification presents a number of challenges. Commonly, as citrullination of histone H3 is required for NET formation, the presence of this modified histone along with DNA is considered to be a hallmark of NETs. Here, we describe and validate a novel assay for the quantification of NETs based on the detection of citrullinated histone H3 bound to DNA (CitH3DNA binding assay). Using this assay, we investigated the effect of phorbol 12-myristate 13-acetate (PMA) on NET formation by neutrophils isolated from the bone marrow of control and myeloma-bearing mice. We found that PMA induced citrullination of histone H3, an increase in the level of CitH3DNA, and NET formation in neutrophils from both tumor-free and myeloma-bearing mice. The levels of CitH3DNA in the NET fractions, as measured by our assay directly correlated with the citrullination of histone H3 in neutrophils, as detected by Western blotting, and were significantly higher in PMA-stimulated compared to unstimulated neutrophils. Neutrophils from tumor-bearing mice produced more NETs than those from tumor-free counterparts following stimulation with PMA. The increase in NET production correlated with significantly higher histone H3 citrullination levels and increased measurements of CitH3DNA. Thus, our data indicate that bone marrow neutrophils from myeloma-bearing hosts are prone to NET formation.

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Section: Discussionmentioning

confidence: 99%

Quantification of Citrullinated Histone H3 Bound DNA for Detection of Neutrophil Extracellular Traps

Lin

Leso

et al. 2020

Cancers

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“…The plasma level of DNA-elastase was measured by using a modified sandwich ELISA as previously described (33). In brief, a 96-well plate was coated with 5 μg/mL anti-NE monoclonal antibody (ab68672, Abcam, MA, USA), at 4°C overnight.…”

Section: Methodsmentioning

confidence: 99%

IL-1β Blockade Attenuates Thrombosis in a Neutrophil Extracellular Trap-Dependent Breast Cancer Model

et al. 2019

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Cancer patients are at increased risk of developing thrombosis, comorbidity that has been associated with increased neutrophil counts and the formation of neutrophil extracellular traps (NETs). Interleukin-1β (IL-1β) modulates the expression of granulocyte colony-stimulating factor (G-CSF), a cytokine that promotes cancer-associated neutrophilia and NET generation. Herein, we combined a murine breast cancer model with a flow-restriction thrombosis model to evaluate whether the IL-1β blockade could interfere with cancer-associated thrombosis. Mice bearing metastatic 4T1 tumors exhibited high neutrophil counts as well as elevated expression of G-CSF and IL-1β in their tumors. On the other hand, mice bearing non-metastatic 67NR tumors showed no elevation in neutrophil counts and displayed low expression levels of G-CSF and IL-1β in their tumors. 4T1 tumor-bearing mice but not 67NR tumor-bearing mice exhibited a NET-dependent prothrombotic state. Pharmacological blockade of IL-1 receptor (IL-1R) decreased the primary growth of 4T1 tumors and reduced the systemic levels of myeloperoxidase, cell-free DNA (cfDNA) and G-CSF, without interfering with the neutrophil counts. Most remarkably, the blockade of IL-1R abolished the prothrombotic state observed in 4T1 tumor-bearing mice. Overall, our results demonstrate that IL-1β might be a feasible target to attenuate cancer-associated thrombosis, particularly in cancer types that rely on increased G-CSF production and involvement of NET formation.

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“…The plasma level of MPO-DNA was measured by ELISA as described previously [ 14 ]. In brief, quantitative detection of MPO-DNA was performed using a “sandwich” ELISA with anti-MPO monoclonal antibody (Merck Millipore Corp., catalog no.…”

Section: Methodsmentioning

confidence: 99%

Plasma myeloperoxidase-conjugated DNA level predicts outcomes and organ dysfunction in patients with septic shock

et al. 2018

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BackgroundRecent studies have suggested that excessive formation of neutrophil extracellular traps (NETs) plays a critical role in the pathogenesis of sepsis. Although elevation of the plasma level of cell-free DNA (cf-DNA) has been reported in sepsis patients, there has been little direct measurement of circulating free NETs such as myeloperoxidase-conjugated DNA (MPO-DNA). The objectives of this study were to detect NETs in the bloodstream of patients with septic shock, and to assess the correlations of circulating NET levels with organ dysfunction, disease severity, and mortality.MethodsFifty-five patients with septic shock admitted to the intensive care units (ICUs) of 35 Japanese hospitals were studied. Septic shock was diagnosed according to the 1997 definition of the American College of Chest Physicians/Society of Critical Care Medicine. To detect circulating NETs, plasma levels of MPO-DNA and cf-DNA were measured by sandwich enzyme-linked immunosorbent assay and by fluorometric assay on days 1, 3, and 7 after the onset of septic shock. Physiological and mortality data were collected from the clinical database.ResultsOn days 1, 3, and 7, the patients showed a marked increase in plasma MPO-DNA levels compared with healthy volunteers, whereas the plasma cf-DNA level was only increased significantly on day 1 and then decreased rapidly. A high MPO-DNA level on days 3 and 7 were associated with 28-day mortality.On days 3 and 7, the MPO-DNA levels were inversely correlated with both the mean arterial pressure and the PaO2/FIO2 ratio, whereas the cf-DNA level was not correlated with either parameter. There was a positive correlation between the plasma MPO-DNA level and the sepsis-related organ failure assessment score on days 3 and 7. Neither cf-DNA nor MPO-DNA levels were correlated with the disseminated intravascular coagulation (DIC) score or the platelet count.ConclusionThe increase in circulating MPO-DNA in patients with septic shock indicates acceleration of NET formation in the early stages of sepsis. High MPO-DNA levels are associated with the severity of organ dysfunction and 28-day mortality due to septic shock, but not with the DIC score. These results suggest that excessive NET formation contributes to the pathogenesis of septic shock.Electronic supplementary materialThe online version of this article (10.1186/s13054-018-2109-7) contains supplementary material, which is available to authorized users.

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Sandwich ELISA for Circulating Myeloperoxidase- and Neutrophil Elastase-DNA Complexes Released from Neutrophil Extracellular Traps

Cited by 29 publications

References 9 publications

Quantification of Citrullinated Histone H3 Bound DNA for Detection of Neutrophil Extracellular Traps

Quantification of Citrullinated Histone H3 Bound DNA for Detection of Neutrophil Extracellular Traps

IL-1β Blockade Attenuates Thrombosis in a Neutrophil Extracellular Trap-Dependent Breast Cancer Model

Plasma myeloperoxidase-conjugated DNA level predicts outcomes and organ dysfunction in patients with septic shock

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