Naoshi Takeyama

Neutrophils are the principal trouper of the innate immune system. Activated neutrophils undergo a noble cell death termed NETosis and release a mesh-like structure called neutrophil extracellular traps (NETs) as a part of their defensive strategy against microbial pathogen attack. This web-like architecture includes a DNA backbone embedded with antimicrobial proteins like myeloperoxidase (MPO), neutrophil elastase (NE), histones and deploys in the entrapment and clearance of encountered pathogens. Thus NETs play an inevitable beneficial role in the host’s protection. However, recent accumulated evidence shows that dysregulated and enhanced NET formation has various pathological aspects including the promotion of sepsis, pulmonary, cardiovascular, hepatic, nephrological, thrombotic, autoimmune, pregnancy, and cancer diseases, and the list is increasing gradually. In this review, we summarize the NET-mediated pathophysiology of different diseases and focus on some updated potential therapeutic approaches against NETs.

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

Background: The purpose of this study is to investigate the time course of syndecan-1 (Syn-1) plasma levels, the correlation between Syn-1 and organ damage development, and the associations of Syn-1 level with cumulative fluid balance and ventilator-free days (VFD) in patients with septic shock. Methods: We collected blood samples from 38 patients with septic shock upon their admission to ICU and for the first 7 days of their stay. Syn-1 plasma level, acute respiratory distress syndrome (ARDS), other organ damage, VFD, and cumulative fluid balance were assessed daily. Results: Over the course of 7 days, Syn-1 plasma levels increased significantly more in patients with ARDS than in those without ARDS. Patients with high levels of Syn-1 in the 72 h after ICU admission had significantly higher cumulative fluid balance, lower PaO₂/FiO₂, and fewer VFD than patients with low levels of Syn-1. Syn-1 levels did not correlate with sequential organ failure assessment score or with APACHE II score. Conclusions: In our cohort of patients with septic shock, higher circulating level of Syn-1 of cardinal glycocalyx component is associated with more ARDS, cumulative positive fluid balance, and fewer VFD. Measurement of Syn-1 levels in patients with septic shock might be useful for predicting patients at high risk of ARDS.

Introduction: Acute respiratory response syndrome (ARDS) leads to increased permeability of the endothelial-epithelial barrier, which in turn promotes edema formation and hypoxemic respiratory failure. Although activated neutrophils are thought to play a significant role in mediating ARDS, at present the contribution of neutrophil extracellular traps (NETs) to lung endothelial barrier function is unclear. Methods: To clarify their role, we co-cultured in vitro NETs induced by phorbol myristate acetate (PMA)–activated neutrophils with lung endothelial cell monolayers and examined the barrier function of lung endothelial cells by immunofluorescence microscopy and albumin permeability in a double-chamber culture method. Results: Co-culture with stimulated neutrophils increased the albumin permeability of the human pulmonary artery endothelial cell (HPAEC) monolayer and altered cytoskeleton F-actin and vascular endothelial-cadherin in cell-cell junctions. Hyperpermeability to albumin and histological alterations were prevented by inhibition of NET formation with peptidyl arginine deiminase inhibitor or a neutrophil elastase inhibitor and were also prevented by increased degradation of NET structure with DNase. Conclusion: This in vitro experiment shows that altered HPAEC barrier function and increased albumin permeability are caused by the direct effect of PMA-induced NETs and their components. NET formation may be involved in the increased vascular permeability of the lung, which is a common feature in ARDS of various etiologies. These insights may help generate novel approaches for medical interventions.

Arsenic, a major environmental toxicant and pollutant, is a global public health concern. Among its many adverse effects, arsenic is immunotoxic, but its effects on human neutrophil functions are not yet well-defined. In this study, we aimed to evaluate the in vitro effects of acute low-dose NaAsO₂ exposure on human polymorphonuclear neutrophils (PMNs) for 12 h on the following innate defense mechanisms: formation of neutrophil extracellular traps (NETs), production of reactive oxygen species (ROS), and phagocytosis. Phorbol myristate acetate (PMA) was added to induce NETs formation, which was quantified by measuring cell-free extracellular DNA (cf-DNA), myeloperoxidase-conjugated (MPO)-DNA and neutrophil elastase-conjugated (NE)-DNA, and confirmed by immunofluorescence labeling and imaging. Extracellular bactericidal activity by NETs was evaluated by co-culturing Escherichia coli and PMNs in the presence of a phagocytic inhibitor. Levels of NETs in the culture medium after PMA stimulation was significantly lower in PMNs pre-exposed to arsenic than those not exposed to arsenic. Immunofluorescence staining and extracellular bactericidal activity by NETs revealed similar results. Phagocytosis and ROS production by PMNs were also significantly reduced by arsenic pre-exposure. Together, our findings provide new insights in arsenic immunotoxicity and suggest how it increases susceptibility to infectious diseases in humans.

Background: Elderly patients have a blunted host response, which may influence vital signs and clinical outcomes of sepsis. This study was aimed to investigate whether the associations between the vital signs and mortality are different in elderly and non-elderly patients with sepsis. Methods: This was a retrospective observational study. A Japanese multicenter sepsis cohort (FORECAST, n = 1148) was used for the discovery analyses. Significant discovery results were tested for replication using two validation cohorts of sepsis (JAAMSR, Japan, n = 624; SPH, Canada, n = 1004). Patients were categorized into elderly and non-elderly groups (age ≥ 75 or < 75 years). We tested for association between vital signs (body temperature [BT], heart rate, mean arterial pressure, systolic blood pressure, and respiratory rate) and 90-day in-hospital mortality (primary outcome). Results: In the discovery cohort, non-elderly patients with BT < 36.0 °C had significantly increased 90-day mortality (P = 0.025, adjusted hazard ratio 1.70, 95% CI 1.07–2.71). In the validation cohorts, non-elderly patients with BT < 36.0 °C had significantly increased mortality (JAAMSR, P = 0.0024, adjusted hazard ratio 2.05, 95% CI 1.29–3.26; SPH, P = 0.029, adjusted hazard ratio 1.36, 95% CI 1.03–1.80). These differences were not observed in elderly patients in the three cohorts. Associations between the other four vital signs and mortality were not different in elderly and non-elderly patients. The interaction of age and hypothermia/fever was significant (P < 0.05). Conclusions: In septic patients, we found mortality in non-elderly sepsis patients was increased with hypothermia and decreased with fever. However, mortality in elderly patients was not associated with BT. These results illuminate the difference in the inflammatory response of the elderly compared to non-elderly sepsis patients.

Background: Diagnosing sepsis remains difficult because it is not a single disease but a syndrome with various pathogen- and host factor-associated symptoms. Sepsis-3 was established to improve risk stratification among patients with infection based on organ failures, but it has been still controversial compared with previous definitions. Therefore, we aimed to describe characteristics of patients who met sepsis-2 (severe sepsis) and sepsis-3 definitions. Methods: This was a multicenter, prospective cohort study conducted by 22 intensive care units (ICUs) in Japan. Adult patients (≥ 16 years) with newly suspected infection from December 2017 to May 2018 were included. Those without infection at final diagnosis were excluded. Patient’s characteristics and outcomes were described according to whether they met each definition or not. Results: In total, 618 patients with suspected infection were admitted to 22 ICUs during the study, of whom 530 (85.8%) met the sepsis-2 definition and 569 (92.1%) met the sepsis-3 definition. The two groups comprised different individuals, and 501 (81.1%) patients met both definitions. In-hospital mortality of study population was 19.1%. In-hospital mortality among patients with sepsis-2 and sepsis-3 patients was comparable (21.7% and 19.8%, respectively). Patients exclusively identified with sepsis-2 or sepsis-3 had a lower mortality (17.2% vs. 4.4%, respectively). No patients died if they did not meet any definitions. Patients who met sepsis-3 shock definition had higher in-hospital mortality than those who met sepsis-2 shock definition. Conclusions: Most patients with infection admitted to ICU meet sepsis-2 and sepsis-3 criteria. However, in-hospital mortality did not occur if patients did not meet any criteria. Better criteria might be developed by better selection and combination of elements in both definitions. Trial registration: UMIN000027452

Background: Predisposing conditions and risk modifiers instead of causes and risk factors have recently been used as alternatives to identify patients at a risk of acute respiratory distress syndrome (ARDS). However, data regarding risk modifiers among patients with non-pulmonary sepsis is rare. Methods: We conducted a secondary analysis of the multicenter, prospective, Focused Outcomes Research in Emergency Care in Acute Respiratory Distress Syndrome, Sepsis and Trauma (FORECAST) cohort study that was conducted in 59 intensive care units (ICUs) in Japan during January 2016–March 2017. Adult patients with severe sepsis caused by non-pulmonary infection were included, and the primary outcome was having ARDS, defined as meeting the Berlin definition on the first or fourth day of screening. Multivariate logistic regression modeling was used to identify risk modifiers associated with ARDS, and odds ratios (ORs) and their 95% confidence intervals were reported. The following explanatory variables were then assessed: age, sex, admission source, body mass index, smoking status, congestive heart failure, chronic obstructive pulmonary disease, diabetes mellitus, steroid use, statin use, infection site, septic shock, and acute physiology and chronic health evaluation (APACHE) II score. Results: After applying inclusion and exclusion criteria, 594 patients with non-pulmonary sepsis were enrolled, among whom 85 (14.3%) had ARDS. Septic shock was diagnosed in 80% of patients with ARDS and 66% of those without ARDS (p = 0.01). APACHE II scores were higher in patients with ARDS [26 (22–33)] than in those without ARDS [21 (16–28), p < 0.01]. In the multivariate logistic regression model, the following were independently associated with ARDS: ICU admission source [OR, 1.89 (1.06–3.40) for emergency department compared with hospital wards], smoking status [OR, 0.18 (0.06–0.59) for current smoking compared with never smoked], infection site [OR, 2.39 (1.04–5.40) for soft tissue infection compared with abdominal infection], and APACHE II score [OR, 1.08 (1.05–1.12) for higher compared with lower score]. Conclusions: Soft tissue infection, ICU admission from an emergency department, and a higher APACHE II score appear to be the risk modifiers of ARDS in patients with non-pulmonary sepsis.