key: cord-0955498-eu0azqfu
authors: Lee, Yun Young; Park, Hee Ho; Park, Wooram; Kim, Hyelim; Jang, Jong Geol; Hong, Kyung Soo; Lee, Jae-Young; Seo, Hee Seung; Na, Dong Hee; Kim, Tae-Hyung; Choy, Young Bin; Ahn, June Hong; Lee, Wonhwa; Park, Chun Gwon
title: Long-acting nanoparticulate DNase-1 for effective suppression of SARS-CoV-2-mediated neutrophil activities and cytokine storm
date: 2020-10-23
journal: Biomaterials
DOI: 10.1016/j.biomaterials.2020.120389
sha: cf1771a5b5cb2c9d9226d1bfecbe5d7dea9eca74
doc_id: 955498
cord_uid: eu0azqfu

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus not previously identified in humans. Globally, the number of confirmed cases and mortality rates of coronavirus disease 2019 (COVID-19) have risen dramatically. Currently, there are no FDA-approved antiviral drugs and there is an urgency to develop treatment strategies that can effectively suppress SARS-CoV-2-mediated cytokine storms, acute respiratory distress syndrome (ARDS), and sepsis. As symptoms progress in patients with SARS-CoV-2 sepsis, elevated amounts of cell-free DNA (cfDNA) are produced, which in turn induce multiple organ failure in these patients. Furthermore, plasma levels of DNase-1 are markedly reduced in SARS-CoV-2 sepsis patients. In this study, we generated recombinant DNase-1-coated polydopamine-poly(ethylene glycol) nanoparticulates (named long-acting DNase-1), and hypothesized that exogenous administration of long-acting DNase-1 may suppress SARS-CoV-2-mediated neutrophil activities and the cytokine storm. Our findings suggest that exogenously administered long-acting nanoparticulate DNase-1 can effectively reduce cfDNA levels and neutrophil activities and may be used as a potential therapeutic intervention for life-threatening SARS-CoV-2-mediated illnesses.

There is a massive concern regarding the outbreak of coronavirus disease 2019 caused by the highly infectious and lethal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1] . The COVID-19 pandemic is a severe threat to public health across the globe. Current statistics report approximately 250,000 deaths and more than 3,500,000 confirmed cases in 180 countries worldwide [2] , but the situation is highly fluid and the numbers continue to rise. Experts are forecasting hundreds of thousands of mortalities worldwide.

SARS-CoV-2 infections are associated with both impaired adaptive immune responses and uncontrolled inflammatory innate immune responses, which lead to local and systemic tissue damage [1] . The radical immune reactions cause septic shock which lead to increase in the mortality rates of patients [1] . Thus, there is an urgent need to develop new treatment strategies that can suppress the progression of SARS-CoV-2-induced sepsis. Many research groups and global pharmaceutical companies have been conducting intense preclinical and phase I trials of potential SARS-CoV-2 treatments [3] [4] [5] . However, most of these drugs have not been developed for the actual treatment of SARS-CoV-2, but rather were developed for the treatment of other viruses, such as Ebola virus, human immunodeficiency virus (HIV), severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, Influenza A virus, and ZIKA virus [6] . Various clinical studies have shown that suppression of cytokine release syndrome (CRS) and respiratory damage are substantial hurdles needed to be overcome in severely affected patients [2, 7] . Recently, Genentech has announced FDA approval of phase III clinical trial for Tocilizumab (Actemra) to treat hospitalized patients with severe SARS-CoV-2 pneumonia (NCT04320615) [8] .

J o u r n a l P r e -p r o o f However, even after the successful resolution of the virus infection, patients may not exhibit full recovery from the damage and may progress to more severe disease, such as acute respiratory distress syndrome (ARDS) and sepsis, or in worse cases, death. Currently, the World Health Organization (WHO) does not recommend corticosteroids for the treatment of patients with COVID-19, as they may exacerbate SARS-CoV-2-associated acute respiratory infections [9] . Since acute complications and fatalities are associated with the virus, new strategies are urgently needed to address the rapid respiratory-related inflammatory responses.

Neutrophil extracellular traps (NETs) are reticular structures consisting of a DNA backbone and a number of functional proteins that are formed through a process known as NETosis [10, 11] . NETs exert anti-infective activity by capturing pathogenic microorganisms (including viruses), preventing their spread, and by inactivating pathogenic factors. However, it has been reported that immune responses caused by NETosis may also induce tissue damage [12] . SARS-CoV-2 infections may impair adaptive immune responses as well as induce uncontrolled inflammatory innate immune responses, which cause local and systemic tissue damage. These phenomena are typically associated with elevated levels of NETosis markers, such as cell-free DNA (cfDNA), myeloperoxidase (MPO), and neutrophil elastase (NE). These factors correlate with increased disease severity and poor clinical outcome. It is particularly challenging to treat patients that exhibit high levels of NETosis. Consistent with this, an approach that inhibits NETosis has significantly increased the survival in pulmonary sepsis [13, 14] .

In the current study, we sought to determine if NETosis markers were actually increased in patients infected with SARS-CoV-2 and whether suppressing NETs could be used as a possible treatment approach for acute respiratory infections associated with J o u r n a l P r e -p r o o f COVID-19. We analyzed NETosis makers in the blood of patients with COVID-19 to determine the association of NETosis with the acute immune response triggered by SARS-CoV-2. We showed that an intravenous administration of DNase-1-coated polydopamine-poly (ethylene glycol) nanoparticulates, named long-acting DNase-1 (Scheme 1), effectively inhibited NETosis factors in blood samples of patients with COVID-19 and also improve survival in a sepsis model. Currently, there are no FDA-approved therapeutic agents available for treating patients infected with SARS-CoV-2. Monitoring and modulation of NETosis are novel strategies in the diagnosis and treatment of patients with COVID-19 and may ultimately contribute to the development of therapeutic agents.

Poly (lactic-co-glycolic acid) (PLGA; lactic acid:glycolic acid = 1:1, viscosity 0.55-0.75 DL/g, Part#: B6013-2P was purchased from Durect Corporation (AL, USA). Poly(vinyl alcohol) (PVA), Tris buffer (pH 8.5) and polydopamine (PD) were obtained from Sigma-Aldrich (PA, USA). Poly (ethylene glycol) (PEG, 4 arm-amine termini, HCL salt) was provided by JenKem, (TX, USA). Dichloromethane (DCM) and DNase-1 were purchased from Daejung (Seoul, South Korea) and Roche (Basel, Switzerland), respectively.

Long-acting DNase-1 was prepared as previously described [15, 16] . Primary nanoparticles J o u r n a l P r e -p r o o f were prepared with the FDA-approved polymer, PLGA, via a conventional single emulsification method (Scheme 1). To prepare bare nanoparticles (PLGA NP), 400 mg PLGA was dissolved in 5 mL DCM and then poured into a 20 mL solution of 1% PVA. The resulting polymer solution was sonicated for 10 min at 40% amplitude with 1 sec on and 1 sec off using a Q700 sonicator (Qsonica, LLC, Newtown, CT, USA). The prepared emulsions were poured into 5 mL DI water and residual DCM was allowed to evaporate overnight at room temperature with magnetic stirring. The PLGA NPs (400 mg) were resuspended in 20 mL 10 mM, pH 8.5 Tris buffer and coated with a bio-adhesive made of polydopamine (PD, 100 mg dopamine in 1 mL water) with vigorous stirring at 4 for 3 h. The bio ℃ -adhesive nanoparticles (PLGA-PD NPs) were collected by centrifugation at 17,000 rpm for 20 min, resuspended in 5 mL Tris buffer (10 mM, pH 8.5) containing 50 mg DNase-1 and 50 mg poly(ethylene glycol), and stirred at 4 ℃ for 3 h. The resulting DNase-1-coated NPs were then collected and washed with DI water to prepare PLGA-PD-PEG-DNase-1 NPs (referred to as long-acting DNase-1).

The long-acting DNase-1 was diluted in Milli-Q water and a drop of the solution was placed on a copper grid followed by drying in a vacuum chamber. Upon drying, the grid was carefully placed on a sample holder for analysis using a JEM-7500F (Akishima, Japan) scanning electron microscope (SEM). The size and zeta potential of the particles were 

Whole blood was collected from patients admitted at Yeungnam University Medical Center after they were diagnosed with the SARS-CoV-2 infection at a public health center in Daegu, 

Complete blood counts were obtained from the patients' venous blood samples. The laboratory findings were analyzed within 24 hours after admission. Neutrophil count was performed using a Sysmex XE-2100 Automated Hematology System.

Freshly isolated neutrophils (1 × 10 5 cells) were stimulated to generate NETs by incubating with 25 nM phorbol-myristate acetate (PMA, Sigma-Aldrich, MO, USA) or control media (RPMI 1640 supplemented with glutamine, penicillin, and streptomycin) and evaluated using a fluorometric technique as previously described [18] . NET production was measured as arbitrary fluorescent units (AFUs).

To quantify the release of granule matrix proteins upon degranulation in peripheral blood mononuclear cells (PBMCs)of SARS-CoV-2-infected patients and mice, plasma were analyzed using a human MPO ELISA kit (BMS2038INST, Invitrogen) and mouse J o u r n a l P r e -p r o o f myeloperoxidase ELISA kit (MBS700747, MyBioSource), respectively.

Cit-histone H3 concentrations in cell culture media or septic patient sera were determined (fluorescence emission, Em) was then measured using a fluorometer.

PBMCs were isolated on a Percoll (pH 8.5-9.5; Sigma-Aldrich, UK) density gradient as previously described [19] . The PBMCs (95 % purity and 97 % viable according to trypan blue exclusion) were resuspended in RPMI 1640 media (Sigma-Aldrich). Neutrophils were isolated using discontinuous density gradient centrifugation on 1-step Polymorphs (Axis-Shield, Oslo, Norway). To increase purity, the neutrophil population was further purified using CD45 antibody-conjugated magnetic beads and magnetic-activated cell sorting (MACS). Viability of the neutrophils was generally > 95 % as assessed by trypan blue dye exclusion. To verify the effect of long-acting DNase-1 on inhibition of cytokine production, neutrophils were isolated from SARS-CoV-2-infected patients and treated with long-acting DNase-1. Long-acting DNase-1 was also administrated septic CLP mice. Supernatants were used for cytokine analyses using ELISA and the cell lysates were used for analysis of NF-kB activity.

Preparation of nuclear extracts and TransAM assays were performed as previously described [20] . The activity of individual NF-κB subunits was determined using an ELISA-based NF-κB Family Transcription Factor Assay Kit (43296; Active Motif, Carlsbad, CA, USA).

Briefly, nuclear extracts (2 µg) were incubated in a 96-well plate, which was coated with NF-κB consensus oligonucleotides. The captured complexes were incubated with specific NF-κB primary Abs and subsequently detected using HRP-conjugated secondary Abs included with the kit. Finally, the optical density (OD) at 450 nm was measured using a Tecan Spark microplate reader (Tecan, Austria GmbH, Austria).

Serum levels of inflammatory cytokines IL-1β, IL-6, IFN-γ and TNF-α and LDH were J o u r n a l P r e -p r o o f

The CLP-induced septic mouse model was prepared as previously described [21] . Briefly, a 2-cm midline incision was made to expose the cecum and adjoining intestine. The cecum was then tightly ligated using a 3.0-silk suture 5.0 mm from the cecal tip, punctured with a 22gauge needle, and then gently squeezed to extrude feces from the perforation site. The cecum was then returned to the peritoneal cavity and the laparotomy site sutured using 4.0-silk. For sham operations, the cecum of animals was surgically exposed but not ligated or punctured and then returned to the abdominal cavity.

To analyze the neutrophils and macrophages that migrated into the peritoneal cavity, CD45- 

Male C57BL/6 mice underwent CLP and were then intravenously administered free DNase-1 J o u r n a l P r e -p r o o f especially for those who have progressed to sepsis, undergo lymphopenia and thus have reduced numbers of lymphocytes [22, 23] . In addition, increases in absolute neutrophil numbers and neutrophil-to-lymphocyte ratios are also usually associated with the severity of diseases [24] . Therefore, we evaluated the neutrophils in SARS-CoV-2-infected patients and observed elevated neutrophil numbers (Fig. 1A) . Absolute neutrophil counts were also seen in in COVID-19 patients with sepsis (Fig. 1B) . Noticeably, the level of activated NET formation (Fig. 1C) and expression of MPO were significantly increased in these patients ( Fig. 1D) . It has been reported that excessive amounts of NETosis lead to a hyperinflammatory response [25] , which subsequently attributes to organ damage and multiple organ failure [26] . In most cases, elderly patients and those individuals with underlying conditions progress toward sepsis [27, 28] . Computed tomography (CT) imaging revealed that patients with abnormally high numbers of neutrophils had severe lung tissue damage, indicating an increase in the severity of septic symptoms (Fig. 1E) . The diagnosis of pneumonia in COVID-19 patients with sepsis was verified based on lactate dehydrogenase (LDH) levels. LDH levels in the COVID-19 sepsis group were significantly increased compared with those in COVID-19 patients without sepsis (Fig. S1 ). in the 20 COVID-19 patients with sepsis ( Fig. 2A) . Differences were also observed for DNase-1 levels among the groups. For the normal group, the median DNase-1 was 2.11

(1.59-2.77) µg/mL compared to 3.11 (2.08-6.78) µg/mL for the COVID-19 patients without sepsis and 0.97 (0.52-1.44) µg/mL for the COVID-19 patients with sepsis (Fig. 2B) (Fig. 2C) .

We evaluated the expression levels of histone H3 and Cit-histone H3 in the plasma of patients with COVID-19 using western blot analysis (Fig. 2D) . As shown in Fig. 2C and 2D , Cithistone H3 levels were increased in the plasma samples of COVID-19 patients with sepsis compared to those without sepsis and healthy volunteers.

The levels of cfDNA, DNase-1, and Cit-histone H3 were re-analyzed in the 80 patients with COVID-19 according to patient end outcome (n = 69 survived and n = 11 deceased). The differences in the levels of cfDNA, DNase-1, and Cit-histone H3 were much more pronounced in the deceased group of patients. Unexpectedly high levels of cfDNA ( Fig.   2E ) and significantly reduced levels of DNase-1 (Fig. 2F) were observed, as well as increased levels of Cit-histone H3 (Fig. 2G) were observed, especially in the deceased patient group. Treatment is often ineffective for COVID-19 patients with severe sepsis symptoms, even when conventional antiviral drugs against other virus are administered [24] .

Having confirmed the abnormal levels of factors related to NETosis and reduced amounts of endogenous DNase-1 in COVID-19 patients, we then sought to leverage the use of exogenous DNase-1 to suppress the NETosis of neutrophils in effort to compensate for the loss of DNase-1 in patients with COVID-19. Due to the extremely short half-life of DNase-1 in blood [29] , we prepared DNase-1-coated nanoparticles as previously described [15, 16] to serve as long-acting DNase-1 (Scheme 1 and Fig. 3 ) since immobilization of DNase-1 on the surface of nanoparticles can enhance the stability of DNase-1 in the blood and thereby improving activity of the DNase-1. Mussel-inspired polydopamine chemistry was utilized to effectively immobilize DNase-1 on the nanoparticle surface [30, 31] . SEM analysis showed that PLGA-NPs have a spherical shape with a size of about 170 nm (Fig. 3A) . When the PLGA NP surface was modified with polydopamine and coated with PEG and DNase-1, it was observed that the particle size gradually increased to approximately 220 nm (Fig. 3B-D) .

The shape of the particles was spherical even after DNase-1 was coated. The surface charge of the nanoparticles was -19.6 mV for PLGA NPs, -24.5 mV for PLGA-PD NPs, -0.2 mV for PLGA-PD-PEG NPs, and -12.0 mV for PLGA-PD-PEG-DNase-1 NPs, respectively (Fig. 3E ).

DNA digestion was tested to confirm the activity of DNase-1 immobilized on the nanoparticle surface (Fig. 3F) . The long-acting DNase-1 effectively degraded DNA at concentrations > 0.25 mg/mL. In vitro release test was performed to evaluate the stability of DNase-1 bound on nanoparticles. As shown in Fig. S2 , DNase-1 released less than 20% from long-acting DNase-1 for 72 h. Additionally, when the activity of long-acting DNase-1 over time was tested in vitro, long-acting DNase-1 stably degrades DNA for up to 36 h (Fig. S3) .

To demonstrate the effects of DNase-1 on DNA degradation, we treated the plasma of COVID-19 patients with sepsis with the free DNase-1 and long-acting DNase-1. The results showed that both forms of DNase-1 significantly reduced cfDNA levels (Fig. 4A) and that exposure of DNase-1 to the plasma of COVID-19 patients with sepsis increased the activity of DNase-1 (Fig. 4B) . We also observed markedly reduced NET levels, MPO activity, and NE levels in neutrophils of COVID-19 patients with sepsis upon treatment of the DNase-1 formulations (Fig. 4C-E) . ARDS and sepsis are common immunopathological phenomena and the leading causes of death for patients infected with SARS-Cov-2 [32, 33] . One of the primary mechanisms for ARDS and sepsis in SARS-CoV-2 infection is the cytokine storm [1] , which is a deadly uncontrollable systemic inflammatory response resulting from the release of large amounts of pro-inflammatory cytokines and chemokines by immune effector cells [34, 35] . Therefore, the effects of DNase-1 on the activation of NF-κB and secretion of cytokines from neutrophils were evaluated. The results showed that the activity of NF-κB and secretion of cytokines IL-1β, IL-6, IFN-γ, and TNF-α were reduced slightly by treatment with free DNase-1 and further decreased by treatment with long-acting DNase-1 (Fig. 4F-J) .

Having confirmed anti-septic activity and reduction in NETosis of neutrophils by treatment DNase-1, CLP-treated mice were intravenously injected with phosphate-buffered saline, dexamethasone, long-acting DNase-1, or dexamethasone + long-acting DNase-1 at 12 h and 24 h post CLP-treatment (Fig. S4) . In the experiment, 2.5 mg/kg of dexamethasone was used [36] . When no dexamethasone or DNase-1 was given, all of the CLP-treated mice died within DNase-1 group (Fig. 5B) . Having inducing an anti-septic effect using the long-acting DNase-1, we then aimed to determine whether this treatment could specifically reduce the infiltration of Ly6G + neutrophils to peritoneal cavity. Increased numbers of Ly6G + neutrophils were observed in the control group, but free DNase-1 and long-acting DNase-1 treatment led to a substantial decrease in the number of infiltrating Ly6G + neutrophils (Fig. 5C) . Previously, it was reported that Ly6G-expressing (Ly6G + ) neutrophils increase secretion of various cytokines and chemokines. They capture and destroy invading microorganisms, through phagocytosis and intracellular degradation, thereby releasing granules and forming neutrophil extracellular traps (NETs) after detecting pathogens [37] . It is considered that abundant lunginfiltrating neutrophils in acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) also participate as mediators of inflammation. We also confirmed that the longacting DNase-1, which acts on the lungs, substantially reduced the morphological changes caused by CLP, including pulmonary edema, hemorrhage, alveolar collapse, and inflammatory cell infiltration (Fig. 5D) . To further support the acting of long acting DNase-1 on the lungs, we evaluated the biodistribution of nanoparticulate DNase-1 (Fig. S5) . Mice J o u r n a l P r e -p r o o f were intravenously injected with fluorescently-labeled free DNase-1 or long-acting DNase-1 and analyzed at 6 h, 12 h, and 24 h post treatment. Unsurprisingly, the long acting of DNase-1 was predominantly accumulated in the liver and kidney due to the mononuclear phagocytic system (MPS) [38] [39] [40] . Interestingly, the long acting of DNase-1 was accumulated in the lung and was cleared after 24 h, suggesting sufficient time for biological activity in vivo. As for the free DNase-1, some accumulation in the lung was observed, but it was soon cleared after 12 h.

We also verified the effect of the DNase-1 formulations on the degradation of DNA in the plasma using the CLP septic mouse model. Similar to the plasma of COVID-19

patients with sepsis, the long-acting DNase-1 significantly reduced cfDNA levels and increased the activity of the DNase-1 ( Fig. S6A and B) . Additionally, significant reductions in NET, MPO activity, NE levels, and neutrophil numbers were observed upon treatment with the long-acting DNase-1 (Fig. S6C-F) .

Next, we attempted to elucidate the effect of the long-acting DNase-1 on the regulation of inflammatory responses during sepsis. Systemic inflammation associated with sepsis often causes multiple organ failure [41] with the kidney and liver being the major organs targeted [42] . CLP treatment significantly increased serum levels of hepatic injury markers aspartate transaminase (AST) and alanine transferase (ALT), renal injury markers creatinine and blood urea nitrogen (BUN), the tissue injury marker lactate dehydrogenase (LDH), and the pneumonia and sepsis marker C-reactive protein (CRP) (Fig. S7) . Serum levels of these makers were significantly reduced by treatment with the long-acting DNase-1.

J o u r n a l P r e -p r o o f

The complex pathophysiology of sepsis is characterized by multiple organ failure caused by hyper inflammation and CRS. The organ failure is accompanied by an immunosuppressive state, which results in a failure to return to normal homeostasis. These events have hampered the development of therapeutic agents that can effectively treat the complex process of sepsis.

It has been reported that SARS-CoV-2 infection can activate both innate and adaptive immune responses [43] . Similar uncontrollable innate inflammatory responses and impaired adaptive immune responses have been shown to be induced by SARS-CoV-2, which results in increased tissue damage, both locally and systemically [44] . In patients with severe COVID-19, lymphopenia has also been observed with a collective and representative characteristic accompanied by an increased ratio of neutrophils and increase in the absolute neutrophil count [22, 23] . However, these attributes have not been observed in patients with mild COVID-19 disease. These phenomena are most often associated with increased disease severity and therefore the prognosis is usually a poor clinical outcome.

In our current findings, elevated levels of NETosis markers were observed, such as cfDNA [45] , MPO [46] , and NE [25] . These are characteristic features of patients critically ill with COVID-19 [47] . It is particularly challenging to treat patients with high levels of NETosis, which is why it is linked to high morbidity and mortality. Therefore, an innovative NETosis-targeting agent that is able to reduce the level of NETosis in the blood and prevent CoV-2 is not recommended highlights the urgent need for an alternative mean of treating SARS-CoV-2-infected individuals that can effectively suppress hyperinflammation-mediated organ damage that is observed in critically ill patients [48] .

Here, we report the effects of exogenous long-acting nanoparticulate DNase-1 on resolving inflammatory responses and reducing organ damage in a CLP-induced septic mouse model. Immobilization of the enzyme on the nanoparticle surface greatly improves the stability of the enzyme [49] . In previous study, DNase-1 coated nanoparticles increased plasma enzyme concentrations compared to native DNase-1 [16] . This means that the short half-life of DNase-1 can be improved by the immobilization on the nanoparticles. Thus, we adopted the DNase-1 coated nanoparticles as long-acting DNase-1 in this study. Indeed, longacting DNase-1 showed long-term enzymatic activity in vitro (Fig. S3) , and when injected into the body, DNase-1 coated nanoparticles were expelled through kidney and liver more slowly than native DNase-1 (Fig. S5) .

We also performed a comparative study using blood samples from patients with COVID-19 and demonstrated the suppression of neutrophil activity and cytokine levels.

Through the administration of long-acting DNase-1 in the septic mouse model, we demonstrated that DNase-1 could effectively reduce sepsis-associated NETosis factors. Our results demonstrated that the long-acting DNase-1 treatment significantly reduced markers cfDNA, NET, MPO, and NE (Fig. S6) in the CLP-induced septic mice. Notably, the longacting DNase-1 conferred a 50% survival rate for the CLP-treated septic mice at 96 h post procedure (Fig. 5) for SARS-CoV-2 infection. We found that treatment with long-acting DNase-1 significantly decreased NETosis according to levels of markers cfDNA, NET, MPO, and NE (Fig. 4) . NF-κB activation and cytokine levels are known to be associated with induced septic inflammation responses. As expected, our results showed that the long-acting DNase-1 inhibited NF-κB activation and cytokine levels. It should be noted that because the CLP- 

All data obtained from this study are included in the article or uploaded as supplementary information. The data that support the findings of this research are available from the corresponding authors upon reasonable request.

The authors declare that they have no competing interests. (Cit-histone H3) was analyzed by western blot. (E-F) Concentration of cfDNA, DNase-1, and histone H3 in SARS-CoV-2 patients. Patients were grouped according to survival or death (E-G). Statistics were calculated using a two-tailed unpaired t-test. Samples in the colored area (patients with sepsis) were excluded in the survival group for statistical analysis (E-G). *** P < 0.001. μm. The experiment was performed at least three times with replicates. Statistical analysis was performed using a two-tailed unpaired t-test. The log-rank (Mantel-Cox) test was used for statistical analysis of survival. Data are presented as mean ± SEM. * P < 0.05, *** P < 0.001.

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Physicochemical characterization of long-acting nanoparticulate DNase-1

Scale bar: 500 nm. The long-acting DNase-1 possessed a smooth spherical shape with an average size of approximately 200 nm. (E) Surface charges of PLGA-NPs, PLGA-PD-NPs, PLGA-PD-PEG NPs, and PLGA-PD-PEG-DNase-1 NPs (long-acting DNase-1). The long-acting DNase-1 had a negative charge of −12 mV. (F) DNA digestion capability of long

A-B) SARS-CoV-2 Sepsis patient plasma was incubated with free DNase-1 (100 units) or long-acting DNase-1 (100 units) for 1 h (each group n = 10). (A) cfDNA was isolated from the plasma, and concentration of the cfDNA was measured. (B) DNase-1 activity was quantified and compared between PBS, free DNase-1, or long-acting DNase-1. (C-E) Purified PBMCs from SARS-CoV-2 Sepsis patient whole blood cultured with free DNase-1 or long-acting DNase-1 for 6 h. NET was characterized by measuring the concentration of myeloperoxidase (MPO) and neutrophil elastase (NE). (C) NET ratio of SARS-CoV-2 Sepsis patient PBMCs was suppressed after free DNase-1 or long-acting DNase-1 treatment. (D) Free DNase-1 or longacting DNase-1 reduced the MPO activity in SARS-CoV-2 Sepsis patient PBMCs. (E) Concentration of NE in PBMC cultured media was decreased by free DNase-1 or long-acting DNase-1. (F) Binding activity of NF-κB p65 in PBMC cultured with PBS

DNase-1 (100 units, 6 h)-, or long-acting DNase-1 (100 units, 6 h)-treated PBMC of SAR-CoV-2 patients. The experiment was performed with replicates at least three times. Statistical analysis was performed using a two-tailed unpaired t-test