2C5-surface modified nanocarriers targeting neutrophil extracellular traps

Background: Neutrophil extracellular traps (NETs) are composed of processed chromatin bound to granular and selected cytoplasmic proteins and released by neutrophils. NETs consist of smooth filaments composed of stacked nucleosomes. Fully hydrated NETs have a cloud-like appearance and occupy a space 10-15-fold larger than the volume of the cells they originate from. DNases are the enzymes that cleave extracellular DNA including NETs. Together with their protective role in microbial infections, NETs are involved in multiple pathological processes and represent key events in a variety of pathologies including cancer, autoimmunity, and cardiovascular disease. Sites of NETs concentration are dangerous for the host if the process of NETs formation becomes chronic or the mechanism of NETs removal does not work. NETosis has been linked to the development of periodontitis, cystic fibrosis, type 2 diabetes, COVID-19 or rheumatoid arthritis as well as cancer progression. Purpose(s): Thus, the destruction of NETs is of primary significance in many pathologies. In our approach, we are focusing on mimicking one of the natural mechanisms of destroying excessive NETs by delivering deoxyribonuclease I to the specific site of pathological NETs accumulation by modifying the nanoparticles using an anti-nucleosome monoclonal antibody (2C5). The antibody is specific to nucleosomes and can recognize histones in NETs. DNase I is U.S. Food and Drug Administration (FDA)-approved active component and is commonly used in therapeutic methods of modern medicine for cystic fibrosis to clear extracellular DNA fibers in the lungs and systemic lupus erythematosus. Recent findings have also shown the effectiveness of DNase I in the digestion of NETs. However, the low serum stability and fast deactivation by environmental stimuli have been considered as the limiting factors for clinical applications of DNase I, which can be overcome by its targeted specific delivery in pharmaceutical nanocarriers. Method(s): In this study, we generate NETs in vitro using human neutrophils and HL-60 cells differentiated into granulocyte-like cells. We used interleukin-8, lipopolysaccharide from E.Coli (LPS), phorbol myristate acetate (PMA), and calcium ionophore A23187 (CI) to generate the NETs. We confirmed the specificity of 2C5 toward NETs by ELISA, which showed that it binds to NETs with the specificity like that for purified nucleohistone substrate. We further utilized that feature to create two delivery systems (liposomes and micelles) for DNAse I enzyme to destroy NETs, which was confirmed by staining NETs with SYTOX Green dye and followed by flow cytometric measurements and microscopic images. Conclusion(s): Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies. Also, we proved that our carrier can successfully deliver DNase to NETs to provide their degradation.

Evaluation and comparison of NETosis biomarkers in sepsis and COVID-19 patients

Background: Neutrophils are involved in the defense of the body against pathogens through the formation neutrophil extracellular trap, a mechanism called NETosis. These pathogens can be fungi, bacteria, viruses or other microorganisms. However, neutrophils through NETs have a double-edged sword activity and can also be harmful. Aim(s): This study aims to evaluate biomarkers of NETosis in two different patient populations admitted to the intensive care unit, i.e. COVID-19 and sepsis patients. A control population of matched subjects have been included. Method(s): Among the individuals admitted to the ICU and included in this study, 46 were sepsis patients and 22 were COVID-19 patients. 48 controls were included. Nucleosome histone H3.1, nucleosome citrullinated histone H3R8, free citrullinated histone (citrullinated at R2, R8 and R17), neutrophil elastase an myeloperoxidase were measured. Blood samples were taken at the admission to the intensive care unit. The different groups were compared using ordinary two-way ANOVA with a Tukey's multiple comparison on log-transformed data. Result(s): A significant difference in the levels of Nu.H3.1 and NE was observed between sepsis and COVID-19 subjects. All NETosis parameters differs in ICU patients versus controls. A positive correlation was found between SOFA and APCHE-II score and Nu.H3.1 in sepsis patients. No positive correlation was observed in COVID-19 patients. Normalization of NETosis parameters according to the neutrophil count improves the sensitivity of Nu.H3.1 to discriminate between sepsis and COVID-19 patients. The other parameters were also influenced but to a lesser extent. NE and Nu.H3.1 correlates well in sepsis patients while it is not the case in COVID-19 patients. Conclusion(s): Nu.H3.1 appears to be an interesting marker of NETosis in sepsis and COVID-19 patients. Its correlation with NE in sepsis patients reveals that NE is important in generating circulating nucleosomes while its weak association in COVID-19 suggest different patterns between the diseases.

Neutrophil extracellular traps have auto-catabolic activity and produce mononucleosome-associated circulating DNA.

BACKGROUND: As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA. METHODS: We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114). RESULTS: Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); "ex vivo" NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI. CONCLUSIONS: Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA.

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions. These vRNPs depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.