Severe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as well as microbial sepsis, has been found to be associated with significant mortality rates.
They share certain pathological features such as coagulopathy, hyper inflammation, and immune dysfunction that can in turn lead to organ failure. Sepsis associated with secondary infection is also reported to be common in patients with severe SARS-CoV-2 infections. However, the treatment options are not clearly understood.
Cell-free chromatin has been found to circulate in SARS-CoV-2 patients and microbial sepsis that can be linked to coagulopathy, hyper inflammation, and immune dysfunction in murine models. Mice that lack DNase I and DNase I L3 have been found to develop severe sepsis following sterile or microbial challenges. Also, chronic autoimmunity and severe thermal injury are associated with low DNA and NET clearance activity. However, the effect of acute infection on the DNA clearance mechanism is still unknown. Previous studies also indicated that T cell death along with actin release can be associated with increased risk for thrombosis in murine models of fungal sepsis.
A new study published in the pre-print server medRxiv* involved activity-based proteomic profiling of patients who had severe SARS-CoV-2 infections to understand the impact of DNA clearance on mortality.
About the study
The study involved the collection of peripheral blood samples from healthy individuals, sepsis samples from sepsis patients, and respiratory samples from SARS-CoV-2 positive patients. Assessment of epidemiological and demographic parameters, clinical course, medical history, morbidity, and quality of life during hospitalization of coronavirus disease 2019 (COVID-19) patients was carried out. Immunological, molecular, and virological phenotyping was also performed.
The study also included 12-week-old female mice and wild-type Candida albicans that were cultured overnight in yeast extract peptone dextrose medium with shaking at 190 RPM at 37 degrees centigrade.
Histology and immunofluorescence imaging of extracted mouse spleen and COVID-19 patient's spleen was carried out. This was followed by the isolation of neutrophils from peripheral blood. Thereafter plasmid DNA degradation assay, DNA quantification, and NET degradation quantification took place followed by western blot analysis. Finally, the samples were prepared for proteomic analysis followed by liquid chromatography-mass spectrometry.
The results indicated that the splenic T cell death was associated with the release of cell-free actin and increased thrombosis. Assays to degrade plasmid DNA and dissolve neutrophil extracellular traps (NETs) indicated that plasmid DNA was degraded by DNA I while degradation of NET was carried out by other plasma factors.
The results further reported that 30 percent of SARS-CoV-2 patients and 60 percent microbial sepsis patients showed reduced DNase activity as compared to healthy donors. High levels of plasma actin in microbial sepsis and SARS-CoV-2 patients were found to be associated with low DNase activity while low NETase activity was found to be associated with most of the microbial sepsis and SARS-CoV-2 patients.
Low DNase activity was shown to be associated with high levels of orosomucoid 1 and 2 (ORM1; OMR2) peptides while low NETase activity was associated with high levels of complement cascade factors (C1QA, C1QB, C1QC) and apolipoproteins such as APOB and APOE. Both the average ORM1; ORM2 levels and actin levels were found to decrease over time from higher levels in surviving patients while high levels persisted in the case of non-survivors. Moreover, they were reported to be positively correlated with average C-reactive protein (CRP) levels in all the patients.
The results also indicated that low and medium DNase activity was mostly associated with non-survivors and both DNase activity as well as NETase activity had an impact on several clinical parameters. Low and medium DNase activity exhibited higher procalcitonin (PCT), CRP, and creatinine levels along with lower lipase levels. Furthermore, these patients showed higher oxygen requirements and lower lung function recovery.
Low NETse activity was found to be associated with low neutrophil chemokine pro-platelet basic protein (PPBP) and high protease inhibitor alpha-2-macroglobulin (A2M) while high NETase activity was found to be associated with high complement component 4 binding protein beta (C4BPB). Also, low NETase activity was associated with non-survivors.
The NETase coefficient was found to be higher in survivors as compared to deceased patients. Also, patients who had below 60 NETase coefficients were found to have a 15-fold higher mortality rate. Treatment with DNase I or anti-histone antibodies showed changes in several apolipoproteins as well as immunoglobulin heavy constant gamma 4 (IGHG4) which were directly associated with DNase and NETase activity. Therefore, both these treatments could block T cell death in the spleen, actin release, and hypothermia.
The current study, therefore, demonstrates the impact of extracellular DNA clearance pathways in the survival of patients with severe SARS-CoV-2 infection. The correlations between DNA clearance and apolipoproteins may suggest a link between DNA clearance and lipid metabolism.
The study is of importance as it helps in the identification of pathologically relevant markers in samples that are otherwise difficult to distinguish, that is, multiple activity tracing in large samples, and identification of high-risk individuals in a cohort of severely affected COVID-19 patients, which sometimes is difficult to be determined by conventional clinical parameters.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.