In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls

Abstract

Schizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated the in vitro response of ten human skin fibroblast (HSF) lines to five DNA probes containing different amounts of a GC-rich marker (the ribosomal repeat) and a DNA oxidation marker (8-oxodG) including sz-cfDNA and healthy control c-cfDNA (hc-cfDNA) probes. After 1 h, 3 h, and 24 h of incubation, the expression of 6 protein genes responsible for cfDNA transport into the cell (EEA1 and HMGB1) and the recognition of cytosolic DNA (TLR9, AIM2, STING and RIG-I) was analyzed at the transcriptional (RT-qPCR) and protein level (flow cytometry and fluorescence microscopy). Additionally, we analyzed changes in the RNA amount of 32 genes (RT-qPCR), which had been previously associated with different cellular responses to cell-free DNA with different characteristics. Adding sz-cfDNA and hc-cfDNA to the HSF medium in equal amounts (50 ng/mL) blocked endocytosis and stimulated TLR9 and STING gene expression while blocking RIG-I and AIM2 expression. Sz-cfDNA and hc-cfDNA, compared to gDNA, demonstrated much stronger stimulated transcription of genes that control cell proliferation, cytokine synthesis, apoptosis, autophagy, and mitochondrial biogenesis. No significant difference was observed in the response of the cells to sz-cfDNA and hc-cfDNA. Sz-cfDNA and hc-cfDNA showed similarly high biological activity towards HSFs, stimulating the gene activity of TLR9 and STING DNA sensor proteins and blocking the activity of the AIM2 protein gene. Since the sz-cfDNA content in the patients’ blood is several times higher than the hc-cfDNA content, sz-cfDNA may upregulate pro-inflammatory cytokines in schizophrenia.

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls—Part 2: Adaptive Response

“…The length of DNA fragments varied from 6 up to 10 kb. The cfDNA samples contained 1–2% shorter DNA fragments (200 b) [ 29 ].…”

“…In this work, we continued the in vitro study of the bioactivity of cfDNA obtained from the plasma of schizophrenia patients and healthy controls. Earlier, we showed considerable activation of DNA sensors upon the exposure to sz-cfDNA or hc-cfDNA for the HSF [ 29 ]. The activation of the sensors launched synthesis of pro-inflammatory cytokines.…”

“…Due to the altered GC-ratio and the levels of methylation and oxidation compared to intracellular DNA, cfDNA acquires advanced capacities to govern the functional activity of the body’s cells [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. In our previous study on cultured skin fibroblasts in vitro, we showed that cfDNA isolated from human blood plasma stimulated the known DNA-sensors, which control the signaling pathways modulating the immune response [ 29 ].…”

“…The present study is Part 2 of that research to continue the in vitro investigation of biological activity of cfDNA obtained from plasma of a schizophrenia patient (sz-cfDNA) and healthy controls (hc-cfDNA). In contrast to the previous study [ 29 ], we explored the response in two cell types: cultured human skin fibroblasts (HSFs) and peripheral blood lymphocytes (PBL). Since the cfDNA samples isolated from blood plasma had been shown to contain oxidized DNA fragments [ 17 , 29 ], we supposed that hc-cfDNA and sz-cfDNA could induce the adaptive response in cultured cells.…”

“…In contrast to the previous study [ 29 ], we explored the response in two cell types: cultured human skin fibroblasts (HSFs) and peripheral blood lymphocytes (PBL). Since the cfDNA samples isolated from blood plasma had been shown to contain oxidized DNA fragments [ 17 , 29 ], we supposed that hc-cfDNA and sz-cfDNA could induce the adaptive response in cultured cells.…”