Elucidating the Biological Effects of Cell-Free DNA (cfDNA) Extracted from Septic Mice: In Vitro and In Vivo Investigations and Mechanistic Insights

cfDNA is known to be a beneficial biomarker for the diagnosis, treatment response, and prognosis of sepsis. Previous cell line studies have shown that cfDNA in septic mice triggers inflammatory responses and tissue damage, and the mechanism occurs by binding to toll-like receptors (TLRs). Meanwhile, reactive oxidative species (ROS) and nitric oxide (NO) are increased in sepsis, which is known to be associated with organ failure and poor prognosis of sepsis through immunoredox imbalance. This study investigates the occurrence and mechanisms of inflammatory responses when cfDNA from septic mice is administered to macrophages (RAW 264.7 cells) and normal ICR mice aged 7-9 weeks. In addition, cfDNA-activated genes and their pathways are investigated and studied. Tumor necrosis factor-alpha (TNF-α) measured 24 hours after administration of 100, 200, and 400 ng/mL of sepsis-derived cfDNA to healthy mice was significantly higher in the 400 ng/mL group than in the control group. Furthermore, in macrophage cell line experiments, total ROS, NO, and catalase concentrations measured at 24 hours after cfDNA administration were significantly increased compared to the control group. In the meantime, cell viability studies revealed that the viability of the group treated with cfDNA from septic mice was much lower than that of the control group treated with normal human cfDNA. Cytokine levels such as granulocyte colony-stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-σ), interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF) were significantly higher in the 5 ng/mL group when compared to the 1 ng/mL group, but levels of IL-1β and IL-6 were not significantly higher. Finally, we measured the expression levels of several oxidative stress genes and receptor (TLR) genes and found that 46 cytokines & chemokines genes, namely II23a, Cxc10, XcI1, Csf1, Tnfsf10, Tnf, II10, Tgfb2, Ltb, II15, CcI22, Cd70, Cc15, Lta, CxcI11, Cntf, Ctf1, Bmp6, II11, II22, II24, II27, CxcI12, Ccl7, Tnfsf11, Gapdh, Hprt, Ppia, RpI13a, Tbp, Spp1, Cd40lg, Cxcl3, Cxcl1, lfng, Thpo, Ppbp, II5, II13, Fasl, Ccl11, Bmp4, Ccl4, Cxc19, Adipoq and Tnfrs11b and 18 oxidative stress genes, namely Sod3, Gpx1, Ucp2, Krt1, Cygp, II19, Rag2, Epx, Hspa1a, Psmb5, Prdx2, Txn1, Ngb, Nox4, Ercc2, Ncf2, Noxo1, and Ptgs1 were expressed at various levels, and 13 receptor genes, namely Sod3, Gpx1, Cd80, NfkbiL1, Tlr3, II2, Eif2ak2, II6ra, Hmgb1, Chuk, Tirap, Tradd, and Mapk8ip3, were also expressed at various levels. cfDNA from septic mice and healthy humans exhibit different immunoredox responses through immunoredox-related gene pathways.