AUTHOR=Schnepppfister Mike , Wang Yue , Zhong Chen , Huey Tori , El-Shewy Hesham , Kao Yichu , Scalea Joseph R. , Morinelli Thomas A. , Zhai Yuan , van der Windt Dirk J. 
  
TITLE=Beyond a Biomarker: Investigations of a Proinflammatory Role for Cell-Free DNA in Liver Transplant Ischemia and Reperfusion
  
JOURNAL=Transplant International
  
VOLUME=Volume 39 - 2026
  
YEAR=2026
  
URL=https://www.frontierspartnerships.org/journals/transplant-international/articles/10.3389/ti.2026.15340
  
DOI=10.3389/ti.2026.15340
  
ISSN=1432-2277
  
ABSTRACT=Donor-derived cell-free DNA (dd-cfDNA) is a biomarker for rejection after organ transplantation. We hypothesized that high release of cfDNA immediately after liver transplant also has a biologic role in inflammation in ischemia and reperfusion injury (IRI). To investigate this concept, C57BL/6 mice were subjected to 90 min in situ liver ischemia. After 6 h reperfusion, cfDNA was purified from serum and used to stimulate macrophages in vitro, which resulted in production of high levels of inflammatory cytokines TNFα and IL-6, and chemokine CXCL10. Enzymatic degradation of cfDNA by DNase I inhibited these inflammatory responses (e.g., TNFα: DNase I 48.1 ± 37.4 vs. untreated 1,030 ± 206 pg/mL, p = 0.0001). cfDNA from netosis-deficient PAD4KO mice was found to be equally pro-inflammatory compared to wild type cfDNA (TNFα: PAD4KO 1048 ± 199 vs. wild-type 1,162 ± 150 pg/mL, p = 0.64), indicating its mechanism is not dependent on neutrophils undergoing netosis. Next, a single dose of DNase I was added to the perfusate during rat liver normothermic machine perfusion (NMP) to significantly reduce perfusate cfDNA levels (384 ± 132 to 129 ± 18 ng/mL, p = 0.026). In conclusion, our data suggest that cfDNA can have pro-inflammatory effects during liver IRI beyond being a biomarker. DNase I may be a promising therapeutic intervention during NMP to reduce the graft’s inflammatory propensity prior to implantation.