Mitochondrial DNA via recipient TLR9 acts as a potent first-hit in murine transfusion-related acute lung injury (TRALI).

Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related mortality. Though the precise mechanism is not fully understood, a two-hit model is widely accepted, involving both a predisposing patient condition and the transfusion itself. Mitochondrial damage-associated molecular patterns (mtDAMPs), such as mitochondrial DNA (mtDNA) and N-formylated peptides (NFPs), are elevated in trauma patients and stored blood products, and have been implicated in adverse transfusion outcomes, prompting us to investigated whether mtDAMPs could serve as a priming "first-hit" in TRALI. Using a murine model, we found that injection of purified mitochondria followed by a monoclonal anti-major histocompatibility complex class I (MHC class I) antibody (34-1-2s) induced significantly greater lung injury compared to the isotype control. This was evidenced by increased pulmonary edema, elevated plasma macrophage inflammatory protein-2 (MIP-2), enhanced neutrophil lung infiltration, hypothermia, and respiratory distress. Similar effects were observed using a Toll-Like Receptor 9 agonist (ODN 2395), purified mtDNA, and a synthetic NFP (WKYMVm), agonist of Formyl Peptide Receptor (FPR). Notably, a TLR9 antagonist blocked the mtDAMP-induced TRALI response, whereas two FPR antagonists did not, underscoring a key role for mtDNA and TLR9 signaling in disease priming. These findings suggest that mtDAMPs, particularly mtDNA, present in both transfusion products and recipient plasma may predispose patients to antibody-mediated TRALI. Targeting mtDAMPs or their receptors may offer a novel therapeutic strategy to mitigate TRALI risk.