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

Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related mortality. Though the precise mechanism is not fully understood, a 2-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 patients who have experienced trauma, and stored blood products, and have been implicated in adverse transfusion outcomes, prompting us to investigate 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 antibody (34-1-2s) induced significantly greater lung injury compared with the isotype control. This was evidenced by increased pulmonary edema, elevated plasma macrophage inflammatory protein 2, enhanced neutrophil lung infiltration, hypothermia, and respiratory distress. Similar effects were observed using a Toll-like receptor 9 (TLR9) agonist (oligonucleotide 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 2 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.