The TLR9-CXCL2 complex orchestrates mitochondrial DNA-driven regulatory B cell dysfunction in acute lung injury

Acute lung injury (ALI)/Acute respiratory distress syndrome (ARDS) is a lethal condition driven by unresolved inflammation. Circulating mitochondrial DNA (mtDNA) acts as a potent damage-associated molecular pattern, yet its direct role in disrupting adaptive immune checkpoints remains undefined. We integrated clinical data from ARDS patients with mechanistic studies in murine models, employing single-cell RNA sequencing, confocal imaging, and genetic approaches to dissect how mtDNA influences B cell-mediated immunoregulation. In ARDS patients, circulating mtDNA levels inversely correlated with protective IL-10 + regulatory B cells (Bregs). In mice, exogenous mtDNA exacerbated lung injury and cell-intrinsically reprogrammed Bregs toward an inflammatory phenotype. Mechanistically, mtDNA was internalized via the B cell receptor (BCR) into endosomal compartments, where it engaged TLR9 and promoted formation of a previously undescribed TLR9-CXCL2 complex. This interaction drove MyD88-NF-κB signaling and suppressed IL-10 and TGF-β1 expression. TLR9 inhibition reversed this maladaptive reprogramming. Adoptive transfer of healthy Bregs restored lung integrity and immune balance, and single-cell transcriptomics revealed that Breg therapy reshaped the pulmonary B cell repertoire toward a reparative state. We define a pathogenic loop wherein mtDNA subverts Breg-mediated tolerance through a BCR-TLR9-CXCL2 axis in B cells. This axis represents a potential therapeutic target for ALI.