Macrophage-associated TLR9 signaling contributes to chronic cardiac allograft fibrosis via macrophage-to-myofibroblast transition and paracrine fibroblast activation.

Chronic allograft rejection (CAR) is characterized by progressive fibrosis and remains a major cause of long-term graft failure after transplantation. Here, we investigated the contribution of recipient TLR9 signaling to macrophage-associated fibrotic remodeling in CAR and evaluated pharmacological modulation of this pathway. Using murine heterotopic heart transplantation models of acute and chronic rejection, combined with single-cell RNA sequencing, global TLR9-deficient recipients, and primary cell co-culture systems, we found that macrophages with high TLR9 pathway activity were enriched in the chronic rejection microenvironment. Although TLR9 deficiency did not significantly affect acute rejection survival, it markedly reduced collagen deposition, vascular remodeling features visible on H&E staining, and prolonged graft survival in chronic rejection. Mechanistically, TLR9 activation was associated with macrophage-to-myofibroblast transition via the TGF-β1/Smad3 axis and enhanced IL-6 secretion, which activated resident fibroblasts through Stat3 signaling in a paracrine manner. Hydroxychloroquine (HCQ), an endolysosomal immunomodulatory agent that can interfere with nucleic-acid sensing pathways including endosomal TLR signaling, suppressed macrophage transition and fibroblast activation, attenuated graft fibrosis, and improved survival in vivo. These findings identify recipient TLR9 signaling as an important regulator of macrophage-associated chronic cardiac allograft fibrotic remodeling and support further investigation of this pathway as a therapeutic target.