*Purpose: Vascularized composite tissue allotransplantation (VCA) ‘s wide application has been impeded by deleterious side-effect of current immunosuppressive regimens. This study is aimed to determine whether targeting Myeloid differentiation primary response 88 (MyD88) dependent innate immune response reduces VCA associated ischemia and reperfusion (I/R) response and delay VCA graft rejection in a mouse hind-limb transplantation model.

*Methods: Right hind-limbs from B6 (isografts) or BALB/c (allografts) mice were orthotopically transplanted to either amputated WT B6 mice or mice deficient of Myd88 alone (Myd88-/-) or Myd88 and TRIF (double knockout mice) on B6 background. Graft rejection, survivals, post-transplant systemic responses were monitored and the RNA expression of signal pathway downstream inflammatory factors in the graft skin and muscle were performed at 7 days after VCA. Phenotypic analysis of infiltrating cells in graft muscle were performed using Flow-Cytometry. Additionally, graft histology was examined by H&E staining.

*Results: Abrogating Myd88 in recipient significantly reduced immediate post-transplant local and systemic inflammation, as manifested by edema and BUN levels, in both Myd88-/- or double ko hind-limb isografts and allografts. Moreover, recipient MyD88 deficiency significantly delayed rejection process in the full MHC-mismatched VCA allografts (from 7 days to 15 days post-transplant). MyD88-/- recipients have significantly reduced graft-Infiltrating cells on day 7 after VCA. Specifically, phenotypic analysis showed marked reduction of neutrophils, macrophages and CD8 T cells in the Myd88-/- allograft, compared with Myd88+/+ allograft, which accompanied by downregulated expression of proinflammatory cytokines (TNFa, IL-6, IL-1) in the skin and skeletal muscles of Myd88 ko limb allografts. In stark contrast, there was a significant increase in the frequency of regulatory T cells (CD4+CD25+Foxp3+) homing in the limb Myd88-/- allografts . Interestingly, abrogating Myd88 significantly upregulated expression of amphiregulin a ligand of the epidermal growth factor receptor in the muscle of Myd88-/- allografts.

*Conclusions: Inhibition of innate immunity by genetic depletion of MyD88 in recipients significantly delayed limb allografts rejection and promoted generation and accumulation of Tregs in the limb graft limb. Our results for the first time revealed that Myd88 deletion upregulated expression of muscular amphiregulin, a tissue factor associated with enhancing Treg function and tissue repair.

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