While graft injury following cold ischemia reperfusion (IR) can accelerate graft rejection, mechanisms are incompletely understood. Evidence indicates that posttransplant IR injury is associated with upregulation of TNFa, which can initiate necroptosis, a form of inflammatory cell death characterized by RIPK3-dependent phosphorylation of MLKL and release of HMGB1. We transplanted WT H-2b hearts exposed to 8h of cold ischemia (CI) or control 0.5h CI into allogeneic BALB/c recipients treated with CTLA4Ig. Those exposed to 8h CI underwent rejection at 42+/- 8 days (n=7) while 0.5h CI controls survived >60 d (p<0.05). Sera obtained at 48h contained significantly more TNFa in the 8h CI recipients. Analysis of graft tissue at 48h showed positive staining for pMLKL in the 8h CI hearts but not the controls, together implicating necroptosis. Recent reports showed that TNF-driven cellular death drive the development of multi-organ inflammation in mice deficient in Sharpin, a molecule required for post-translational linear ubiquitin chain modifications; Sharpin^-/- cells are more susceptible to TNF-mediated apoptosis and necroptosis. To gain insight into whether enhanced cellular death accelerate graft rejection, we repeated the transplants using Sharpin^-/- donors exposed to 8h CI and observed rejection at 17±4 days (n=7, p<0.05 vs. WT 8h CI). The deubiquitylase CYLD is required for propagating signals to elicit TNF-dependent cell death. To test whether accelerated rejection of Sharpin^-/- allografts is linked to enhanced cell death, we transplanted Sharpin^-/-Cyld^-/- hearts into allogeneic recipients. These allografts were rejected 32.5 ±4 days (n=6, p<0.05 vs Sharpin^-/-), consistent with the hypothesis that sensitivity to TNF-mediated cell death leads to accelerated graft rejection. Transplantation of 8h CI Cyld^-/- allografts also delayed rejection when compared to 8h CI Cyld^+/+ allografts (p<0.05). Furthermore, pharmacological inhibition of the RIPK1 kinase that acts downstream of CYLD to induce death, had the same effect as absent Cyld; it prolonged survival of WT hearts exposed to 8h CI (p<0.05 vs controls). In translational studies, urine samples obtained from kidney transplant patients with delayed graft function (DGF) showed significantly higher HMGB1 levels vs. those without DGF (p<0.05, n=10-15/group). Together, these unique findings support the hypothesis that IR injury is linked to allograft rejection through necroptosis.

CITATION INFORMATION: Ang R, Heeger P, Ting A. Necroptotic Cellular Death Modulates TNFα Mediated Inflammation and Cardiac Allograft Rejection in Mice. Am J Transplant. 2017;17 (suppl 3).

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