Reduced cyclooxygenase 2 levels after hypoxia exposure result in the loss of immunoprivilege of allogeneic mesenchymal stem cells

Allogenic mesenchymal stem cells (MSCs) from young and healthy donors have demonstrated the potential to treat multiple degenerative disorders. However, recent pre-clinical and clinical studies report that immunogenicity and poor survival of transplanted MSCs impairs the efficacy of cells for regenerative applications. According to these studies, initially immunoprivileged under in vitro conditions, MSCs are targeted by the host immune system after transplantation in the ischemic tissues. We performed in vitro and in vivo (rat myocardial infarction [MI] model) studies using bone-marrow derived MSCs to elucidate the mechanisms responsible for the switch of MSCs from immunoprivileged to immunogenic state, under ischemic conditions. MSCs were cultured under normal or hypoxic conditions (0.4%O2, 24hr), and the levels of immunosuppressive molecules cyclooxygenase-2 (COX2, rate limiting enzyme for PGE2 synthesis) and prostaglandin E2 (PGE2) were assessed using western blot and ELISA, respectively. Our results show that the levels of COX2 and PGE2 decreased in MSCs following exposure to hypoxia. Activation of immune response was evaluated using flow cytometry analysis after co-culturing leukocytes with MSCs lacking, or over-expressing COX2. We also found that proteasome-mediated degradation of COX2 in hypoxic MSCs is responsible for PGE2 decrease and loss of immunoprivilege of MSCs. Finally, when COX2 over-expressing MSCs were transplanted in rat MI model we found that maintaining COX2 levels improved their survival in vivo. While investigating the mechanisms involved in COX2 degradation, we found that interaction of COX2 with COP9 signalosome subunit 5 (CSN5) under normoxia prevents its degradation by the proteasome. On the other hand, hypoxia mediated decrease in the levels of CSN5 and, its reduced binding to COX2, makes COX2 protein susceptible to proteasome-mediated degradation. This subsequently causes PGE2 downregulation and loss of immunoprivilege of MSCs. Our results provide novel mechanistic evidence that PGE2 is downregulated in hypoxic MSCs causing post-transplantation rejection of allogeneic MSCs. This suggests that new strategies that target CSN5-COX2 signaling may improve survival and utility of transplanted allogeneic MSCs in the ischemic heart.