Date of Award

2019

Embargo Period

5-8-2069

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Satish N. Nadig

Second Advisor

Carl Atkinson

Third Advisor

Laura Kasman

Fourth Advisor

Chrystal Paulos

Fifth Advisor

Shikhar Mehrotra

Sixth Advisor

Craig Beeson

Abstract

Background: In cardiac transplantation, microvascular endothelial cells (mECs) are central to an allograft’s immunogenicity. Early insults associated with the transplant process, including donor brain death, cold ischemia, and reperfusion injury, increase mEC immunogenicity. As antigen-presenting cells, mECs interact with recipient alloreactive memory T-cells and promote responses leading to allograft rejection. Thus, modulating mEC immunogenicity is of great interest to achieve transplant tolerance. Recent studies have shown that modulating mitochondrial physiology in immune cells alters their phenotype. However, whether this has an impact on mECs has not been described. In this research project, we assessed whether modulating aspects of mitochondrial physiology could reduce mEC immunogenicity and alter mEC-T cell interaction. Approach: In the first aim, using a cold storage-warm reperfusion model, we evaluated mEC mitochondrial bioenergetics during the first 24 hours post-reperfusion through gene microarrays and Seahorse flux assays. We also inhibited mEC mitochondrial permeability transition pore (mPTP) opening with an inhibitor, NIM811, to modulate mitochondrial bioenergetics post-reperfusion and assessed mEC immunogenicity in co-culture systems with allogeneic T-cells. In the second aim, using genetic and pharmacologic approaches, we promoted mEC mitochondrial fusion/inhibited fission prior to cold storage and investigated mEC immunogenicity as well as the downstream T-cell response in vitro. Additionally, using a donor brain-dead mouse model, we pre-treated donor Balb/c hearts with M1/Mdivi1, a fusion promoter/fission inhibitor, followed by heterotopic heart transplant into C57BL/6 recipients and assessed cardiac injury, T-cell infiltrate characteristics, and allograft survival. Results: In the first aim, mEC mitochondrial bioenergetics was compromised during the first 12 hours post-reperfusion due to mPTP opening. Inhibiting mPTP opening post-reperfusion reduced mEC immunogenicity to allogeneic T-cells, decreased mEC expression of VCAM-1, MHC-I, and MHC-I machinery proteins. In the second aim, skewing mitochondrial dynamics from fission to fusion reduces mEC immunogenicity to allogeneic T-cells, decreased mEC expression of VCAM-1, MHC-I, Ki-67, and increased PD-L1 expression. Also, promoting fusion/inhibiting fission in mEC altered co-cultured T-cells’ phenotype, decreasing their memory response, proliferation and intracellular production of cytotoxic proteins. In vivo, pre-treating donor hearts with M1/Mdivi1 reduced cardiac injury, decreased infiltrating T-cells’ intracellular production of cytotoxic proteins, and prolonged cardiac allograft survival. Conclusions: Modulating mitochondrial physiology in donor mECs can mitigate recipient T-cell response and improve cardiac transplant outcome.

Rights

All rights reserved. Copyright is held by the author.

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Available for download on Wednesday, May 08, 2069

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