Ischemia reperfusion injury (IRI) is a pathological process affecting solid organ allografts and occurs when a transplanted organ subjected to prolonged disruption of blood flow undergoes tissue damage following surgical implantation. IRI frequently occurs among deceased donors and predisposes to chronic antibody- mediated rejection (CABMR), decreased graft survival, and worsened patient outcomes. This condition has no effective medical treatments and is especially relevant to veteran transplant recipients who undergo higher rates of deceased donor transplants and are thus at risk for developing IRI and its related complications. In recognition of the significant lifetime health burden and costs incurred by IRI to veteran patients and to the VA Healthcare System, respectively, recent federal mandates have established mechanisms to systemically improve care for veteran recipients of solid organ transplants, in particular renal transplants. This mandate will increase the number of transplanting VA centers, institute systems-based changes to improve pre- and post-operative access to care, and promote basic research to improve allograft survival. In this context, we submit this transplant immunology application to explore a role for Hedgehog (Hh) signaling in CABMR, an IRI-associated complication in solid organ transplantation. Complement (C') are immune proteins involved in host defense that are pathologically activated on endothelial cells (ECs) during IRI, a process we have found selectively expands T peripheral helper (TPH cells), a recently discovered CD4+ T cell subset specialized in provision of B cell help. Hh signaling is a widely studied pathway regulating wound healing by eliciting vascular cell proliferation. We reasoned that Hh ligands are released by C'-injured ECs during IRI as a signal for autologous wound repair, but concurrently these same ligands could expand alloimmune TPH cells that go on to promote alloantibody responses and CABMR-related pathologies. To test this notion, we used humanized models in vitro and in vivo, and we used prospectively collected patient samples to increase the clinical relevance of our findings. Our data showed that IRI-treated ECs produced Hh ligands in a C'-dependent manner that selectively activated T peripheral helper (TPH) cells, a newly described T cell subset specialized in provision of B cell help. Mechanistically, Hh induced ZFYVE21, a novel Rab5 effector we discovered, to elicit Akt-mediated NRLP3 inflammasomes in TPH cells. This resulted in IL-18 release and IL-18-mediated expansion of IL-18R1+TPH cells that promoted CABMR-like pathologies including vascular inflammation, alloAb production, and vasculopathy. These exciting preliminary data address an important and prevalent clinical problem that is a focus area of recent federal initiatives at the VA. From these data, we hypothesize that Hedgehog ligands released by C'-injured EC during IRI activate CD4+ TPH cells to elicit CABMR-associated pathologies. We will explore this hypothesis in 2 non- interdependent Aims.
In Aim 1, we will employ biochemical and functional assays to identify mechanisms(s) by which Hh agonism activates a ZFYVE21-Akt-Casp1 axis to elicit inflammasome-dependent expansion of TPH cells. We propose cutting edge approaches including phosphoproteomic analyses to facilitate mechanistic discovery.
In Aim 2, we will test the relevance of ZFYVE21-Akt-Casp1 signaling in vivo using 2 humanized mouse models of CABMR, and we will test the clinical relevance of lead molecule(s) using prospectively collected patient specimens. Our studies explore Hedgehog signaling as a new pathway that may be feasibly modulated in clinical settings with recently FDA-approved agents to putatively block IRI-associated complications like CABMR in solid organ transplantation. As such, findings from these studies may be relevant for improving allograft survival in veteran patients who are at risk for IRI and its associated complications.

Public Health Relevance

Ischemia reperfusion injury (IRI) predisposes to chronic antibody-mediated rejection (CABMR) and worsened outcomes for veteran transplant recipients. Complement (C?) are immune proteins that injure endothelial cells (EC) during IRI, and Hedgehog (Hh) is a mitogenic pathway promoting wound healing. We found that graft EC injured by C? during IRI elaborated Hh ligands that selectively expanded T peripheral helper (TPH) cells, a newly discovered T cell subset involved in B cell help, to promote CABMR. We explore mechanisms by which EC-derived Hh ligands assemble TPH cell intrinsic NLRP3 inflammasomes to mediate expansion of these cells. We then test salient molecule(s) in two humanized mouse models and assess their clinical relevance in patient specimens. This work implicates Hh signaling as a new druggable pathway to block IRI-induced complications and to improve graft survival in veteran transplant patients.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Special Emphasis Panel (ZRD1)
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VA Connecticut Healthcare System
West Haven
United States
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