Targeting Ischemia/Reperfusion Stress to Inhibit Cytomegalovirus Reactivation After Lung Transplant - PROJECT SUMMARY Cytomegalovirus (CMV) is the most common opportunistic infection in lung transplant recipients, with the highest risk seen in seronegative recipients (R-) of seropositive organ donors (D+). While anti-rejection immunosuppression (IS) has been implicated as a primary cause of CMV reactivation, our studies have newly suggested otherwise. We have recently shown that CMV reactivation occurs independent of IS and seemingly independent of the allo-immune response following a D+/R- kidney transplant in mice. Treatment with a regimen of clinically relevant IS, while failing to induce CMV reactivation in latently infected mice (no transplant), does permit subsequent viral replication and dissemination following transplant, despite inhibition of the host rejection response and inflammation. These findings suggest that ischemia/reperfusion (I/R) stress inherent in a transplant procedure due to organ preservation and revascularization is necessary and sufficient to induce reactivation. However, molecular mechanisms underlying I/R stress mediated transcriptional reactivation from latency in the grafts remain elusive. The goal for this study is to identify key molecular pathways responsible for I/R stress- mediated CMV reactivation and to develop effective therapeutics to prevent both I/R injury and CMV reactivation following lung transplant. In our preliminary studies using a mouse model of syngeneic D+/R- lung transplant, we observed that expression of MCMV immediate early (IE) genes is induced within 48 hours of transplant surgery, followed by increased viral DNA replication in the lung grafts and dissemination to peripheral organs by day 14 post-transplant. We revealed that pre-transplant depletion of donor alveolar macrophages (AMs) decreased viral load in transplanted lungs and salivary glands, suggesting that AMs are primary sites of MCMV latency and reactivation, consistent with the findings from HCMV studies. We have further showed that Myeloid differentiation primary response 88 (Myd88) was critical in mediating transplant induced I/R injury, likely via activation of transcription factors (TFs) NF-kB, AP-1 and XBP-1. These TFs are shown to bind to MIEP and promote MCMV/HCMV reactivation. Thus, we hypothesize that I/R stress mediates Myd88 dependent activation of TFs in viral harboring donor lung cells (e.g. AMs); leading to reactivation of latent CMV in the transplanted lungs. Thus, tissue/cell specific inhibition of Myd88 pathways in latently infected lung donors may prevent viral reactivation. We will use a preclinical mouse model of lung transplantation to test our hypotheses. In Aim 1, we will 1) investigate how donor macrophage-derived Myd88 regulate expression of viral IE genes and viral DNA replication by utilizing genetically modified time-specific myeloid Myd88 knockout mice and 2) delineate impact of Myd88 on I/R stress mediated signaling pathways in the lung cells using single cells RNA analysis. In Aim 2, we will determine the therapeutic potentials of tissue/cell-specific targeting of Myd88 in mitigating I/R stress and MCMV reactivation and dissemination leveraging our novel nanocarrier delivery system. Data generated from this study will provide proof of concept for developing novel therapeutics for further clinical translation.
