Purpose: Cytomegalovirus (CMV) is the most common  opportunistic infection in lung transplant recipients, with the highest risk seen in CMV seronegative recipients (R-) of seropositive organs (D+). This study aims to explore key molecular pathways responsible for transplant-mediated CMV reactivation by using single-cell RNA sequencing (scRNA-seq) in a mouse model of syngeneic D+/R- lung transplantation.

Methods: Left lungs from naïve (D-) mice or MCMV latently infected (D+) mice were transplanted to syngeneic R- recipients, while the contralateral right lungs served as controls. Lung tissues from the transplants at day 2 post-transplant (POD) or controls were harvested for viral RNA analysis and single cell isolation. CD31+ and CD45+ enriched single cell suspensions were processed using 10X Genomics, followed by sequencing. The sequenced data were processed using Cell Ranger pipeline and Seurat. Additional transplants were treated with small molecule Myd88 inhibitor to determine whether innate immunity plays a role in transplant induced CMV reactivation.

Results: MCMV IE-1 mRNA transcripts were significantly upregulated at POD 2, but were not detectable in the controls, indicating that transcriptional activation is induced in the early phase of transplant and ischemia/reperfusion (I/R) injury associated with the transplant procedures is an important contributing factor. In addition, viral DNA amplification was detected in the salivary glands of D+/R- recipients at POD14, confirming that transplant of a D+ graft results in viral reactivation and dissemination. These data support the mouse lung transplant model as a viable approach to study mechanisms of CMV reactivation.  Data from scRNAseq analysis showed distinct lung and immune cell clusters in lung transplants and controls. There were minimal differences in transcriptome landscapes between D- and D+ lungs prior to transplant (controls). In contrast, the differences were prominent between lungs from D-/R- transplant vs D+/R- lung grafts in the myeloid populations [e.g. neutrophils, macrophages (Macs)] and endothelial cells. Gene expression of Macs showed more differences between transplant lungs and control lungs than any endothelial cells or neutrophils. Pathway analysis of differentially expressed genes for myeloid cell types showed several significant immune response pathways including MIF regulation of innate immunity and communication between innate and adaptive immune cells.  Expression of Myd88 and c-fos were increased in the lung grafts regardless of latent MCMV infection. Furthermore, our preliminary study using a Myd88 inhibitor showed a trend of CMV viral load reduction in lung grafts.

Conclusions: Early I/R injury following lung transplant resulted significant alterations of transcriptome landscape unique to different cell types. Myd88/AP-1 plays a critical role in CMV reactivation; thus targeting Myd88 has a great therapeutic potential to prevent I/R injury-mediated CMV reactivation.