Single-cell Profiling Reveals The Role Of Intrahepatic Macrophages In Liver Transplantation

Part 1: A simplified one-stitch sleeve technique for hepatic arterial reconstruction of orthotopic liver transplantation using 3-Dimensional-printed instruments in ratPurpose: End-to-end sleeve anastomosis is the most common technique for hepatic rearterialization in rat orthotopic liver transplantation(OLT).However,current technical variants of the sleeve technique are hampered by the high rate of anastomosis bleeding due to mismatched vascular lumen diameter and insufficient overlap.We describe a simplified one-stitch sleeve technique that includes artery selection and suture technique,which eases the surgical procedure,efficiently inhibits backflow bleeding,and achieves a high patency rate and good survival.Materials and Methods: The simplified one-stitch sleeve technique was used for the artery anastomosis in syngeneic and allogeneic OLT models using Dark Agouti(DA)and Lewis(LEW)rat strains.OLT without re-arterialization served as controls.The sleeve technique using graft common hepatic artery(CHA)as receiving artery requires the selection of feeding artery according to artery diameter,which only consisted of a guiding suture by everting the donor's artery wall to get a sufficient overlap between graft and recipient artery to prevent backflow bleeding.Results: The CHA is nearly double the diameter of the proper hepatic artery(PHA).Notably,the HA shows a significantly larger diameter in LEW than in DA.In the rearterialized group(n=35),the operation time was 113.5�5.67min(median,SD),the anhepatic phase lasted 12.18�1.49 min,and the artery reconstruction required 5.11�1.05 min.The patency rate of the artery was 96%.The long-term survival rate was 89.3% in the re-arterialization cohort compared to 57.14% observed in the nonarterialization cohort,which was also reflected in histology not distinguished from the normal liver and rapid recovery of the body weight and hepatic function.Conclusions: This modified one stitch sleeve anastomosis proved to be a physiological and feasible method to reconstruct the hepatic artery because of its high patency rate,time-saving,and efficiency in avoiding backflow bleeding.A relative diameter match between receiving and feeding artery will ease the re-arterialization.Notably,a prolonged overlap between the connecting vessels was critical to prevent backflow bleeding.3D-printed cuffs and instruments facilitated the LT process and shorted the an-hepatic time.Part 2: Single-cell profiling reveals strain-specific differences in rat intrahepatic macrophage inflammatory potentialPurpose: The degree of liver allograft rejection is known to be strain-specific,and while the transplantation is accepted without rejection in some strains,it leads to acute rejection in others.We present a comprehensive map of the healthy rat liver at a single-cell resolution to shed light on the hepatic cellular landscape and build the foundation for strain-based liver transplant experiments.We aim to unravel the cellular and molecular mechanisms contributing to rats ' strain-specific rejection by comparing the single-cell liver maps of the baseline DA and LEW rat strains.Method: We generated single-cell transcriptomic maps of the livers of healthy DA and LEW rat strains using single-cell RNA sequencing,covering three male samples from each strain.To clean the technical effects from our data and identify critical biological sources of variation,we developed an analysis workflow using a matrix factorization method,called varimax PCA,that enabled us to identify factors representing cell-type identities and strain-specific variations that could be further interpreted.Results: The varimax-based analysis pipeline guided the detailed annotation of the singlecell map of the rat liver and provided a foundation to identify the cellular and molecular sources of variability between DA and LEW strains.Using this approach,two main strain-specific factors were identified,representing variation within the hepatocyte and macrophage populations.Interpretation of the macrophage-related factor highlighted the enrichment of pro-inflammatory signals in LEW myeloid cells,suggesting that the LEW rat's baseline hepatic microenvironment is more pro-inflammatory than the DA strain.Conclusion:Taken together,we provide the first examination of the healthy rat liver by singlecell transcriptomics and uncover critical insights into strain-specific differences in this valuable model animal.Part 3: The potential role of intrahepatic macrophage in acute cellular rejection after liver transplantationPurpose: A subset of LT patients develop spontaneous operational tolerance after immunosuppression withdrawal and are commonly referred to as operational tolerant individuals.By deciphering the cellular and molecular aspects involved in ACR,we will be able to inform the design of new interventions that promote graft acceptance in the absence of IS therapy while preserving host immunocompetence in transplanted patients.Further understanding of phenotypic and functional changes in macrophage populations post-LT can help elucidate key signals and immune cells involved in progression to ACR or tolerance.Method: Here,we employ a refined OLT protocol to comprehensively characterize the dynamic changes in the liver cellular landscape post-LT during ACR at multiple postoperative days(PODs)by combining this relevant model with immunohistochemistry and flow cytometry.Results: Rats in the ACR group(DA->LEW)progressed to fatal ACR within 12 days following transplantation,with marked increase in liver enzymes,RAI,and pronounced hepatocyte damage.Immunohistochemical analysis of hepatic liver tissue following LT showed a significantly higher frequency of CD3+,CD8+ and CD68+ cells in ACR animals than in syngeneic rats.Immune profiling using flow cytometry showed that the macrophages isolated from ACR animals significantly downregulated CD86 expression at POD 3.These results were functionally validated by performing ex vivo LPS stimulation experiments followed by intracellular cytokine staining to measure myeloid cell inflammatory response.We found that LEW intrahepatic myeloid cells(CD45+CD68+CD11b+)secreted overall more TNF-? in response to LPS stimulation compared to DA,suggesting that hepatic myeloid cells may play a role in mediating differential response to liver transplants among these rat strains.Conclusion: LT showed a significantly higher frequency of CD3+,CD8+ and CD68+ cells in the liver,the phenotype of macrophages might play a vital role in the acute rejection after LT.