Immunological consequences of cross‐species infection of porcine endothelial cells with human cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) infection or reactivationrepresents one of the most important infectious disease problems in allotransplantation. Transmission and activation of porcine cytomegalovirus in pig‐to‐baboon xenotransplantation is associated with xenograft injury and possibly an increased incidence of consumptive coagulopathy. However, stringent protocols used in the rearing of pig donors practically exclude a risk of porcine CMV. In contrast, in pig‐to‐human xenotransplantation, the porcine endothelium will inevitably be exposed to human pathogens such as HCMV, and the consequences of potential HCMV cross‐species infection of porcine xenografts, in particular endothelial injury and immune activation, remain to be elucidated. Since it is of outmost importance to anticipate any detrimental consequence of this contact, the susceptibility of porcine endothelial cells (pEC) to HCMV infection and its effect on cell surface molecule expression and human leukocyte recruitment was evaluated.Clinical and laboratory HCMV strains, including an EC propagated (TB40/E) and two fibroblast‐adapted (AD169 and TB40/F) strains, as well as UV‐inactivated, or heparin‐treated controls were inoculated to a panel of pEC stemming from different vascular beds.Infection at various MOI was analyzed by PCR, immediate early (IE), early and late antigen expression. To determine HCMV cytopathogenicity, morphological changes were monitored using live cell imaging and apoptosis was analyzed by flow cytometry. The capability of pEC to produce infectious virus was quantified by focus expansion assays. After inoculation, cell surface receptor expression was studied by flow cytometry on pEC bulk cultures and also differentially on IE‐positive (infected) and IE‐negative (bystander) pEC. Adhesion of human PBMC was tested on HCMV infected pEC monolayers and cell‐free pEC supernatants were analyzed for their cytokine content, chemotactic activity, and stimulatory effect on resting secondary pEC cultures.All viral strains infected pEC with differences in the infection efficiency (percentage of IE‐positive cells) and the kinetics of cytopathology. Moreover, differences in susceptibility of pEC derived from distinct vascular beds were observed. HCMV underwent a complete replication cycle in about 5% of the infected pEC. Comparing the permissiveness of pEC to human aortic EC (HAEC) revealed differences in strain susceptibility and lower rates of late antigen expression in pEC. Finally, HCMV‐infected pEC were demonstrated to release viral progeny with a lower efficiency than infected HAEC.Cell‐surface expression of E‐selectin and VCAM‐1 was upregulated not only in the IE‐positive fractions (infected) of HCMV‐inoculated cultures but also in the IE‐negative fractions, presumably by bystander effects. In contrast, porcine MHC class I expression was downregulated in IE‐positive, and upregulated in IE‐negative cells. The receptor alterations in the IE‐negative fraction were mediated by pEC‐derived soluble factors and consequently both porcine IL‐6 and IL‐8 were detected in the supernatants. The increased adhesion receptor expression was paralleled by enhanced human leukocyte chemotaxis and adhesion to infected pEC cultures. Pretreatment of HCMV with heparin blocking viral entry, but not UV‐inactivation blocking viral replication, prevented adhesion‐receptor modulation, and reversed the increased adhesion and chemotaxis.In summary, HCMV may productively infect pECfollowing pig‐to‐human solid organ transplantation and may alter and activate the porcine endothelium, rendering the xenograft more susceptible to human leukocyte recruitment and rejection. Therefore, understanding the mechanisms of cross‐species HCMV infection and finding strategies to render pEC resistant to HCMV infection willbe essential for xenotransplantation to become a clinical reality.