Porcine IL‐6, IL‐1β, and TNF‐α regulate the expression of pro‐inflammatory‐related genes and tissue factor in human umbilical vein endothelial cells

Abstract

Whether porcine cytokines are induced after pig-to-primate xenotransplantation and activate human cells remains unknown. First, we investigated the regulation of porcine IL-6, IFN-γ, IL-1β, and TNF-α in xenotransplantation using an in vitro model in which porcine aortic endothelial cells (PAECs) and porcine peripheral blood mononuclear cells (PBMCs) were stimulated with human serum. Downstream cytokines/chemokines were monitored. Pro-inflammatory cytokines (IL-6, IFN-γ, and IL-1β) and chemokines (IL-8, MCP-1, and CXCL2) were upregulated in the both cell types. TNF-α was induced 10-fold in PAECs, but not in PBMCs. Then, we assessed the role of porcine IL-6, IFN-γ, IL-1β, and TNF-α in xenotransplantation using western blotting and real-time PCR. Human umbilical vein endothelial cells (HUVECs) were selected as the target cells. Signaling pathways and downstream genes, such as those related to adhesion, inflammation, and coagulation, and chemokines were investigated. Porcine IL-1β and TNF-α significantly activated NF-κB and P38, and STAT3 was activated by porcine IL-6 in HUVECs. The adhesion genes (E-selectin, VCAM-1, and ICAM-1), inflammatory cytokines (IL-6, IL-1β, and TNF-α), chemokines (MCP-1 and IL-8), and the pro-coagulation gene (tissue factor) were upregulated by porcine IL-1β and TNF-α. Porcine IL-6 increased the expression of ICAM-1, IL-6, MCP-1, and tissue factor, but decreased IL-8 expression slightly. Surprisingly, porcine IFN-γ could not activate STAT1 or regulate the expression of any of the above genes in HUVECs. In conclusion, these findings suggest that porcine IL-6, IL-1β, and TNF-α activate HUVECs and regulate downstream genes expression, which may promote inflammation and coagulation response after xenotransplantation.

Potential pathological role of pro‐inflammatory cytokines (IL‐6, TNF‐α, and IL‐17) in xenotransplantation

“…Both of the two groups were treated with tocilizumab, and these data suggested that the combination of (a) a graft from several genes genetically modified pig, (b) an effective immunosuppressive regimen, and (c) anti‐inflammatory therapy (eg, tocilizumab and etanercept) would prevent coagulation dysfunction effectively. Moreover, IL‐6 induced the expression of TF in both PAECs and HUVECs . Collectively, although we can reasonably predict that blockade of IL‐6 signaling might be beneficial for inhibiting coagulation dysfunction in xenotransplantation, we cannot reach definite conclusions of IL‐6 in xenotransplantation according to these emerging data.…”

“…In 2017, Iwase and his colleagues demonstrated that the baboon received kidney graft from GTKO/CD46/CD55/EPCR/TFPI/CD47 survived about 8 months while the baboon received graft from GTKO/CD46/TBM died of consumptive coagulopathy at 12 days under the same treatment of immunosuppressive regimens including tocilizumab and etanercept, which suggested that CD55 and anti‐inflammatory therapy (eg, tocilizumab and etanercept) were important for coagulation dysregulation (Table ) . Moreover, TNF‐α increased TF expression in both PAECs and HUVECs . Collectively, blockade of TNF‐α might suppress coagulation dysfunction in xenotransplantation.…”

“…Most papers reported that blockade of IL‐17 or TNF‐α could increase the survival of xenograft . The possible reasons are as following: IL‐17 and TNF‐α could not only activate NF‐κB and MAPKs pathways, but also induce the production of various pro‐inflammatory and pro‐coagulation genes, in both human and porcine endothelial cells . Mounting studies indicated that inhibited or impaired NF‐κB and MAPKs activation with immunosuppressive drugs could lead a reduction in allograft rejection after transplantation .…”

Selective inhibition of cyclooxygenase‐2 protects porcine aortic endothelial cells from human antibody‐mediated complement‐dependent cytotoxicity

“…Human serum activated NF‐κB signaling in PAECs . We wondered if activation of NF‐κB signaling was responsible for the induction of COX‐2 expression.…”

“…Human serum activated NF-κB signaling in PAECs. [49][50][51] We won- Figure 3C) and by about 50% at protein level ( Figure 3D). Along with decreased COX-2 expression, p52 processing and phosphorylation of p65 were consistently attenuated by NF-κB inhibitors ( Figure 3D).…”