Human IL-6, IL-17, IL-1β, and TNF-α Differently Regulate the Expression of Pro-inflammatory Related Genes, Tissue Factor, and Swine Leukocyte Antigen Class I in Porcine Aortic Endothelial Cells

Background: Pro-inflammatory cytokines play important pathological effects in various diseases and allotransplantation; however, their pathological role in xenotransplantation remains elusive. In pig-to-human cell or organ transplantation, whether porcine cells or organs are activated by human cytokines or not as an important question needs to be investigated.

Methods: We investigated the effect of human IL-6, IFN-γ, IL-17, IL-1β, and TNF-α in xenotransplantation using several in vitro models and porcine aortic endothelial cells (PAECs) as target cells. The downstream signaling pathways activated by these cytokines were studied with Western blotting, the regulation of the pro-inflammatory related genes and pro-coagulation factor were assessed using real-time PCR or enzyme-linked immunosorbent assay, and swine leukocyte antigen (SLA) class I and SLA class II DR were analyzed by flow cytometry.

Results: We found that NF-κB and mitogen-activated protein kinases (MAPKs) were activated by recombinant human IL-17 (rhIL-17), rhIL-1β, and rhTNF-α, while rhIL-6 activated signal transducer and activator of transcription 3 (STAT3) in PAECs. The adhesion molecules (E-selectin, VCAM-1, and ICAM-1), pro-inflammatory gene (IL-6), chemokines (IL-8 and MCP-1), and the pro-coagulation factor (tissue factor) were induced by rhIL-17, rhIL-1β, and rhTNF-α, while rhIL-6 only increased the expression of MCP-1 and tissue factor. Using flow cytometry analysis, SLA class I was upregulated in PAECs after exposure to rhIL-1β and rhTNF-α, but not rhIL-6 or rhIL-17, whereas SLA class II DR could not be induced by rhIL-6, rhIL-17, rhIL-1β, or rhTNF-α, although it could by recombinant porcine IFN-γ (rpIFN-γ). Although activation of PAECs by rhIL-17 alone was not strong, rhIL-17 combined with rhTNF-α amplified the expression of E-selectin, IL-6, and IL-8. Unexpectedly, we found that tocilizumab, a humanized anti-human IL-6 receptor antibody, could not block rhIL-6-mediated STAT3 activation in PAECs. Human IFN-γ could not activate STAT1 or induce the downstream gene expression in PAECs, which was consistent with a previous report.

Conclusion: In conclusion, our data suggest that human IL-6, IL-17, IL-1β, and TNF-α significantly activate PAECs and are likely to promote inflammation and coagulation reaction in response to xenograft.