Hanchao Gao1,2, Lu Liu1,2, Yanli Zhao1, Hidetaka Hara3, Pengfei Chen1,2, Jia Xu1, Jia Tang4, Ling Wei1, Zesong Li1, David K C Cooper3, Zhiming Cai1, Lisha Mou1. 1. Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, China. 2. Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. 3. Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, Birmingham, AL, USA. 4. Medical Genetics Center, Jiangmen Maternity and Child health Care Hospital, Jiangmen, China.
Abstract
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.
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 humanIL-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 humanIL-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-humanIL-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 humanIL-6, IL-17, IL-1β, and TNF-α significantly activate PAECs and are likely to promote inflammation and coagulation reaction in response to xenograft.
Authors: Juan Li; Hidetaka Hara; Yi Wang; Charles Esmon; David K C Cooper; Hayato Iwase Journal: J Inflamm (Lond) Date: 2019-05-28 Impact factor: 4.981
Authors: Hidetaka Hara; Hayato Iwase; Huy Nguyen; Yuko Miyagawa; Kasinath Kuravi; Jeremy B Foote; Will Eyestone; Carol Phelps; David Ayares; David K C Cooper Journal: Cytokine Date: 2021-06-04 Impact factor: 3.861