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Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University

Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine

Tumor necrosis factor (TNF) and Toll-like receptor (TLR)3/TLR4 activation trigger necroptotic cell death through downstream signaling complex containing receptor-interacting protein kinase 1 (RIPK1), RIPK3, and pseudokinase mixed lineage kinase-domain-like (MLKL). However, the regulation of necroptotic signaling pathway is far less investigated. Here we showed that c-Jun N-terminal kinases (JNK1 and JNK2) displayed kinase-dependent and -independent functions in regulating TNF- and TLRs-mediated necroptosis. We found that RIPK1 and RIPK3 promoted cell-death-independent JNK activation in macrophages, which contributed to pro-inflammatory cytokines production. Meanwhile, blocking the kinase activity of JNK dramatically reduced TNF and TLRs-induced necroptotic cell death. Consistently, inhibition of JNK activity protected mice from TNF-induced death and Staphylococcus aureus-mediated lung damage. However, depletion of JNK protein using siRNA sensitized macrophages to necroptosis that was triggered by LPS or poly I:C but still inhibited TNF-induced necroptosis. Mechanistic studies revealed that RIPK1 recruited JNK to the necrosome complex and their kinase activity was required for necrosome formation and the phosphorylation of MLKL in TNF- and TLRs-induced necroptosis. Loss of JNK protein consistently suppressed the phosphorylation of MLKL and necrosome formation in TNF-triggered necroptosis, but differentially promoted the phosphorylation of MLKL and necrosome formation in poly I:C-triggered necroptosis by promoting the oligomeration of TRIF. In conclusion, our findings define a differential role for JNK in regulating TNF- and TLRs-mediated necroptosis by their kinase or scaffolding activities.

c-Jun N-terminal kinases differentially regulate TNF- and TLRs-mediated necroptosis through their kinase-dependent and -independent activities.

The major limitation of organ transplantation is the shortage of available organs from deceased human donors which leads to the deaths of thousands of patients each year. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection and coagulation dysfunction are two major hurdles for the successful survival of pig xenografts in primate recipients. Pro-inflammatory cytokines, such as IL-6, TNF-α, and IL-17, play important roles in many diseases and in allotransplantation. However, the pathological roles of these pro-inflammatory cytokines in xenotransplantation remain unclear. Here, we briefly review the signaling transduction and expression regulation of IL-6, TNF-α, and IL-17 and evaluate their potential pathological roles in in vitro and in vivo models of xenotransplantation. We found that IL-6, TNF-α, and IL-17 were induced in most in vitro or in vivo xenotransplantation model. Blockade of these cytokines using gene modification, antibody, or inhibitor had different effects in xenotransplantation. Inhibition of IL-6 signaling with tocilizumab decreased CRP but did not increase xenograft survival. The one possible reason is that tocilizumab can not suppress IL-6 signaling in porcine cells or organs. Other drugs which inhibit IL-6 signaling need to be investigated in xenotransplantation model. Inhibition of TNF-α was beneficial for the survival of xenografts in pig-to-mouse, rat, or NHP models. Blockade of IL-17 using a neutralizing antibody also increased xenograft survival in several animal models. However, the role of IL-17 in the pig-to-NHP xenotransplantation model remains unclear and needs to be further investigated. Moreover, blockade of TNF-α and IL-6 together has got a better effect in pig-to-baboon kidney xenotransplantation. Blockade two or even more cytokines together might get better effect in suppressing xenograft rejection. Better understanding the role of these cytokines in xenotransplantation will be beneficial for choosing better immunosuppressive strategy or producing genetic modification pig.

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

Microbiota plays an important role in immunoglobulin A (IgA) nephropathy (IgAN); however, the pathogenesis, early diagnosis, and treatment of IgAN remain unclear. The aim of the present study was to develop a preliminary model based on saliva‑specific microbes and clinical indicators to facilitate the early diagnosis of IgAN and obtain insights into its treatment. The microbial profile of the saliva of 28 IgAN patients and 25 healthy control subjects was investigated using high‑throughput sequencing and bioinformatics analyses of the V4 region in microbial 16S rRNA genes. IgAN patients and healthy subjects did not differ significantly in α‑diversity indices (Chao1 and Shannon index) or phylum composition. At the genus level, however, Granulicatella was significantly less abundant in healthy individuals than in IgAN patients, while Prevotella and Veillonella were significantly more abundant in the healthy subjects than in IgAN patients (P<0.05 and P<0.01, respectively). Correlation analysis between biochemical indicators and operational taxonomic units (OTUs) revealed that the glomerular filtration rate was positively correlated with OTU86 and OTU287 at P<0.05, positively correlated with OTU165 at P<0.001, and negatively correlated with OTU455 at P<0.05. The serum creatinine index was negatively correlated with OTU287 at P<0.05 and negatively correlated with OTU165 at P<0.001. The pathological changes were positively correlated with OTU255 at P<0.05, OTU200 at P<0.01, and OTU455 and OTU75 at P<0.001, and negatively correlated with OTU86, OTU287, and OTU788 at P<0.05 and with OTU165 at P<0.01. The differences between Chinese IgAN patients and healthy subjects in terms of OTUs and biochemical indicators were analyzed and a mathematical model to facilitate the clinical diagnosis of IgAN was established.

Hanchao Gao

Department of Nephrology,
Department of Medical Laboratory

Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Hanchao Gao

Department of Nephrology,Department of Medical Laboratory

Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Department of Nephrology,
Department of Medical Laboratory