Naked Caspase-3 siRNA Amplifies Inflammation via TLR and PKR Signaling in a Porcine Renal Auto-transplantation Model

Abstract

Event Abstract Back to Event Naked Caspase-3 siRNA Amplifies Inflammation via TLR and PKR Signaling in a Porcine Renal Auto-transplantation Model Cheng Yang1, 2, Long Li1, 2, Yinjia Xue1, 2, Zitong Zhao1, 2, Tian Zhao1, 2, Yichen Jia1, 2, Ruiming Rong1, 2, Ming Xu1, 2, Michael L. Nicholson3, Tongyu Zhu1, 2* and Bin Yang3, 4* 1 Zhongshan Hospital, Fudan University, China 2 Shanghai Key Laboratory of Organ Transplantation, China 3 Leicester General Hospital, University Hospitals of Leicester, Transplant Group, Department of Infection, Immunity and Inflammation, University of Leicester, United Kingdom 4 Affiliated Hospital of Nantong University, University of Nantong, Department of Nephrology, China Background. Small interfering RNA (siRNA) has the potential to elicit innate immune responses and trigger interferon responses like long, double-stranded RNA. In our previous study, naked caspase-3 siRNA infused into the renal artery during cold preservation was effective, but did not protect the auto-transplant kidneys in a porcine model, with increased inflammation and apoptosis. Therefore, there is a warranty to further elucidate whether the siRNA activates innate immune responses and which signaling pathways are involved. Materials and Methods. The left kidney was retrieved from mini pigs and infused by University of Wisconsin solution with/without 0.3 mg caspase-3 siRNA into the renal artery with the renal artery and vein clamped for 24-h cold storage. After right nephrectomy, the left kidney was auto-transplanted into the right for 48-h without siRNA systemic treatment. The protein expression was detected by western blotting, while the mRNA expression was detection by qPCR. Results. The protein level of toll like receptor (TLR)-3 and TLR-7, as well as their main adapters, TRIF and MyD88, was up-regulated by the siRNA in the auto-transplant kidneys. Furthermore, the mRNA level of inflammatory transcription factors, NF-κB and c-Jun, was also increased, which resulted in the enhanced mRNA expression of pro-inflammatory cytokines, including IL-1β and IL-6, TNF-α and interferon (IFN)-α, β and γ. The protein of PKR, an non-TLR RNA sensor, was also up-regulated by the siRNA, but RIG-1 was not affected. Conclusion. Naked caspase-3 siRNA administered into the kidney activated TLR- and PKR-mediated innate immune responses, and then amplified inflammation, which provided valuable evidence to guide future pre-clinic studies. Figure 1 Acknowledgements This study was supported by the UK-China Fellowship for Excellence, Department for Business Innovation and Skills; University Hospitals of Leicester NHS Trust, UK, and National Natural Science Foundation of China (81170689, 81270832, 81270833); Science and Technology Commission of Shanghai Municipality (12ZR1405500); Zhongshan Hospital, Fudan University, China.