Innate immunity activation involved in unprotected porcine auto-transplant kidneys preserved by naked caspase-3 siRNA

Abstract

BackgroundThe naked caspase-3 small interfering RNA (siRNA) infused into the renal artery during cold preservation was effective, but did not protect auto-transplant porcine kidneys with increased inflammation and apoptosis in our previous study. The mechanisms involved, in particular, whether siRNA or complementary systemic feedback eliciting innate immune responses are worthy to be further investigated.MethodsThe protein and mRNA expression of innate immunity-related molecules were detected by western blotting and quantitative PCR in the tissues previously collected from 48 h auto-transplant kidneys. The donor kidneys were retrieved from mini pigs and cold preserved by University of Wisconsin solution with/without 0.3 mg caspase-3 siRNA for 24 h.ResultsThe protein level of Toll like receptor (TLR) 3, TLR7, and their main adapters, TRIF and MyD88, was up-regulated in the siRNA preserved auto-transplant kidneys. The mRNA level of NF-κB and c-Jun was increased, as well as pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α and interferon (IFN)-α, β and γ. In addition, the non-TLR RNA sensor PKR protein, but not RIG1, was also increased in the siRNA preserved auto-transplant kidneys.ConclusionsThe activation of innate immunity with amplified inflammatory responses in the caspase-3 siRNA preserved auto-transplant kidneys are associated with increased TLR3, TLR7 and PKR, which might be due to complementary systemic feedback, although persistent actions initiated by short-acting caspase-3 siRNA cannot be completely ruled out. These results provided valuable evidence to guide future siRNA design and pre-clinic studies.

MyD88-silenced dendritic cells induce T-cell hyporesponsiveness and promote Th2 polarization in vivo

“…MyD88 mRNA transcription was significantly downregulated in MyD88-silenced DCs, and the expression of MyD88 protein was also remarkably reduced. As reported, toll-like receptors (TLRs) are a family of molecules that activate the innate immune response and modulate adaptive immunity [11,12]. Emerging studies have shown that TLRs are key regulators of both the innate and adaptive immune responses.…”

siRNA-Induced RNAi Therapy in Acute Kidney Injury

“…Based on this second type of off-target RNAi effects, our group further investigated the mechanism of how short-acting caspase-3 siRNA impaired posttransplanted kidneys. The results suggested that the amplified inflammatory responses in caspase-3 siRNA preserved autotransplant kidneys were associated with TLR3, TLR7, and PKR activation, which may be due to systemic compensative responses, although persistent actions initiated by short-acting caspase-3 siRNA cannot be completely excluded [54]. Other studies have also indicated that the horseshoe-like structure of TLR3 facilitates dsRNA recognition [55,56].…”

“…For instance, the apoptosis of inflammatory cells is associated with inflammation clearance and tissue remodeling, whereas the apoptosis of renal parenchymal cells is link to tubular atrophy and renal fibrosis. Therefore, using the genetic material such as siRNA targeting specific cell at particular time frame is crucial to achieve high efficacy of treatment in AKI and also avoid site-effects [12,54].…”

“…Choosing appropriate siRNA carriers has to consider the safety, effectiveness, ease of manufacturing, off-target effects [12], and innate immune responses. Of course, the efficacy of siRNA is still a most important factor dominating the selection of its carriers [54].…”

Emerging Therapies Targeting Intra-Organ Inflammation in Transplantation

“…Caspase 3 is a major enzyme that cleaves, dimerizes and therefore activates as part of the apoptosis cascade and its gene expression has shown to increase following IRI . While delivering naked caspase 3 siRNA to isolated porcine kidney resulted in less apoptotic cells after IRI, it not only failed to show protection but also augmented inflammatory response and renal tissue damage when treated kidneys were auto‐transplanted . These studies reflect the difficulty of applying siRNA in clinic, not only with regard to its delivery, specificity and stability, but also in predicting its behavior in vivo and the possibility of it activating innate immunity which can adversely affect tolerance induction.…”