Negative feedback circuit for toll like receptor-8 activation in human embryonic Kidney 293 using outer membrane vesicle delivered bi-specific siRNA

Anurag Adhikari, Birendra Prasad Gupta, Krishna Das Manandhar, Shravan Kumar Mishra, Hari Krishna Saiju, Rajendra Maan Shrestha, Nawneet Mishra, Shishir Sharma

TLR8 assists in antiviral approach by producing Type 1 INF via MyD88 dependent IRF7 pathway. However, over expression of INFα/β molecule poses threat by developing tolerance in chronic infection cases and enhancing inflammatory response. Here we report a bi-specific siRNA based complex which differentially activates and silences the TLR8 and MYD88 respectively in a negatively regulated fashion. Outer membrane vesicle from Escherichia coli used for siRNA delivery was observed more efficient when attached with invasive protein Ail along with OmpA (P < 0.001) in HEK293-TLR8 cell line. siRNA complexed with p19 protein was efficient in activating TLR8, confirmed by the increment of INFβ molecules (P < 0.001) in HEK293-TLR8 compared to its counterpart. Fusion of lipid bilayer of endosomal compartment was significant at pH 4.5 when fusogenic peptides (diINF-7) were incubated in membrane vesicle, thus facilitating the escape of siRNA complex to the host cytoplasm in order to silence MyD88 transcript (P < 0.001). We investigated the activation of TLR8 by bi-specific si-RNA for the production of INFβ. In the same setting we showed that bi-specific si-RNA was able to silence MyD88 transcript in a delayed manner. For the cases of auto immune disease and inflammation where over activation of endosomal TLRs poses serious threat, bi specific siRNA could be used as negative feedback controlled system.