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

## TL;DR

This study presents a bi-specific siRNA system that activates TLR8 and silences MyD88 in a controlled way using bacterial vesicles, potentially offering a new approach for managing immune-related diseases.

## Contribution

A novel bi-specific siRNA delivery system using outer membrane vesicles for negative feedback regulation of TLR8 and MyD88.

## Key findings

- Outer membrane vesicles with invasive protein Ail and OmpA efficiently deliver siRNA to HEK293-TLR8 cells.
- siRNA complexed with p19 activates TLR8, increasing INFβ production significantly.
- Fusogenic peptides enable siRNA escape from endosomes to silence MyD88 transcripts.

## Abstract

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.

The online version of this article (doi:10.1186/s12865-015-0109-9) contains supplementary material, which is available to authorized users.