Abstract
The innate immune system senses viral and bacterial ribonucleic acid (RNA) via pattern recognition receptors (PRR) leading to subsequent activation of the immune system. One group of RNA sensors is formed by endosomal/lysosomal Toll-like receptors (TLR) such as TLR7 and TLR8. During viral or bacterial infection, immunostimulatory RNA is part of the pathogen reaching the endosomal/lysosomal compartment after cellular uptake. Synthetic single-stranded or double-stranded oligoribonucleotides (ORN) can mimic RNA from pathogens and are widely used as activating ligands for TLR7 and TLR8. However, one limitation in the use of synthetic ORN driven immune stimulation is the need for transfection reagents for RNA delivery into cells. Here we demonstrate that the conjugation of cholesterol to a double-stranded version of immunostimulatory RNA40 strongly enhanced RNA uptake into monocytes and plasmacytoid dendritic cells when compared to naked RNA. Cholesterol-conjugated RNA (RNA-chol) formed nanoparticles that were superior to RNA-liposomes complexes in regard to induction of type I interferon from human and murine plasmacytoid dendritic cells as well as proinflammatory cytokine production (e.g. TNF-α, IL12p70 or IL-6) in human monocytes. Furthermore, the RNA40-chol induced cytokines in human monocyte cultures supported TH1 and TFH cell differentiation underscoring a strong adjuvant function of RNA-chol nanoparticles for adaptive immune responses. In summary, cholesterol-conjugated immunostimulatory RNA forms nanoparticles and functions as a potent immune adjuvant in human and murine immune cells. It further simplifies the use of immunostimulatory RNA by avoiding the need for liposomal transfection reagents.
Highlights
The synthetic immunostimulatory toll-like receptor 7- (TLR7) and TLR8-ligand (TLR8L) ribonucleic acid 40 (RNA40), which is a single-stranded GU-rich sequence from the U5 region of HIV-1 RNA, and its potential to act as adjuvant or for immunotherapy has already been described [1]
Since ssRNA40 could rapidly be digested by cellular RNases, we analyzed the immunostimulatory potential of cholesterol-conjugated dsRNA40, which should be more resistant against degradation by RNases
Immunostimulation of the innate immune system with subsequent activation and tuning of the adaptive immune response relies on activation of germline-encoded pattern recognition receptors (PRR) by pathogen-associated molecular patterns (PAMP)
Summary
The synthetic immunostimulatory toll-like receptor 7- (TLR7) and TLR8-ligand (TLR8L) ribonucleic acid 40 (RNA40), which is a single-stranded (ss) GU-rich sequence from the U5 region of HIV-1 RNA, and its potential to act as adjuvant or for immunotherapy has already been described [1]. A major drawback of this method is the additional step of complex formation, which has to be performed prior to adding the TLRL to target cells. For this reason, developing TLRL that can be directly added to and activate target cells is highly desirable. We investigated the potential of a cholesterolconjugated dsRNA [based on the sequence of the TLR7/TLR8 ligand RNA40 [1]] to form nano- or microparticles and activate TLR7 and TLR8. We analyzed cellular uptake and the immunostimulatory potential of cholesterol-conjugated dsRNA40 by murine Flt3L-DC, human pDC and human monocytes and analyzed the contribution of RN40-chol-induced immunostimulation to T cell differentiation
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