Delivery with polycations extends the immunostimulant Ribomunyl into a potent antiviral Toll-like receptor 7/8 agonist.

BACKGROUND: Upper respiratory tract infection is a frequent cause of morbidity worldwide. Although the course of infection is usually mild, it is responsible for enormous social and economic costs. Immunostimulation with bacterial extracts consisting of ribosomal RNA and proteoglycans, such as Ribomunyl, was introduced into the clinic in the 1980s as a new treatment concept, but did not achieve widespread application. Ribomunyl has been proposed to activate innate immunity, but the contribution of its RNA content as well as its antiviral potential has not been studied. METHODS: Peripheral blood mononuclear cells from healthy donors and immune cells from adenoids were incubated with Ribomunyl either by itself or formulated in a complex with cationic polypeptides such as poly-l-arginine or protamine, and induction of cytokines was quantified by ELISA. RESULTS: Ribomunyl in complex with either poly-l-arginine or protamine, but not on its own, was able to strongly induce interferon-α (P<0.01) and interleukin-12 (P<0.01) in peripheral blood mononuclear cells, whereas induced tumour necrosis factor-α and interleukin-6 levels were independent of the formulation. Comparable results were obtained in immune cells from adenoids, suggesting efficacy also in virus-affected tissue. Cell sorting, RNase digests and selective receptor expression show that the RNA in Ribomunyl acts as an agonist of Toll-like receptor (TLR)7 and TLR8. CONCLUSIONS: Ribomunyl is, in principle, able to potently induce antiviral interferon-α and interleukin-12 via TLR7 and TLR8, respectively, but only when formulated in a complex with cationic polypeptides.

Monocyte-mediated inhibition of TLR9-dependent IFN-α induction in plasmacytoid dendritic cells questions bacterial DNA as the active ingredient of bacterial lysates.

Bacterial DNA contains unmethylated CpG dinucleotides and is a potent ligand for TLR9. Bacterial DNA has been claimed the active ingredient in bacterial lysates used for immunotherapy. Whereas the detection of viral DNA by TLR9 expressed in plasmacytoid dendritic cells (PDCs) with subsequent IFN-α production is well defined, the role of bacterial DNA during microbial infection is less clear. In fact, IFN-α is not a hallmark of antibacterial immune responses. Unlike in mice, TLR9 expression in humans is restricted to PDCs and B cells; thus, conclusions from murine models of infection have limitations. In this study, we demonstrate that lysates of heat-killed Escherichia coli containing bacterial DNA induced IFN-α in isolated PDCs but not in the mixed cell populations of human PBMCs. Depletion of monocytes restored IFN-α secretion by PDCs within PBMCs. We found that monocyte-derived IL-10 and PGs contribute to monocyte-mediated inhibition of IFN-α release in PDCs. We conclude that human PDCs can be stimulated by bacterial DNA via TLR9; however, in the physiological context of mixed-cell populations, PDC activation is blocked by factors released from monocytes stimulated in parallel by other components of bacterial lysates such as LPS. This functional repression of PDCs by concomitantly stimulated monocytes avoids production of antiviral IFN-α during bacterial infection and thus explains how the innate immune system is enabled to distinguish bacterial from viral CpG DNA and thus to elicit the appropriate responses despite the presence of CpG DNA in both types of infection.

Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA.

TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9-mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN-alpha and TNF-alpha in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN-alpha in PDC; IFN-alpha was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN-alpha in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte-derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self-DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self-DNA even if DNA is presented in the condensed form of a complex.