Recognition of Specified RNA Modifications by the Innate Immune System.

Microbial nucleic acids have been described as important activators of human innate immune responses by triggering so-called pattern recognition receptors (PRRs) that are expressed on innate immune cells, including plasmacytoid dendritic cells and monocytes. Although host and microbial nucleic acids share pronounced chemical and structural similarities, they significantly differ in their posttranscriptional modification profile, allowing the host to discriminate between self and nonself. In this regard, ribose 2'-O-methylation has been discovered as suppressor of RNA-induced PRR activation. Although 2'-O-methylation occurs with higher frequencies in eukaryotic than in prokaryotic RNA, the immunosuppressive properties of 2'-O-methylated nucleotides may be misused by certain bacteria as immune evasion mechanism. In the course of identifying inhibitory RNA modifications, our groups have synthesized and comparatively analyzed a series of differentially modified RNAs, so-called modivariants, for their immune stimulatory capacities. In this chapter, we will detail the protocols for the design and synthesis of RNA modivariants by molecular cut-and-paste techniques (referred to as molecular surgery) and describe testing of their immune stimulatory properties upon transfection into peripheral blood mononuclear cells.

2'-O-Methylation within Bacterial RNA Acts as Suppressor of TLR7/TLR8 Activation in Human Innate Immune Cells.

Microbial RNA is an important stimulator of innate immune responses. Differences in posttranscriptional RNA modification profiles enable the immune system to discriminate between self and non-self nucleic acids. This principle may be exploited by certain bacteria to circumvent immune cell activation. In this regard, 2'-O-methylation of Escherichia coli tRNATyr at position 18 (Gm18) has recently been described to inhibit TLR7-mediated IFN-α production in human plasmacytoid dendritic cells (pDCs). Extending these findings, we now demonstrate that Gm18 also potently inhibits TLR7-independent human monocyte activation by RNA derived from a variety of bacterial strains. The half minimal inhibitory concentration values were similar to those found for IFN-α inhibition in pDCs. Mechanistically, 2'-O-methylated RNA impaired upstream signalling events, including MAP kinase and NFx03BA;B activation. Our results suggest that antagonizing effects of Gm18-modified RNA are due to competition with stimulatory RNA for receptor binding. The antagonistic effect was specific for RNA because the small molecule TLR7/8 agonist R848 was not inhibited. Despite the striking phenotype in human cells, 2'-O-methylated RNA did not interfere with TLR13 activation by bacterial 23S rRNA in murine DC and BMDM. Thus, we identify here Gm18 in E. coli tRNA(Tyr) as a universal suppressor of innate immune activation in the human but not the murine system.

A modified dinucleotide motif specifies tRNA recognition by TLR7.

RNA can function as a pathogen-associated molecular pattern (PAMP) whose recognition by the innate immune system alerts the body to an impending microbial infection. The recognition of tRNA as either self or nonself RNA by TLR7 depends on its modification patterns. In particular, it is known that the presence of a ribose methylated guanosine at position 18, which is overrepresented in self-RNA, antagonizes an immune response. Here, we report that recognition extends to the next downstream nucleotide and the effectively recognized molecular detail is actually a methylated dinucleotide. The most efficient nucleobases combination of this motif includes two purines, while pyrimidines diminish the effect of ribose methylation. The constraints of this motif stay intact when transposed to other parts of the tRNA. The results argue against a fixed orientation of the tRNA during interaction with TLR7 and, rather, suggest a processive type of inspection.

Body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via Toll-like receptor signaling.

Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the underlying signaling pathways are poorly understood. Here, we analyzed the effects of ex vivo-derived exosomes on monocytic cell differentiation/activation using THP-1 cells as model. We isolated exosomes from various body fluids such as amniotic fluid, liver cirrhosis ascites, and malignant ascites of ovarian cancer patients. We observed that exosomes were internalized by THP-1 cells and induced the production of IL-1ß, TNF-α, and IL-6. Analysis of the signaling pathways revealed a fast triggering of NFκB and a delayed activation of STAT3. Pharmacologic and antibody-blocking experiments showed that the initial production of IL-6 was instrumental for subsequent activation of STAT3. Importantly, triggering of cell signaling was not a unique property of tumor exosomes but was also observed with exosomes of noncancerous origin. Exosomal signaling was TLR-dependent as the knockdown of Toll-like receptor 2 (TLR2) and TLR4 blocked NFκB and STAT3 activation. Similar results were obtained with TLR-neutralizing antibodies. Exosomes also triggered the release of cytokines from mouse bone marrow-derived dendritic cells or macrophages. This process was MyD88-dependent, further supporting a role of TLR signaling. Our results suggest that exosomes trigger TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. This process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases.

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity.

Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells. As a notable exception, tRNA(Tyr) from Escherichia coli was not immunostimulatory, as were all tested eukaryotic tRNAs. However, the unmodified, 5'-unphosphorylated in vitro transcript of tRNA(Tyr) induced IFN-α, thus revealing posttranscriptional modifications as a factor suppressing immunostimulation. Using a molecular surgery approach based on catalytic DNA, a panel of tRNA(Tyr) variants featuring differential modification patterns was examined. Out of seven modifications present in this tRNA, 2'-O-methylated G(m)18 was identified as necessary and sufficient to suppress immunostimulation. Transplantation of this modification into the scaffold of yeast tRNA(Phe) also resulted in blocked immunostimulation. Moreover, an RNA preparation of an E. coli trmH mutant that lacks G(m)18 2'-O-methyltransferase activity was significantly more stimulatory than the wild-type sample. The experiments identify the single methyl group on the 2'-oxygen of G(m)18 as a natural modification in native tRNA that, beyond its primary structural role, has acquired a secondary function as an antagonist of TLR7.