Corrigendum: Inflammatory response against Staphylococcus aureus via intracellular sensing of nucleic acids in keratinocytes.

[This corrects the article DOI: 10.3389/fimmu.2022.828626.].

Inflammatory Response Against Staphylococcus aureus via Intracellular Sensing of Nucleic Acids in Keratinocytes.

Staphylococcus aureus is one of the clinically most relevant pathogens causing infections. Humans are often exposed to S. aureus. In approximately one-third of the healthy population it can be found on the skin either for long or short periods as colonizing "commensals", without inducing infections or an inflammatory immune response. While tolerating S. aureus seems to be limited to certain individuals and time periods in most cases, Staphylococcus epidermidis is tolerated permanently on the skin of almost all individuals without activating overwhelming skin inflammation. To investigate this, we co-cultured a keratinocyte cell line (HaCaT) with viable S. aureus or S. epidermidis to study the differences in the immune activation. S. aureus activated keratinocytes depicted by a profound IL-6 and IL-8 response, whereas S. epidermidis did not. Our data indicate that internalization of S. aureus and the subsequent intracellular sensing of bacterial nucleic acid may be essential for initiating inflammatory response in keratinocytes. Internalized dsRNA activates IL-6 and IL-8 release, but not TNF-α or IFNs by human keratinocytes. This is a non-specific effect of dsRNA, which can be induced using Poly(I:C), as well as RNA from S. aureus and S. epidermidis. However, only viable S. aureus were able to induce this response as these bacteria and not S. epidermidis were actively internalized by HaCaT. The stimulatory effect of S. aureus seems to be independent of the TLR3, -7 and -8 pathways.

Assessment of SARS-CoV-2 Infection among Healthcare Workers of a German COVID-19 Treatment Center.

To date, more than 160 million people have been infected with COVID-19 worldwide. In the present study, we investigated the history of SARS-CoV-2 infection among 3067 healthcare workers (HCW) in a German COVID-19 treatment center during the early phase of the pandemic (July 2020) based on the seroprevalence of SARS-CoV-2 antibodies and self-reported previous PCR results. The results demonstrate a low prevalence of SARS-CoV-2 infection (n = 107 [3.5%]) with no increased risk for employees with a high level of patient exposure in general or working in COVID-19-confined areas in particular. This suggests that the local hygiene standards implemented in our hospital during the first wave of COVID-19 pandemic were effective in preventing patient-to-HCW transmission. No evidence for highly mobile staff serving as a vector for SARS-CoV-2 transmission could be found. In addition, impairment of smell and/or taste was strongly associated with SARS-CoV-2 history.

Comparative evaluation of the effect of different growth media on in vitro sensitivity to azithromycin in multi-drug resistant Pseudomonas aeruginosa isolated from cystic fibrosis patients.

Long-term treatment with azithromycin is a therapeutic option in Cystic Fibrosis (CF) patients chronically infected with P. aeruginosa. It was recently shown that azithromycin has direct antimicrobial activity when P. aeruginosa isolates are tested in Roswell Park Memorial Institute medium supplemented with fetal calf serum (RPMI 1640/FCS) by broth microdilution. We now investigated whether (i) azithromycin might also be active against multidrug resistant (MDR) P. aeruginosa isolated from CF patients and (ii) how in vitro sensitivity assays perform in synthetic cystic fibrosis sputum medium (SCFM), a medium that mimics the particular CF airway environment. In 17 (59%) out of 29 MDR P. aeruginosa CF isolates MICs for azithromycin ranged between 0.25 and 8 µg/ml and 12 isolates (41%) showed a MIC ≥512 µg/ml when measured in RPMI/FCS. In contrast, MICs were ≥ 256 µg/ml for all P. aeruginosa MDR isolates when tested in either SCFM or in conventional cation-adjusted Mueller Hinton Broth. High MIC values observed in CF adapted medium SCFM for both PAO1 and MDR P. aeruginosa CF isolates, as opposed to findings in RPMI, argue against routine azithromycin MIC testing of CF isolates.

Bacterial tRNA 2'-O-methylation is dynamically regulated under stress conditions and modulates innate immune response.

RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2'-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.

Neutrophil extracellular trap-associated RNA and LL37 enable self-amplifying inflammation in psoriasis.

Psoriasis is an inflammatory skin disease with strong neutrophil (PMN) infiltration and high levels of the antimicrobial peptide, LL37. LL37 in complex with DNA and RNA is thought to initiate disease exacerbation via plasmacytoid dendritic cells. However, the source of nucleic acids supposed to start this initial inflammatory event remains unknown. We show here that primary murine and human PMNs mount a fulminant and self-propagating neutrophil extracellular trap (NET) and cytokine response, but independently of the canonical NET component, DNA. Unexpectedly, RNA, which is abundant in NETs and psoriatic but not healthy skin, in complex with LL37 triggered TLR8/TLR13-mediated cytokine and NET release by PMNs in vitro and in vivo. Transfer of NETs to naive human PMNs prompts additional NET release, promoting further inflammation. Our study thus uncovers a self-propagating vicious cycle contributing to chronic inflammation in psoriasis, and NET-associated RNA (naRNA) as a physiologically relevant NET component.

Molecular characterization of carbapenem-resistant Acinetobacter baumannii using WGS revealed missed transmission events in Germany from 2012-15.

BACKGROUND: Infection and colonization with multi-resistant Acinetobacter baumannii causes therapeutic and economic problems in the nosocomial setting. Due to the sensitivity issue of screening schemes for A. baumannii, it is difficult to implement adequate transmission prevention measures. The high discriminatory power of WGS for transmission-chain analysis provides us with the necessary tool to study and identify transmission events. We retrospectively sequenced and analysed 39 A. baumannii isolates from 2012-15 to search for possible missed transmission events. METHODS: Molecular typing by WGS was performed for non-repetitive (n=39) carbapenem-resistant A. baumannii. Retrospective assessment of patient records was performed to investigate and confirm possible transmission events. RESULTS: Between July 2012 and September 2015, A. baumannii was isolated from 268 patients, of which 16% (42/268) were carbapenem resistant. Thirty-nine of these isolates were recoverable and sequenced. Fifteen percent (6/39) of these were resistant to all antibiotics tested. Most isolates belong to the circulating IC2 clonal type. SNP analysis revealed four potential outbreak clusters. Two of these clusters showed high concordance with the local spatio-temporal epidemiology, suggesting that transmission events were very likely. CONCLUSIONS: Our data suggest that there were two independent transmission events, which would have been missed by conventional MLST owing to high clonality. The routine implementation of WGS can optimize surveillance and initiation of suitable containment measures. In addition, emerging resistance to salvage therapy is a major therapeutic problem and should be monitored closely.

2'-O-methylation within prokaryotic and eukaryotic tRNA inhibits innate immune activation by endosomal Toll-like receptors but does not affect recognition of whole organisms.

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2'-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA-induced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm3 in S. cereviase, and CRISPR/Cas9-induced knockout of TARBP1 in H. sapiens results in loss of Gm18 within tRNA. Lack of Gm18 across the kingdoms resulted in increased immunostimulation of peripheral blood mononuclear cells when activated by tRNA preparations. In E. coli, lack of 2'-O-methyltransferase trmH also enhanced immune stimulatory properties by whole cellular RNA. In contrast, lack of Gm18 in yeasts and human cells did not affect immunostimulation by whole RNA preparations. When using live E. coli bacteria, lack of trmH did not affect overall immune stimulation although we detected a defined TLR8/RNA-dependent gene expression signature upon E. coli infection. Together, these results demonstrate that Gm18 is a global immune inhibitory tRNA modification across the kingdoms and contributes to tRNA recognition by innate immune cells, but as an individual modification has insufficient potency to modulate recognition of the investigated microorganisms.

Crucial Role of Nucleic Acid Sensing via Endosomal Toll-Like Receptors for the Defense of Streptococcus pyogenes in vitro and in vivo.

Streptococcus pyogenes is a major human pathogen causing a variety of diseases ranging from common pharyngitis to life-threatening soft tissue infections and sepsis. Microbial nucleic acids, especially bacterial RNA, have recently been recognized as a major group of pathogen-associated molecular patterns (PAMPs) involved in the detection of Streptococcus pyogenes via endosomal Toll-like receptors (TLRs) in vitro. However, the individual contribution and cooperation between TLRs as well as cell-type and strain specific differences in dependency on nucleic acid detection during S. pyogenes infection in vitro have not been clarified in detail. Moreover, the role of particularly bacterial RNA for the defense of S. pyogenes infection in vivo remains poorly defined. In this study, we report that in all investigated innate immune cells involved in the resolution of bacterial infections, including murine macrophages, dendritic cells and neutrophils, recognition of S. pyogenes strain ATCC12344 is almost completely dependent on nucleic acid sensing via endosomal TLRs at lower MOIs, whereas at higher MOIs, detection via TLR2 plays an additional, yet redundant role. We further demonstrate that different S. pyogenes strains display a considerable inter-strain variability with respect to their nucleic acid dependent recognition. Moreover, TLR13-dependent recognition of S. pyogenes RNA is largely non-redundant in bone marrow-derived macrophages (BMDMs), but less relevant in neutrophils and bone marrow-derived myeloid dendritic cells (BMDCs) for the induction of an innate immune response in vitro. In vivo, we show that a loss of nucleic acid sensing blunts early recognition of S. pyogenes, leading to a reduced local containment of the bacterial infection with subsequent pronounced systemic inflammation at later time points. Thus, our results argue for a crucial role of nucleic acid sensing via endosomal TLRs in defense of S. pyogenes infection both in vitro and in vivo.

RNA Modifications Modulate Activation of Innate Toll-Like Receptors.

Self/foreign discrimination by the innate immune system depends on receptors that identify molecular patterns as associated to pathogens. Among others, this group includes endosomal Toll-like receptors, among which Toll-like receptors (TLR) 3, 7, 8, and 13 recognize and discriminate mammalian from microbial, potentially pathogen-associated, RNA. One of the discriminatory principles is the recognition of endogenous RNA modifications. Previous work has identified a couple of RNA modifications that impede activation of TLR signaling when incorporated in synthetic RNA molecules. Of note, work that is more recent has now shown that RNA modifications in their naturally occurring context can have immune-modulatory functions: Gm, a naturally occurring ribose-methylation within tRNA resulted in a lack of TLR7 stimulation and within a defined sequence context acted as antagonist. Additional RNA modifications with immune-modulatory functions have now been identified and recent work also indicates that RNA modifications within the context of whole prokaryotic or eukaryotic cells are indeed used for immune-modulation. This review will discuss new findings and developments in the field of immune-modulatory RNA modifications.

Double methylation of tRNA-U54 to 2'-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7.

Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2'-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNALys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNALys3 were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2'-O-methylthymidine (m5Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2'O, as shown by the fact that neither Um54 nor m5U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.

From macrophage to osteoclast - How metabolism determines function and activity.

Osteoclasts are specialised cells that resorb bone and develop from the monocyte/macrophage lineage. While there is a wealth of information on the regulation of macrophage function through metabolic activity, the connection between osteoclast differentiation and metabolism is less well understood. Recent data show that mitochondria participate in switching macrophages from an inflammatory phenotype towards differentiation into osteoclasts. Additionally, it was found that reactive oxygen species (ROS) actively take place in osteoclast differentiation by acting as secondary signalling molecules. Bone resorption is an energy demanding process and differentiating osteoclasts triggers the biogenesis of mitochondria. In addition, the activity of specific OXPHOS components of macrophages and osteoclasts is differentially regulated. This review summarises our knowledge on macrophage-mediated inflammation, its impact on a cell's metabolic activity and its effect on osteoclast differentiation.

The SiLuDrain Trial: a prospective randomized controlled trial comparing standard versus silver-impregnated lumbar drains.

OBJECTIVECatheter-associated cerebrospinal fluid (CSF) infection remains a serious event, especially for patients in neurocritical care units. The use of external ventricular drain (EVD) catheters impregnated with antimicrobial substances has led to a significant reduction of infection rates. This study was undertaken to compare the use of antimicrobial, silver-impregnated external lumbar drains (si-ELDs) and conventional ELDs.METHODSPatients with an indication for ELD placement were randomized to receive either a conventional or an si-ELD catheter. Regular assessment for CSF infections and device-related complications was performed. Neurosurgeons placing the ELD rated the usability and handling of the catheter on a 6-item ordinal performance scale (range: 1, very bad, to 5, very good). All microorganisms isolated in this study were tested for silver-susceptibility via a catheter-roll method.RESULTSA total of 48 patients were enrolled in the trial. The si-ELD catheters showed a nonsignificantly lower infection rate compared to conventional ones (4.2% vs 16.7%, p = 0.16). The majority of infections were caused by Staphylococcus species. Device-related complications occurred significantly less often with silver-impregnated-catheters than with conventional ones (8.3% vs 37.5%, p = 0.02). The usability was rated significantly better for si-ELDs (p = 0.003). Antimicrobial susceptibility was shown for si-ELDs against various Staphylococcus spp., but Candida parapsilosis and Escherichia coli were not affected by this antimicrobial agent.CONCLUSIONSSilver-impregnated ELD catheters, which could potentially reduce the number of CSF infections, show significantly better properties in regard to handling and fewer device-related complications. Whether they are superior to antibiotic-impregnated catheters or a clinical regimen involving antibiotic prophylaxis remains to be proven.Clinical trial registration no.: DRKS00013513 (Deutsches Register Klinischer Studien).

Influence of Pasteurella multocida Toxin on the differentiation of dendritic cells into osteoclasts.

Dendritic cells (DC) are antigen-presenting cells that connect the innate and adaptive immune system to ensure an efficient immune response during the course of an infection. Recently, DC came into the spotlight as a potential source of osteoclast progenitors, especially under (auto)inflammatory conditions. The virulence factor Pasteurella multocida Toxin (PMT) causes atrophic rhinitis in pigs, a disease characterised by a severe reduction of nasal bone. Our group and others have shown the potential of PMT in mediating differentiation of monocytes/macrophages into bone-resorbing osteoclasts. However, whether DC are target cells for PMT-induced osteoclast differentiation, is currently unknown. Using different murine DC model systems, we investigated the ability of PMT to induce osteoclast formation in DC. Similar to our previous observations in macrophages, PMT was endocytosed by DC and triggered intracellular deamidation of residue Q209 of the Gq alpha subunit. Still, PMT failed to induce prolonged secretion of osteoclastogenic cytokines and osteoclast formation; instead PMT-treated DC secreted interleukin-12 (IL-12), an inhibitor of osteoclastogenesis. In this study, we show that in comparison to bone marrow-derived macrophages, PMT induces maturation of DC through increased expression of the activation markers CD80 and CD86. As maturation of DC prevents their transdifferentiation into osteoclasts, we hypothesize that PMT, a potent osteoclastogenic toxin, fails to trigger osteoclastogenesis in DC due to its effect on DC maturation and IL-12 production.

Interaction and Mutual Activation of Different Innate Immune Cells Is Necessary to Kill and Clear Hepatitis C Virus-Infected Cells.

Innate immune cells can sense hepatitis C virus (HCV)-infected cells and respond with anti-viral actions including secretion of interferons (IFNs). In previous studies, the response of individual innate immune cells against HCV was analyzed in detail. We hypothesized that interaction of multiple innate immune cells increases the magnitude of the immune response and eventually leads to clearance of HCV-infected cells. To investigate this, we co-cultured Huh-7 HCV subgenomic replicon (SGR) cells with peripheral blood mononuclear cells (PBMCs). We confirm secretion of IFNα by plasmacytoid dendritic cells (pDCs) and IFNγ by natural killer (NK) cells in the co-culture setup. Moreover, we observed that also monocytes contribute to the anti-viral response. Flow cytometry and ImageStream analysis demonstrated that monocytes take up material from HCV SGR cells in co-culture with PBMCs. Preceding the uptake, PBMCs caused apoptosis of HCV SGR cells by tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression on NK cells. We observed that only the interplay of monocytes, pDCs, and NK cells resulted in efficient clearance of HCV SGR cells, while these cell populations alone did not kill HCV SGR cells. Despite similar TRAIL receptor expression on Huh-7 control cells and HCV SGR cells, HCV activated PBMCs specifically killed HCV SGR cells and did not target Huh-7 control cells. Finally, we showed that HCV replicating cells per se are sensitive toward TRAIL-induced apoptosis. Our results highlight the importance of the interplay of different innate immune cells to initiate an efficient, rapid, and specific response against HCV-infected cells.

Identification of an optimized 2'-O-methylated trinucleotide RNA motif inhibiting Toll-like receptors 7 and 8.

Bacterial RNA serves an important function as activator of the innate immune system. In humans bacterial RNA is sensed by the endosomal receptors TLR7 and TLR8. Differences in the posttranscriptional modification profile of prokaryotic when compared with eukaryotic RNA allow innate immune cells to discriminate between "host" and "foreign" RNA. Ribose 2'-O-methylation is of particular importance and has been reported to antagonize TLR7/8 activation. Yet, the exact sequence context in which 2'-O-methylation has to occur to mediate its inhibitory activity remains largely undefined. On the basis of a naturally occurring 2'-O-methylated RNA sequence, we performed a systematic permutation of the methylated nucleotide as well as adjacent bases and hereby identify two minimal trinucleotide motifs within a 9-mer oligoribonucleotide that are necessary and sufficient to antagonize TLR7 and TLR8 activation, respectively. Given the growing interest in the development of inhibitors of nucleic acid-sensing TLRs for therapeutic purposes, these results will facilitate the rational design of such antagonists in the future.

Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7.

A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern-recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single-stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccines have been reported to exhibit synergistic effects. Here, we describe a concept to chemically combine three therapeutic functions in one RNA bioconjugate. This consists in the simultaneous TLR7 stimulation by ssRNA and smTLRa as well as the therapeutic function of the RNA itself, e.g., as a vaccinating or knockdown agent. We have hence synthesized bioconjugates of mRNA and siRNA containing covalently attached smTLRa and tested their function in TLR7 stimulation. Strikingly, the bioconjugates displayed decreased rather than synergistically increased stimulation. The decrease was distinct from the antagonistic action of an siRNA bearing a Gm motive, as observed by direct comparison of the effects in the presence of otherwise stimulatory RNA. In summary, these investigations showed that TRL7 activation can be impeded by bioconjugation of small molecules to RNA.

Evaluation of antibiotic resistance to orally administrable antibiotics in staphylococcal bone and joint infections in one of the largest university hospitals in Germany: is there a role for fusidic acid?

Bone and joint infections (BJIs) are often difficult to treat. Staphylococcus spp. is the major pathogen causing these infections, which is often associated with biofilm formation on prosthetic materials. Therapeutic measures are complex, ranging from surgical intervention to initial intravenous and supportive long-term oral antibiotic therapy. The options for oral antimicrobial therapy are limited, mainly due to the resistance profile of the causative pathogen and the unfavourable pharmacodynamic and pharmacokinetic properties of most antibiotics in biofilm. Data analysis over a 5-year period was performed on staphylococci isolated from BJI patients in the Orthopaedic Department of the University Hospital Heidelberg (Heidelberg, Germany) to assess the plausibility of fusidic acid (FA)-based alternative oral treatment regimens. Six percent of BJIs were caused by meticillin-resistant Staphylococcus aureus (MRSA), and multiresistance was common. Over 75% of MRSA in BJIs were resistant to the commonly used rifampicin (RIF)-based combinations. Resistance to FA-based combinations was high. However, over 80% were susceptible to the combination RIF+FA. In coagulase-negative staphylococci, resistance to RIF-based combinations was similar to FA-based combinations. Almost two-thirds of the isolates tested were susceptible to RIF+FA. These data suggest FA as a possible option as a substitution for RIF or as a combination companion in case of resistance or unavailability.

Inhibition of Histone Deacetylases Permits Lipopolysaccharide-Mediated Secretion of Bioactive IL-1ß via a Caspase-1-Independent Mechanism.

Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1ß processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1ß maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1ß secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1ß, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1ß cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1ß by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1ß, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications.

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.