Nonreducing Sugar Scaffold Enables the Development of Immunomodulatory TLR4-specific LPS Mimetics with Picomolar Potency.

Innate immune defense mechanisms against infection and cancer encompass the modulation of pattern recognition receptor (PRR)-mediated inflammation, including upregulation of various transcription factors and the activation of pro-inflammatory pathways important for immune surveillance. Dysfunction of PRRs-mediated signaling has been implicated in cancer and autoimmune diseases, while the overactivation of PRRs-driven responses during infection can lead to devastating consequences such as acute lung injury or sepsis. We used crystal structure-based design to develop immunomodulatory lipopolysaccharide (LPS) mimetics targeting one of the ubiquitous PRRs, toll-like receptor 4 (TLR4). Taking advantage of an exo-anomeric conformation and specific molecular shape of synthetic nonreducing ß,ß-diglucosamine, which was investigated by NMR, we developed two sets of Lipid A mimicking glycolipids capable of either potently activating innate immune responses or inhibiting pro-inflammatory signaling. Stereoselective 1,1'-glycosylation towards fully orthogonally protected nonreducing GlcNß(1↔1')ßGlcN followed by stepwise assembly of differently functionalised phosphorylated glycolipids provided biologically active molecules that were evaluated for their ability to trigger or to inhibit cellular innate immune responses. Two LPS mimetics, identified as potent TLR4-specific inducers of the intracellular signaling pathways, serve as vaccine adjuvant- and immunotherapy candidates, while anionic glycolipids with TLR4-inhibitory potential hold therapeutic promise for the management of acute or chronic inflammation.

Exploring the human archaeome: its relevance for health and disease, and its complex interplay with the human immune system.

This Review aims to coalesce existing knowledge on the human archaeome, a less-studied yet critical non-bacterial component of the human microbiome, with a focus on its interaction with the immune system. Despite a largely bacteria-centric focus in microbiome research, archaea present unique challenges and opportunities for understanding human health. We examine the archaeal distribution across different human body sites, such as the lower gastrointestinal tract (LGT), upper aerodigestive tract (UAT), urogenital tract (UGT), and skin. Variability in archaeal composition exists between sites; methanogens dominate the LGT, while Nitrososphaeria are prevalent on the skin and UAT. Archaea have yet to be classified as pathogens but show associations with conditions such as refractory sinusitis and vaginosis. In the LGT, methanogenic archaea play critical metabolic roles by converting bacterial end-products into methane, correlating with various health conditions, including obesity and certain cancers. Finally, this work looks at the complex interactions between archaea and the human immune system at the molecular level. Recent research has illuminated the roles of specific archaeal molecules, such as RNA and glycerolipids, in stimulating immune responses via innate immune receptors like Toll-like receptor 8 (TLR8) and 'C-type lectin domain family 4 member E' (CLEC4E; also known as MINCLE). Additionally, metabolic by-products of archaea, specifically methane, have demonstrated immunomodulatory effects through anti-inflammatory and anti-oxidative pathways. Despite these advancements, the mechanistic underpinnings of how archaea influence immune activity remain a fertile area for further investigation.

Investigating pyroptosis as a mechanism of L. major cell-to-cell spread in the human BLaER1 infection model.

Leishmania is the causative agent of the tropical neglected disease leishmaniasis and infects macrophages as its definitive host cell. In order to sustain and propagate infections, Leishmania parasites have to complete cycles of exit and re-infection. Yet, the mechanism driving the parasite spread to other cells remains unclear. Recent studies reported pro-inflammatory monocytes as replicative niche of Leishmania major and showed prolonged expression of IL-1ß at the site of infection, indicating an activation of the NLRP3 inflammasome and pointing toward pyroptosis as a possible mechanism of parasite spread. To address the species-specific inflammasome activation of human cells, we characterized the BLaER1 monocytes as a model for L. major infection. We found that BLaER1 monocytes support infection and activation by Leishmania parasites to the same extent as primary human macrophages. Harnessing the possibilities of this infection model, we first showed that BLaER1 GSDMD-/- cells, which carry a deletion of the pore-forming protein gasdermin D, are more resistant to pyroptotic cell death and, concomitantly, display a strongly delayed release of intracellular parasite. Using that knockout in a co-incubation assay in comparison with wild-type BLaER1 cells, we demonstrate that impairment of the pyroptosis pathway leads to lower rates of parasite spread to new host cells, thus, implicating pyroptotic cell death as a possible exit mechanism of L. major in pro-inflammatory microenvironments.

Recurrent Phases of Strict Protein Limitation Inhibit Tumor Growth and Restore Lifespan in A Drosophila Intestinal Cancer Model.

Diets that restrict caloric or protein intake offer a variety of benefits, including decreasing the incidence of cancer. However, whether such diets pose a substantial therapeutic benefit as auxiliary cancer treatments remains unclear. We determined the effects of severe protein depletion on tumorigenesis in a Drosophila melanogaster intestinal tumor model, using a human RAF gain-of-function allele. Severe and continuous protein restriction significantly reduced tumor growth but resulted in premature death. Therefore, we developed a diet in which short periods of severe protein restriction alternated cyclically with periods of complete feeding. This nutritional regime reduced tumor mass, restored gut functionality, and rescued the lifespan of oncogene-expressing flies to the levels observed in healthy flies on a continuous, fully nutritious diet. Furthermore, this diet reduced the chemotherapy-induced stem cell activity associated with tumor recurrence. Transcriptome analysis revealed long-lasting changes in the expression of key genes involved in multiple major developmental signaling pathways. Overall, the data suggest that recurrent severe protein depletion effectively mimics the health benefits of continuous protein restriction, without undesired nutritional shortcomings. This provides seminal insights into the mechanisms of the memory effect required to maintain the positive effects of protein restriction throughout the phases of a full diet. Finally, the repetitive form of strict protein restriction is an ideal strategy for adjuvant cancer therapy that is useful in many tumor contexts.

Exploring Species-Specificity in TLR4/MD-2 Inhibition with Amphiphilic Lipid A Mimicking Glycolipids.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex is a key receptor of the innate immune system and a major driver of inflammation that is responsible for the multifaceted defense response to Gram-negative infections. However, dysfunction in the tightly regulated mechanisms of TLR4-mediated signaling leads to the uncontrolled upregulation of local and systemic inflammation, often resulting in acute or chronic disease. Therefore, the TLR4/MD-2 receptor complex is an attractive target for the design and development of anti-inflammatory therapies which aim to control the unrestrained activation of TLR4-mediated signaling. Complex structure-activity relationships and species-specificity behind ligand recognition by the TLR4/MD-2 complex complicate the development of MD-2-specific TLR4 antagonists. The restriction of the conformational flexibility of the disaccharide polar head group is one of the key structural features of the newly developed lipid A-mimicking glycophospholipids, which are potential inhibitors of TLR4-mediated inflammation. Since phosphorylation has a crucial influence on MD-2-ligand interaction, glycolipids with variable numbers and positioning of phosphate groups were synthesized and evaluated for their ability to inhibit TLR4-mediated pro-inflammatory signaling in human and murine immune cells. A bis-phosphorylated glycolipid was found to have nanomolar antagonist activity on human TLR4 while acting as a partial agonist on murine TLR4. The glycolipid inhibited mTLR4/MD-2-mediated cytokine release, acting as an antagonist in the presence of lipopolysaccharide (LPS), but at the same time induced low-level cytokine production.

The lncRNA LUCAT1 is elevated in inflammatory disease and restrains inflammation by regulating the splicing and stability of NR4A2.

The nuclear long non-coding RNA LUCAT1 has previously been identified as a negative feedback regulator of type I interferon and inflammatory cytokine expression in human myeloid cells. Here, we define the mechanistic basis for the suppression of inflammatory gene expression by LUCAT1. Using comprehensive identification of RNA-binding proteins by mass spectrometry as well as RNA immunoprecipitation, we identified proteins important in processing and alternative splicing of mRNAs as LUCAT1-binding proteins. These included heterogeneous nuclear ribonucleoprotein C, M, and A2B1. Consistent with this finding, cells lacking LUCAT1 have altered splicing of selected immune genes. In particular, upon lipopolysaccharide stimulation, the splicing of the nuclear receptor 4A2 (NR4A2) gene was particularly affected. As a consequence, expression of NR4A2 was reduced and delayed in cells lacking LUCAT1. NR4A2-deficient cells had elevated expression of immune genes. These observations suggest that LUCAT1 is induced to control the splicing and stability of NR4A2, which is in part responsible for the anti-inflammatory effect of LUCAT1. Furthermore, we analyzed a large cohort of patients with inflammatory bowel disease as well as asthma and chronic obstructive pulmonary disease. In these patients, LUCAT1 levels were elevated and in both diseases, positively correlated with disease severity. Collectively, these studies define a key molecular mechanism of LUCAT1-dependent immune regulation through post-transcriptional regulation of mRNAs highlighting its role in the regulation of inflammatory disease.

Lipid A Mimetics Based on Unnatural Disaccharide Scaffold as Potent TLR4 Agonists for Prospective Immunotherapeutics and Adjuvants.

TLR4 is a key pattern recognition receptor that can sense pathogen- and danger- associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro-inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species-specific, and barely predictable structure-activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan-based lipid A-mimicking molecules that can activate human and murine TLR4-mediated signaling with picomolar affinity. Exploiting crystal structure - based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible ß(1→6)-linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non-reducing ßGlcN(1↔1')ßGlcN scaffold. Excellent stereoselectivity in a challenging ß,ß-1,1' glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of ß,ß-1,1'-linked diglucosamine-derived glycolipids entailing multiple long-chain (R)-3- acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D-molecular shape and conformational rigidity of unnatural ß,ß-1,1'-linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4-mediated signaling in a species-independent manner.

TLR8 is activated by 5'-methylthioinosine, a Plasmodium falciparum-derived intermediate of the purine salvage pathway.

The innate immune recognition of the malaria-causing pathogen Plasmodium falciparum (P. falciparum) is not fully explored. Here, we identify the nucleoside 5'-methylthioinosine (MTI), a Plasmodium-specific intermediate of the purine salvage pathway, as a pathogen-derived Toll-like receptor 8 (TLR8) agonist. Co-incubation of MTI with the TLR8 enhancer poly(dT) as well as synthetic or P. falciparum-derived RNA strongly increase its stimulatory activity. Of note, MTI generated from methylthioadenosine (MTA) by P. falciparum lysates activates TLR8 when MTI metabolism is inhibited by immucillin targeting the purine nucleoside phosphorylase (PfPNP). Importantly, P. falciparum-infected red blood cells incubated with MTI or cultivated with MTA and immucillin lead to TLR8-dependent interleukin-6 (IL-6) production in human monocytes. Our data demonstrate that the nucleoside MTI is a natural human TLR8 ligand with possible in vivo relevance for innate sensing of P. falciparum.

Therapeutic Targeting of TLR4 for Inflammation, Infection, and Cancer: A Perspective for Disaccharide Lipid A Mimetics.

The Toll-like receptor 4 (TLR4) signaling pathway plays a central role in the prompt defense against infectious challenge and provides immediate response to Gram-negative bacterial infection. The TLR4/MD-2 complex can sense and respond to various pathogen-associated molecular patterns (PAMPs) with bacterial lipopolysaccharide (LPS) being the most potent and the most frequently occurring activator of the TLR4-mediated inflammation. TLR4 is believed to be both a friend and foe since improperly regulated TLR4 signaling can result in the overactivation of immune responses leading to sepsis, acute lung injury, or pathologic chronic inflammation involved in cancer and autoimmune disease. TLR4 is also considered a legitimate target for vaccine adjuvant development since its activation can boost the adaptive immune responses. The dual action of the TLR4 complex justifies the efforts in the development of both TLR4 antagonists as antisepsis drug candidates or remedies for chronic inflammatory diseases and TLR4 agonists as vaccine adjuvants or immunotherapeutics. In this review, we provide a brief overview of the biochemical evidences for possible pharmacologic applications of TLR4 ligands as therapeutics and report our systematic studies on the design, synthesis, and immunobiological evaluation of carbohydrate-based TLR4 antagonists with nanomolar affinity for MD-2 as well as disaccharide-based TLR4 agonists with picomolar affinity for the TLR4/MD-2 complex.

WNT6/ACC2-induced storage of triacylglycerols in macrophages is exploited by Mycobacterium tuberculosis.

In view of emerging drug-resistant tuberculosis (TB), host-directed adjunct therapies are urgently needed to improve treatment outcomes with currently available anti-TB therapies. One approach is to interfere with the formation of lipid-laden "foamy" macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis (Mtb). Here, we provide evidence that Wnt family member 6 (WNT6), a ligand of the evolutionarily conserved Wingless/Integrase 1 (WNT) signaling pathway, promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase 2 (ACC2) during pulmonary TB. Using genetic and pharmacological approaches, we demonstrated that lack of functional WNT6 or ACC2 significantly reduced intracellular triacylglycerol (TAG) levels and Mtb survival in macrophages. Moreover, treatment of Mtb-infected mice with a combination of a pharmacological ACC2 inhibitor and the anti-TB drug isoniazid (INH) reduced lung TAG and cytokine levels, as well as lung weights, compared with treatment with INH alone. This combination also reduced Mtb bacterial numbers and the size of mononuclear cell infiltrates in livers of infected mice. In summary, our findings demonstrate that Mtb exploits WNT6/ACC2-induced storage of TAGs in macrophages to facilitate its intracellular survival, a finding that opens new perspectives for host-directed adjunctive treatment of pulmonary TB.

Tailored Modulation of Cellular Pro-inflammatory Responses With Disaccharide Lipid A Mimetics.

Pro-inflammatory signaling mediated by Toll-like receptor 4 (TLR4)/myeloid differentiation-2 (MD-2) complex plays a crucial role in the instantaneous protection against infectious challenge and largely contributes to recovery from Gram-negative infection. Activation of TLR4 also boosts the adaptive immunity which is implemented in the development of vaccine adjuvants by application of minimally toxic TLR4 activating ligands. The modulation of pro-inflammatory responses via the TLR4 signaling pathway was found beneficial for management of acute and chronic inflammatory disorders including asthma, allergy, arthritis, Alzheimer disease pathology, sepsis, and cancer. The TLR4/MD-2 complex can recognize the terminal motif of Gram-negative bacterial lipopolysaccharide (LPS)-a glycophospholipid lipid A. Although immense progress in understanding the molecular basis of LPS-induced TLR4-mediated signaling has been achieved, gradual, and predictable TLR4 activation by structurally defined ligands has not yet been attained. We report on controllable modulation of cellular pro-inflammatory responses by application of novel synthetic glycolipids-disaccharide-based lipid A mimetics (DLAMs) having picomolar affinity for TLR4/MD-2. Using crystal structure inspired design we have developed endotoxin mimetics where the inherently flexible ß(1 → 6)-linked diglucosamine backbone of lipid A is replaced by a conformationally restricted α,α-(1↔1)-linked disaccharide scaffold. The tertiary structure of the disaccharide skeleton of DLAMs mirrors the 3-dimensional shape of TLR4/MD-2 bound E. coli lipid A. Due to exceptional conformational rigidity of the sugar scaffold, the specific 3D organization of DLAM must be preserved upon interaction with proteins. These structural factors along with specific acylation and phosphorylation pattern can ensure picomolar affinity for TLR4 and permit efficient dimerization of TLR4/MD-2/DLAM complexes. Since the binding pose of lipid A in the binding pocket of MD-2 (±180°) is crucial for the expression of biological activity, the chemical structure of DLAMs was designed to permit a predefined binding orientation in the binding groove of MD-2, which ensured tailored and species-independent (human and mice) TLR4 activation. Manipulating phosphorylation and acylation pattern at the sugar moiety facing the secondary dimerization interface allowed for adjustable modulation of the TLR4-mediated signaling. Tailored modulation of cellular pro-inflammatory responses by distinct modifications of the molecular structure of DLAMs was attained in primary human and mouse immune cells, lung epithelial cells and TLR4 transfected HEK293 cells.

Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways.

Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, we established a matching Drosophila model. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. We show that these structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. Our findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes.

Corrigendum: Lipopolysaccharide Recognition in the Crossroads of TLR4 and Caspase-4/11 Mediated Inflammatory Pathways.

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Driver mutations in major lung cancer oncogenes can be analyzed in Drosophila models

Lung cancer remains the leading cause of cancer-associated mortality. Despite recent promising achievements, the overall prognosis remains very poor. In order to integrate the advantages of adapted, transgenic animal models with a high-throughput procedure on the one hand and compliance with the 3R principles on the other hand, we have established and evaluated appropriate Drosophila models. To achieve this goal, we ectopically expressed oncogenes representing the most important driver mutations exclusively in the airway system. These oncogenes were either the human oncogenes or the corresponding Drosophila orthologs. We concentrated on two complementary read-out systems, 1) early larval lethality and 2) quantification of concurrently expressed GFP as a proxy for tumor mass. We could show that ectopic expression of EgfrCA, RasV12, Raf, Rolled (MAPK), PI3K92E, Alk, Akt and Arm can induce early lethality. Thus, they can be used in a straight-forward high-throughput screening approach and can replace mouse models to a considerable extent. Moreover, we could also show that measurement of tumor mass by a concurrently expressed marker (GFP) can be used to detect positive treatment results. Our results show that our Drosophila system provides a superb in vivo inver­tebrate screening system amenable to high-throughput approaches and thus effectively complements the toolbox for the development of novel anti-lung cancer treatments, while complying with the 3R principles.

Lipopolysaccharide Recognition in the Crossroads of TLR4 and Caspase-4/11 Mediated Inflammatory Pathways.

The innate immune response to lipopolysaccharide is essential for host defense against Gram-negative bacteria. In response to bacterial infection, the TLR4/MD-2 complex that is expressed on the surface of macrophages, monocytes, dendritic, and epithelial cells senses picomolar concentrations of endotoxic LPS and triggers the production of various pro-inflammatory mediators. In addition, LPS from extracellular bacteria which is either endocytosed or transfected into the cytosol of host cells or cytosolic LPS produced by intracellular bacteria is recognized by cytosolic proteases caspase-4/11 and hosts guanylate binding proteins that are involved in the assembly and activation of the NLRP3 inflammasome. All these events result in the initiation of pro-inflammatory signaling cascades directed at bacterial eradication. However, TLR4-mediated signaling and caspase-4/11-induced pyroptosis are largely involved in the pathogenesis of chronic and acute inflammation. Both extra- and intracellular LPS receptors-TLR4/MD-2 complex and caspase-4/11, respectively-are able to directly bind the lipid A motif of LPS. Whereas the structural basis of lipid A recognition by the TLR4 complex is profoundly studied and well understood, the atomic mechanism of LPS/lipid A interaction with caspase-4/11 is largely unknown. Here we describe the LPS-induced TLR4 and caspase-4/11 mediated signaling pathways and their cross-talk and scrutinize specific structural features of the lipid A motif of diverse LPS variants that have been reported to activate caspase-4/11 or to induce caspase-4/11 mediated activation of NLRP3 inflammasome (either upon transfection of LPS in vitro or upon infection of cell cultures with intracellular bacteria or by LPS as a component of the outer membrane vesicles). Generally, inflammatory caspases show rather similar structural requirements as the TLR4/MD-2 complex, so that a "basic" hexaacylated bisphosphorylated lipid A architecture is sufficient for activation. However, caspase-4/11 can sense and respond to much broader variety of lipid A variants compared to the very "narrow" specificity of TLR4/MD-2 complex as far as the number and the length of lipid chains attached at the diglucosamine backbone of lipid A is concerned. Besides, modification of the lipid A phosphate groups with positively charged appendages such as phosphoethanolamine or aminoarabinose could be essential for the interaction of lipid A/LPS with inflammatory caspases and related proteins.

Complete Genome Sequence of Lactococcus lactis subsp. lactis G121, an Isolate with Allergy-Protective Features Derived from a Farming Environment.

Early childhood exposure to a farming environment has been found to be protective against asthma and other atopic disorders. Here, we report the complete genome sequence of Lactococcus lactis subsp. lactis G121, which was isolated from the kitchen of a farm in Bavaria (Germany) and is recognized for its allergy-protective properties. It could be assembled into one circular chromosome, three circular plasmids, and one linear plasmid.

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.

The Lipid A from the Lipopolysaccharide of the Phototrophic Bacterium Rhodomicrobium vannielii ATCC 17100 Revisited.

The structure of lipid A from lipopolysaccharide (LPS) of Rhodomicrobium vannielii ATCC 17100 (Rv) a phototrophic, budding bacterium was re-investigated using high-resolution mass spectrometry, NMR, and chemical degradation protocols. It was found that the (GlcpN)-disaccharide lipid A backbone was substituted by a GalpA residue that was connected to C-1 of proximal GlcpN. Some of this GalpA residue was ß-eliminated by alkaline de-acylation, which indicated the possibility of the presence of another so far unidentified substituent at C-4 in non-stoichiometric amounts. One Manp residue substituted C-4' of distal GlcpN. The lipid A backbone was acylated by 16:0(3-OH) at C-2 of proximal GlcpN, and by 16:0(3-OH), i17:0(3-OH), or 18:0(3-OH) at C-2' of distal GlcpN. Two acyloxy-acyl moieties that were mainly formed by 14:0(3-O-14:0) and 16:0(3-O-22:1) occupied the distal GlcpN of lipid A. Genes that were possibly involved in the modification of Rv lipid A were compared with bacterial genes of known function. The biological activity was tested at the model of human mononuclear cells (MNC), showing that Rv lipid A alone does not significantly stimulate MNC. At low concentrations of toxic Escherichia coli O111:B4 LPS, pre-incubation with Rv lipid A resulted in a substantial reduction of activity, but, when higher concentrations of E. coli LPS were used, the stimulatory effect was increased.

An EGFR-Induced Drosophila Lung Tumor Model Identifies Alternative Combination Treatments.

Lung cancer is the leading cause of cancer-associated mortality. Mutations in the EGFR gene are among the most important inducers of lung tumor development, but success of personalized therapies is still limited because of toxicity or developing resistances. We expressed constitutively active EGFR (EGFRCA) exclusively in the airway system of Drosophila melanogaster and performed comprehensive phenotyping. Ectopic expression of EGFRCA induced massive hyper- and metaplasia, leading to early death. We used the lethal phenotype as a readout and screened a library of FDA-approved compounds and found that among the 1,000 compounds, only the tyrosine kinase inhibitors (TKI) afatinib, gefitinib, and ibrutinib rescued lethality in a whole-animal screening approach. Furthermore, we screened the library in the presence of a subtherapeutic afatinib dose and identified bazedoxifene as a synergistically acting compound that rescues EGFR-induced lethality. Our findings highlight the potential of Drosophila-based whole-animal screening approaches not only to identify specific EGFR inhibitors but also to discover compounds that act synergistically with known TKIs. Moreover, we showed that targeting the EGFR together with STAT-signaling is a promising strategy for lung tumor treatment.

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.