Role of the Receptor for Advanced Glycation End-products (RAGE) in the Immune Sensing of Nucleic Acids

Nucleic acid recognition is an important mechanism that enables the innate immune system to detect both microbial infection and tissue damage. To minimize the recognition of self-derived nucleic acids, all nucleic acid sensing signaling receptors are sequestered away from the cell surface and are activated either in the cytoplasm or in endosomes. In endosomes, nucleic acid sensing relies on members of the tolllike receptor (TLR) family. In conditions of infection or damage, however, the immune system must allow recognition of extracellular nucleic acids. But, how these are sensed and internalized is not yet completely understood. Entry of nucleic acids has long been considered to rely on microbe or cell debris uptake into cells before release of their contents including the nucleic acids. It is now clear that free extracellular nucleic acids exist, which can be detected and internalized thanks to extracellular receptors. The receptor for advanced glycation end-products (RAGE) is a multiligand cell surface receptor that has been studied the past twenty years for its role in development of a plethora of inflammation states such as those occurring during microbial infection, sterile injury, neurodegeneration, auto-immunity and cancer. RAGE was shown to trigger inflammatory signals, promote immune cell maturation, proliferation and motility thereby sustaining and exaggerating the inflammatory state. To do so, RAGE senses heterogeneous types of molecules that accumulate during such inflammatory conditions and which often can trigger expression of RAGE itself. These ligands include advanced glycation end-products (AGEs), amyloid fibrils such as amyloid-β, members of the S100 protein family and high-mobility group box 1 (HMGB1). Data presented in this thesis introduces nucleic acids as new class of ligands for RAGE. First, data demonstrate the binding of DNA to RAGE extracellular domain and localizes this binding to the V and C1 immunoglobulin-like domains. Biochemical assays and crystallography analysis show that DNA binds with RAGE through interaction between the DNA phosphate backbone and a positively charged aminoacid surface present on RAGE V-C1 domain. Flow cytometry and confocal microscopy experiments show that stimulatory DNA, a specific activator of the endosomal DNA receptor TLR9, is recruited at the surface of cells expressing RAGE and thereby internalized more efficiently. Thus, RAGE expression increases subsequent TLR9 activation and downstream NFκB activity. Since DNA binds to RAGE in a base-unspecific manner, a potential interaction of RNA with RAGE is further analyzed in the second part of this thesis. Results first prove that single stranded RNA (ssRNA) indeed binds to RAGE. Comparing DNA and RNA binding to RAGE, competing assays show that RNA binds to RAGE at a similar site than DNA. Confocal microscopy and flow cytometry experiments show that RAGE expression at the cell surface recruits RNA and promotes internalization that can be abrogated by truncation of RAGE V-C1 domain. As for TLR9, RAGE expression increases the activation of the RNA-specific receptors, TLR7, TLR8 and TLR13. Confirming these results, RAGE deficiency strikingly reduces the activation of bone marrow cells upon stimulation with a TLR13-specifc RNA agonist. Deeper analysis of mechanisms involving RAGE in TLR-dependent RNA-sensing show that the effect of RAGE relies on actin polymerization and dynamin-dependent cell internalization. Furthermore, truncation of RAGE intracellular signaling domain indicates that direct RAGE downstream signaling is negligible. Finally, study of the effect of RAGE on double stranded RNA (dsRNA) sensing presents contradictory results. Indeed, although TLR3-specific dsRNA binds to RAGE efficiently, RAGE expression inhibits TLR3 activation. Surprisingly, upon cell stimulation with a dsRNA synthetic analog, poly (I:C), RAGE expression increases immune activation, indicating a possible role for RAGE in cytosolic RNA sensing. Together, these results illustrate RAGE as a pivotal membrane receptor for nucleic acids. Hence, data presented in this thesis indicates that RAGE is an integral part of the endosomal nucleic acid sensing system and calls for further analysis of the role of RAGE in cytosolic nucleic acid sensing and potentially non-coding RNA-mediated cell-to-cell communication

HMGB1, IL-1alpha, IL-33 and S100 proteins: dual-function alarmins

Our immune system is based on the close collaboration of the innate and adaptive immune systems for the rapid detection of any threats to the host. Recognition of pathogen-derived molecules is entrusted to specific germline-encoded signaling receptors. The same receptors have now also emerged as efficient detectors of misplaced or altered self-molecules that signal tissue damage and cell death following, for example, disruption of the blood supply and subsequent hypoxia. Many types of endogenous molecules have been shown to provoke such sterile inflammatory states when released from dying cells. However, a group of proteins referred to as alarmins have both intracellular and extracellular functions which have been the subject of intense research. Indeed, alarmins can either exert beneficial cell housekeeping functions, leading to tissue repair, or provoke deleterious uncontrolled inflammation. This group of proteins includes the high-mobility group box 1 protein (HMGB1), interleukin (IL)-1alpha, IL-33 and the Ca2+-binding S100 proteins. These dual-function proteins share conserved regulatory mechanisms, such as secretory routes, post-translational modifications and enzymatic processing, that govern their extracellular functions in time and space. Release of alarmins from mesenchymal cells is a highly relevant mechanism by which immune cells can be alerted of tissue damage, and alarmins play a key role in the development of acute or chronic inflammatory diseases and in cancer development

Necroptosis, pyroptosis and apoptosis: an intricate game of cell death

Cell death is a fundamental physiological process in all living organisms. Its roles extend from embryonic development, organ maintenance, and aging to the coordination of immune responses and autoimmunity. In recent years, our understanding of the mechanisms orchestrating cellular death and its consequences on immunity and homeostasis has increased substantially. Different modalities of what has become known as \u27programmed cell death\u27 have been described, and some key players in these processes have been identified. We have learned more about the intricacies that fine tune the activity of common players and ultimately shape the different types of cell death. These studies have highlighted the complex mechanisms tipping the balance between different cell fates. Here, we summarize the latest discoveries in the three most well understood modalities of cell death, namely, apoptosis, necroptosis, and pyroptosis, highlighting common and unique pathways and their effect on the surrounding cells and the organism as a whole

RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo

eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition

RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α

The sspecific NLRP3 antagonist IFM-514 decreases fibrosis and inflammation in experimental murine non-alcoholic steatohepatitis

Background and Aims: Activation of the inflammasome NLRP3 (NOD-, LRR- and pyrin domain containing 3) contributes to the development of non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH). Therefore, this study explored the therapeutic effects of a novel and selective NLRP3 antagonist in a murine dietary model of NASH. Methods: Groups of 12-week-old ApoE-/- mice were fed ad lib for 7 weeks with a methionine/choline deficient (MCD) and western diet (WD). After 3 weeks of diet-induced injury, mice were injected i. p. with the NLRP3 antagonist IFM-514 (100 mg/kg body weight) or vehicle (0.5% carmellose) every day, 5 days/week for a further 4 weeks. Several markers of inflammation, fibrosis and steatosis were evaluated. Whole transcriptome sequencing and panel RNA expression analysis (NanoString) were performed. Results: IFM-514 inhibited IL-1β production in mice challenged with 20 mg/kg lipopolysaccharide, and in mouse and human inflammatory cells in vitro. IFM-514 inhibited hepatic inflammation in the in vivo non-alcoholic steatohepatitis model assessed by H&E staining and in the hepatic gene expression of inflammasome-related proinflammatory cytokines. This effect was associated with significant reduction in caspase-1 activation. Similarly, IFM-514 was efficacious in vivo in MDC-fed ApoE-/- mice, markedly reducing portal pressure, Sirius red staining and 4-hydroxyproline content compared to vehicle-treated mice. Moreover, IFM-514 significantly reduced hepatic steatosis in MCD-fed ApoE-/- mice, as evidenced by NAFLD scores, oil red O staining, hepatic triglycerides and gene expression. In WD treated animals, similar trends in inflammation and fibrosis were observed, although not sufficient IFM-514 levels were reached. Conclusion: Overall, IFM-514 reduced liver inflammation and fibrosis, with mild effects on liver steatosis in experimental murine NASH. Blocking of NLRP3 may be an attractive therapeutic approach for NASH patients