Bertheloot, Damien: Role of the Receptor for Advanced Glycation End-products (RAGE) in the Immune Sensing of Nucleic Acids. - Bonn, 2016. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Damien Bertheloot}},
title = {Role of the Receptor for Advanced Glycation End-products (RAGE) in the Immune Sensing of Nucleic Acids},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2016,
month = sep,

note = {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.},

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