Double-stranded RNA sensing links antiviral innate immunity with pulmonary endothelial barrier integrity

Giorgia Ciliberti (Mannheim)1, S. Tual-Chalot (Newcastle)2, M. Polycarpou-Schwarz (Mannheim)1, G. Petrakis (Thessaloniki)3, M. Amponsah-Offeh (Mannheim)1, A. Gatsiou (Newcastle)2, S. Dimmeler (Frankfurt am Main)4, K. Stellos (Mannheim)1

1Medizinische Fakultät Mannheim der Universität Heidelberg Abteilung für Herz- Kreislauf-Forschung Mannheim, Deutschland; 2Faculty of Medical Sciences, Vascular Biology and Medicine Theme Bioscience Insitute Newcastle, Großbritannien; 3Medical School, Aristotle University of Thessaloniki Pathology Department Thessaloniki, Griechenland; 4Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland



Long double-stranded RNAs (dsRNAs) are recognised as pathogen-associated molecular patterns by the cytosolic RNA innate immune sensors, inducing antiviral innate immune responses. RNA sensing by the RNA innate immune sensors is determined by the enzyme adenosine deaminase acting on RNA-1 (ADAR1) which catalyzes the deamination of adenosine residues in double-stranded RNA molecules, a process called adenosine-to-inosine RNA editing. Whether double-stranded RNA sensing may affect endothelial barrier integrity remains unknown.



Genetic deletion strategies were employed by crossing mice carrying a conditional (floxed) Adar1Ifh1 or Tlr3 allele with a tamoxifen-inducible VE-Cadherin-CreERT2 mouse line. Primary human and murine vascular EC culture assays, dsRNA metabolism studies, gene-silencing and expression techniques, immunohistochemistry and confocal microscopy were used to assess the EC-specific ADAR1-MDA5 axis effects.



Inducible endothelial ADAR1 ablation in adult mice triggered a sudden premature death within 8-12 days after the first tamoxifen injection. Post-mortem autopsy and histopathology examination revealed the presence of large exudate pleural effusions compressing the lungs as well as the formation of pulmonary oedema and extravasation of albumin in lung parenchyma, respectively. Mechanistically, loss of ADAR1 resulted into the dissociation of β-catenin from VE-cadherin at EC junctions, translocation of β-catenin into the nucleus and endocytosis of VE-cadherin in murine pulmonary ECs. Next, we studied how ADAR1 controls pulmonary endothelial junctional integrity. Previous works suggested that the lack of inosines in dsRNAs may render endogenous dsRNAs as “viral/non-self” leading to the aberrant activation of RNA innate immunity and antiviral interferon responses. Surprisingly, we found that ablation of ADAR1 triggered a rapid accumulation of long dsRNAs in pulmonary EC cytoplasm inducing the activation of the RNA innate immune sensor MDA5. Induction of MDA5 signaling resulted into the dissociation of β-catenin from VE-cadherin at EC junctions, while co-ablation of ADAR1 and the MDA5 in mice was sufficient to restore the integrity of the ADAR1-deficient endothelium and fully rescue the premature lethality. Co-ablation of ADAR1 and another dsRNA sensor, TLR3, increased the lifespan of the mice only for few days failing to rescue the lethal phenotype of endothelial ADAR1-deficient mice, indicating that MDA5 is the main dsRNA sensor in pulmonary ECs. In human ECs, silencing of endothelial ADAR1 led to a striking accumulation of cytoplasmic long dsRNAs and loss of junctional β-catenin and VE-cadherin, while co-silencing of ADAR1 and MDA5 restored endothelial integrity.



Double-stranded RNA sensing by MDA5 links antiviral innate immunity with pulmonary endothelial barrier integrity, a mechanism that may be involved in the development of noncardiogenic pulmonary oedema in respiratory viral infections.

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