The Regulation of Occludin mRNA by miR193b-5p in Influenza Induced ARDS
CCCF ePoster library. Vaswani C. 11/12/19; 283436; EP76
Mr. Chirag Vaswani
Mr. Chirag Vaswani
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Topic: Basic or Translational Science

Vaswani, C.M.1,2*; Plant, P. J.2, Ektesabi, A. M. 4; Varkouhi, A. K.2; Teixeira, A. P. M.2; Watts, T. H.3; Moraes, T. J.4, dos Santos, C.1,2,4,6#.
1Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON, CA. 2Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, ON, CA. 3Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, ON, CA. 4Institute of Medical Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, CA.5Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, ON, CA. 6Ottawa Hospital Research Institute and the University of Ottawa, Ottawa, ON, CA.


Introduction: The leading cause of mortality in critically ill patients from seasonal and pandemic influenza strains is ARDS.1,2 Emerging evidence suggests host-derived cellular miRNAs play a critical role in lung injury, alveolar-capillary membrane integrity, and host responses to viral infections.3–6 We have recently identified a role for miRNA-193b in the innate immune response to bacteria and the regulation of tight-junction protein (occludin) in experimental models of pneumonia. Here, we advance current knowledge by exploiting the role of this microRNA in barrier function, viral infection and host anti-viral responses in a murine model of H1N1.
Methods: Wild type mice (C57Bl/6J, 10 - 14 weeks) were randomized to infection with H1N1 virus (A/PR/8/34) treated a miR193b-5p inhibitor (INH) versus placebo delivered on day 4 post-infection. Body weights and temperatures were collected daily. Lungs were harvested on day 8 to assess for: histology, bronchoalveolar lavage fluid cell counts and differential, membrane permeability, and viral load. In vitro, Beas2b cells were infected with H1N1 and treated with or without miR-193b-5p inhibitor (INH) or mimic. Cells were transfected with the siRNA against occludin or scrambled control. The expression of miR-193b and putative target was evaluated by qRT and digital droplet PCR. 
Results: Intranasal infection with H1N1 results in increased pulmonary inflammation, lung edema, increased levels of miR193b-5p (20-fold) and decreased expression of occludin (>50%) that peak at day 5 days post-infection. Reporter construct demonstrates miR-193b binds specifically to the 3'UTR of occludin. Inhibition of miR193b-5p mitigates H1N1-induced lung injury, edema formation, viral load, and anti-viral Interferon β (IFNb) and Interferon Regulated Genes expression. In vitro, silencing of occludin results in increased viral load and dysregulation of the host antiviral response.
Conclusion: MiR-193b-5p plays a critical role in regulation of occludin, tight junction function and virus induced lung injury. Inhibition of miR-193b-5p results in decreased lung injury, inflammation, and viral load.


1. Dawood, F. S. et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: A modelling study. Lancet Infect. Dis. 12, 687–695 (2012).
2. Luyt, C. édouard, Combes, A., Trouillet, J. L., Nieszkowska, A. & Chastre, J. Virus-induced acute respiratory distress syndrome: Epidemiology, management and outcome. Press. Medicale 40, e561–e568 (2011).
3. Short, K. R., Kroeze, E. J. B. V., Fouchier, R. A. M. & Kuiken, T. Pathogenesis of influenza-induced acute respiratory distress syndrome. Lancet Infect. Dis. 14, 57–69 (2014).
4. Benedicto, I. et al. The Tight Junction-Associated Protein Occludin Is Required for a Postbinding Step in Hepatitis C Virus Entry and Infection. J. Virol. 83, 8012–8020 (2009).
5. Torres-Flores, J. M. & Arias, C. F. Tight junctions go viral! Viruses 7, 5145–5154 (2015).
6. Short, K. R. et al. Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions. Eur. Respir. J. 47, 954–966 (2016).

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