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Circulating Exosomes from Human Lung Transplant Recipients Having Respiratory Viral Infections Contain Nucleic Acids and Activate Signaling Pathways CGAS/STING and RIG-1

Authors
  • Bansal, S.1
  • Limaye, A.2
  • Bharat, A.3
  • Bremner, R.1
  • Smith, M.1
  • Omar, A.1
  • Mohanakumar, T.1
  • 1 Norton Thoracic Institute, St. Joseph's Hospital and Medical Center, Phoenix, AZ
  • 2 Div Allergy and Infectious Diseases, University of Washington, Seattle, WA
  • 3 Department of Surgery, Northwestern Feinberg School of Medicine, Chicago, IL
Type
Published Article
Journal
The Journal of Heart and Lung Transplantation
Publisher
Published by Elsevier Inc.
Publication Date
Mar 30, 2020
Volume
39
Issue
4
Identifiers
DOI: 10.1016/j.healun.2020.01.986
PMCID: PMC7195427
Source
PubMed Central
License
Unknown

Abstract

Purpose Exosomes are membrane bound vesicles are released by cells into body fluids. Our laboratory demonstrated the presence of circulating exosomes with lung self-antigens (Collagen-V and K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Since respiratory viral infections (RVI) is a risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant, we postulated that RVI can lead to induction of exosomes with self-antigens containing viral DNA/RNA capable of activating innate immune signaling via cGAS/STING and RIG1 pathways, a mechanism leading to immune activation resulting in CLAD. Methods Exosomes were isolated using ultracentrifugation. Size (50-200nm) was determined using nanosight. DNA and RNA were isolated using kits and quantified on the Nanodrop. Libraries were generated using Kapa Biosystem's library kit. The raw Illumina 2x150bp pair-end reads were checked on FastQC and were aligned to the human and viral genome build from CHIPseeker Database. Validation was done using antibodies and primers for respiratory syncytial virus, coronavirus, and rhinovirus. To determine the role of exosomes to induce cell signaling and endoplasmic stress (ER), airway epithelial cells (KCC266), and Hep2 cells were incubated with exosomes from LTxRs with RVI or stable. Results Viral nucleic acid sequences were noted in higher levels in exosomes from LTxRs with RVI in comparison to stable. Comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in RVI LTxRs. Further, we identified upregulation of proteins associated with cGAS/STING and RIG1 (MAVS, MDA5, IFNβ) and ER stress (PERK, ATF4 and BiP) in KCC266 and Hep2 cells incubated with exosomes from LTxRs with RVI, but not stable. In contrast, exosomes from stable LTxRs had 91 sequences for MAP kinase and cell death signaling pathways. Conclusion We conclude that LTxRs diagnosed with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling and ER stress. This can lead to activate innate immune signaling via cGAS/STING and RIG1 pathways resulting in immune responses to viral and donor antigens resulting in CLAD.

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