Circulating exosomes from human lung transplant recipients with respiratory viral infections contain nucleic acids potential to activate innate immune signaling (cGAS/STING and RIG-1 Pathways)

Abstract

Abstract Exosomes are 50–200nm size vesicles released by cells into the body fluids. We reported the presence of circulating exosomes with lung self-antigens (SAgs) (Collagen-V,K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Respiratory viral infections (RVI) is a known risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant. We postulated immune mechanism by which RVI increases risk for CLAD is by induction of exosomes with SAgs containing viral DNA/RNA capable of activating innate immune signaling via DNA/RNA sensing. Exosomes were isolated using ultracentrifugation. Exosomal DNA and RNA were used to generate libraries using Kapa Biosystem’s kit. Illumina 2×150bp pair-end reads were checked on FastQC, aligned to human/viral genome from CHIP seeker Database. Role of exosomes inducing cell signaling in human airway epithelial cells (KCC266) and Hep2 were analyzed by incubating exosomes from LTxRs with RVI or stable. Viral nucleic acid sequences were higher in exosomes from LTxRs with RVI while comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in exosomes from RVI. We observed upregulation of proteins associated with cGAS/STING and RIG1 in KCC266 and Hep2 cells (STING; 1.5, cGAS; 1.9, pIRF3; 2.5, pTBK; 3.1, MAVS; 1.7 and IFNβ; 3.8 fold respectively) incubated with exosomes from LTxRs with RVI. We conclude that LTxRs with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling. This can lead to activation of innate immune signaling resulting in adaptive immune responses to viral and donor antigens resulting in CLAD.