Circulating Exosomes from Lung Transplant Recipients Diagnosed with Bronchiolitis Obliterans Syndrome Induces Immune Responses in C57BL/6 Mice Leading to Obliterative Airway Disease and miRNA 155 Plays an Essential Role in Immune Activation

Abstract

Purpose MicroRNAs (miRNAs) are single-strand RNAs that are 18-25 nucleotides in length. Genes of miRNAs suppress newly synthesized proteins by causing miRNA degradation or inhibiting miRNA translation into proteins. MicroRNA-155 (miR155) has also been implicated as a positive regulator of inflammatory cytokine production especially IL17. miR155 is a pivotal regulator of the immune system and we determined the role of miR155 in immune activation of C57BL/6 and miR155 knock out (KO) mice leading to development of Abs and induction of exosomes with lung self-antigens (SAgs) following immunization with circulating exosomes isolated from human lung transplant recipients (LTxRs). Methods C57BL/6 and miR155 KO mice were immunized with exosomes isolated from stable and bronchiolitis obliterans syndrome (BOS) without Freund's adjuvant (20ug/injection, at 1,6,15 and 21 days). After last injection, mice were sacrificed, lungs were analyzed by H&E and trichrome staining. Blood samples collected at days 2,7,16 and 26, were analyzed for the development of Abs to K-alpha 1 Tubulin (KA1T), Collagen V (Col-V) and Col-I (control) by ELISA. Results Cellular immune responses to KA1T, Col-V, and Col-I were enumerated using ELISPOT. Circulating exosomes were isolated and analyzed for lung SAgs by western blot. Our results demonstrate development of Abs to lung SAgs with increased frequencies of IFN-γ and IL-17-producing cells with reduction of IL-10 producing cells (stable vs BOS) against lung-SAgs-following immunization of wild type C57BL/6 animals with BOS exosomes but not exosomes from stable LTxRs. Histological analysis of lungs demonstrated obliterative airway disease (OAD) following immunization with exosomes isolated from BOS but not stable. In contrast, immunization of miR155 KO mice did not result in Abs to lung SAgs, cellular immune response to lung SAgs, nor induction of circulating exosomes with lung SAgs (control vs stable and BOS). These animals also did not develop OAD lesions in the lungs. Conclusion We conclude that circulating exosomes plays a pivotal role in immune activation leading to chronic rejection and miR155 exerts an obligatory role in the pathogenesis of chronic rejection following human lung transplantation.