Original Pre-Clinical Science
Restrictive allograft syndrome vs bronchiolitis obliterans syndrome: Immunological and molecular characterization of circulating exosomes

https://doi.org/10.1016/j.healun.2021.09.001Get rights and content

Background

Chronic lung allograft dysfunction in lung transplant recipients (LTxRs) has 2 phenotypes: obstructive bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Our goal was to define distinct immunologic markers of exosomes from LTxRs with BOS or RAS.

Methods

Plasma was collected from LTxRs with BOS (n = 18), RAS (n = 13), and from stable LTxRs (n = 5). Antibodies to lung self-antigens (SAgs) were determined by ELISA. Exosomes were isolated by ultracentrifugation. Donor specific antibodies to HLA were quantified using Luminex. Exosomes were characterized for lung SAgs, transcription factors, 20S proteasome, HLA class I and II, and polymeric immunoglobulin receptor protein using western blot. Exosome miRNA was analyzed using NanoString. The exosome-induced immune response was determined in mice.

Results

LTxRs with RAS, but not BOS, had donor specific antibodies at diagnosis. CIITA, NFkB, polymeric immunoglobulin receptor protein, 20S proteasome, HLA-DQ, and HLA-DR were significantly higher in RAS exosomes than in BOS exosomes. RAS plasma had high levels of proinflammatory cytokines and distinct exosomal miRNA. Immunization of C57BL/6 mice with RAS exosomes showed severe inflammation and peribronchial fibrosis, whereas BOS exosomes induced patchy inflammation and fibrosis.

Conclusion

LTxRs with BOS or RAS had exosomes with distinct molecular and immunologic profiles. RAS samples had a higher concentration of proinflammatory factors, HLA class II, lung SAgs, and antibodies to HLA class II molecules, indicating severe allograft injury. Mice immunized with RAS exosomes developed lesions in airways, pleura, interlobular septum, and alveoli, whereas BOS exosomes induced mild to patchy inflammation with lung fibrosis.

Section snippets

Study population

We retrospectively analyzed 18 LTxRs with BOS, 13 with RAS and 5 time-matched stable LTxRs (controls). All patients underwent LTx at St. Joseph's Hospital and Medical Center, Phoenix, Arizona, had plasma available, and consented to participate. The Institutional Review Board approved this study (IRB# PHXB16-0027-10-18). Patient demographics, transplant, and laboratory data were collected via chart abstraction (Table S1). CLAD (BOS or RAS) was diagnosed by spirometry and radiographic findings

Lung transplant recipients

Patient demographics including age, sex, ethnicity, and underlying diagnosis were not significantly different between LTxRs diagnosed with RAS or BOS (Table S1). All patients had sufficiently long survival to have a certain CLAD phenotype (BOS/RAS) and an impact on the analysis (i.e., 1 year). Samples selected for BOS and RAS patients are at the time of diagnosis. All BOS samples selected for the current study fall within 1-3 years of transplant after the diagnosis of BOS (>1 year of

Discussion

De novo development of DSA has been associated with risk for developing CLAD.20 Recent data suggests that not only de novo DSA but also antibody-mediated rejection is potential risk factors for the RAS phenotype of CLAD.21,22 LTxRs with persistent DSAs are also at higher risk for the RAS phenotype of CLAD.23 Studies have demonstrated an association of RAS with DSAs against mismatched donor HLA class II molecules, especially HLA-DQ.24,25 In this study, we demonstrated that exosomes isolated from

Author contributions

Concept and design: TM; Acquisition, analysis, or interpretation of data: SB ST, AA and TM; Drafting/writing of the manuscript: SB, ST and TM; Technical or material support: CP, SP, AA, ST, RMB, MAS.

Funding sources

This work was supported by grants from the National Institutes of Health AI123034, HL056643, HL092514 (TM).

Declaration of competing interest

The authors declare no conflict of interest. All authors have reviewed and approved the manuscript and have contributed in a substantial and intellectual manner to the work.

Acknowledgments

We would like to thank Kristina Sanborn for processing the blood samples. We also would like to acknowledge Billie Glasscock and Kristina Nally for their assistance in preparing this manuscript. We would like to thank Kiran Girdhar, Icahn School of Medicine, for helping with Statistical analysis.

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