Restrictive allograft syndrome vs bronchiolitis obliterans syndrome: Immunological and molecular characterization of circulating exosomes

Abstract

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.

Keywords: bronchiolitis obliterans syndrome; exosomes; lung transplantation; restrictive allograft syndrome.

Figure 1:

(A) Western blot of exosomal proteins, transcription factors CIITA, NFkB, 20S proteasome and PIGR, from LTxRs with stable LTxRs (S1-S3), or RAS (R1-R4) and BOS (B1-B4). (B) Densitometry and statistical analysis of western blots for stable n=5, RAS n=13 and BOS n=14. CD9 in the figure is used to normalize the blots for CIITA, NFkB, PIGR and 20S proteasome. All the graphs are represented as dot plots with Mean (Longest horizontal line) and SD (Vertical Bar Line). *p<0.05, **p<0.01 and ***p<0.001. BOS, bronchiolitis obliterans syndrome; RAS, restrictive allograft syndrome

Figure 2:

(A) Western blot of exosomal proteins HLA-DQ, HLA-DR and class I (W6/32) from LTxRs with stable LTxRs (S1-S3), or RAS (R1-R4) and BOS (B1-B4). (B) Densitometry and statistical analysis of western blots for stable n=5, RAS n=13 and BOS n=14. CD9 given in the figure is used to normalize the blots for HLA-DQ, HLA-DR and W6/32. All the graphs are represented as dot plots with Mean (Longest horizontal line) and SD (Vertical Bar Line). p<0.05, **p<0.01 and ***p<0.001. BOS, bronchiolitis obliterans syndrome; RAS, restrictive allograft syndrome.

Figure 3:

(A) Western blot of exosomal proteins, lung SAgs (Col-V, Kα1T), from LTxRs with stable LTxRs (S1-S3), or RAS (R1-R4) and BOS (B1-B4). (B) Densitometry and statistical analysis of western blots for stable n=5, RAS n=13 and BOS n=14. CD9 given in the figure is used to normalize the blots for Col-V and Kα1 Tubulin. All the graphs are represented as dot plots with Mean (Longest horizontal line) and SD (Vertical Bar Line).*p<0.05, **p<0.01 and ***p<0.001. BOS, bronchiolitis obliterans syndrome; RAS, restrictive allograft syndrome.

Figure 4:

Plasma cytokine levels from LTxRs without CLAD (stable) or diagnosed with BOS or RAS. LTxRs with RAS have higher levels of inflammatory cytokines IL-2R (p=0.0001) and MIG (p=0.0104) compared to LTxRs diagnosed with BOS. Analyzed by Luminex assay. . All the graphs are represented as dot plots with Mean (Longest horizontal line) and SD (Vertical Bar Line). *p<0.05, **p<0.01 and ***p<0.001. BOS, bronchiolitis obliterans syndrome; RAS, restrictive allograft syndrome.

Figure 5:

(A) Heat map for miRNA profiling from RAS and BOS exosomes. (B) Nested plot for miRNAs higher in BOS samples. (C) Plot for miRNAs present in higher amounts in RAS samples. Only significantly differentially expressed miRNAs are shown. All the graphs represented as Nested Plots with Mean and SD (Vertical Bar Line). *p<0.05, **p<0.01 and ***p<0.001. *p<0.05, **p<0.01 and ***p<0.001. BOS, bronchiolitis obliterans syndrome; miRNA, micro RNA; RAS, restrictive allograft syndrome.

Figure 6:

(A) ELISA of serum lung SAg Col-V at D7 and D14 from mice immunized with exosomes isolated from LTxRs with RAS or BOS. (B) EliSPOT values for cytokine producing cells. Graphs in A and B are represented as dot plot with bar, dots as individual values with Mean (represented by Bar height) and SD (Vertical Bar Line).*p<0.05, **p<0.01 and ***p<0.001. (C Trichrome staining of lungs from mice immunized with exosomes isolated from LTxRs with BOS or RAS. Interstitial and inflammatory infiltrates and fibrosis was more prominent in mice injected with LTxRs with RAS compared to mice injected with exosomes from BOS LTxRs. Specific lesions in lungs are marked with red arrows. Images were obtained on a Leica microscope at 40X, and morphometric analysis was performed using Aperio ImageScope software (Leica). Scale bar = 60μm. Lungs from mice immunized with RAS exosomes have inflammation which is predominantly mononuclear and not acute. Based on morphology inflammation is predominantly due to macrophages and lymphocytes and not mediated by neutrophils. BOS, bronchiolitis obliterans syndrome; RAS, restrictive allograft syndrome.