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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: J Heart Lung Transplant. 2020 Sep 28;39(12):1476–1490. doi: 10.1016/j.healun.2020.09.012

MicroRNA-206 Antagomir Enriched Extracellular Vesicles Attenuate Lung Ischemia-Reperfusion Injury Via CXCL1 Regulation in Alveolar Epithelial Cells

Jun Cai 1, Ricardo Gehrau 2, Zhenxiao Tu 3, Victoria Leroy 1, Gang Su 1, Junyi Shang 1, Valeria R Mas 4, Amir Emtiazjoo 5, Andres Pelaez 5, Carl Atkinson 6, Tiago Machuca 1, Gilbert R Upchurch Jr 1, Ashish K Sharma 1,5
PMCID: PMC7704771  NIHMSID: NIHMS1632953  PMID: 33067103

Abstract

Background:

Our hypothesis is that the immunomodulatory capacities of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) can be enhanced by specific microRNAs to effectively attenuate post-transplant lung ischemia-reperfusion (IR) injury.

Methods:

Expression of miR-206 was analyzed in BAL fluid of patients on days 0 and 1 post-lung transplantation. Lung IR injury was evaluated in C57BL/6 mice using a left lung hilar-ligation model with or without treatment with EVs or antagomiR-206 enriched EVs. Murine lung tissue was used for microRNA microarray hybridization analysis, and cytokine expression, lung injury and edema were evaluated. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by enriched EVs against lung IR injury. In vitro studies analyzed type II epithelial cell activation after co-cultures with EVs.

Results:

A significant upregulation of miR-206 was observed in BAL fluid on day 1 compared to day 0 in post-lung transplant patients, and in murine lungs after IR injury compared to sham. Treatment with antagomiR-206-enriched EVs attenuated lung dysfunction, injury and edema compared to treatment with EVs alone after murine lung IR injury. Enriched EVs reduced lung injury and neutrophil infiltration as well as improved allograft oxygenation after murine orthotopic lung transplantation. Enriched EVs significantly decreased proinflammatory cytokines, especially epithelial cell-dependent CXCL1 expression, in the in vivo and in vitro IR injury models.

Conclusion:

EVs can be used as biomimetic nanovehicles for protective immunomodulation by enriching them with antagomiR-206, to mitigate epithelial cell activation and neutrophil infiltration in lungs after IR injury.

Introduction

Despite advances in surgical management and immuno-suppression, outcomes for lung transplantation continue to be dismal for solid organ transplants as approximately 60% of transplant patients die within 5 years (1–3). Ischemia-reperfusion (IR) injury causes significant mortality and morbidity in the early post-operative period and primary graft dysfunction is reported to be an independent predictive factor for the development and progression of bronchiolitis obliterans syndrome, which is the most common cause of death after lung transplantation (4). Currently no therapeutic agents are clinically available to prevent IR injury, and treatment strategies are limited to maintaining oxygenation and lung function.

Recent studies have demonstrated that extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) exhibit immunomodulatory capabilities to mitigate pulmonary inflammation including lung ischemia-reperfusion injury (5–7). MSCs release extracellular vesicles (EVs), such as exosomes and microvesicles, which immunomodulate target cells by intercellular interactions or paracrine mechanisms and transfer proteins, RNA, and/or lipids to initiate cellular signaling cascade events (8, 9). EVs originating from MSCs are small vesicles that carry membrane and cytoplasmic constituents of the cells from which they originate and have been shown to shuttle mRNA and microRNAs (miRNAs), thereby playing a critical role in the continuum model of stem cell biology (10).

miRNAs are noncoding, small RNAs (21–23 nt) that bind to the 3’ UTR region of specific mRNA targets, and are potent regulators of diverse biologic and pathophysiologic processes i.e. inflammation and tissue injury (11–13). EVs have been reported to shuttle specific patterns of miRNAs to modulate cell signaling and biological responses and have been implicated in dysregulation of gene expression in signaling pathways associated with lung injury and human lung transplantation (7, 12, 14, 15). However, the role of specific miRNAs in modulating pulmonary inflammation and IR injury and its correlation with MSC-derived EVs remains to be defined.

In this study, we delineated the role of antagomiR-206 enriched EVs for protection against pulmonary inflammation and IR injury using human BAL samples, two murine experimental IR injury models as well as in vitro studies. Using the information from miRNA microarray, we observed that the immunomodulation of EVs can be enhanced by using specific inhibition of miR-206 (antagomiR-206), to target epithelial cell-specific chemokine CXCL1, that mitigates transendothelial migration of neutrophils and lung IR injury.

Materials and Methods (detailed Methods are described in the supplement)

Lung IR injury model

An in vivo hilar ligation model of lung IR was used, as previously described (16, 17).

Microarray analysis of miR expression

Murine left lung tissue was harvested after IR or respective controls (sham) and RNA purity quality control analysis was performed with previous established parameters (18).

Donation after circulatory death and orthotopic lung transplantation

Lungs were harvested from donation after circulatory death (DCD) donors, as previously described (19). Lung transplants were performed utilizing cuff techniques into C57Bl/6 recipient mice, as previously described (20).

Results

Differentially expressed microRNAs after lung IR injury

The expression of miR-206 was significantly increased in the BAL fluid of post-lung transplant patients on day 1 compared to day 0 (Fig. 1A). Pairwise comparison analyses was performed in murine lung tissue using the in vivo murine hilar ligation model of IR injury to identify differentially expressed miRNA profiles. Intensity value analysis of hybridized microarrays detected 449 miRNA probesets (Supplementary Table S1). Briefly, 74 miRNA probesets were observed to be significantly differentially expressed between IR compared to sham. Of those, 34 miRNAs were found to be upregulated and 40 miRNAs were down regulated after lung IR injury (Table 1). The highest upregulation was observed in miR-206 expression that showed ~50-fold increase after IR compared to sham (Fig. 1B–C).

Figure 1.

Figure 1.

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Identification of miRNAs involved in lung IR injury. A. Increased expression of miR-206 in BAL fluid of patients on day 1 post-lung transplantation compared to day 0 was observed. n=4-5/group; *p=0.007. B. Total RNA obtained from lung tissue of WT mice from the hilar ligation model of IR injury was analyzed by miRNA microarray. Hierarchical cluster analysis of lung IR injury compared to sham is shown by heat map for the highly regulated miRNAs. Each column represents the respective sample, and each row represents a single miR probe. C. The most significant increase was observed in miR-206 expression in the lung tissue after IR compared to sham. n=5/group; #p=0.006.

Table 1.

Pairwise comparison analysis results of miRNA microarray.

Probe Set miRNA ID IRI Mean (Log2) SHAM Mean (Log2) IRI vs. Sham† p-value (α-level: 0.01) q-value* FDR*
20500414 mmu-miR-206-3p 7.17 1.51 50.8 0.00694 2.91E-02 2.9
20506730 mmu-miR-669f-5p 6.33 4.48 3.6 0.00211 1.56E-02 1.6
20500895 mmu-miR-23a-5p 7.69 6.20 2.8 0.00028 7.41E-03 0.7
20515417 mmu-miR-3082-5p 5.88 4.44 2.7 0.00011 5.56E-03 0.6
20501101 mmu-miR-139-5p 9.80 8.41 2.6 0.00000 2.73E-04 0.0
20515427 mmu-miR-466m-5p 6.23 4.92 2.5 0.00281 1.75E-02 1.8
20510757 mmu-miR-669m-5p 6.23 4.92 2.5 0.00281 1.75E-02 1.8
20500906 mmu-miR-27a-5p 7.50 6.25 2.4 0.00142 1.20E-02 1.2
20500243 mmu-miR-23b-5p 4.69 3.45 2.4 0.00766 3.05E-02 3.1
20519081 mmu-miR-3473b 11.14 10.04 2.1 0.00658 2.86E-02 2.9
20506724 mmu-miR-466i-5p 5.89 4.86 2.0 0.00255 1.72E-02 1.7
20511664 mmu-miR-2137 12.35 11.37 2.0 0.00206 1.56E-02 1.6
20500392 mmu-miR-195a-3p 7.31 6.33 2.0 0.00019 5.82E-03 0.6
20505730 mmu-miR-574-5p 7.69 6.76 1.9 0.00019 5.82E-03 0.6
20528551 mmu-miR-7666-3p 6.49 5.57 1.9 0.00071 1.03E-02 1.0
20520386 mmu-miR-5130 10.91 10.11 1.7 0.00086 1.03E-02 1.0
20504679 mmu-miR-423 -5p 8.01 7.25 1.7 0.00881 3.15E-02 3.2
20504675 mmu-miR-677-3p 7.45 6.72 1.7 0.00041 7.41E-03 0.7
20529964 mmu-miR-8101 9.69 8.99 1.6 0.00093 1.03E-02 1.0
20501111 mmu-miR-214-3p 11.58 10.89 1.6 0.00989 3.25E-02 3.2
20510740 mmu-miR-1941 -5p 7.08 6.42 1.6 0.00630 2.78E-02 2.8
20525826 mmu-miR-6932-3p 7.02 6.40 1.5 0.00962 3.20E-02 3.2
20501122 mmu-miR-320-3p 12.14 11.52 1.5 0.00711 2.91E-02 2.9
20500276 mmu-miR-132-3p 9.51 8.92 1.5 0.00081 1.03E-02 1.0
20500960 mmu-miR-330-3p 7.49 6.90 1.5 0.00487 2.28E-02 2.3
20525896 mmu-miR-6966-3p 6.51 5.99 1.4 0.00350 1.97E-02 2.0
20519047 mmu-miR-3960 13.22 12.75 1.4 0.00902 3.15E-02 3.2
20500995 mmu-miR-342-5p 7.60 7.19 1.3 0.00344 1.97E-02 2.0
20504680 mmu-miR-423 -3p 8.59 8.21 1.3 0.00361 1.97E-02 2.0
20501145 mmu-miR-125b-1 -3p 6.14 5.76 1.3 0.00296 1.80E-02 1.8
20504737 mmu-miR-709 14.95 14.62 1.3 0.00135 1.20E-02 1.2
20501104 mmu-miR-200c-3p 13.60 13.31 1.2 0.00454 2.28E-02 2.3
20500952 mmu-miR-328-3p 7.67 7.45 1.2 0.00907 3.15E-02 3.2
20500380 mmu-miR-24-3p 15.35 15.22 1.1 0.00812 3.07E-02 3.1
20500896 mmu-miR-23a-3p 14.98 15.08 −1.1 0.00324 1.92E-02 1.9
20500308 mmu-miR-152-3p 11.95 12.18 −1.2 0.00477 2.28E-02 2.3
20500429 mmu-miR-143-3p 14.12 14.36 −1.2 0.00144 1.20E-02 1.2
20500249 mmu-miR-30a-5p 13.66 13.93 −1.2 0.00264 1.74E-02 1.7
20500916 mmu-miR-34a-5p 9.65 9.99 −1.3 0.00822 3.07E-02 3.1
20500253 mmu-miR-99a-5p 12.20 12.54 −1.3 0.00041 7.41E-03 0.7
20500964 mmu-miR-331-3p 6.02 6.44 −1.3 0.00947 3.20E-02 3.2
20501129 mmu-miR-221 -3p 11.97 12.41 −1.4 0.00180 1.41E-02 1.4
20500397 mmu-miR-199a-5p 10.03 10.47 −1.4 0.00103 1.08E-02 1.1
20500860 mmu-miR-192-5p 8.61 9.07 −1.4 0.00115 1.11E-02 1.1
20500246 mmu-miR-27b-3p 12.70 13.17 −1.4 0.00107 1.08E-02 1.1
20504677 mmu-miR-497-5p 10.49 11.03 −1.4 0.00416 2.20E-02 2.2
20501094 mmu-miR-25-3p 10.19 10.77 −1.5 0.00518 2.33E-02 2.3
20504624 mmu-miR-1843a-5p 6.54 7.14 −1.5 0.00125 1.17E-02 1.2
20500292 mmu-miR-141-3p 9.24 9.86 −1.5 0.00036 7.41E-03 0.7
20519054 mmu-miR-1843b-5p 6.51 7.19 −1.6 0.00796 3.07E-02 3.1
20500903 mmu-miR-29a-3p 12.60 13.30 −1.6 0.00498 2.28E-02 2.3
20500272 mmu-miR-130a-3p 10.81 11.51 −1.6 0.00009 5.56E-03 0.6
20504702 mmu-miR-146b-5p 8.96 9.66 −1.6 0.00885 3.15E-02 3.2
20500289 mmu-miR-140-5p 7.74 8.47 −1.7 0.00493 2.28E-02 2.3
20500852 mmu-miR-19b-3p 10.55 11.29 −1.7 0.00365 1.97E-02 2.0
20500251 mmu-miR-30b-5p 11.73 12.49 −1.7 0.00814 3.07E-02 3.1
20500882 mmu-miR-15a-5p 10.04 10.82 −1.7 0.00093 1.03E-02 1.0
20501109 mmu-miR-181a-1-3p 5.41 6.19 −1.7 0.00043 7.41E-03 0.7
20500859 mmu-miR-148a-3p 7.07 7.86 −1.7 0.00462 2.28E-02 2.3
20500866 mmu-miR-200a-3p 10.89 11.73 −1.8 0.00009 5.56E-03 0.6
20500909 mmu-miR-31-3p 5.83 6.74 −1.9 0.00718 2.91E-02 2.9
20501017 mmu-miR-350-3p 6.03 6.98 −1.9 0.00471 2.28E-02 2.3
20500430 mmu-miR-30e-5p 9.41 10.36 −1.9 0.00243 1.69E-02 1.7
20500905 mmu-miR-29c-3p 7.32 8.36 −2.1 0.00705 2.91E-02 2.9
20505770 mmu-miR-872-3p 5.91 7.06 −2.2 0.00019 5.82E-03 0.6
20501135 mmu-miR-199b-5p 7.32 8.49 −2.2 0.00031 7.41E-03 0.7
20519050 mmu-miR-28c 5.26 6.44 −2.3 0.00951 3.20E-02 3.2
20500650 mmu-miR-301a-3p 5.88 7.11 −2.4 0.00244 1.69E-02 1.7
20500248 mmu-miR-29b-3p 6.08 7.45 −2.6 0.00080 1.03E-02 1.0
20500655 mmu-miR-34b-5p 6.35 7.75 −2.6 0.00068 1.03E-02 1.0
20501792 mmu-miR-411 -5p 4.13 5.58 −2.7 0.00867 3.15E-02 3.2
20504643 mmu-miR-1298-5p 4.33 5.85 −2.9 0.00007 5.56E-03 0.6
20504717 mmu-miR-455-5p 3.72 5.69 −3.9 0.00177 1.41E-02 1.4
20501204 mmu-miR-362-3p 2.85 4.97 −4.4 0.00090 1.03E-02 1.0
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†

Fold-change calculation method: 2^ (IRI - Sham).

*

False Discovery Rate (FDR) calculated, as percent value from estimated q-values was controlled fewer than 5% for significance purpose.

Ontology analysis of miRNAs after lung IR injury was performed using IPA (Ingenuity Pathway Analysis) that identified 63 miRNAs represented families, 9 of those (miR-130a-3p, −141-3p, −148b-3p, −16-5p, −199a-5p, −29b-3p, −30c-5p, −34a-5p, −669m-5p) represented by at least two different miRNAs (Table 2). Analysis of cellular toxic functions using IPA database was performed and disease and disorder analysis was restricted to immunological disease, inflammatory disease, inflammatory response, and connective tissue disorders to illustrate the relevant molecular process of lung IR injury. In total, 26 miRNAs revealed functional relevance with those selected disease and disorders (Table 3). All selected disease and disorders revealed significant association with the miRNA profile (Fig. 2A). Interestingly, 9 miRNAs were observed to be common among those disease and disorders (Fig. 2B).

Table 2.

Differentially expressed miRNAs after lung IR injury

miRNA ID MiRNA family symbol IRI vs. Sham (Fold Change)
mmu-miR-206-3p miR-1 (and other miRNAs w/seed GGAAUGU) 50.8
mmu-miR-99a-5p miR-100-5p (and other miRNAs w/seed ACCCGUA) −1.3
mmu-miR-125b-1-3p miR-125b-1-3p (miRNAs w/seed CGGGUUA) 1.3
mmu-miR-1298-5p miR-1298-5p (and other miRNAs w/seed UCAUUCG) −2.9
mmu-miR-130a-3p miR-130a-3p (and other miRNAs w/seed AGUGCAA) −1.6
mmu-miR-301a-3p −2.4
mmu-miR-132-3p miR-132-3p (and other miRNAs w/seed AACAGUC) 1.5
mmu-miR-139-5p miR-139-5p (miRNAs w/seed CUACAGU) 2.6
mmu-miR-140-5p miR-140-5p (and other miRNAs w/seed AGUGGUU) −1.7
mmu-miR-141-3p miR-141-3p (and other miRNAs w/seed AACACUG) −1.5
mmu-miR-200a-3p −1.8
mmu-miR-143-3p miR-143-3p (and other miRNAs w/seed GAGAUGA) −1.2
mmu-miR-146b-5p miR-146a-5p (and other miRNAs w/seed GAGAACU) −1.6
mmu-miR-152-3p miR-148b-3p (and other miRNAs w/seed CAGUGCA) −1.2
mmu-miR-148a-3p −1.7
mmu-miR-497-5p miR-16-5p (and other miRNAs w/seed AGCAGCA) −1.5
mmu-miR-15a-5p −1.7
mmu-miR-181a-1-3p miR-181a-1-3p (and other miRNAs w/seed CCAUCGA) −1.7
mmu-miR-1843a-5p miR-1843-5p (and other miRNAs w/seed AUGGAGG) −1.5
mmu-miR-1843b-5p miR-1843b-5p (miRNAs w/seed UGGAGGU) −1.6
mmu-miR-192-5p miR-192-5p (and other miRNAs w/seed UGACCUA) −1.4
mmu-miR-1941-5p miR-1941-5p (miRNAs w/seed GGGAGAU) 1.6
mmu-miR-195a-3p miR-195-3p (and other miRNAs w/seed CAAUAUU) 2.0
mmu-miR-199a-5p miR-199a-5p (and other miRNAs w/seed CCAGUGU) −1.4
mmu-miR-199b-5p −2.2
mmu-miR-19b-3p miR-19b-3p (and other miRNAs w/seed GUGCAAA) −1.7
mmu-miR-200c-3p miR-200b-3p (and other miRNAs w/seed AAUACUG) 1.2
mmu-miR-214-3p miR-214-3p (and other miRNAs w/seed CAGCAGG) 1.6
mmu-miR-221-3p miR-221-3p (and other miRNAs w/seed GCUACAU) −1.4
mmu-miR-23a-3p miR-23a-3p (and other miRNAs w/seed UCACAUU) −1.1
mmu-miR-23a-5p miR-23a-5p (and other miRNAs w/seed GGGUUCC) 2.8
mmu-miR-23b-5p miR-23b-5p (miRNAs w/seed GGUUCCU) 2.4
mmu-miR-24-3p miR-24-3p (and other miRNAs w/seed GGCUCAG) 1.1
mmu-miR-6932-3p miR-26b-3p (and other miRNAs w/seed CUGUUCU) 1.5
mmu-miR-27b-3p miR-27a-3p (and other miRNAs w/seed UCACAGU) −1.4
mmu-miR-27a-5p miR-27a-5p (miRNAs w/seed GGGCUUA) 2.4
mmu-miR-28c mir-28 −2.3
mmu-miR-29a-3p miR-29b-3p (and other miRNAs w/seed AGCACCA) −1.6
mmu-miR-29c-3p −2.1
mmu-miR-29b-3p −2.6
mmu-miR-3082-5p miR-3082-5p (and other miRNAs w/seed ACAGAGU) 2.7
mmu-miR-30a-5p miR-30c-5p (and other miRNAs w/seed GUAAACA) −1.2
mmu-miR-30b-5p −1.7
mmu-miR-30e-5p −1.9
mmu-miR-31-3p miR-31-3p (and other miRNAs w/seed GCUAUGC) −1.9
mmu-miR-320-3p miR-320b (and other miRNAs w/seed AAAGCUG) 1.5
mmu-miR-328-3p miR-328-3p (and other miRNAs w/seed UGGCCCU) 1.2
mmu-miR-362-3p miR-329-3p (and other miRNAs w/seed ACACACC) −4.4
mmu-miR-330-3p miR-330-3p (and other miRNAs w/seed CAAAGCA) 1.5
mmu-miR-331-3p miR-331-3p (miRNAs w/seed CCCCUGG) −1.3
mmu-miR-342-5p miR-342-5p (and other miRNAs w/seed GGGGUGC) 1.3
mmu-miR-3473b mir-3473 2.1
mmu-miR-34a-5p miR-34a-5p (and other miRNAs w/seed GGCAGUG) −1.3
mmu-miR-34b-5p −2.6
mmu-miR-350-3p miR-350 (and other miRNAs w/seed UCACAAA) −1.9
mmu-miR-3960 mir-3960 1.4
mmu-miR-411-5p miR-411-5p (and other miRNAs w/seed AGUAGAC) −2.7
mmu-miR-423-3p miR-423-3p (miRNAs w/seed GCUCGGU) 1.3
mmu-miR-423-5p miR-423-5p (and other miRNAs w/seed GAGGGGC) 1.7
mmu-miR-455-5p miR-455-5p (and other miRNAs w/seed AUGUGCC) −3.9
mmu-miR-466i-5p miR-466d-5p (and other miRNAs w/seed GUGUGUG) 2.0
mmu-miR-574-5p miR-574-5p (miRNAs w/seed GAGUGUG) 1.9
mmu-miR-669f-5p miR-669a-5p (and other miRNAs w/seed GUUGUGU) 3.6
mmu-miR-669m-5p miR-669m-5p (and other miRNAs w/seed GUGUGCA) 2.5
mmu-miR-466m-5p 2.5
mmu-miR-677-3p miR-677-3p (miRNAs w/seed AAGCCAG) 1.7
mmu-miR-6966-3p miR-6966-3p (miRNAs w/seed GCUGUGU) 1.4
mmu-miR-7666-3p miR-7666-3p (miRNAs w/seed AUGCAGC) 1.9
mmu-miR-872-3p miR-872-3p (miRNAs w/seed GAACUAU) −2.2
mmu-miR-25-3p miR-92a-3p (and other miRNAs w/seed AUUGCAC) −1.5
mmu-miR-2137 Mir2137 2.0
mmu-miR-5130 Mir5130 1.7
mmu-miR-709 Mir709 1.3
mmu-miR-8101 Mir8101 1.6
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Table 3.

Altered miRNAs during lung IR injury associated with selected disease and disorders.

Disease and Disorder miRNA
Immunological Disease miR-140-5p, miR-24-3p, miR-30c-5p, miR-29b-3p, miR-143-3p, miR-148b-3p, miR-92a-3p, miR-192-5p, miR-16-5p, miR-221-3p, miR-130a-3p, miR-19b-3p, miR-342-5p, miR-141-3p, miR-132-3p
Inflammatory Disease miR-140-5p, miR-146a-5p, miR-100-5p, miR-1, miR-199a-5p, miR-24-3p, miR-30c-5p, miR-23a-3p, miR-29b-3p, miR-27a-3p, miR-143-3p, miR-200b-3p, miR-92a-3p, miR-34a-5p, miR-423-3p, miR-423-5p, miR-16-5p, miR-320b, miR-19b-3p, miR-221-3p, miR-130a-3p, miR-141-3p
Inflammatory Response miR-140-5p, miR-100-5p, miR-199a-5p, miR-30c-5p, miR-23a-3p, miR-27a-3p, miR-29b-3p, miR-143-3p, miR-200b-3p, miR-92a-3p, miR-423-3p, miR-423-5p, miR-16-5p, miR-320b, miR-19b-3p, miR-221-3p, miR-130a-3p
Connective Tissue Disorders miR-140-5p, miR-146a-5p, miR-1, miR-199a-5p, miR-30c-5p, miR-24-3p, miR-29b-3p, miR-143-3p, miR-92a-3p, miR-34a-5p, miR-423-3p, miR-16-5p, miR-423-5p, miR-19b-3p, miR-141-3p
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Figure 2.

Figure 2.

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Correlative analysis of molecular processses associated with altered profile of miRNAs after lung IR injury. A. Biological interpretation of diseases and disorders after Ingenuity Pathway Analysis depicting cellular processes associated with lung IR injury. Threshold value (orange line) indicate -log for a p-value of 0.05. B. List of common miRNAs that are altered after lung IR injury and associated with relevant diseases and disorders are depicted.

Pulmonary dysfunction after IR is attenuated by MSC-derived EVs

Since the expression of miR-206 was highly upregulated in human lung transplants and murine lung IR injury model, we used EVs from MSCs transfected with miR-206 mimic or inhibitor in the in vivo mouse model (Fig. 3A). The mean size (131 ±1.5 nm) and particle concentration of EVs were calculated by the Nanoparticle Tracking Analysis software (Supplementary Figure S1). We observed a significant increase in miR-206 expression in EVs from MSCs transfected with miR-206 mimic compared to mimic controls, as well as a significant decrease in miR-206 expression in EVs from MSCs transfected with miR-206 inhibitor compared to inhibitor controls (Supplementary Figure S2). Significant pulmonary dysfunction occurred after IR in WT mice compared to sham as indicated by increased airway resistance (1.82±0.07 vs. 0.54±0.03 cm H2O/μ/sec) and pulmonary artery pressure (14.7±0.5 vs. 5.3±0.1 cm H2O) as well as decreased pulmonary compliance (2.7±0.2 vs. 7.3±0.2 μl/cm H2O) (Fig. 3B–D). Lungs of mice treated with EVs alone or EVs transfected with miR-206 mimic were protected after IR compared to untreated mice as shown by significantly decreased airway resistance (1.11 ±0.03 and 1.09±0.05 vs. 1.82±0.07 cm H2O/μ/sec) and pulmonary artery pressure (9.78±0.3 and 9.73±0.2 cm H2O vs. 14.7±0.5 cm H2O) as well as increased pulmonary compliance (4.2±0.1 and 3.7±0.2 vs. 2.7±0.2 μl/cm H2O). Importantly, mice treated with EVs+antagomir-206 demonstrated further protection against lung IR injury compared to treatment with EVs+mimic miR-206, as observed by decreased airway resistance (0.65±0.03 vs. 1.09±0.05 cm H2O/μ/sec) and pulmonary artery pressure (7.3±0.3 vs. 9.73 ±0.2 cm H2O) as well as decreased pulmonary compliance (5.8±0.18 vs. 3.7±0.28 μl/cm H2O). In addition, sham mice treated with EVs+antagomir-206 did not affect the lung function parameters compared to untreated shams (data not shown). These results show that EVs with antagomir-206 can effectively mitigate lung dysfunction after IR that is significantly better than treatment with EVs alone.

Figure 3.

Figure 3.

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EVs enriched with antagomiR-206 effectively attenuate pulmonary dysfunction after IR injury. A. Schematic of murine lung IR protocol where pulmonary function and injury were measured in WT mice after sham surgery or IR injury. B-D. Significant lung dysfunction occurred after IR as demonstrated by increased airway resistance and pulmonary artery (PA) pressure as well as decreased pulmonary compliance compared to sham controls. Pretreatment with EVs+miR-206 inhibitor (Inh.; antagomiR-206) resulted in significantly reduced lung dysfunction after IR compared to EVs or EVs+miR-206 mimic. n=10/group; *p<0.05 vs. sham; #p<0.05 vs. IR, §p<0.05 vs. IR+EVs or EVs+miR-206 mimic.

Lung injury and inflammation after IR is effectively attenuated by treatment with EVs enriched with antagomiR-206

To determine the protective role of antagomiR-206 enriched EVs on lung injury and edema after IR, neutrophil infiltration in lung tissue, myeloperoxidase (MPO) levels in bronchoalveolar lavage fluid, lung wet/dry ratios and alteration in microvascular permeability were measured (Fig. 4). A marked increase in neutrophil infiltration and MPO levels occurred in WT mice after IR that was blocked by treatment with EVs or EVs+miR-206 mimic after IR compared to untreated mice (Fig. 4A–C). Treatment with antagomiR-206 enriched EVs further attenuated neutrophil infiltration in lung tissue as well as MPO levels in BAL fluid compared to EVs+miR-206 mimic after lung IR injury. Similarly, increased pulmonary edema and microvascular permeability were also reduced after treatment with antagomiR-206 enriched EVs compared to EVs+miR-206 mimic (Fig. 4D–E). Cytokine/chemokine expression were measured to evaluate the effect of EVs+miR-206 inhibition on lung inflammation. We observed a significant decrease in proinflammatory cytokine (IL-17, TNF-α, HMGB1, MCP-1, IL-6, Ml P-1α, RANTES) production after treatment with EVs or EVs+miR-206 mimic in WT mice undergoing IR compared to IR alone (Fig. 5A). Treatment with EVs+antagomiR-206 also attenuated the proinflammatory cytokine production that was comparable to EVs+miR-206 mimic. Importantly, we observed an enhanced and significant reduction in CXCL1 expression after treatment with EVs+antagomiR-206 compared to EVs+miR-206 mimic (Fig. 5B). The expression of keratinocyte growth factor (KGF), prostaglandin E2 (PGE2) and IL-10 was increased in the BAL fluid of mice treated with EVs or EVs+mimic miR-206 after IR compared to IR alone (Fig. 5C). The expression of PGE2 was significantly increased after treatment with EVs+antagomiR-206 compared to treatment with EVs+miR-206 mimic. These results confirm that EVs with antagomiR-206 can effectively attenuate lung inflammation, edema and neutrophil infiltration and activation after IR that is associated with enhanced mitigation of CXCL1 and increased expression of PGE2.

Figure 4.

Figure 4.

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EVs enriched with miR-206 antagomiR decrease neutrophil infiltration and activation as well as lung edema and microvascular permeability after IR injury. A. Representative images showing neutrophil immunostaining in lung sections. Neutrophils are stained red and sections are counterstained with hematoxylin. Scale bars indicate 50pm. B. The number of neutrophils per high power field (HPF) was quantified from immunostained sections. Neutrophil infiltration was significantly attenuated after IR in mice treated with EVs+miR-206 inhibitor (antagomiR-206) compared to EVs or EVs+miR-206 mimic treated mice. C. Myeloperoxidase (MPO) levels in BAL fluid were significantly decreased after IR in mice treated with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. D. Pulmonary edema (lung wet/dry weight) was significantly decreased after IR in mice treated with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. E. Pulmonary vascular permeability was significantly elevated in lungs after IR injury, which was attenuated by treatment with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. n=5-8/group. *p<0.05 vs. sham; #p<0.05 vs. IR, §p<0.05 vs. IR+EVs or EVs+miR-206 mimic.

Figure 5.

Figure 5.

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Lung inflammation after IR is attenuated by antagomiR-206 enriched EVs. A. Proinflammatory cytokine levels (IL-17, TNF-α, HMGB1, MCP-1, IL-6, MIP-1α and RANTES) were significantly attenuated in BAL fluid after IR in mice treated with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. B-C. Expression of CXCL1 was significantly decreased, and prostaglandin E2 (PGE2) was significantly increased, after treatment with EVs+miR-206 mimic. EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic alone. n=5/group; *p<0.05 vs. sham; #p<0.05 vs. IR, §p<0.05 vs. IR+EVs or EVs+miR-206 mimic.

Post-transplant lung injury is significantly ameliorated by EVs enriched with antagomiR-206 in recipients of DCD donors

We have previously shown that EV therapy of DCD donors lungs during preservation enhanced lung endothelial barrier function and the integrity of donor lungs (7, 21). Here we build upon these studies with EVs enriched with antagomiR-206 and determine the impact of DCD therapy on post-transplant outcomes. We treated DCD donors prior to and after static cold storage with EVs enriched with antagomiR-206 or normal saline (NS) to evaluate the impact on lung IR injury (Fig. 6A). Similar to the hilar ligation model, histological analysis showed that EVs+miR-206 inhibitor had a significant reduction in lung IR injury compared to EVs+miR-206 mimic after 24hrs post-transplantation (Supplementary Fig. 3A). There was a trend towards increase in Pa02 levels in EVs+miR-206 inhibitor treated lungs compared to EVs+miR-206 mimic, but it was not significant (Supplementary Fig. 3B). Further analyses focused on protective phenotype of antagomiR-206 enriched EVs after 6hrs and 24hrs post-transplant. Assessment of DCD lungs at the end of cold storage showed that administration of EVs had no detrimental effect on lung pathology or inflammation (Fig. 6B). Post transplantation NS treated DCD lungs had widespread diffuse red blood cell accumulation in the alveolar space, neutrophil infiltration and evidence of alveolar fibrin deposition. Semi-quantitative histological analysis of enriched EV treated DCD lungs shows a significant reduction in injury (Fig. 6C and D) and reduced lung infiltration of neutrophils at 6hrs and 24 hrs post-transplantation, compared to NS controls (Fig. 6E). Analysis of graft oxygenation showed no significant differences between groups after 6hrs, but antagomiR-206 enriched EV treated DCD lungs had improved oxygenation after 24hrs post-transplantation (Fig. 6F). Finally, we observed a significant decrease in proinflammatory cytokine (CXCL1, IL-17, MIP-1α, and IL-6) production and lower alveolar albumin concentrations (indicative of reduced lung edema) after treatment with antagomiR-206 enriched EVs after 24hrs post-transplant (Fig. 6G).

Figure 6.

Figure 6.

Figure 6.

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EVs enriched with antagomir-206 reduce lung allograft IR injury and inflammation in recipients of DCD lungs. A. Balb/c donor mice were treated with EVs enriched with antagomir-206 (EV-miR-206) or normal saline (NS) immediately upon DCD induction and immediately prior to transplantation (Tx). B. Examination of treated DCD lungs following cold storage but prior to transplantation demonstrated that no significant differences could be seen between treated and untreated lungs. C. Representative histological images 6hrs and 24hrs post transplantation demonstrate decreased diffuse red blood cell alveolar accumulation, neutrophil infiltration, and fibrin deposition in EV-miR206 treated mice compared to untreated controls. n=4-7/group. Scale bars as marked. D. Recipients receiving DCD lungs from EV-miR206 treated donor mice had a significant reduction in IR injury as determined by semi-quantitative histological assessment of injury at both 6hrs and 24hrs, as compared to controls. #p=0.006, ##p=0.002. E. Neutrophil numbers were determined by image analysis of computerized randomly generated high-power fields. Transplanted lungs from DCD donors showed marked diffuse neutrophil infiltration and a significant decrease in neutrophil numbers in EV-miR206 treated recipients as compared to NS controls. #p=0.006, ##p=0.001. F. Arterial blood partial pressure of oxygen (PaO2) was not significantly different between EV+miR-206 inhibitor treated and control mice at 6 hrs, but PaO2 was significantly improved in EV-miR206 treated lungs after 24 hrs post-transplantation; ##p=0.01. G. Analysis of BAL at 24 hrs post-transplantation demonstrated that DCD treatment with EV-miR206 significantly reduced lung edema as demonstrated by albumin levels, and significantly reduced pro-inflammatory cytokine expression; ##p<0.001.

EVs with antagomiR-206 attenuates CXCL1 production by alveolar type II epithelial cells

We have previously shown that macrophage-produced TNF-α, iNKT cell-dependent IL-17A and alveolar type II epithelial cell-dependent CXCL1 production mediate lung IR injury (16, 22). In this study, we investigated the role of miR-206 antagonism delivered via EVs, in the mitigation of these pro-inflammatory mediators using hypoxia/reoxygenation (HR) as an in vitro surrogate model of IR. HR significantly increased the expression of IL-17A by iNKT cells that were markedly decreased by co-culturing these cells with either EVs or EVs+miR-206 mimic (Fig. 7A). However, EVs+miR-206 inhibitor did not offer any additional mitigation of IL-17A production compared to EVs+miR-206 mimic. Similarly, HR-exposed MH-S macrophage cells induced a significant increase in TNF-α production that was inhibited by co-culturing with either EVs or EVs+miR-206 mimic (Fig. 7B). However, EVs+antagomir-206 did not offer any additional attenuation of TNF-α production compared to EVs+miR-206 mimic. HR significantly increased the expression of CXCL1 by MLE12 cells which was markedly decreased by co-culture with either EVs or EVs+miR-206 mimic (Fig. 1C). Importantly, co-culture of HR-exposed MLE12 cells with EVs+miR-206 inhibitor further mitigated CXCL1 production compared to EVs+miR-206 mimic. These results signify that EVs with anatgomiR-206 enhances inhibition of epithelial cell-dependent CXCL1 production thereby confirming similar in vivo results, and demonstrate the immunomodulatory functions of enriched EVs on iNKT cells and macrophages to be miR-206 independent, whereas enhanced inhibition of epithelial cells is miR-206 dependent (Fig. 7D).

Figure 7.

Figure 7.

Figure 7.

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EVs enriched with antagomiR-206 inhibit epithelial cell activation after HR. A. HR-exposed iNKT cells produced increased levels of IL-17 compared to normoxia (Norm), and was significantly attenuated by treatment with EVs or EVs+miR-206 mimic. No additional protection was offered by treatment with EVs+miR-206 inhibitor. B. MH-S (alveolar macrophages; AMs) cells were exposed to HR and demonstrated a significant increase in TNF-α production compared to normoxia which was significantly attenuated by treatment with EVs or EVs+mimic miR-206. EVs+miR-206 inhibitor did not offer additional attenuation of TNF-α levels. C. HR-exposed MLE12 (alveolar epithelial; ATM) cells increased the CXCL1 secretion that was significantly attenuated by co-cultures with EVs+miR-206 inhibitor compared to EVs or EVs+miR-206 mimic. D. Schematic depicting the actions of antagomiR-206 enriched EVs on the lung microenvironment following IR injury leading to decreased ATM cell-secreted CXCL1 and subsequent neutrophil (PMN) infiltration. n=8/group; *p<0.05 vs. sham; #p<0.05 vs. IR, §p<0.05 vs. IR+EVs or EVs+miR-206 mimic.

Discussion

This study establishes that expression of specific miRNAs is altered during early lung IR injury, and that antagonism of miR-206 coupled with the immunomodulatory properties of EVs derived from MSCs can be used as an effective tool for mitigating experimental pulmonary inflammation, edema and injury after IR. The results pinpoint three important new findings. First, increased miR-206 expression can be an early predictor and/or biomarker of lung IR injury as observed in human BAL samples of post-lung transplant patients and confirmed in our experimental murine models. Secondly, EVs enriched with miR-206 inhibitor offers significant protection against pulmonary inflammation and injury in two experimental murine models of IR injury. Finally, the miR-206 mediated inhibition was primarily dependent on modulation of alveolar type II epithelial cell activation and CXCL1 production. These results demonstrate that miR-206 enriched EVs offer a viable therapeutic strategy to mitigate post-transplant IR injury.

Micro RNAs (miRNAs) are a class of approximately 22 nucleotide-long, non-coding RNAs predominantly involved in the regulation of target gene expression, mainly at post transcriptional level, and have been demonstrated to play a pivotal role in regulating diverse physiological and pathological processes (23). Recent studies have demonstrated that dysregulated miRNA expression is associated with rejection after organ transplantation, including patients with chronic lung allograft dysfunction (24–26). The upregulation of pro-fibrotic mediators (i.e. miR-199 family, miR-21) correlates with the pathophysiology observed in chronic inflammation and fibrosis in lung transplant patients. Alternatively, downregulation of anti-inflammatory miRs i.e. miR-146 has been shown to be associated with increased expression of NF-kB and biosynthesis of pro-inflammatory cytokines i.e. IL-1 β, IL-6, CXCL1, and TNF-α expression (27).

However, the precise role of miR-206, which is a multifunctional molecule capable of regulating pro-inflammatory response via regulating NF-kB, MMPs and cytokine secretion in various disease processes including pulmonary infectious pathologies, has not been described in post-transplant lung IR injury (28, 29). miR-206 has been shown to be involved in the regulation of alveolar air-blood barrier permeability by regulating connexin-43 expression in sepsis-induced acute lung injury (30). miR-206 is also tightly correlated with interleukin 17a (IL17a) gene that expresses IL-17, a potent cytokine mediating pro-inflammatory responses in lung IR injury (31).

EVs can be classified as exosomes (originating from multivesicular bodies, 10–100 nm) or microvesicles (derived from plasma membrane, 100–1000 nm) and carry specific markers, such as CD44, β1 -integrins, CD73, and CD81, in addition to surface markers of their cells of origin (8). EVs have a long circulating half-life, the intrinsic ability to target tissues, biocompatibility, and minimal or no inherent toxicity issues and thus can be used as drug delivery vehicles as they offers important advantages relative to other nanoparticulate drug delivery systems, such as liposomes and polymeric nanoparticles (32). Recent studies have shown that MSC-derived EVs given during ex vivo lung perfusion mediate the transfer of hyaluronan and change the expression of genes involved in resolution of inflammation and oxidative stress to improve lung IR injury (33). EVs are efficient biovectors for small RNAs therapies because they are natural vectors of molecular cargoes (e.g., pre-miRNAs, miRNAs) and therefore can be delivered to target cells under different pathophysiological contexts (34, 35). Anti-apoptotic miRNAs i.e. miR-21-5p have been shown to mediate the protective effects of MSC-derived EVs against lung IR injury (36). Our study demonstrates that further enhancement of EV-mediated protection by enriching them with specific miRNAs offers a synergistic approach to alleviate lung IR injury.

Although specific miRNAs may act as master regulators of tissue-specific inflammation, however the potential challenges of using anti-miR administration limits its clinical utility for long-term administration. The use of single antagomiR or protectomiR is likely to have limited benefit in a complex, multifactorial disease processes like organ transplantation injury and may have unintended off-target effects. Therefore, we chose to administer EVs, which can inhibit inflammatory signaling pathways via its multiple inherent properties involving keratinocyte, vascular endothelial, and human growth factors (37, 38), prostaglandin E2 (39), and indoleamine-2,3-dioxygenase (40), and act as a unique platform to deliver specific miRs. This approach of using modified EVs is a multifaceted therapeutic strategy that combines the beneficial properties of EVs and acts as delivery platform for specific antagomiRs (miR-206, miR-23a, miR-27a) or protectomiRs (miR-221, miR-29a, miR-146b) to immunomodulate immune cells i.e. iNKT cells and alveolar macrophages as well as resident lung cells i.e. alveolar type II epithelial cells. The current in vivo and in vitro results demonstrate miR-206 regulates CXCL1 secretion, a potent chemokine preferentially secreted after lung IR injury by alveolar type II epithelial cells but not by macrophages or iNKT cells, as shown by our previous studies (22, 41). However, the relative contribution of other cells i.e. endothelial cells and neutrophils were not analyzed in this study.

It is plausible that the isolation and characterization of clinical grade enriched EVs can be challenging as ultracentrifugation inadvertently contains a heterogeneous population of EVs, including microvesicles and exosomes with varied contents. The relative contribution of these microparticles and their detailed contents i.e. mitochondrial DNA and long non-coding RNAs should be further delineated in lung IR injury. The use of modified EVs with a combination of specific antagomiRs and protectomiRs offers a multi-faceted therapeutic option against lung IR injury and likely mitigate chronic rejection, that may involve multiple administration of engineered EVs containing mimics and inhibitors of relevant and highly regulated miRNAs. Such strategies harnessing the regulatory transcriptional effects via multiple miRs may provide a useful tool via dampening proinflammatory mechanistic pathways by targeting not only epithelial cell-derived CXCL1 to block neutrophil infiltration, but also inhibiting HMGB1 and IL-17 signaling by macrophages and iNKT cells, respectively. Simultaneous use of anti-inflammatory miRNAs i.e. miR-146b can be used to up-regulate signaling pathways involving IL-10 and TGF-β through regulatory T cells and M2 macrophages.

In summary, our results demonstrate that EVs can be used as biomimetic nanovehicles where agonists and/or antagonists of tissue-injury specific and relevant miRNAs can be delivered to effectively target multiple cell populations involved in lung IR injury. These preclinical observations provide a molecular framework for the generation of engineered EVs that can not only themselves provide protection against pulmonary inflammation but also act as a pivotal platform to deliver molecular signatures i.e. miRNAs for the effective immunomodulation of organ transplant injury.

Supplementary Material

1

Acknowledgment:

The authors thank Vanessa Scheuble, BS and Jessica Cobb, PhD for assistance with IRB study and collection of human lung transplant samples. We thank Dr. Irving Kron, University of Virginia, for his support and guidance for this project.

Funding: David and Kim Raab Foundation for Stem Cell Research in Lung Transplantation (AKS), National Institute of Health R01 DK080074, R01 DK109581, RO1 DK22682 (VRM) and RO1 HL140470-0181 (CA).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Meeting: Accepted for the International Society for Heart and Lung Transplantation Conference, 2020

Disclosure: None

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