Downregulation of Tumor Suppressor Gene LKB1 During Severe Primary Graft Dysfunction After Human Lung Transplantation: Implication for the Development of Chronic Lung Allograft Dysfunction

Abstract

Background.

Severe primary graft dysfunction (PGD) after lung transplantation (LTx) is a significant risk factor for the development of bronchiolitis obliterans syndrome (BOS). Recent data from our group demonstrated that small extracellular vesicles (sEVs) isolated from the plasma of LTx recipients with BOS have reduced levels of tumor suppressor gene liver kinase B1 (LKB1) and promote epithelial-to-mesenchymal transition (EMT) and fibrosis. Here, we hypothesized that early inflammatory responses associated with severe PGD (PGD2/3) can downregulate LKB1 levels in sEVs, predisposing to the development of chronic lung allograft dysfunction (CLAD).

Methods.

sEVs were isolated from the plasma of human participants by Exosome Isolation Kit followed by 0.20-μm filtration and characterized by NanoSight and immunoblotting analysis. Lung self-antigens (K alpha 1 tubulin, Collagen V), LKB1, nuclear factor kappa B, and EMT markers in sEVs were compared by densitometry analysis between PGD2/3 and no-PGD participants. Neutrophil-derived factors and hypoxia/reperfusion effects on LKB1 levels and EMT were analyzed in vitro using quantitative real-time polymerase chain reaction and Western blotting.

Results.

LKB1 was significantly downregulated in PGD2/3 sEVs compared with no-PGD sEVs. Within PGD2/3 participants, lower post-LTx LKB1 was associated with CLAD development. Hypoxia/reperfusion downregulates LKB1 and is associated with markers of EMT in vitro. Finally, lower LKB1 levels in PGD2/3 are associated with increased markers of EMT.

Conclusions.

Our results suggest that in post-LTx recipients with PGD2/3, downregulation of LKB1 protein levels in sEVs is associated with increased EMT markers and may result in the development of CLAD. Our results also suggest that ischemia/reperfusion injury during LTx may promote CLAD through the early downregulation of LKB1.

INTRODUCTION

Primary graft dysfunction (PGD) is a serious complication that can occur within 72 h after lung transplantation (LTx). PGD, which is the major cause of early mortality and morbidity after human LTx, is defined as a severe form of acute lung injury. According to the International Society for Heart and Lung Transplantation, PGD can be graded from 0 to 3, and grades 2 and 3 are considered the forms associated with the most severe respiratory dysfunction.¹ PGD is characterized by acute pulmonary edema associated with bilateral pulmonary infiltrates and hypoxemia in the first 3 postoperative days. Although the underlying patho-physiology mechanisms of PGD remain unknown, there is a general consensus that it is worsened by the ischemia/reperfusion injury (IRI) resulting from graft retrieval, preservation, and implantation.² Recently, it has been reported that IRI can trigger chronic kidney injury by stimulating epithelial-to-mesenchymal transition (EMT) in the kidney canaliculus.^3,4 It has also been reported that donor airway epithelial cells—as well as being a recognized pathological target in LTx—may be a significant source of effector fibroblasts through EMT.⁵

Small extracellular vesicles (sEVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules.⁶ Therefore sEVs can be potential candidates for the assessment of graft status. sEVs are lipid bilayer membrane structures (30–200 nm in diameter) that are involved in cellular communication. In transplantation, levels of (human) donor-specific sEVs in animal models have been shown to be associated with acute rejection of the allograft.^7,8 After solid organ transplantation (heart, lung, and kidney), sEVs can be readily identified in circulation and carry distinct molecular markers reflecting the clinical status of the patient.⁹ Studies from our laboratory have demonstrated the presence of heart-, lung-, and kidney-associated antigens on sEVs surfaces after transplantation, for example, cardiac myosin and vimentin after cardiac transplantation; fibronectin, perlecan, and collagen IV after kidney transplantation; and Collagen V (Col-V) and K alpha 1 tubulin (Kα1T) after LTx, suggesting that sEVs detection can be developed as a biomarker for monitoring allograft rejection after solid organ transplantation.^10–13

The tumor suppressor gene liver kinase B1 (LKB1) is a ubiquitously expressed kinase that activates multiple downstream kinases regulating cell functions, including metabolism, migration, and proliferation.¹⁴ LKB1 is a metabolic sensor that helps maintain adenosine triphosphate levels during intense activity and stress.¹⁵ We showed that LKB1 was downregulated in lung biopsies diagnosed with bronchiolitis obliterans syndrome (BOS) compared with stable after LTx.¹⁶ LKB1 is also downregulated in exosomes and the donor’s lung compared with the recipient’s lung after LTx in mice.¹⁷ Treatment of airway epithelial cell lines with exosomes derived from BOS also downregulated LKB1 compared with cells treated with stable exosomes.¹⁶ The mechanisms resulting in LKB1 downregulation remain unknown at present. Recently, it has been reported that the level of LKB1 decreased in liver IRI.¹⁸ However, whether LKB1 is modulated early after IRI because of LTx is not known.

In this study, we examined the level of LKB1 in circulating sEVs isolated from LTx recipients (LTxRs) diagnosed with PGD. Our study provided novel evidence that LKB1 expression is downregulated in sEVs isolated from severe PGD compared with no-PGD and that lower levels are associated with the development of chronic lung allograft dysfunction (CLAD). We also found that hypoxia/reperfusion reduced LKB1 expression in the airway epithelium and upregulated markers of EMT.

MATERIALS AND METHODS

Patient Population

Thirty LTxRs (15 with PGD grade 2/3 and 15 time-matched stables) and 24 serial samples (before LTx n = 12] and during PGD2/3 [n = 12] after LTx) were analyzed in the current study. Informed consent was obtained from all patients, and this study was approved by the Institutional Review Board (PHXB-16-0027-10-18) at St. Joseph’s Hospital and Medical Center. PGD 2/3 and normal pulmonary function were defined according to the International Society for Heart and Lung Transplant guidelines.¹ No significant differences were observed in the demographics of each group (Tables S1 and S2, SDC, http://links.lww.com/TP/D133). Immunosuppressive regimens consisted of tacrolimus, mycophenolate, and prednisone. Plasma was collected within 72 h after LTx from the recipient for both PGD and no-PGD patients (Table S1, SDC, http://links.lww.com/TP/D133), and these samples were stored at −80 °C until analysis.

Isolation and Characterization of Exosomes

Current isolation techniques classify sEVs according to size, density, and the presence of surface antigens, not based on their origin.¹⁹ We isolated circulating sEVs from both PGD 2/3 and no-PGD after LTx using a modified kit method, as reported in our earlier publication.¹⁶ sEVs were characterized by NanoSight and Western blot. The protein concentrations of sEVs were measured by a Bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The sEVs used in this report had a size of <200 nm and were considered sEVs, in agreement with the recommendation made by the nomenclature committee on exosomes.²⁰

Western Blot Analysis

Western blotting was performed on total protein extracts from sEVs. Radioimmunoprecipitation assay lysis buffer with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO) was used to determine the total protein fraction. Protein concentrations in the sEVs were determined using a Bicinchoninic acid protein assay kit (Thermo Fisher Scientific) to standardize for quantification. Twenty micrograms of total lysates were diluted 1:1 in radio-immunoprecipitation assay SDS-PAGE sample buffer, loaded onto polyacrylamide gels, and blotted onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% nonfat milk in PBS (pH 7.6) and 0.2% Tween-20 for 1 h and then incubated overnight with primary antibodies: mouse monoclonal anti-LKB1 (1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti–Col-V (1:1000; Abcam, Cambridge, United Kingdom), mouse monoclonal anti-Ka1Tubulin (Kα1T) (1:1000; Santa Cruz Biotechnology), rabbit polyclonal anti-nuclear factor kappa B (NF-κB) (1:1000; Cell Signaling Technology), rabbit polyclonal anti-E-Cadherin (1:1000; Cell Signaling Technology), mouse monoclonal anti-vimentin (1:1000), and rabbit polyclonal anti-α-SMA (1:1000; Abcam). After washing in PBS Tween-20, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:1000; Cell Signaling Technology), and the immunoblots were visualized using electrochemiluminescence detection kits (Pierce, Rockford, IL). Mouse anti-GAPDH antibody (1:5000; Santa Cruz Biotechnology) was used as the internal control. Semiquantitation of the bands was performed using Image J software.

Cell Culture

Human bronchial epithelial cell line (BEAS-2B) cells immortalized human bronchial epithelial cells, and the human primary bronchial epithelial cell (HPBEC) lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA). BEAS-2B cells were grown in an alpha-MEM complete medium containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin. HPBEC were grown in complete airway epithelial cell serum-free basal medium (ATCC-PCS-300–030) supplemented with bronchial/tracheal cell growth kit (ATCC-PCS-300–040) and gentamicin/amphotericin B solution (ATCC-PCS-999–025).²¹ Recently, the BEAS-2B cell line has been claimed as a mesenchymal cell line.²² Therefore, we also used the HPBEC cell line in addition to the BEAS-2B cell line in most of our experiments in this study. The cells were maintained in a 10 cm petri dish in a 5% CO₂ and 37 °C humidified environment. The cells from passages 2 to 7 were used throughout the experiments at an initial seeding density of 6 to 7 × 10⁴ cells/cm² unless otherwise mentioned. For small interfering RNA delivery (LKB1; Santa Cruz Biotechnology [sc-35816]), 2 × 10⁵ cells were grown in a 6-well plate for 24 h in an antibiotic-free medium, and 80 Pico moles of small interfering RNA were transfected using Lipofectamine 2000 (Invitrogen, NY). For exposure to hypoxemic reperfusion (H/R), culture dishes were placed in a humidified, sealed hypoxic chamber that was purged with 95% N₂, 0.1% O₂, and 5% CO₂ for 25 min to establish hypoxia. The chamber was then placed in a cell culture incubator for 3 h. Reoxygenation was achieved by removing the plates from the hypoxic chamber and placing them in a normoxic, humidified incubator (37 °C, 5% CO₂, and 95% O₂) for 1 h.

A549 Cell Culture and Overexpression of LKB1 Generation of LKB1 Expressing Stable Lines

Retroviral vectors, pBABE or Pbabe-LKB1, were obtained from Addgene (Cambridge, MA). A549 cells were transfected with viral plasmids using Lipofactamine-LTx. Retroviral particles were retrieved from media super-natants after 48 h. PBabe or pBabe-LKB1 virus media was filtered through 0.45-μm filters and added to A549 cells with polybrene (10 ug/mL) overnight. After 2 subsequent transductions, A549 cells were selected with 2 μg/mL puromycin for 1 wk before performing experiments.

Quantitative Real-time Polymerase Chain Reaction

BEAS-2B cells were cultured to ~80% confluence in 6-well plates and treated with different doses of human neutrophil elastase (NE; Millipore Sigma, Burlington, MA; 0.1 μg/mL [NE low]; 0.5 μg/mL [NE high]) or with neutrophil extracellular traps (NETs; 50 ng/mL) isolated from phorbol 12-myristate 13-acetate-activated human neutrophils as previously described.²³ Saline vehicle was used as a control. RNA extracts were collected using the QIAzol Lysis Reagent (Qiagen, Germantown, MD) at 30-min, 3, 6, and 24-h time points after dosing. cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative real-time polymerase chain reaction was performed on a StepOnePlus Thermocycler (Applied Biosystems) using TaqMan probes for LKB1 (Hs00176092_m1). GAPDH (Hs02786624_g1) was used as a housekeeping gene for normalization. Fold changes were calculated using the 2-ΔΔCt method.

Immunofluorescence

BEAS-2B cells were seeded at 70 000 cells/well and grown to ~60% confluence in a 48-well tissue culture-treated plate in 2% FBS Roswell Park Memorial Institute. The cells were cultured for 24 h in FBS-free Roswell Park Memorial Institute before treatment (0.1 μg/mL [NE low]; 0.5 μg/mL [NE high]; 50 ng/mL [NETs]). After treatment, the cells were first fixed overnight in 4% PFA and then permeabilized and blocked in a PBS solution containing 5% bovine serum albumin (Research Products International, Mt. Prospect, IL) and 0.1% Triton-X (Sigma-Aldrich). The cells were then incubated with (1:100) monoclonal mouse anti-human LKB1 (Santa Cruz Biotechnology, Dallas, TX) and stained with (1:200) goat anti-mouse Alexa Fluor 488 conjugated secondary antibody (Abcam, Cambridge, United Kingdom). A DAPI (Invitrogen, Carlsbad, CA) counterstain was used to indicate cell nuclei. Immunofluorescence images were collected on the Evos M700 at ×20 magnification. The fluorescent images were analyzed using FIJI software (version 1.54f), and the corrected total cell fluorescence was measured as previously described by McCloy et al²⁴: (corrected total cell fluorescence = integrated density – [area of selected cell × mean fluorescence of background readings]).

Statistical Analysis

Statistical differences between means were analyzed using a paired or unpaired Student t test. A 1-way ANOVA test with post hoc comparisons was used to analyze data with >2 groups. A P value of <0.05 was considered significant. All data analyses were obtained using GraphPad5 Prism (Graph Pad Software, La Jolla, CA).

RESULTS

sEVs From Human LTxRs With Severe PGD Are Characterized by Reduced LKB1 Levels, Increased Markers of Lung Self-antigens, NF-κB, and NE

Recently, we demonstrated that the tumor suppressor gene, LKB1, is downregulated in lung biopsies and the circulating sEVs of LTxRs diagnosed with BOS.¹⁶ Because the severity of PGD is a well-established risk factor for BOS.¹⁶ we asked whether circulating sEVs from PGD2/3 may also display an altered LKB1 expression early after LTx. Our results show that circulating sEVs from participants with PGD2/3 have significantly lower LKB1 expression when compared with those without PGD (P = 0.016; Figure 1A and B). On the contrary, expressions of both lung self-antigens, Col-V and Kα1T, recently implicated in the development of DSA,²⁵ were significantly upregulated (P = 0.002 for Col-V, P = 0.003 for Kα1T) in PGD2/3 compared with without PGD sEVs (Figure 1C and D). Because PGD2/3 is typically associated with inflammation, we also ask whether the reduced LKB1 levels observed were associated with increased inflammatory markers. Our data indicate that the sEV expression of NF-κB (P = 0.01) was significantly higher in PGD2/3 compared with no-PGD (Figure 1E). We also found that a serine protease typically expressed by neutrophils such as NE was increased in the exosome from PGD2/3 sEVs compared with the no-PGD sEVs (Figure 1F). Our results indicate that exosomes from participants with severe PGD have lower LKB1 levels, increased expression of lung self-antigens, and higher markers of inflammation and neutrophil activation compared with no PGD.

FIGURE 1.

LKB1 expression levels are downregulated significantly in circulating sEVs isolated from LTxRs with PGD2/3. A, A representative picture of Western blot analysis of LKB1, Kα1T, Col-V, NF-κB, and NE in circulating sEVs isolated from PGD2/3 and no-PGD. CD9 was used as the sEVs marker and loading control. Densitometry analysis showed significantly decreased levels of LKB1 in the PGD2/3 group compared with no-PGD (B), increased levels of lung antigens Kα1T (C), increased levels of Col-V (D), increased levels of NF-κB (E), and increased levels of NE (F) in PGD2/3 compared with sEVs isolated from no PGD. Col-V, Collagen V; Kα1T, Kα1 tubulin; LKB1, liver kinase B1; LTxR, lung transplant recipient; NE, neutrophil elastase; NF-κB, nuclear factor kappa B; PGD, primary graft dysfunction; sEV, small extracellular vesicle.

Reduced Levels of LKB1 in Severe PGD Are Associated With CLAD Development

The mechanisms linking the early inflammatory events typical of severe PGD with the increased risk for CLAD development are not entirely understood. Given that our previous study¹⁶ indicated that LKB1 levels are associated with BOS, we asked whether lower LKB1 levels in PGD2/3 participants were associated with the development of BOS. Interestingly, we noticed that, although all participants before LTx had comparable levels of LKB1, only severe PGD with early lower LKB1 content developed CLAD (Figure 2). These data suggest that early lower levels of sEVs LKB1 in severe PGD are associated with an increased risk of developing CLAD.

FIGURE 2.

Downregulation of LKB1 after LTx is a risk factor for CLAD development. A, A representative picture of Western blot analysis of LKB1 in serial circulating sEVs isolated from before lung transplantation and after lung transplantation. CD9 was used as the exosome marker and loading control. B, Densitometry analysis showed significant downregulation of LKB1 expression in sEVs isolated from LTxR who developed CLAD compared with those who did not develop CLAD. Values are mean ± SD; all values are representative of at least 3 independent experiments. CLAD, chronic lung allograft dysfunction; LKB1, liver kinase B1; LT, lung transplant; LTxR, LT recipient; sEV, small extracellular vesicle.

NE and NETs Do Not Downregulate LKB1 in Airway Epithelium

Based on the observation that severe PGD has an exosome signature characterized by low level of LKB1 and high levels of NE, we asked whether this serine protease, typically released during neutrophil degranulation, may play a role in reducing LKB1 expression in the lung epithelium. Our results suggest that is not the case since, as shown in Figure 3A, NE did not significantly modulate LKB1 transcript in BEAS2B cells up to 24 h. LKB1 protein levels, measured by immunofluorescence after NE treatment, also did not change (Figure 3C). In addition to degranulation, NE can also be released by neutrophils in the contest of NETs in a process defined by NETosis.²⁶ Because NETosis has been shown to play a role in the pathogenesis of PGD,^27,28 we then asked whether the downregulation of LKB1 observed in severe PGD participants could be caused by NETs. Our results show that, similarly to purified NE, NETs did not significantly modulate LKB1 expression both at transcriptional and protein levels (Figure 3B–D). In conclusion, although sEVs from severe PGD have significantly higher NE and lower LKB1 levels, our data do not suggest a causative link for NE or NETs in reducing LKB1 expression.

FIGURE 3.

LKB1 expression in BEAS-2B cells after NE and NETs co-culture. A and B, qRT-PCR analysis showing LKB1 fold change in BEAS-2B cells cultured, for the time indicated, in the presence of human neutrophil elastase (0.1 μg/mL [NE low], 0.5 μg/mL [NE high]) (A) or of isolated NETs (50 ng/mL) (B). Saline was used as a control. Data are presented as bar graph showing the mean of 3 independent experiments ± SD. C and D, Representative immunofluorescence and corresponding CTCF quantification of LKB1 in BEAS-2B treated for 3 h in the presence of human NE (0.1 μg/mL [NE low], 0.5 μg/mL [NE high]) (A) or of isolated NETs (50 ng/mL) (B). Saline was used as a control. Data are presented as a bar graph showing the mean of 3 independent experiments ± SD. BEAS-2B, human bronchial epithelial cell line; CTCF, corrected total cell fluorescence; LKB1, liver kinase B1; NE, neutrophil elastase; NET, neutrophil extracellular trap; qRT-PCR, quantitative real-time polymerase chain reaction.

Hypoxia/Reperfusion Injury Downregulates LKB1 Expression and Upregulated EMT Markers

Because ischemia/reperfusion is one of the main drivers in PGD pathogenesis,²³ we ask whether the cellular stress associated with hypoxia and reperfusion can induce LKB1 downregulation and EMT.

Human airway cell lines, BEAS-2B and HPBEC, were grown in hypoxic conditions for 3 h followed by 1 h of reperfusion before measuring their level of LKB1 and EMT markers (vimentin and E-cadherin). Densitometric analysis showed significant downregulation of LKB1 (P = 0.009), E-cadherin (P = 0.02), and upregulation of vimentin (P = 0.03) in H/R injury compared with normoxia in BEAS-2B cells (Figure 4A and B). We also found significant downregulation of LKB1 (P = 0.001), E-cadherin (P = 0.04), and upregulation of vimentin (P = 0.03) in HPBEC cells (Figure 4A and C). We also check the kinetic of the EMT markers in BEAS-2B cells. Our results showed similar expression of E-cadherin and vimentin in 1 h, 2 h, and 3 h after H/R injury (Figure S1, SDC, http://links.lww.com/TP/D133).

FIGURE 4.

Downregulation of LKB1 during H/R-induced EMT but overexpression of LKB1 can rescue the cells from EMT. A, Western blot analysis showing the effect of H/R injury on LKB1, E-cadherin, and vimentin, expression in HPBEC, and BEAS-2B cells. GAPDH was used as an internal loading control. Densitometry analysis showed a significant decrease of LKB1 and E-cadherin and a significant increase of vimentin expression in HPBEC (B) and BEAS-2B cells (C). D, Western blot analysis showed forced overexpression of LKB1 increased E-cadherin and decreased vimentin expression in A549 cells. Densitometry analysis showed H/R injury decreased E-cadherin and increased vimentin expression in A549-pBABE cells (E), but overexpression of LKB1 (A549-pBABE-LKB1) rescues A549 cells from EMT during H/R injury (F). Values are mean ± SD; all values are representative of at least 3 independent experiments. BEAS-2B, human bronchial epithelial cell line; EMT, epithelial-to-mesenchymal transition; HPBEC, human primary bronchial epithelial cell; H/R, hypoxemic reperfusion; LKB1, liver kinase B1.

To evaluate whether the forced overexpression of LKB1 protects from H/R-induced EMT, we transfected A549 cells (inactivated LKB1) with viral plasmids using Lipofactamine-LTx. A549-pBABE-LKB1 cells and A549-pBABE cells were then exposed to 3 hypoxia/reperfusion. Our results showed that H/R significantly decreased E-cadherin (P = 0.013) and increased vimentin expression (P = 0.019) in A549-pBABE cells compared with normoxic conditions (Figure 4D and E). However, overexpression of LKB1 significantly protected A549 cells (A549-pBABE-LKB1) from the hypoxia reperfusion activation of EMT, restoring E-cadherin and vimentin to normoxic levels (Figure 4D and F).

Both Vimentin and α-SMA Were Upregulated in PGD2/3 With Low Levels of LKB1 Compared With PGD2/3 With High Levels of LKB1

EMT markers were compared between PGD2/3 participants with low (LKB1-LE) versus high (LKB1-HE) levels of LKB1. Western blot analysis showed both vimentin (P = 0.03) and α-SMA (P = 0.04) expression upregulated in LKB1-LE sEVs (Figure 5A and B).

FIGURE 5.

Low levels of LKB1 expression induced EMT in PGD2/3. A, A representative picture of Western blot analysis of LKB1, vimentin, and α-SMA in circulating exosomes isolated from PGD2/3 (negative or low expressed LKB1) and PGD2/3 (positive or high expressed LKB1). CD9 was used as the exosome marker and loading control. Densitometry analysis showed significantly increased levels of vimentin (B) and α-SMA (C) in PGD2/3 (negative or low expressed LKB1) exosomes compared with PGD2/3 (positive or high expressed LKB1) exosomes. Values are mean ± SD; all values are representative of at least 3 independent experiments. EMT, epithelial-to-mesenchymal transition; LKB1, liver kinase B1; PGD, primary graft dysfunction; α-SMA, alpha-smooth muscle actin.

DISCUSSION

In this study, we explored how LKB1 is modulated during PGD and what the consequences of this early modulation could be for the development of CLAD.

Our results obtained in a small cohort of LTxRs show for the first time that LKB1 expression is downregulated in the circulatory sEVs derived from severe PGD compared with no PGD. In LTx, both allo- and autoimmunity, particularly to lung self-antigens (Kα1T and Col-V), have been implicated in rejection and chronic graft dysfunction. Furthermore, autoantibodies have been shown to increase the risk for PGD.²⁹ Along with downregulated levels of LKB1, here we found increased expression of Kα1T and Col-V in PGD2/3 compared with no-PGD sEVs. Although the direct relationship between lung self-antigens and LKB1 levels was not studied here, our data suggest that they are both expressions of the IRI severity.

Our research also indicates that individuals with severe PGD who experience early downregulation of LKB1 are more likely to be diagnosed with CLAD at the time of observation. This suggests that early post-LTx LKB1 downregulation may increase the risk of developing CLAD.

The mechanisms of LKB1 downregulation in inflammatory conditions unrelated to cancer are currently not well defined.³⁰ In LTx, the severity of the acute inflammatory response to IRI is usually associated with graft neutrophilia, as confirmed in our study by the higher levels of NE observed in severe PGD participants. Neutrophils play a pathogenic role in PGD through the aberrant release of proteases and NETs with cytotoxic effects within the lung graft.²⁷ However, more recent work indicates that NETs or NETs deprived of their histone components may have biological effects unrelated to cytotoxicity, such as increasing the expression of NF-κB or inducing cytokine production.³¹ Given the association between high levels of NE and low levels of LKB1 observed in severe PGD participants, we hypothesized that NE, in its isolated form or the contest of NETs, could directly reduce LKB1 expression. However, our in vitro findings did not support this interpretation, suggesting that neutrophil activation and LKB1 downregulation are likely 2 independent manifestations of IRI. During the transplantation process, the donor’s lungs undergo a period of ischemia followed by reperfusion when the new lungs are implanted. Therefore, here we asked whether LKB1 downregulation could be directly dependent on H/R-induced cellular stress. Our findings show, in 2 different airway epithelial cell lines, that the exposure to 3 h of hypoxia followed by 1 h of reperfusion was sufficient to downregulate LKB1 level. Moreover, the H/R-induced downregulation of LKB1 also triggered cellular changes consistent with EMT as shown by the reduction in E-cadherin and the parallel increase in vimentin levels. Interestingly, the forced overexpression of LKB1 in transfected A549 cells restored EMT markers to normoxic levels, suggesting that the LKB1-dependent EMT changes triggered by H/R can be reversed by increasing LKB1 expression.

Recent work has shown that EMT may underlie the dysfunctional airway repair processes that lead to BOS.³² LKB1 can activate AMPK, a regulator of metabolism and cell growth,³³ and AMPK plays an inhibitory role in EMT development. Our previous study demonstrated significantly downregulated LKB1 expression in the lung biopsies diagnosed with BOS after LTx compared with stable biopsies.¹⁶ In the current study, we found that sEVs from participants with severe PGD have downregulation of LKB1 and increased markers of EMT. Overall, our data suggest that an early downregulation of LKB1 in severe PGD might link IRI with the development of CLAD by increasing the level of EMT.

Understanding the precise role of LKB1 in these processes could help develop strategies to prevent or mitigate fibrotic changes associated with CLAD. Moreover, LKB1 is a key regulator of cellular energy metabolism by activating the AMP-activated protein kinase pathway. By promoting energy homeostasis and reducing oxidative stress, LKB1 could protect against cellular damage and inflammation contributing to PGD and CLAD.

This study has some limitations. First, we did not differentiate between donor-derived versus recipient-derived sEVs. Although previous studies from our group suggest that LKB1 sEVs originated from the donor,³⁴ our current approach cannot exclude a possible contribution for recipient-derived LKB1. The small number of participants enrolled in this study is another limitation. Larger studies will be required to define the potential role of early sEVs LKB1 levels as a biomarker for increased risk of CLAD. Our in vitro results indicate that H/R downregulated LKB1 expression and induced EMT markers. However, the precise molecular mechanism triggered by H/R leading to LKB1 downregulation needs further evaluation. Finally, our in vitro data indicate that H/R is able to drive EMT by downregulating LKB1. Whether increasing LKB1 levels can be a therapeutic approach to attenuate IRI and BOS development should be thoroughly investigated in preclinical models of LTx-related IRI.

In conclusion, this study showed that LKB1 is downregulated in severe PGD compared with no PGD after LTx. H/R can significantly inhibit LKB1 expression in airway epithelium, and the downregulation of LKB1 is associated with increased markers of EMT. Overexpression of LKB1 protects cells from EMT induced by H/R.

These results suggest that LKB1 downregulation in response to severe ischemia/reperfusion injury may play an important role in the pathogenesis of CLAD and set the foundation for future investigations exploring the potential role of LKB1 as a biomarker and therapeutic target for CLAD.

Supplementary Material

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