Zehong Guan

Protein expression profiling predicts graft performance in clinical ex vivo lung perfusion.

OBJECTIVES To study the impact of ex vivo lung perfusion (EVLP) on cytokines, chemokines, and growth factors and their correlation with graft performance either during perfusion or after transplantation. BACKGROUND EVLP is a modern technique that preserves lungs on normothermia in a metabolically active state. The identification of biomarkers during clinical EVLP can contribute to the safe expansion of the donor pool. METHODS High-risk brain death donors and donors after cardiac death underwent 4 to 6 hours EVLP. Using a multiplex magnetic bead array assay, we evaluated analytes in perfusate samples collected at 1 hour and 4 hours of EVLP. Donor lungs were divided into 3 groups: (I) Control: bilateral transplantation with good early outcome [absence of primary graft dysfunction- (PGD) grade 3]; (II) PGD3: bilateral transplantation with PGD grade 3 anytime within 72 hours; (III) Declined: lungs unsuitable for transplantation after EVLP. RESULTS Of 50 cases included in this study, 27 were in Control group, 7 in PGD3, and 16 in Declined. From a total of 51 analytes, 34 were measurable in perfusates. The best marker to differentiate declined lungs from control lungs was stem cell growth factor -β [P < 0.001, AUC (area under the curve) = 0.86] at 1 hour. The best markers to differentiate PGD3 cases from controls were interleukin-8 (P < 0.001, AUC = 0.93) and growth-regulated oncogene-α (P = 0.001, AUC = 0.89) at 4 hours of EVLP. CONCLUSIONS Perfusate protein expression during EVLP can differentiate lungs with good outcome from lungs PGD3 after transplantation. These perfusate biomarkers can be potentially used for more precise donor lung selection improving the outcomes of transplantation.

Lentivirus IL‐10 Gene Therapy Down‐Regulates IL‐17 and Attenuates Mouse Orthotopic Lung Allograft Rejection

The purpose of the study was to examine the effect of lentivirus‐mediated IL‐10 gene therapy to target lung allograft rejection in a mouse orthotopic left lung transplantation model. IL‐10 may regulate posttransplant immunity mediated by IL‐17. Lentivirus‐mediated trans‐airway luciferase gene transfer to the donor lung resulted in persistent luciferase activity up to 6 months posttransplant in the isograft (B6 to B6); luciferase activity decreased in minor‐mismatched allograft lungs (B10 to B6) in association with moderate rejection. Fully MHC‐mismatched allograft transplantation (BALB/c to B6) resulted in severe rejection and complete loss of luciferase activity. In minor‐mismatched allografts, IL‐10‐encoding lentivirus gene therapy reduced the acute rejection score compared with the lentivirus‐luciferase control at posttransplant day 28 (3.0 ± 0.6 vs. 2.0 ± 0.6 (mean ± SD); p = 0.025; n = 6/group). IL‐10 gene therapy also significantly reduced gene expression of IL‐17, IL‐23, and retinoic acid‐related orphan receptor (ROR)‐γt without affecting levels of IL‐12 and interferon‐γ (IFN‐γ). Cells expressing IL‐17 were dramatically reduced in the allograft lung. In conclusion, lentivirus‐mediated IL‐10 gene therapy significantly reduced expression of IL‐17 and other associated genes in the transplanted allograft lung and attenuated posttransplant immune responses after orthotopic lung transplantation.

Stromal activation and formation of lymphoid-like stroma in chronic lung allograft dysfunction.

Background. Lymphoid neogenesis is associated with the development of chronic lung allograft dysfunction (CLAD). Activation of stromal resident cells may be an important mechanism of lymphoid neogenesis. Methods. Twenty CLAD lungs explanted for retransplantation were immunohistochemically examined for lymphoid neogenesis, ectopic lymphoid chemokines, and dendritic cells (DCs). Formation of peripheral lymph node addressin (PNAd)+ high endothelial venule (HEV)-like vessels was examined in 134 transbronchial biopsies taken over 2 years posttransplant from 20 consecutive lung transplant recipients. Results. CLAD lungs were characterized by higher grades of CXCL12 in alveolar (P=0.002) and airway epithelial cells (P=0.001), CCL21+ lymph vessels (P=0.01), and infiltration of DC-specific intercellular adhesion molecule-grabbing nonintegrin+ immature DCs (P=0.056) than normal control lungs. Activation of stromal resident cells in CLAD lungs was highlighted by formation of lymphoid-like stroma including expression of CCL21 and CXCL13, fibroblastic reticular-like cells and DC-specific lysosome-associated membrane protein+ mature DCs in association with a significantly larger number of lymphoid aggregates (P<0.001) with lymphangitc distribution compared with normal lungs. A larger number of PNAd+ HEV-like vessels were also observed outside of lymphoid aggregates with a lymphangitic distribution (P<0.001). HEV-like vessels in transbronchial biopsies were more graded in lungs that eventually developed CLAD (n=7) than those that did not (n=13) by 3 years after transplantation (P=0.001). Conclusion. Lymphoid neogenesis associated with CLAD accompanies activation of stromal resident cells and formation of lymphoid-like stroma. Induction of PNAd+ HEV-like vessels occurs before the manifestation of CLAD.

The role of the endothelin-1 pathway as a biomarker for donor lung assessment in clinical ex vivo lung perfusion

BACKGROUND Normothermic ex vivo lung perfusion (EVLP) is a preservation technique that allows reassessment of donor lungs before transplantation. We hypothesized that the endothelin-1 (ET-1) axis would be associated with donor lung performance during EVLP and recipient outcomes after transplantation. METHODS ET-1, Big ET-1, endothelin-converting enzyme (ECE), and nitric oxide (NO) metabolites were quantified in the perfusates of donor lungs enrolled in a clinical EVLP trial. Lungs were divided into 3 groups: (I) Control: bilateral transplantation with good early outcomes defined as absence of primary graft dysfunction (PGD) Grade 3 (PGD3) ; (II) PGD3: bilateral lung transplantation with PGD3 any time within 72 hours; and (III) Declined: lungs rejected after EVLP. RESULTS There were 25 lungs in Group I, 7 in Group II, and 16 in Group III. At 1 and 4 hours of EVLP, the perfusates of Declined lungs had significantly higher levels of ET-1 (3.1 ± 2.1 vs. 1.8±2.3 pg/ml, p = 0.01; 2.7 ± 2.2 vs. 1.3 ± 1.1 pg/ml, p = 0.007) and Big ET-1 (15.8 ± 14.2 vs. 7.0 ± 6.5 pg/ml, p = 0.001; 31.7 ± 17.4 vs. 19.4 ± 9.5 pg/ml, p = 0.007) compared with Controls. Nitric oxide metabolite concentrations were significantly higher in Declined and PGD3 lungs than in Controls. For cases of donation after cardiac death, PGD3 and Declined lungs had higher ET-1 and Big ET-1 levels at 4 hours of perfusion compared with Controls. At this time point, Big ET-1 had excellent accuracy to distinguish PGD3 (96%) and Declined (92%) from Control lungs. CONCLUSIONS In donation after cardiac death lungs, perfusate ET-1 and Big ET-1 are potential predictors of lung function during EVLP and after lung transplantation. They were also associated with non-use of lungs after EVLP and thus could represent useful biomarkers to improve the accuracy of donor lungs selection.

Human α1-antitrypsin improves early post-transplant lung function: Pre-clinical studies in a pig lung transplant model

BACKGROUND The translation of novel drugs in lung transplantation is challenged by different physiologic conditions between small animals and humans. Large-animal models provide important pre-clinical evidence and the next step that best informs clinical trials. In the present study, we used a pig lung transplant model to determine whether human α1-antitrypsin (A1AT), a medication shown to prevent pulmonary ischemia-reperfusion injury in rats, could attenuate reperfusion injury after prolonged hypothermic preservation in a large-animal lung transplant model. METHODS Donor lungs were preserved for 24 hours at 4°C, followed by lung transplantation. In a randomized and blinded fashion, intravenous A1AT (240 mg/kg; n = 5) or human albumin (n = 5) was administered to the recipient before reperfusion. Allograft gas exchange function and lung mechanics were monitored during a 4-hour reperfusion period. Microscopic lung injury, inflammatory response, coagulation activity, and cell death were assessed. RESULTS Pulmonary gas exchange was significantly better during the 4-hour reperfusion period in the A1AT group. Treatment with A1AT improved static pulmonary compliance and significantly reduced pulmonary edema and lung permeability. A1AT treatment inhibited inflammatory mediators in the circulation, with reduced activation of nuclear factor-κB and inflammasome, reduced formation of thrombin-antithrombin complex in plasma, and reduced apoptosis in the allografts. CONCLUSIONS Administration of human A1AT before reperfusion in recipients improved immediate post-transplant lung function in pigs. A large-animal survival model should be considered to support further advancement toward a clinical trial of A1AT to prevent primary graft dysfunction in lung transplantation.

Halofuginone treatment reduces interleukin-17A and ameliorates features of chronic lung allograft dysfunction in a mouse orthotopic lung transplant model.

BACKGROUND Increasing evidence suggests that interleukin (IL)-17A plays an important role in chronic lung allograft dysfunction (CLAD), characterized by airway and lung parenchymal fibrosis, after lung transplantation. Halofuginone is a plant derivative that has been shown to inhibit Th17 differentiation. The purpose of this study was to examine the effect of halofuginone on CLAD development using a minor alloantigen‒mismatched mouse orthotopic lung transplant model. METHODS C57BL/6 recipient mice received an orthotopic left lung transplant from C57BL/10 donors, mismatched for minor antigens. Lung transplant recipients received daily intraperitoneal injections of 2.5 μg halofuginone or vehicle alone. Lung grafts were assessed on Days 7, 14, and 28 post-transplant. RESULTS Compared with control mice, on Day 28 post-transplant, lung grafts of mice treated with halofuginone showed a significant reduction in the percentage of obliterated airways (6.8 ± 4.7% vs 52.5 ± 13.8%, p < 0.01), as well as significantly reduced parenchymal fibrosis (5.5 ± 2.3% vs 35.9 ± 10.9%, p < 0.05). Immunofluorescent staining for IL-17A demonstrated a decreased number and frequency of IL-17A‒positive cells in halofuginone-treated lung grafts on Day 28, as compared with controls. Halofuginone treatment also decreased IL-17A and IL-22 transcripts at Day 14, transforming growth factor-β1 and matrix metalloproteinase-2 transcripts at Days 14 and 28. CONCLUSION The beneficial effect of halofuginone on development of airway and lung parenchymal fibrosis in the mouse lung transplant model highlights the important role of IL-17A in CLAD and merits further pre-clinical and clinical studies.

Annexin V homodimer protects against ischemia reperfusion–induced acute lung injury in lung transplantation

OBJECTIVE We hypothesized that administration of a homodimer of recombinant annexin V, diannexin, could shield phosphatidylserine on the endothelium, and inhibit leukocyte and platelet adhesion, thereby potentially reducing ischemia reperfusion injury (IRI) in lung transplantation. This hypothesis was tested using a rat syngeneic single left-lung transplant model. METHODS Rats were randomly assigned to receive diannexin (DN group; n = 10) or normal saline (control group; n = 10). Diannexin (1000 μg/kg) was administered to the donor lung in the pulmonary flush solution, and to the recipient intravenously, 5 minutes after initiation of reperfusion. Grafts were reperfused for 2 hours. RESULTS The transplanted grafts in the DN group performed significantly better in gas exchange with higher partial pressure of oxygen (control group: 179 ± 121 vs DN group: 330 ± 54 mm Hg; P = .007) and lower partial pressure of carbon dioxide (control: 55.1 ± 26 vs DN: 34.2 ± 11 mm Hg; P = .04), as well as lower peak airway pressure (control: 20.5 ± 8.5 vs DN: 12.0 ± 7.9 cm H2O; P = .035) after 2 hours of reperfusion. Wet-to-dry lung weight ratio (P = .054), and alveolar fibrin deposition score (P = .04), were reduced in the DN group. Caspase-cleaved cytokeratin 18 in plasma (a marker of epithelial apoptosis) was significantly reduced in the DN group (P = .013). Furthermore, gene-expression levels of proinflammatory cytokines in the transplanted graft, including interleukin-6 (P = .04) and macrophage inflammatory protein 2 (P = .03) were significantly decreased in the DN group. CONCLUSIONS A homodimer of recombinant annexin V reduced ischemia reperfusion injury in a lung transplant animal model, by reducing cell death and tissue inflammation.

Rejection of tracheal allograft by intrapulmonary lymphoid neogenesis in the absence of secondary lymphoid organs.

Background Obliterative bronchiolitis after lung transplantation is associated with intrapulmonary lymphoid neogenesis. The purpose of this study was to examine the role of lymphoid neogenesis, especially its relationship with secondary lymphoid organs (SLOs) in allograft airway rejection. Methods A murine intrapulmonary tracheal transplant model and a conventional subcutaneous tracheal transplant model were tested using wild-type control mice and splenectomized lymphotoxin &agr; knockout (LT−/−) mice deficient in SLOs as recipients. Results In both subcutaneous and intrapulmonary tracheal transplant models using wild-type animals, tracheal isografts remained open without rejection, whereas allografts showed progressive luminal obliteration after transplantation. Lymphoid neogenesis containing alloreactive T cells was observed in the lungs, which received an intrapulmonary tracheal allograft. Despite a lack of SLOs, intrapulmonary allografts in splenectomized LT−/− mice were rejected and obliterated by day 28, but the rejection of subcutaneous allografts was significantly delayed. Extensive lymphoid neogenesis was observed in the lungs of both intrapulmonary and subcutaneous allograft LT−/− recipients. Increased proliferation of CD4+ T cells and B220+ B cells was observed in the lungs but not in the thymus or bone marrow. Conclusions Intrapulmonary lymphoid neogenesis is capable of mounting alloimmune responses without SLOs. Tracheal allograft rejection occurs as efficiently as in wild-type animals when it is placed in the lungs. Tracheal allograft rejection in the subcutaneous tissue occurs in a delayed manner without SLO in association with intrapulmonary lymphoid neogenesis.

Inhibition of regulated necrosis attenuates receptor-interacting protein kinase 1–mediated ischemia-reperfusion injury after lung transplantation

BACKGROUND Increasing evidence indicates that regulated necrosis plays a critical role during cell death caused by ischemia-reperfusion (IR) injury. Necroptosis is one form of regulated necrosis. Necrostatin-1 (Nec-1), an inhibitor of receptor-interacting protein kinase 1 (RIPK1), is known to reduce necroptosis. We investigated the effect of Nec-1 treatment on IR-induced lung injury in a rat lung transplant model. METHODS Lewis rats were divided into 4 groups (n = 6 each): (1) Control (no treatment), (2) Donor treatment (D), (3) Recipient treatment (R), and (4) Donor plus Recipient treatment (D+R) groups. Donor lungs were flushed and preserved for 18 hours at 4ºC before transplantation. Recipient animals underwent a left single lung transplant. After 2 hours of reperfusion, we assessed the physiologic function, cytokine expression, pathway activation, and the extent of necrosis. RESULTS Pulmonary gas exchange in D+R group was significantly better than in the other 3 groups (p = 0.003). Lung edema was significantly lower in the D+R group compared with the Control group (p = 0.006). The expression of interleukin-6 in lung tissue and plasma was significantly reduced in the D+R group compared with the Control group (p = 0.036). The percentage of necrotic cells in D+R group was significantly lower than in the Control and D groups (p = 0.01), indicating Nec-1inhibited regulated necrosis. CONCLUSIONS The administration of Nec-1 to both donor and recipient improved graft function after lung transplantation through the reduction of necroptosis. The inhibition of regulated necrosis appears to be a promising strategy to attenuate IR lung injury after lung transplantation.