H. Oishi

Impact of CLAD Phenotype on Survival After Lung Retransplantation: A Multicenter Study

Chronic lung allograft dysfunction (CLAD) remains a major problem after lung transplantation with no definitive treatment except redo lung transplantation (re‐LTx) in selected candidates. However, CLAD is not a homogeneous entity and different phenotypes exist. Therefore, we aimed to evaluate the effect of CLAD phenotypes on survival after re‐LTx for CLAD. Patients who underwent re‐LTx for respiratory failure secondary to CLAD in four LTx centers between 2003 and 2013 were included in this retrospective analysis. Bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD) were distinguished using pulmonary function, radiology and explant lung histopathology. Patient variables pre‐ and post‐re‐LTx were collected and analyzed. A total of 143 patients underwent re‐LTx for CLAD resulting in 94 BOS (66%) and 49 rCLAD (34%) patients. Unadjusted and adjusted survival after re‐LTx for rCLAD was worse compared to BOS (HR = 2.60, 1.59–4.24; p < 0.0001 and HR = 2.61, 1.51–4.51; p = 0.0006, respectively). Patients waiting at home prior to re‐LTx experienced better survival compared to hospitalized patients (HR 0.40; 0.23–0.72; p = 0.0022). Patients with rCLAD redeveloped CLAD earlier and were more likely to redevelop rCLAD. Survival after re‐LTx for rCLAD is worse compared to BOS. Consequently, re‐LTx for rCLAD should be critically discussed, particularly when additional peri‐operative risk factors are present.

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.

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.

A novel combined ex vivo and in vivo lentiviral interleukin-10 gene delivery strategy at the time of transplantation decreases chronic lung allograft rejection in mice

Objective Our objective was to develop a rapid‐onset and durable gene‐delivery strategy that is applicable at the time of transplant to determine its effects on both acute rejection and chronic lung allograft fibrosis using a mouse orthotopic lung transplant model. Methods C57BL/6 mice received an orthotopic left lung transplant from syngeneic donors or C57BL/10 donors. By using syngeneic lung transplantation, we established a novel gene transfer protocol with lentivirus luciferase intrabronchial administration to the donor lung ex vivo before transplantation. This strategy was applied in allogeneic lung transplantation with lentivirus engineering expression of human interleukin‐10 or lentivirus luciferase (control). Results Bioluminescent imaging revealed that the highest early transgene expression was achieved when lentivirus luciferase was administered both directly into the donor lung graft ex vivo before implantation and subsequently to the recipient in vivo daily on post‐transplant days 1 to 4, compared with post‐transplant in vivo administration only (days 0 to 4). Our previous work with adenoviral interleukin‐10 gene therapy indicates that early interleukin‐10 expression in the allograft is desirable. Therefore, we selected the combined protocol for human interleukin‐10 encoding lentiviral vector therapy. In the allogeneic transplant setting, ex vivo and in vivo human interleukin‐10 encoding lentiviral vector therapy reduced acute rejection grade (2.0 vs 3.0, P < .05) at day 28. The percentage of fibrotic obliterated airways was reduced in the human interleukin‐10 encoding lentiviral vector–treated group (10.9% ± 7.7% vs 40.9% ± 9.3%, P < .05). Conclusions Delivery of lentiviral interleukin‐10 gene therapy, using a novel combined ex vivo and in vivo delivery strategy, significantly improves acute and chronic rejection in the mouse lung transplant model.

Pentraxin 3 deficiency enhances features of chronic rejection in a mouse orthotopic lung transplantation model

Chronic lung allograft dysfunction (CLAD) is a serious complication after lung transplantation and thought to represent chronic rejection. Increased expression of Pentraxin 3 (PTX3), an acute phase protein, was associated with worse outcome in lung transplant patients. To determine the role of recipient PTX3 in development of chronic rejection, we used a minor alloantigen-mismatched murine orthotopic single lung transplant model. Male C57BL/10 mice were used as donors. Male PTX3 knockout (KO) mice and their wild type (WT) littermates on 129/SvEv/C57BL6/J background were used as recipients. In KO recipients, 7/13 grafted lungs were consolidated without volume recovery on CT scan, while only 2/9 WT mice showed similar graft consolidation. For grafts where lung volume could be reliably analyzed by CT scan, the lung volume recovery was significantly reduced in KO mice compared to WT. Interstitial inflammation, parenchymal fibrosis and bronchiolitis obliterans scores were significantly higher in KO mice. Presence of myofibroblasts and lymphoid aggregation was significantly enhanced in the grafts of PTX3 KO recipients. Recipient PTX3 deficiency enhanced chronic rejection-like lesions by promoting a fibrotic process in the airways and lung parenchyma. The underlying mechanisms and potential protective role of exogenous PTX3 as a therapy should be further explored.