Orthotopic Tracheal Transplantation Research Articles

Iron Overload-induced Ferroptosis as a Target for Protection against Obliterative Bronchiolitis after Orthotopic Tracheal Transplantation in Mice.

The major complication of Obliterative Bronchiolitis (OB) is characterized by epithelial cell loss, fibrosis, and luminal occlusion of the terminal small airways, which limits the long-term survival of the recipient after lung transplantation. However, the underlying mechanisms are still not fully clarified. This research aims to investigate whether iron overload-induced ferroptosis is involved in OB development and provide a new target for OB prevention. Allograft orthotopic tracheal transplantation in mice was applied in our study. Ferrostatin-1 and deferoxamine were administrated to inhibit ferroptosis and get rid of ferric iron, while iron dextran was used to induce an iron overload condition in the recipient. The histological examination, luminal occlusion rate, collagen deposition, iron level, ferroptosis marker (GPX4, PTGS2), and mitochondrial morphological changes of the graft were evaluated in mice. Our research indicated that ferroptosis and iron overload contribute to OB development, while ferroptosis inhibition and iron chelator could reverse the changes. Iron overload exacerbated OB development after orthotopic tracheal transplantation via promoting ferroptosis. Overall, this research demonstrated that iron overload-induced ferroptosis is involved in OB, which may be a potential therapeutic target for OB after lung transplantation.

3D-bioprinted cell-laden hydrogel with anti-inflammatory and anti-bacterial activities for tracheal cartilage regeneration and restoration

Despite the notable advances in tissue-engineered tracheal cartilage (TETC), there remain several challenges that need to be addressed, such as uneven cell distribution for cartilage formation, customized C-shaped tracheal morphology, local inflammatory reactions, and infections. To overcome these challenges, this study proposed the addition of icariin (ICA) and chitosan (CS) into a gelatin methacryloyl (GelMA) hydrogel to develop a new ICA/CS/GelMA hydrogel with anti-inflammatory and anti-bacterial properties, and three-dimensional (3D)-bioprinting feasibility. The aim of this study was to construct a TETC, a customized C-shaped cartilage structure, with uniform chondrocyte distribution as well as anti-inflammatory and anti-bacterial functions. Our results confirmed that ICA/CS/GelMA hydrogel provides desirable rheological properties, suitable printability, favorable biocompatibility, and simulated microenvironments for chondrogenesis. Moreover, the addition of ICA stimulated chondrocyte proliferation, extracellular matrix synthesis, and anti-inflammatory ability, while the encapsulation of CS enhanced the hydrogels’ anti-bacterial ability. All these led to the formation of an enhanced TETC after submuscular implantation and an elevated survival rate of experimental rabbits after orthotopic tracheal transplantation. This study provides a reliable cell-laden hydrogel with anti-inflammatory and anti-bacterial activities, suitable printability, and significant advancements in in vivo cartilage regeneration and in situ tracheal cartilage restoration.

Critical Care and Postoperative Management of the First Human Long Segment Orthotopic Tracheal Transplantation.

The recipient required reconstruction of a long segment tracheal defect from a previous prolonged intubation. A male donor was chosen for a female recipient to enable analysis of the reepithelialization kinetics using fluorescence in situ hybridization to analyze the source of the new ciliated epithelium. Transplant ICU at the Mount Sinai Hospital, New York, NY. The female recipient was previously intubated for an asthma exacerbation and subsequently developed long segment tracheal stenosis and failed conventional management including dilatation, stenting, and six major surgical procedures rendering her chronically tracheostomy-dependent. The male donor suffered a massive subarachnoid hemorrhage and was subsequently pronounced brain dead. Organ procurement occurred after obtaining appropriate consent from the patient's family. The patient received a deceased donor tracheal allograft that included the thyroid gland, parathyroid glands, and the muscularis of the cervical and thoracic esophagus. Triple therapy immunosuppression (tacrolimus, mycophenolate mofetil, and a corticosteroid taper) was maintained. The patient was initially managed postoperatively with deep sedation on ventilator via armored/reinforced endotracheal tube placed through a small tracheostomy located along the superior tracheal anastomosis. Serial bronchoscopies were performed for graft assessment, pulmonary toilet, and biopsies, which initially showed acute inflammatory changes but no features of acute allograft rejection. A euthyroid state was maintained but hypercalcemia developed. The ICU management of this first long segment orthotopic tracheal transplant required a multidisciplinary approach involving critical care, otolaryngology, transplant surgery, interventional pulmonary, endocrinology, 1:1 nursing throughout the recipient's transplant ICU stay, and respiratory therapy that resulted in the successful establishment of a viable tracheal airway and heralded the end of chronic tracheostomy dependence.

Skin-derived epithelial lining facilitates orthotopic tracheal transplantation by protecting the tracheal cartilage and inhibiting granulation hyperplasia.

Long-segment tracheal defects caused by tumours, inflammation or trauma can cause serious damage to the quality of life of patients. Although many novel neotracheas have been constructed, the therapeutic effect of orthotopic transplantation was compromised mainly because of the lack of an epithelial lining in those neotracheas. In this study, we aimed to investigate the therapeutic function of skin-derived epithelial lining for orthotopic tracheal transplantation. Strips of auricular cartilage with fixed interval were interrupted sutured on a silicone tube to mimic the cartilage rings of the native trachea. Neotrachea in the with epithelium group retained the unilateral skin as the epithelial lining in the lumen, whereas the neotrachea in the without epithelium group consisted solely of cartilage strips. After revascularized in the sternohyoid muscle, 2-cm-long tracheal defects were made and were reconstructed using these neotracheas. Our results showed that the skin-derived epithelial lining simultaneously protected the engineered tracheal cartilage and inhibited granulation hyperplasia in the tracheal lumen; further, compared with the without epithelium group, the group with epithelium showed a marked improvement in the tracheal lumen patency and the survival rate of rabbits. Our study provides a critical cue for improvements in the repair of tracheal defects via skin-derived epithelial lining and may significantly advance the clinical translation of tissue-engineered trachea.

Macrophage Lysosomal Alkalinization Drives Invasive Aspergillosis in a Mouse Cystic Fibrosis Model of Airway Transplantation.

Cystic fibrosis (CF) lung transplant recipients (LTRs) exhibit a disproportionately high rate of life-threatening invasive aspergillosis (IA). Loss of the cystic fibrosis transmembrane conductance regulator (CFTR-/-) in macrophages (mφs) has been associated with lyosomal alkalinization. We hypothesize that this alkalinization would persist in the iron-laden post-transplant microenvironment increasing the risk of IA. To investigate our hypothesis, we developed a murine CF orthotopic tracheal transplant (OTT) model. Iron levels were detected by immunofluorescence staining and colorimetric assays. Aspergillus fumigatus (Af) invasion was evaluated by Grocott methenamine silver staining. Phagocytosis and killing of Af conidia were examined by flow cytometry and confocal microscopy. pH and lysosomal acidification were measured by LysoSensorTM and LysotrackerTM, respectively. Af was more invasive in the CF airway transplant recipient compared to the WT recipient (p < 0.05). CFTR-/- mφs were alkaline at baseline, a characteristic that was increased with iron-overload. These CFTR-/- mφs were unable to phagocytose and kill Af conidia (p < 0.001). Poly(lactic-co-glycolic acid) (PLGA) nanoparticles acidified lysosomes, restoring the CFTR-/- mφs’ ability to clear conidia. Our results suggest that CFTR-/- mφs’ alkalinization interacts with the iron-loaded transplant microenvironment, decreasing the CF-mφs’ ability to kill Af conidia, which may explain the increased risk of IA. Therapeutic pH modulation after transplantation could decrease the risk of IA.

Bioinspired carbon monoxide delivery using artificial blood attenuates the progression of obliterative bronchiolitis via suppression of macrophage activation by IL-17A

Carbon monoxide (CO) is expected to attenuate the progression of obliterative bronchiolitis (OB), which is a serious complication after lung transplantation. However, issues in terms of feasible exogenous CO supply, such as continuousness and safety, remain unsolved. Here, we applied nano red blood cells, namely hemoglobin vesicles (Hb-V), as a CO cargo based on the biomimetic concept and investigated the therapeutic potential of CO-loaded Hb-V on OB in orthotopic tracheal transplant model mice. The CO-loaded Hb-V was comprised of negatively charged liposomes encapsulating carbonylhemoglobin with a size of ca. 220 nm. The results of histological evaluation showed that allograft luminal occlusion and fibrosis were significantly ameliorated by treatment with CO-loaded Hb-V compared to treatment with saline, cyclosporine, and Hb-V. The therapeutic effects of CO-loaded Hb-V on OB were due to the suppression of M1 macrophage activation in tracheal allografts, resulting from decreased IL-17A production. Furthermore, the expression of TNF-α and TGF-β in tracheal allografts was decreased by CO-loaded Hb-V treatment but not saline and Hb-V treatment, indicating that CO liberated from CO-loaded Hb-V inhibits epithelial-mesenchymal transition. These findings suggest that CO-loaded Hb-V exerts strong therapeutic efficacy against OB via the regulation of macrophage activation by IL-17A and TGF-β-driven epithelial-mesenchymal transition.

High CO2 Levels Impair Lung Wound Healing.

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-κB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-κB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, CXCL12 transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.

Nlrp3 Inflammasome Inhibitor MCC950 Ameliorates Obliterative Bronchiolitis by Inhibiting Th1/Th17 Response and Promoting Treg Response After Orthotopic Tracheal Transplantation in Mice.

Obliterative bronchiolitis (OB) remains the major complication limiting long-term survival of patients after lung transplantation. We aimed to explore the effects of the selective NACHT, LRR, and PYD domains-containing protein 3 (Nlrp3) inflammasome inhibitor MCC950 on the pathogenesis of OB. Mouse orthotopic tracheal transplants were performed to mimic OB. MCC950 (50 mg/kg) or saline was intraperitoneally injected daily. The luminal occlusion rate and collagen deposition were evaluated by hematoxylin and eosin and Masson's trichrome staining, respectively. Infiltration of CD4+, CD8+ T cells, and neutrophils was detected with immunohistochemical staining. The frequencies of T helper 1 cell (Th1), T helper 17 cell (Th17), and regulatory T cells (Treg) were measured by flow cytometry. Cytokine levels were measured by ELISA kits. MCC950 treatment significantly inhibited Nlrp3 inflammasome activation after allogeneic tracheal transplant and markedly decreased the luminal occlusion rate and collagen deposition in the allograft. The numbers of infiltrating CD4+, CD8+ T cells, and neutrophils in the allograft were also significantly reduced by MCC950 treatment. MCC950 dramatically decreased the frequencies of Th1/Th17 cells and the levels of interferon gamma/interleukin (IL)-17A and increased the Treg cell frequencies and IL-10 level; however, these effects were abolished by the addition of IL-1β and IL-18 both in vitro and in vivo. OB was also rescued by the addition of IL-1β and/or IL-18. Blocking Nlrp3 inflammasome activation with MCC950 ameliorates OB lesions. The mechanistic analysis showed that MCC950 regulated the balance of Th1/Th17 and Treg cells and that this process is partially mediated by inhibition of IL-1β and IL-18. Therefore, targeting the Nlrp3 inflammasome is a promising strategy for controlling OB after lung transplantation.

IPSC-derived MSC therapy induces immune tolerance and supports long-term graft survival in mouse orthotopic tracheal transplants

BackgroundLung transplantation is a life-saving surgical replacement of diseased lungs in patients with end-stage respiratory malfunctions. Despite remarkable short-term recovery, long-term lung survival continues to face several major challenges, including chronic rejection and severe toxic side effects due to global immunosuppression. Stem cell-based immunotherapy has been recognized as a crucial immunoregulatory regimen in various preclinical and clinical studies. Despite initial therapeutic outcomes, conventional stem cells face key limitations. The novel Cymerus™ manufacturing facilitates production of a virtually limitless supply of consistent human induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells, which could play a key role in selective immunosuppression and graft repair during rejection.MethodsHere, we demonstrated the impact of iPSC-derived human MSCs on the development of immune tolerance and long-term graft survival in mouse orthotopic airway allografts. BALB/c → C57BL/6 allografts were reconstituted with iPSC-derived MSCs (2 million/transplant/at d0), and allografts were examined for regulatory T cells (Tregs), oxygenation, microvascular blood flow, airway epithelium, and collagen deposition during rejection.ResultsWe demonstrated that iPSC-derived MSC treatment leads to significant increases in hTSG-6 protein, followed by an upregulation of mouse Tregs and IL-5, IL-10, and IL-15 cytokines, which augments graft microvascular blood flow and oxygenation, and thereby maintained a healthy airway epithelium and prevented the subepithelial deposition of collagen at d90 post transplantation.ConclusionsCollectively, these data confirmed that iPSC-derived MSC-mediated immunosuppression has potential to establish immune tolerance and rescue allograft from sustained hypoxic/ischemic phase, and subsequently limits long-term airway epithelial injury and collagen progression, which therapeutically warrant a study of Cymerus iPSC-derived MSCs as a potential management option for immunosuppression in transplant recipients.

Establishment of a heterotopic tracheal transplantation model in rat for the study of acute rejection after transplantation

Objective The mechanism of acute rejection after tracheal transplantation remains unclear. Orthotopic tracheal transplantation model is technically challenging, and difficult to be widely adopted. In this study, a heterotopic tracheal transplantation model in rat was used to simulate acute rejection after transplantation, establishing a simple but effective animal model. Methods This work was performed from May 2017 to July 2017. SD and Wistar rats were selected for both syngeneic and allogeneic heterotopic tracheal transplantations. The average time and success rate of tracheal transplantation were calculated.Trachea was enclosed with omentum through an abdominal incision. The implants were harvested on postoperative day 1, 3, 7 and 14, hematoxylin and eosin (HE) staining was used to compare the infiltration of inflammatory cells at each time point, descriptive statistics were done afterwards. Results Thirty-one heterotopic tracheal transplantations were performed successfully, and one died of accidental anesthesia. All tracheal grafts from the 8 donors were obtained within 10 min, the procedure of heterotopic tracheal transplantation spent an average of (6.0±1.8) min. There was no graft infection or other failure. The overall success rate was 96.9%. There was much more omentum wrapping the allograft than the isograft. HE staining indicated that infiltration of inflammatory cells in allograft gradually aggravated over time, and the tracheal wall was damaged progressively. Conclusion The heterotopic tracheal transplantation model in rat is simple but effective, has been verified to present typical pathological changes of acute rejection and would be suitable for the study of immune response after transplantation. Key words: Tracheal transplantation; Heterotopic transplantation; Acute rejection; Animal model

MiR-27a-3p downregulation contributes to the development of occlusive bronchiolitis.

The only effective clinical treatment for many end-stage lung diseases is lung transplantation. However, chronic rejection of transplanted lung affects the long-term efficacy of lung transplantation to a large extent, thereby limiting the clinical application of lung transplantation. Occlusive bronchiolitis (OB) is a major cause of chronic functional loss of the transplanted lung. However, the OB pathogenesis remains unclear. Therefore, studying the OB pathogenesis and finding effective intervention methods are highly important. This study analyzed changes in the expression profile of microRNAs and transcription factors in mice with OB after orthotopic tracheal transplantation. miR-27a-3p was upregulated in lung tissue 20days after transplantation. Transcription factor microarray analysis revealed that Smad3 was significantly downregulated. A miRNA-mRNA interaction network was constructed, and specific regulatory effects of miR-27a-3p on Smad3 were found. Smad3 was strongly associated with tumorigenesis and organ fibrosis. Compared with the control group, miR-27a-3p inhibited the epithelial-mesenchymal transformation (EMT) of lung epithelial cells. In addition, miR-27a-3p inhibition promoted the invasion and migration of lung epithelial cells. Dual luciferase reporter gene assay showed that miR-27a-3p can regulate the promoter activity of Smad3. MiR-27a-3p also inhibited the expression of inflammatory cytokines. Western blot results showed that miR-27a-3p can upregulate the E-cadherin expression and downregulate the expression of vimentin, fibronectin, and α-SMA. By studying the OB pathogenesis, we found that inhibition or alteration of the occurrence of EMT may reduce the proportion of chronic rejection of lung transplantation. MiR-27a-3p may also be developed as a new drug for the OB therapy. This finding will provide many possibilities for OB treatment and improve the prognosis of patients with OB.

Pioglitazone Induces Infiltrations of Regulatory T Cells in the Tracheal Graft and Attenuates Allograft Rejection

Purpose Bronchiolitis obliterans (BO) and bronchiolitis obliterans syndrome were recognized as serious complications after lung transplantation. We hypothesized that pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-γ agonist, could prevent airway rejection in established mouse orthotopic tracheal transplant models. Methods Tracheal grafts from wild type BALB/c or C57BL/6 were transplanted to C57BL/6 recipients. Experimental groups were treated with multiple doses of pioglitazone, at total 5mg/kg per day. Histopathologic evaluation of luminal obliteration was blindly reviewed at day 7. Immunohistochemistry for CD3, FoxP3 and real-time PCR analyses for cytokines were performed. Results Allografts treated with pioglitazone revealed a striking reduction of thickening in airway layers (P Conclusion Pioglitazone leads to decreased airway graft luminal occlusion and induced accumulation of Tregs in the graft. These data indicated a strong protective role for pioglitazone against BO. The potential mechanism of this beneficial effect of pioglitazone appears to increase the production of IL-4 and decrease the production of IFN-γ, TNF-α and IL-6.

Role of Toll-like Receptor 4 Expressed by Fibroblasts in Allograft Fibrosis in Mouse Orthotopic Tracheal Transplantation

Development of chronic lung allograft dysfunction involves various alloimmune-independent insults including those mediated by Toll-like receptor (TLR) signaling, which is known to activate alloimmune responses. We hypothesized that TLR signaling may also contribute to the activation of fibroblasts and promoting allograft airway fibrosis. Mouse orthotopic tracheal transplants were conducted between major histocompatibility complex (MHC)-mismatched Balb/c donor and wild-type C3H or C3H-derived TLR4 mutant recipients (nonfunctional TLR4). Immunohistochemistry on day 21 showed significantly smaller alpha-smooth muscle actin (α-SMA)-positive areas in TLR4 mutant recipients than wild-type recipients (P = .01). No difference was found for CD3+ T-cell infiltration. Proliferation of alloreactive T cells derived from the recipient spleen showed no difference between TLR4 mutant and wild-type recipients in a mixed lymphocyte reaction. The effect of TLR4 signaling was examined in primary pulmonary fibroblast cultures both with lipopolysaccharide (LPS) and transforming growth factor (TGF)-β1. Stimulation with LPS significantly increased expression of α-SMA mRNA in wild-type fibroblasts cultured with TGF-β1 compared with the control without LPS (P = .001). Taken together, these findings suggest disruption of TLR signaling leads to reduced activation of fibroblasts without affecting T-cell infiltration and proliferation in this model. TLR4-mediated activation of fibroblasts may be a potentially important mechanism of allograft remodeling.

AhR activation plays a detrimental role in airway epithelial injury after transplantation

Abstract Patients with tracheal tumors, trauma, infection, congenital malformations or injury after prolonged intubation may require partial or complete tracheal resection, and tracheal transplantation will be necessary when the length of resected trachea is longer than 30% in children or 50% in adults. Currently, obliterative bronchiolitis (OB) is a major obstacle that affects allograft function and survival after tracheal transplantation. Th17 polarization and inflammatory cytokine expression is crucially involved in airway transplant rejection, and activation of aryl hydrocarbon receptor (AhR) could modulate Th17 differentiation and inflammatory cytokine production. However, the role of AhR signal pathway in airway epithelial injury, a leading factor of OB development, after transplantation remains unclear. In this study, we performed murine orthotopic tracheal transplants in C57BL/6 wild type mice using BALB/c donors. The levels of IFNγ, IL-6 and IL-17 expression were significantly elevated in allografts, and the airway pseudostratified epithelia changed into flat epithelia with inflammation and fibroblast proliferation in submucosa tissue. Allografts of recipients administered with AhR antagonist (CH-223191) displayed inhibition of IL-17 and IFNγ, and some airway epithelial protection. Significant lamina fibrosis and higher levels of inflammatory cytokine production was observed in allografts of recipients administered with AhR agonist (FICZ). Together, our results demonstrate that AhR activation plays a detrimental role in airway epithelial injury after transplantation, pointing to a new therapeutic avenue for benefitting airway allograft survival through manipulating AhR pathway.

Effects of bone marrow mesenchymal stem cells on experimental bronchiolitis obliterans

Objective Through orthotopic tracheal transplantation model to analyze the therapeutic effect of bone marrow mesenchymal stem cells (BMSCs) on obliterative bronchiolitis (OB). Methods Brown Norway-to-Lewis rats were subjected to orthotopic tracheal transplantation, and intragastically given 15 mg/(kg·d) cyclosporine (CsA) until the day when the animals were ready to be killed. BMSCs single cell suspension liquid [1 ml, (1×106/ml) and normal saline (NS) (1 ml) were injected via the tail vein, respectively. Tracheal grafts were harvested at 7th, 30th, and 60th days post-transplantation. The tracheal luminal obliteration rate and epithelial loss were assessed on sections stained with hematoxylin and eosin. CD4+ and CD8+ T lymphocytes in peripheral blood were aexamined using flow cytometry. The expression of transforming factor-beta1 (TGF-β1) was detected by Western blotting. Results The luminal obliteration in NS, CsA and BMSCs treated groups after 60 days post-transplant were (52.62±10.21)%, (28.52±8.81)% and (21.30±2.27)% respectively, CsA and BMSCs were significantly attenuated (P=0.030, P=0.004); the epithelial loss were (96.10±0.86)%, (93.67±4.04)% and (25.26±1.10)% respectively, BMSCs were protective against the epithelial loss post-transplant compared with NS and CsA group (P=0.009); the percentage of CD8+ T lymphocyte in peripheral blood were (24.13±1.82)%, (49.50±3.03)% and (26.27±0.40)% respectively, which were significantly suppressed by BMSCs compared with NS and CsA group (P=0.007). The relative expression quantity of TGF-β1 were 1.21, 0.77 and 0.36 respectively, which can decrease TGF-β1 expression induced by transplant (P=0.000). Conclusion BMSCs could prevent development of airway occlusion through downregulating the expression of TGF-β1, reducing the CD8+ T lymphocyte infiltration and enhancing epithelial preservation in a orthotopic tracheal transplantation mouse model of OB. Key words: Orthotopic tracheal transplantation; Bone marrow mesenchymal stem cells; Cyclosporine; Lung transplantation; Obliterative bronchiolitis; Rat

FOXP3+ regulatory T cell ameliorates microvasculature in the rejection of mouse orthotopic tracheal transplants

Microvascular loss may be a root cause of chronic rejection in lung transplants, which leads to the bronchiolitis obliterans syndrome. Previous research implicates T regulatory cell (Treg) as a key component of immune modulation, however, Treg has never been examined as a reparative mediator to salvage microvasculature during transplantation. Here, we reconstituted purified Tregs in to allografts, and serially monitored allografts for tissue oxygenation, microvascular perfusion for four weeks. We demonstrated that Tregs reconstitution of allografts significantly improve tissue oxygenation, microvascular flow, epithelial repair, number of CD4+CD25highFOXP3+ Tregs, followed by an upregulation of proinflammatory, angiogenic and regulatory genes, while prevented subepithelial deposition of CD4+T cells at d10, and collagen at d28 post-transplantation. Altogether, these findings concluded that Treg-mediated immunotherapy has potential to preserve microvasculature and rescue allograft from sustained hypoxic/ischemic phase, limits airway tissue remodeling, and therefore may be a useful therapeutic tool to prevent chronic rejection after organ transplantation.

Simultaneously Targeting Myofibroblast Contractility and Extracellular Matrix Cross-Linking as a Therapeutic Concept in Airway Fibrosis.

Fibrosis after solid organ transplantation is considered an irreversible process and remains the major cause of graft dysfunction and death with limited therapies. This remodeling is characterized by aberrant accumulation of contractile myofibroblasts that deposit excessive extracellular matrix (ECM) and increase tissue stiffness. Studies demonstrate, however, that a stiff ECM itself promotes fibroblast-to-myofibroblast differentiation, stimulating further ECM production. This creates a positive feedback loop that perpetuates fibrosis. We hypothesized that simultaneously targeting myofibroblast contractility with relaxin and ECM stiffness with lysyl oxidase inhibitors could break the feedback loop, reversing established fibrosis. To test this, we used the orthotopic tracheal transplantation (OTT) mouse model, which develops robust fibrotic airway remodeling. Mice with established fibrosis were treated with saline, mono-, or combination therapies. Although monotherapies had no effect, combining these agents decreased collagen deposition and promoted re-epithelialization of remodeled airways. Relaxin inhibited myofibroblast differentiation and contraction in a matrix-stiffness-dependent manner through prostaglandin E2 (PGE2 ). Furthermore, the effect of combination therapy was lost in PGE2 receptor knockout and PGE2 -inhibited OTT mice. This study revealed the important synergistic roles of cellular contractility and tissue stiffness in the maintenance of fibrotic tissue and suggests a new therapeutic principle for fibrosis.

Orthotopic tracheal transplantation using human bronchus: an original xenotransplant model of obliterative airway disorder.

Bronchiolitis obliterans syndrome (BOS) is a main cause of allograft dysfunction and mortality after lung transplantation (LTx). A better understanding of BOS pathogenesis is needed to overcome this treatment-refractory complication. Orthotopic tracheal transplantation using human bronchus was performed in Brown Norway (BN) and nude (RNU) rats. Allografts were recovered in both strains at Day 7 (BN7 , n = 6; RNU7 , n = 7) or Day 28 (BN28 , n = 6; RNU28 , n = 6). Immune response of the host against the bronchial graft was assessed. Human samples from BOS patients were used to compare with the histological features of the animal model. Obstruction of the allograft lumen associated with significant infiltration of CD3+ and CD68+ cells was observed in the BN group on Day 28. Immune response from type 1 T-helper cells against the tracheal xenograft was higher in BN animals compared to nude animals on Days 7 and 28 (P < 0.001 and P = 0.035). Xenoreactive antibodies were significantly higher at Day 7 (IgM) and Day 28 (IgG) in the BN group compared to RNU (respectively, 37.6 ± 6.5 vs. 5.8 ± 0.7 mean fluorescence, P = 0.039; and 22.4 ± 3.8 vs. 6.9 ± 1.6 mean fluorescence, P = 0.011). Immunocompetent animals showed a higher infiltration of S100A4+ cells inside the bronchial wall after 28 days, associated with cartilage damage ranging from invasion to complete destruction. In vitro expression of S100A4 by human fibroblasts was higher when stimulated by mononuclear cells (MNCs) from BN rats than from RNU (2.9 ± 0.1 vs. 2.4 ± 0.1 mean fluorescence intensity, P = 0.005). Similarly, S100A4 was highly expressed in response to human MNCs compared to stimulation by T-cell-depleted human MNCs (4.3 ± 0.2 vs. 2.7 ± 0.1 mean fluorescence intensity, P < 0.001). Obliterative bronchiolitis has been induced in a new xenotransplant model in which chronic airway obstruction was associated with immune activation against the xenograft. Cartilage infiltration by S100A4+ cells might be stimulated by T cells.

Apoptosis of the Tracheal Epithelium Can Increase the Number of Recipient Bone Marrow-Derived Myofibroblasts in Allografts and Exacerbate Obliterative Bronchiolitis After Tracheal Transplantation in Mice.

The long-term outcome of lung transplantation is impeded by the development of obliterative bronchiolitis (OB). We sought to investigate the relationship between the apoptosis of tracheal epithelial cells and bone marrow-derived myofibroblasts in the process of OB. The mouse orthotopic tracheal transplant model was established. The allografts and syngrafts were harvested at 1, 2, 3, and 4 weeks after tracheal transplant. The percentage of tracheal lumen occlusion was assayed via histology and morphometry. Immunofluorescence staining was used to detect the apoptotic epithelial cells and recipient-derived myofibroblasts. The expression of SDF-1α and TGF-β and the infiltrations of CD4 and CD8 T cells in the grafts were detected using immunohistochemical staining. We found that there were more apoptotic epithelia in the allograft group than in the syngraft group and that the level of tracheal lumen occlusion was higher at different time points. Moreover, the increase in the apoptosis of the tracheal epithelium occurred earlier than that of occlusion of the tracheal lumen. There were more myofibroblasts derived from the recipient's bone marrow and more CD4 and CD8 T cells in the allografts. The expression of SDF-1α and TGF-β was higher in the epithelium from allografts than in those of the syngrafts. Our study indicated that the apoptotic tracheal epithelia in the OB model might increase the amount of myofibroblasts derived from the recipient's bone marrow. Therapeutic methods aimed at preventing apoptosis of the tracheal epithelium may improve the outcome of lung transplantation.

Cyclosporine Does Not Prevent Microvascular Loss in Transplantation but Can Synergize With a Neutrophil Elastase Inhibitor, Elafin, to Maintain Graft Perfusion During Acute Rejection

The loss of a functional microvascular bed in rejecting solid organ transplants is correlated with fibrotic remodeling and chronic rejection; in lung allografts, this pathology is predicted by bronchoalveolar fluid neutrophilia which suggests a role for polymorphonuclear cells in microcirculatory injury. In a mouse orthotopic tracheal transplant model, cyclosporine, which primarily inhibits T cells, failed as a monotherapy for preventing microvessel rejection and graft ischemia. To target neutrophil action that may be contributing to vascular injury, we examined the effect of a neutrophil elastase inhibitor, elafin, on the microvascular health of transplant tissue. We showed that elafin monotherapy prolonged microvascular perfusion and enhanced tissue oxygenation while diminishing the infiltration of neutrophils and macrophages and decreasing tissue deposition of complement C3 and the membrane attack complex, C5b-9. Elafin was also found to promote angiogenesis through activation of the extracellular signal-regulated kinase (ERK) signaling pathway but was insufficient as a single agent to completely prevent tissue ischemia during acute rejection episodes. However, when combined with cyclosporine, elafin effectively preserved airway microvascular perfusion and oxygenation. The therapeutic strategy of targeting neutrophil elastase activity alongside standard immunosuppression during acute rejection episodes may be an effective approach for preventing the development of irreversible fibrotic remodeling.