Xingan Wang

Hyaluronan Contributes to Bronchiolitis Obliterans Syndrome and Stimulates Lung Allograft Rejection through Activation of Innate Immunity

RATIONALE Although innate immunity is increasingly recognized to contribute to lung allograft rejection, the significance of endogenous innate ligands, such as hyaluronan (HA) fragments, in clinical or experimental lung transplantation is uncertain. OBJECTIVES To determine if HA is associated with clinical bronchiolitis obliterans syndrome (BOS) in lung transplant recipients, and evaluate the effect of low- or high-molecular-weight HA on experimental lung allograft rejection, including dependence on innate signaling pathways or effector cells. METHODS HA concentrations were measured in bronchoalveolar lavage and plasma samples from lung recipients with or without established BOS. BOS and normal lung tissues were assessed for HA localization and expression of HA synthases. Murine orthotopic lung recipients with established tolerance were treated with low- or high-molecular-weight HA under varied experimental conditions, including Toll-like receptor (TLR) 2/4 and myeloid differentiation protein 88 deficiency and neutrophil depletion. MEASUREMENTS AND MAIN RESULTS HA localized within areas of intraluminal small airways fibrosis in BOS lung tissue. Moreover, transcripts for HA synthase enzymes were significantly elevated in BOS versus normal lung tissues and both lavage fluid and plasma HA concentrations were increased in recipients with BOS. Treatment with low-molecular-weight HA abrogated tolerance in murine orthotopic lung recipients in a TLR2/4- and myeloid differentiation protein 88-dependent fashion and drove expansion of alloantigen-specific T lymphocytes. Additionally, TLR-dependent signals stimulated neutrophilia that promoted rejection. In contrast, high-molecular-weight HA attenuated basal allograft inflammation. CONCLUSIONS These data suggest that accumulation of HA could contribute to BOS by directly activating innate immune signaling pathways that promote allograft rejection and neutrophilia.

Increased T Cell Glucose Uptake Reflects Acute Rejection in Lung Grafts

Although T cells are required for acute lung rejection, other graft–infiltrating cells such as neutrophils accumulate in allografts and are also high glucose utilizers. Positron emission tomography (PET) with the glucose probe [18F]fluorodeoxyglucose ([18F]FDG) has been employed to image solid organ acute rejection, but the sources of glucose utilization remain undefined. Using a mouse model of orthotopic lung transplantation, we analyzed glucose probe uptake in the grafts of syngeneic and allogeneic recipients with or without immunosuppression treatment. Pulmonary microPET scans demonstrated significantly higher [18F]FDG uptake in rejecting allografts when compared to transplanted lungs of either immunosuppressed or syngeneic recipients. [18F]FDG uptake was also markedly attenuated following T cell depletion therapy in lung recipients with ongoing acute rejection. Flow cytometric analysis using the fluorescent deoxyglucose analog 2‐NBDG revealed that T cells, and in particular CD8+ T cells, were the largest glucose utilizers in acutely rejecting lung grafts followed by neutrophils and antigen‐presenting cells. These data indicate that imaging modalities tailored toward assessing T cell metabolism may be useful in identifying acute rejection in lung recipients.

Five-year update on the mouse model of orthotopic lung transplantation: Scientific uses, tricks of the trade, and tips for success

It has been 5 years since our team reported the first successful model of orthotopic single lung transplantation in the mouse. There has been great demand for this technique due to the obvious experimental advantages the mouse offers over other large and small animal models of lung transplantation. These include the availability of mouse-specific reagents as well as knockout and transgenic technology. Our laboratory has utilized this mouse model to study both immunological and non-immunological mechanisms of lung transplant physiology while others have focused on models of chronic rejection. It is surprising that despite our initial publication in 2007 only few other laboratories have published data using this model. This is likely due to the technical complexity of the surgical technique and perioperative complications, which can limit recipient survival. As two of the authors (XL and WL) have a combined experience of over 2500 left and right single lung transplants, this review will summarize their experience and delineate tips and tricks necessary for successful transplantation. We will also describe technical advances made since the original description of the model.

Human recombinant apyrase therapy protects against canine pulmonary ischemia-reperfusion injury

BACKGROUND There is accumulating evidence that extracellular adenosine triphosphate (eATP) promotes many of the underlying mechanisms that exacerbate acute lung injury. However, much of these data are from inbred rodent models, indicating the need for further investigation in higher vertebrates to better establish clinical relevance. To this end we evaluated a human recombinant apyrase therapy in a canine warm pulmonary ischemia-reperfusion injury (IRI) model and measured eATP levels in human lung recipients with or without primary lung graft dysfunction (PGD). METHODS Warm ischemia was induced for 90 minutes in the left lung of 14 mongrel dogs. Seven minutes after reperfusion, the apyrase APT102 (1 mg/kg, n = 7) or saline vehicle (n = 7) was injected into the pulmonary artery. Arterial blood gases were obtained every 30 minutes up to 180 minutes after reperfusion. Bronchioalveolar lavage fluid (BALF) was analyzed for eATP concentration, cellularity, and inflammatory mediator accumulation. Thirty bilateral human lung transplant recipients were graded for immediate early PGD and assessed for BALF eATP levels. RESULTS APT102-treated dogs had progressively better lung function and less pulmonary edema during the 3-hour reperfusion period compared with vehicle-treated controls. Protection from IRI was observed, with lower BALF eATP levels, fewer airway leukocytes, and blunted inflammatory mediator expression. Human lung recipients with moderate to severe PGD had significantly higher eATP levels compared with recipients without this injury. CONCLUSIONS Extracellular ATP accumulates in acutely injured canine and human lungs. Strategies that target eATP reduction may help protect lung recipients from IRI.

Imaging mouse lung allograft rejection with 1H MRI

To demonstrate that longitudinal, noninvasive monitoring via MRI can characterize acute cellular rejection in mouse orthotopic lung allografts.

Surgical technique for lung retransplantation in the mouse

Microsurgical cuff techniques for orthotopic vascularized murine lung transplantation have allowed for the design of studies that examine mechanisms contributing to the high failure rate of pulmonary grafts. Here, we provide a detailed technical description of orthotopic lung retransplantation in mice, which we have thus far performed in 144 animals. The total time of the retransplantation procedure is approximately 55 minutes, 20 minutes for donor harvest and 35 minutes for the implantation, with a success rate exceeding 95%. The mouse lung retransplantation model represents a novel and powerful tool to examine how cells that reside in or infiltrate pulmonary grafts shape immune responses.

Eosinophils promote inducible NOS–mediated lung allograft acceptance

Lungs allografts have worse long-term survival compared with other organ transplants. This is most likely due to their unique immunoregulation that may not respond to traditional immunosuppression. For example, local NO generation by inducible NOS (iNOS) is critical for lung allograft acceptance but associates with rejection of other solid organs. The source of NO in accepting lung allografts remains unknown. Here, we report that, unlike the case for other pulmonary processes in which myeloid cells control NO generation, recipient-derived eosinophils play a critical and nonredundant role in iNOS-mediated lung allograft acceptance. Depletion of eosinophils reduces NO levels to that of recipients with global deletion of iNOS and leads to a costimulatory blockade-resistant form of rejection. Furthermore, NO production by eosinophils depends on Th1 polarization by inflammatory mediators, such as IFN-γ and TNF-α. Neutralization of such mediators abrogates eosinophil suppressive capacity. Our data point to what we believe to be a unique and previously unrecognized role of eosinophil polarization in mediating allograft tolerance and put into perspective the use of high-dose eosinophil-ablating corticosteroids after lung transplantation.