Targeting leukocytes, neutrophil extracellular traps and cytokines: A conceptual review to prevent primary graft dysfunction after lung transplantation.

Primary graft dysfunction (PGD) causes early mortality after lung transplantation and may contribute to late graft failure. No effective treatments exist. The pathogenesis of PGD is unclear, although both neutrophils and activated platelets have been implicated. We hypothesized that neutrophil extracellular traps (NETs) contribute to lung injury in PGD in a platelet-dependent manner. To study NETs in experimental models of PGD and in lung transplant patients. Two experimental murine PGD models were studied: hilar clamp and orthotopic lung transplantation after prolonged cold ischemia (OLT-PCI). NETs were assessed by immunofluorescence microscopy and ELISA. Platelet activation was inhibited with aspirin, and NETs were disrupted with DNaseI. NETs were also measured in bronchoalveolar lavage fluid and plasma from lung transplant patients with and without PGD. NETs were increased after either hilar clamp or OLT-PCI compared with surgical control subjects. Activation and intrapulmonary accumulation of platelets were increased in OLT-PCI, and platelet inhibition reduced NETs and lung injury, and improved oxygenation. Disruption of NETs by intrabronchial administration of DNaseI also reduced lung injury and improved oxygenation. In bronchoalveolar lavage fluid from human lung transplant recipients, NETs were more abundant in patients with PGD. NETs accumulate in the lung in both experimental and clinical PGD. In experimental PGD, NET formation is platelet-dependent, and disruption of NETs with DNaseI reduces lung injury. These data are the first description of a pathogenic role for NETs in solid organ transplantation and suggest that NETs are a promising therapeutic target in PGD.

Report of the ISHLT Working Group on Primary Lung Graft Dysfunction Part I: Introduction and Methods

National Heart, Lung, and Blood Institute and American Association for Thoracic Surgery Workshop Report: Identifying collaborative clinical research priorities in lung transplantation

Immunopathogenesis of Primary Graft Dysfunction After Lung Transplantation

Primary graft dysfunction in heart transplantation: How to recognize it, when to institute extracorporeal membrane oxygenation, and outcomes

Donor Fat Embolism and Primary Graft Dysfunction After Lung Transplantation

PurposeIschemia reperfusion injury (IRI) unavoidably occurs during lung transplantation, further contributing to primary graft dysfunction (PGD). Neutrophils are the end effectors of IRI and activated neutrophils release neutrophil extracellular traps (NETs) to further amplify damage. Nevertheless, potential contributions of NETs in IRI remain incompletely understood. This study aimed to explore NET-related gene biomarkers in IRI during lung transplantation.MethodsDifferential expression analysis was applied to identify differentially expressed genes (DEGs) for IRI during lung transplantation based on matrix data (GSE145989, 127003) downloaded from GEO database. The CIBERSORT and weighted gene co-expression network analysis (WGCNA) algorithms were utilized to identify key modules associated with neutrophil infiltration. Moreover, the least absolute shrinkage and selection operator regression and random forest were applied to identify potential NET-associated hub genes. Subsequently, the screened hub genes underwent further validation of an external dataset (GSE18995) and nomogram model. Based on clinical peripheral blood samples, immunofluorescence staining and dsDNA quantification were used to assess NET formation, and ELISA was applied to validate the expression of hub genes.ResultsThirty-eight genes resulted from the intersection between 586 DEGs and 75 brown module genes, primarily enriched in leukocyte migration and NETs formation. Subsequently, four candidate hub genes (FCAR, MMP9, PADI4, and S100A12) were screened out via machine learning algorithms. Validation using an external dataset and nomogram model achieved better predictive value. Substantial NETs formation was demonstrated in IRI, with more pronounced NETs observed in patients with PGD ≥ 2. PADI4, S100A12, and MMP9 were all confirmed to be up-regulated after reperfusion through ELISA, with higher levels of S100A12 in PGD ≥ 2 patients compared with non-PGD patients.ConclusionWe identified three potential NET-related biomarkers for IRI that provide new insights into early detection and potential therapeutic targets of IRI and PGD after lung transplantation.

Frailty and aging-associated syndromes in lung transplant candidates and recipients.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

The respiratory microbiome after lung transplantation: Reflection or driver of respiratory disease?

Primary graft dysfunction caused by ischemia-reperfusion injury is one of the most frequent causes of early morbidity and death after lung transplantation. We hypothesized that the perioperative management with aprotinin decreases the incidence of allograft reperfusion injury and dysfunction after clinical lung transplantation. Lung transplant databases of two transplant centers were used to investigate the incidence of severe post-transplant reperfusion injury (PTRI). We examined data of 142 patients who underwent either single lung (81) or bilateral sequential lung (61) transplantation for COPD, idiopathic pulmonary fibrosis, cystic fibrosis, and miscellaneous lung disorders between 1997 and 2000. Thirty patients were excluded due to heart-lung transplantation or lung transplantation for Eisenmenger's disease, re-transplantation, rejection, or deviation from the standardized triple immunosuppression protocol. The data of remaining 112 patients (control group, 64% single lung, 36% sequential bilateral lung transplants) were compared to the prospectively collected data of 59 lung transplant patients over the last 5 years. All of these 59 patients were managed perioperatively with aprotinin infusion. In addition, Euro-Collins-aprotinin procurement solution (Apt-EC group) was used for 50 donor lungs (58% single lung, 42% sequential bilateral lung transplants). Aprotinin in combination with low-potassium dextran (LPD) flush solution (Apt-LPD group) was used for the procurement of 34 lungs (59% single lung, 41% sequential bilateral lung transplants). The International Society of Heart and Lung Transplantation (ISHLT) grade III injury score was used for the diagnosis of severe PTRI, which is based on a PaO(2)-FIO(2) ratio of less than 200 mmHg. Severe reperfusion injury grade III was observed in 18% of the control group. ECMO support was required in 25% of these patients. The associated mortality rate was 40%. Correlating factors for PTRI were donor age greater than 35 years (45%, p=0.01, mean age 38+/-8) and recipient pulmonary artery systolic pressure greater than 60 mmHg (48%, p<0.05). Lung graft ischemic times (231+/-14 min) and intraoperative techniques (cardiopulmonary bypass in 12%) were not associated with negative outcomes. Despite longer ischemic times (258+/-36 min and 317+/-85 min, respectively) and older donors (42+/-12 years and 46+/-12 years, respectively) in the aprotinin patient groups (Apt-EC and Apt-LPD group), the incidence of PTRI was markedly lower (6% and 9%, respectively). There was no mortality in the Apt-EC group and one patient died in the Apt-LPD group due to PTRI-induced graft failure. Severe PTRI increased short-term morbidity and mortality. The incidence of reperfusion injury was not dependent upon the duration of donor organ ischemia. The use of aprotinin in the perioperative patient management in lung transplantation had strong beneficial effects on the patient outcomes and decreased the incidence of post-transplant ischemia-reperfusion injury significantly.

(107) - Leukocytes Transfer More Severe Rejection from Lung Transplant Recipients with Severe PGD into Humanized Mice and Are Sensitive to T Cell Regulation

Background Primary graft dysfunction (PGD), the major complication associated with lung transplantation in the peri-operative period, is characterized by vascular and alveolar inflammation and results in hypoxemia and lung edema. Up to 30% of recipients develop the most severe form of PGD (Grade 3; PGD3), leading to 50% mortality. Animal studies suggest that neutrophils can contribute to the inflammatory process through the release of neutrophil extracellular traps(NETs). NETs are composed of DNA filaments decorated with granular proteins, which can contribute to vascular occlusion associated with PGD. Our main objective is to correlate NETosis with recipient outcomes. Methods Clinical data and blood samples were prospectively collected from donors and recipients (n=32) pre-, intra- and post-operatively (up to 72hrs). Inflammatory markers inducing NETs’ synthesis (CRP, IL6, IL8) and markers of NETs (myeloperoxidase [MPO], MPO-DNA complexes, cell-free DNA) were quantified by ELISA. When available, histology and immunochemistry techniques were used on donor lung samples collected before transplantation to evaluate the presence of activated neutrophils and NETs. Results In lung samples from which PGD3 developed in recipients, there was a marked increase of vascular occlusion composed of activated neutrophils and NETs before transplantation. Also, in donors and recipients pre- and intra-operatively, circulating levels of inflammatory (CRP, IL6, IL8) and NETosis biomarkers (MPO-DNA, MPO, cfDNA) were up to 4-fold higher in PGD3 recipients compared to non-PGD3. Conclusion Elevation of these NETosis biomarkers might serve to elaborate an algorithm to better delineate the recipients at risk of developing severe PGD.

Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism.