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Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here, we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independently of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.

Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first posttransplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. NK cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. These data support an aberrant NK cell/neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.

Histopathologic lung allograft injuries are putative harbingers for chronic lung allograft dysfunction (CLAD). However, the mechanisms responsible are not well understood. CXCL9 and CXCL10 are potent chemoattractants of mononuclear cells and potential propagators of allograft injury. We hypothesized that these chemokines would be quantifiable in plasma, and would associate with subsequent CLAD development. In this prospective multicenter study, we evaluated 721 plasma samples for CXCL9/CXCL10 levels from 184 participants at the time of transbronchial biopsies during their first-year post-transplantation. We determined the association between plasma chemokines, histopathologic injury, and CLAD risk using Cox proportional hazards models. We also evaluated CXCL9/CXCL10 levels in bronchoalveolar lavage (BAL) fluid and compared plasma to BAL with respect to CLAD risk. Plasma CXCL9/CXCL10 levels were elevated during the injury patterns associated with CLAD, acute rejection, and acute lung injury, with a dose-response relationship between chemokine levels and CLAD risk. Importantly, there were strong interactions between injury and plasma CXCL9/CXCL10, where histopathologic injury associated with CLAD only in the presence of elevated plasma chemokines. We observed similar associations and interactions with BAL CXCL9/CXCL10 levels. Elevated plasma CXCL9/CXCL10 during allograft injury may contribute to CLAD pathogenesis and has potential as a minimally invasive immune monitoring biomarker.

IntroductionTransbronchial lung cryobiopsy (TBLC) has emerged as a promising alternative to surgical lung biopsy for the diagnosis of interstitial lung disease. However, uncertainty remains regarding its overall complications due to a lack of procedural standardization including the size of cryoprobe utilized.MethodsThis is a prospective cohort study of a protocolized transbronchial cryobiopsy program utilizing a 2.4 mm cryoprobe. 201 consecutive subjects were enrolled at a single academic center.ResultsThe average biopsy size was 106.2 ± 39.3 mm2. Complications included a total pneumothorax rate of 4.9% with 3.5% undergoing chest tube placement. Severe bleeding defined by the Nashville Working Group occurred in 0.5% of cases. There were no deaths at 30-days.DiscussionA protocolized transbronchial cryobiopsy program utilizing a 2.4 mm cryoprobe in can achieve a high diagnostic yield with a favorable safety profile.

BackgroundChronic lung allograft dysfunction (CLAD) phenotype determines prognosis and may have therapeutic implications. Despite the clarity achieved by recent consensus statement definitions, their reliance on radiologic interpretation introduces subjectivity. The Center for Computer Vision and Imaging Biomarkers at the University of California, Los Angeles (UCLA) has established protocols for chest high-resolution computed tomography (HRCT)-based computer-aided quantification of both interstitial disease and air-trapping. We applied quantitative image analysis (QIA) at CLAD onset to demonstrate radiographic phenotypes with clinical implications.MethodsWe studied 47 first bilateral lung transplant recipients at UCLA with chest HRCT performed within 90 d of CLAD onset and 47 no-CLAD control HRCTs. QIA determined the proportion of lung volume affected by interstitial disease and air-trapping in total lung capacity and residual volume images, respectively. We compared QIA scores between no-CLAD and CLAD, and between phenotypes. We also assigned radiographic phenotypes based solely on QIA, and compared their survival outcomes.ResultsCLAD onset HRCTs had more lung affected by the interstitial disease (P = 0.003) than no-CLAD controls. Bronchiolitis obliterans syndrome (BOS) cases had lower scores for interstitial disease as compared with probable restrictive allograft syndrome (RAS) (P < 0.0001) and mixed CLAD (P = 0.02) phenotypes. BOS cases had more air-trapping than probable RAS (P < 0.0001). Among phenotypes assigned by QIA, the relative risk of death was greatest for mixed (relative risk [RR] 11.81), followed by RAS (RR 6.27) and BOS (RR 3.15).ConclusionsChest HRCT QIA at CLAD onset appears promising as a method for precise determination of CLAD phenotypes with survival implications.

Despite the common detection of non-donor specific anti-HLA antibodies (non-DSAs) after lung transplantation, their clinical significance remains unclear. In this retrospective single-center cohort study of 325 lung transplant recipients, we evaluated the association between donor-specific HLA antibodies (DSAs) and non-DSAs with subsequent CLAD development. DSAs were detected in 30% of recipients and were associated with increased CLAD risk, with higher HRs for both de novo and high MFI (>5000) DSAs. Non-DSAs were detected in 56% of recipients, and 85% of DSA positive tests had concurrent non-DSAs. In general, non-DSAs did not increase CLAD risk in multivariable models accounting for DSAs. However, non-DSAs in conjunction with high BAL CXCL9 levels were associated with increased CLAD risk. Multivariable proportional hazards models demonstrate the importance of the HLA antibody-CXCL9 interaction: CLAD risk increases when HLA antibodies (both DSAs and non-DSAs) are detected in conjunction with high CXCL9. Conversely, CLAD risk is not increased when HLA antibodies are detected with low CXCL9. This study supports the potential utility of BAL CXCL9 measurement as a biomarker to risk stratify HLA antibodies for future CLAD. The ability to discriminate between high versus low-risk HLA antibodies may improve management by allowing for guided treatment decisions.

Sweet's Syndrome (SS), also known as acute febrile neutrophilic dermatosis, is one of several cutaneous inflammatory disorders classified as neutrophilic dermatoses. Respiratory complications are described in

Bronchoalveolar lavage (BAL) is a key clinical and research tool in lung transplantation (LTx). However, BAL collection and processing are not standardized across LTx centers. This International Society for Heart and Lung Transplantation-supported consensus document on BAL standardization aims to clarify definitions and propose common approaches to improve clinical and research practice standards. The following 9 areas are covered: (1) bronchoscopy procedure and BAL collection, (2) sample handling, (3) sample processing for microbiology, (4) cytology, (5) research, (6) microbiome, (7) sample inventory/tracking, (8) donor bronchoscopy, and (9) pediatric considerations. This consensus document aims to harmonize clinical and research practices for BAL collection and processing in LTx. The overarching goal is to enhance standardization and multicenter collaboration within the international LTx community and enable improvement and development of new BAL-based diagnostics.

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.

Telehealth platforms with remote phlebotomy and biomarker implementation represent a novel paradigm for surveillance after lung transplantation (LT). In a pilot study, we investigated donor-derived cell-free DNA (dd-cfDNA) in plasma using a clinical-grade "next-generation sequencing" assay.Methodsdd-cfDNA levels determined in biorepository venous plasma samples obtained during the lung allograft rejection gene expression observation study, implementing a clinical-grade next-generation sequencing assay. Sixty-nine unique LT patients encompassing 9 LT centers, with associated clinical-histopathologic diagnoses, were examined-allograft infection (n = 26), normal histopathology without infection (n = 30), and acute cellular rejection (ACR; n = 13).Resultsdd-cfDNA in ACR patients were significantly elevated (1.52%; interquartile range [IQR], 0.520-2.2550) compared with the normal stable patients (0.485%; IQR, 0.220-0.790) (P = 0.026). During allograft infection, dd-cfDNA values were not different (0.595; IQR, 0.270-1.170) from normal (P = 0.282) and ACR (P = 0.100). AUC-receiver operator characteristics curve analysis for allograft ACR was 0.717 (95% confidence interval, 0.547-0.887; P = 0.025). At a 0.87% threshold dd-cfDNA-sensitivity = 73.1%, specificity = 52.9%, positive predictive value = 34.1%, and negative predictive value = 85.5%.Conclusionsdd-cfDNA assessment holds promise as a noninvasive biomarker of "allograft injury" with acute rejection following LT while prospective, multicenter studies should further refine utility across the spectrum of allograft rejection and infection.

BACKGROUND: Some interstitial lung disease (ILD) patients develop a progressive fibrosing-ILD phenotype (PF-ILD), with similar persistent lung function decline suggesting common molecular pathways involved. Nintedanib, a tyrosine kinase inhibitor targeting the PDGF, FGF, VEGF and M-CSF pathways, has shown comparable efficacy in idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated ILD (SSc-ILD). We hypothesize that Nintedanib targeted molecular pathways will be augmented to a similar degree across PF-ILD regardless of aetiology. METHODS: We collected explanted lung tissue at the time of lung transplantation from 130 PF-ILD patients (99 (76%) IPF, 14 (11%) SSc-ILD, 17 (13%) other PF-ILD), and wedge biopsies from 200 donor lungs and measured PDGF, FGF, VEGF and M-CSF concentrations by Luminex. FINDINGS: The concentrations of PDGF-AA, PDGF-BB, FGF-2, VEGF and M-CSF were significantly increased in PF-ILD lungs compared to donor lungs (PDGF-AA 93·0 pg/ml [±97·2] vs. 37·5 pg/ml [±35·4], p

BackgroundChronic lung allograft dysfunction (CLAD) is the main limitation to long-term survival after lung transplantation. Because effective therapies are lacking, early identification and mitigation of risk factors is a pragmatic approach to improve outcomes. Acute cellular rejection (ACR) is the most pervasive risk factor for CLAD, but diagnosis requires transbronchial biopsy, which carries risks. We hypothesized that gene expression in the bronchoalveolar lavage (BAL) cell pellet (CP) could replace biopsy and inform on mechanisms of CLAD.MethodsWe performed RNA sequencing on BAL CPs from 219 lung transplant recipients with A-grade ACR (n = 61), lymphocytic bronchiolitis (n = 58), infection (n = 41), or no rejection/infection (n = 59). Differential gene expression was based on absolute fold difference >2.0 and Benjamini-adjusted p-value ≤0.05. We used the Database for Annotation, Visualization and Integrated Discovery Bioinformatics Resource for pathway analyses. For classifier modeling, samples were randomly split into training (n = 154) and testing sets (n = 65). A logistic regression model using recursive feature elimination and 5-fold cross-validation was trained to optimize area under the curve (AUC).ResultsDifferential gene expression identified 72 genes. Enriched pathways included T-cell receptor signaling, natural killer cell-mediated cytotoxicity, and cytokine-cytokine receptor interaction. A 4-gene model (AUC = 0.72) and classification threshold defined in the training set exhibited fair performance in the testing set; accuracy was 76%, specificity 82%, and sensitivity 60%. In addition, classification as ACR was associated with worse CLAD-free survival (hazard ratio = 2.42; 95% confidence interval = 1.29-4.53).ConclusionsBAL CP gene expression during ACR is enriched for immune response pathways and shows promise as a diagnostic tool for ACR, especially ACR that is a precursor of CLAD.

Introduction: We assessed the prognostic utility of circulating levels of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in patients with idiopathic pulmonary fibrosis (IPF) in the IPF-PRO Registry. Methods: MMP and TIMP concentrations were quantified by ELISA in plasma from 300 patients. A Cox proportional hazard regression model was used to assess associations between select MMPs and TIMPs and death and disease progression (absolute decline in forced vital capacity ≥10% predicted, death, or lung transplant). Results: Over a median follow-up of 30.4 months, 98 patients died and 182 patients had disease progression. In unadjusted analyses, higher concentrations of MMPs 2, 3, 8 and 9 and TIMPs 1, 2 and 4 were associated with an increased risk of death. MMPs 2 and 8 and TIMP1 remained associated with death after adjustment for clinical factors. In unadjusted analyses, higher concentrations of MMPs 8 and 9 and TIMPs 1 and 4 were associated with an increased risk of disease progression. MMPs 8 and 9 and TIMP1 remained associated with progression after adjustment for clinical factors. Conclusion: Circulating levels of MMP8 and TIMP1 may provide information on the risk of outcomes in patients with IPF not captured by clinical measures. [ABSTRACT FROM AUTHOR]

BackgroundLung transplantation outcomes remain complicated by bronchiolitis obliterans syndrome (BOS), a major cause of mortality and retransplantation for patients. A variety of factors linking inflammation and BOS have emerged, meriting further exploration of the microbiome as a source of inflammation. In this analysis, we determined features of the pulmonary microbiome associated with BOS susceptibility.MethodsBronchoalveolar lavage (BAL) samples were collected from 25 patients during standard of care bronchoscopies before BOS onset. Microbial DNA was isolated from BAL fluid and prepared for metagenomics shotgun sequencing. Patient microbiomes were phenotyped using k-means clustering and compared to determine effects on BOS-free survival.ResultsClustering identified 3 microbiome phenotypes: Actinobacteria dominant (AD), mixed, and Proteobacteria dominant. AD microbiomes, distinguished by enrichment with Gram-positive organisms, conferred reduced odds and risks for patients to develop acute rejection and BOS compared with non-AD microbiomes. These findings were independent of treatment models. Microbiome findings were correlated with BAL cell counts and polymorphonuclear cell percentages.ConclusionsIn some populations, features of the microbiome may be used to assess BOS susceptibility. Namely, a Gram-positive enriched pulmonary microbiome may predict resilience to BOS.

BACKGROUND:Aspergillus colonization after lung transplant is associated with an increased risk of chronic lung allograft dysfunction (CLAD). We hypothesized that gene expression during Aspergillus colonization could provide clues to CLAD pathogenesis. METHODS:We examined transcriptional profiles in 3- or 6-month surveillance bronchoalveolar lavage fluid cell pellets from recipients with Aspergillus fumigatus colonization (n = 12) and without colonization (n = 10). Among the Aspergillus colonized, we also explored profiles in those who developed CLAD (n = 6) or remained CLAD-free (n = 6). Transcription profiles were assayed with the HG-U133 Plus 2.0 microarray (Affymetrix). Differential gene expression was based on an absolute fold difference of 2.0 or greater and unadjusted P value less than 0.05. We used NIH Database for Annotation, Visualization and Integrated Discovery for functional analyses, with false discovery rates less than 5% considered significant. RESULTS:Aspergillus colonization was associated with differential expression of 489 probe sets, representing 404 unique genes. "Defense response" genes and genes in the "cytokine-cytokine receptor" Kyoto Encyclopedia of Genes and Genomes pathway were notably enriched in this list. Among Aspergillus colonized patients, CLAD development was associated with differential expression of 69 probe sets, representing 64 unique genes. This list was enriched for genes involved in "immune response" and "response to wounding", among others. Notably, both chitinase 3-like-1 and chitotriosidase were associated with progression to CLAD. CONCLUSIONS:Aspergillus colonization is associated with gene expression profiles related to defense responses including cytokine signaling. Epithelial wounding, as well as the innate immune response to chitin that is present in the fungal cell wall, may be key in the link between Aspergillus colonization and CLAD.

BACKGROUND: The long term clinical significance of respiratory infections after lung transplantation remains uncertain. METHODS: In this retrospective single-center cohort study of 441 lung transplant recipients, we formally evaluate the association between respiratory infection and chronic lung allograft dysfunction (CLAD). We furthermore hypothesized that bronchoalveolar lavage fluid (BALF) CXCL9 concentrations are augmented during respiratory infections, and that episodes of infection with elevated BALF CXCL9 are associated with greater CLAD risk. RESULTS: In univariable and multivariable models adjusted for other histopathologic injury patterns, respiratory infection, regardless of the causative organism, was a strong predictor of CLAD development (adjusted HR 1.8 95% CI 1.3-2.6). Elevated BALF CXCL9 concentrations during respiratory infections markedly increased CLAD risk in a dose-response manner. An episode of respiratory infection with CXCL9 concentrations greater than the 25th, 50th, and 75th percentile had adjusted HRs for CLAD of 1.8 (95% CI 1.1-2.8), 2.4 (95% CI 1.4-4.0) and 4.4 (95% CI 2.4-8.0), respectively. CONCLUSIONS: Thus, we demonstrate that respiratory infections, regardless of the causative organism, are strong predictors of CLAD development. We furthermore demonstrate for the first time, the prognostic importance of BALF CXCL9 concentrations during respiratory infections on the risk of subsequent CLAD development.

Lung transplantation is now considered to be a therapeutic option for patients with advanced-stage lung diseases. Unfortunately, due to post-transplant complications, both infectious and noninfectious, it is only a treatment and not a cure. Infections (e.g., bacterial, viral, and fungal) in the immunosuppressed lung transplant recipient are a common cause of mortality post transplant. Infections have more recently been explored as factors contributing to the risk of chronic lung allograft dysfunction (CLAD). Each major class of infection-(1) bacterial (Staphylococcus aureus and Pseudomonas aeruginosa); (2) viral (cytomegalovirus and community-acquired respiratory viruses); and (3) fungal (Aspergillus)-has been associated with the development of CLAD. Mechanistically, the microbe seems to be interacting with the allograft cells, stimulating the induction of chemokines, which recruit recipient leukocytes to the graft. The recipient leukocyte interactions with the microbe further up-regulate chemokines, amplifying the influx of allograft-infiltrating mononuclear cells. These events can promote recipient leukocytes to interact with the allograft, triggering an alloresponse and graft dysfunction. Overall, interactions between the microbe-allograft-host immune system alters chemokine production, which, in part, plays a role in the pathobiology of CLAD and mortality due to CLAD.

Inflammasomes are high-molecular-weight cytosolic complexes that mediate the activation of caspases. There are many inflammasomes, and each is influenced by a unique pattern-recognition receptor response. Two signals are typically involved in the inflammasome pathways. Signal one involves recognition of pathogen-associated molecular patterns (PAMPs), such as LPS or other colonizing/invading microbes, that interact with TLRs, which induce the downstream production of pro-IL-1β. This is followed by signal two, which involves recognition of PAMPs or damage-associated molecular patterns (DAMPs), such as uric acid or ATP, via NLRP3, which leads to caspase-1-dependent cleavage of pro-IL-1β to active IL-1β and pyroptosis. Ultimately, these two signals cause the release of multiple proinflammatory cytokines. Both PAMPs and DAMPs can be liberated by early insults to the allograft, including ischemia/reperfusion injury, infections, and rejection. The consequence of inflammasome activation and IL-1 expression is the upregulation of adhesion molecules and chemokines, which leads to allograft neutrophil sequestration, mononuclear phagocyte recruitment, and T cell activation, all of which are key steps in the continuum from allograft insult to chronic allograft dysfunction.

BackgroundChronic Lung Allograft Dysfunction (CLAD) is the main limitation to long-term survival after lung transplantation. Although CLAD is usually not responsive to treatment, earlier identification may improve treatment prospects.MethodsIn a nested case control study, 1-year post transplant surveillance bronchoalveolar lavage (BAL) fluid samples were obtained from incipient CLAD (n = 9) and CLAD free (n = 8) lung transplant recipients. Incipient CLAD cases were diagnosed with CLAD within 2 years, while controls were free from CLAD for at least 4 years following bronchoscopy. Transcription profiles in the BAL cell pellets were assayed with the HG-U133 Plus 2.0 microarray (Affymetrix). Differential gene expression analysis, based on an absolute fold change (incipient CLAD vs no CLAD) >2.0 and an unadjusted p-value ≤0.05, generated a candidate list containing 55 differentially expressed probe sets (51 up-regulated, 4 down-regulated).ResultsThe cell pellets in incipient CLAD cases were skewed toward immune response pathways, dominated by genes related to recruitment, retention, activation and proliferation of cytotoxic lymphocytes (CD8+ T-cells and natural killer cells). Both hierarchical clustering and a supervised machine learning tool were able to correctly categorize most samples (82.3% and 94.1% respectively) into incipient CLAD and CLAD-free categories.ConclusionsThese findings suggest that a pathobiology, similar to AR, precedes a clinical diagnosis of CLAD. A larger prospective investigation of the BAL cell pellet transcriptome as a biomarker for CLAD risk stratification is warranted.

RationaleSince the pathogenesis of chronic lung allograft dysfunction (CLAD) remains poorly defined with no known effective therapies, the identification and study of key events which increase CLAD risk is a critical step towards improving outcomes. We hypothesized that bronchoalveolar lavage fluid (BALF) CXCR3 ligand concentrations would be augmented during organizing pneumonia (OP) and that episodes of OP with marked chemokine elevations would be associated with significantly higher CLAD risk.MethodsAll transbronchial biopsies (TBBX) from patients who received lung transplantation between 2000 to 2010 were reviewed. BALF concentrations of the CXCR3 ligands (CXCL9, CXCL10 and CXCL11) were compared between episodes of OP and "healthy" biopsies using linear mixed-effects models. The association between CXCR3 ligand concentrations during OP and CLAD risk was evaluated using proportional hazards models with time-dependent covariates.ResultsThere were 1894 bronchoscopies with TBBX evaluated from 441 lung transplant recipients with 169 (9%) episodes of OP and 907 (49%) non-OP histopathologic injuries. 62 (37%) episodes of OP were observed during routine surveillance bronchoscopy. Eight hundred thirty-eight (44%) TBBXs had no histopathology and were classified as "healthy" biopsies. There were marked elevations in BALF CXCR3 ligand concentrations during OP compared with "healthy" biopsies. In multivariable models adjusted for other injury patterns, OP did not significantly increase the risk of CLAD when BAL CXCR3 chemokine concentrations were not taken into account. However, OP with elevated CXCR3 ligands markedly increased CLAD risk in a dose-response manner. An episode of OP with CXCR3 concentrations greater than the 25th, 50th and 75th percentiles had HRs for CLAD of 1.5 (95% CI 1.0-2.3), 1.9 (95% CI 1.2-2.8) and 2.2 (95% CI 1.4-3.4), respectively.ConclusionsThis study identifies OP, a relatively uncommon histopathologic finding after lung transplantation, as a major risk factor for CLAD development when considered in the context of increased allograft expression of interferon-γ inducible ELR- CXC chemokines. We further demonstrate for the first time, the prognostic importance of BALF CXCR3 ligand concentrations during OP on subsequent CLAD risk.

BackgroundCommunity-acquired respiratory virus (CARV) infections occur frequently after lung transplantation and may adversely impact outcomes. We hypothesized that while asymptomatic carriage would not increase the risk of chronic lung allograft dysfunction (CLAD) and graft loss, severe infection would.MethodsAll lung transplant cases between January 2000 and July 2013 performed at our center were reviewed for respiratory viral samples. Each isolation of virus was classified according to clinical level of severity: asymptomatic, symptomatic without pneumonia, and viral pneumonia. Multivariate Cox modeling was used to assess the impact of CARV isolation on progression to CLAD and graft loss.ResultsFour thousand four hundred eight specimens were collected from 563 total patients, with 139 patients producing 324 virus-positive specimens in 245 episodes of CARV infection. Overall, the risk of CLAD was elevated by viral infection (hazard ratio [HR], 1.64; P < 0.01). This risk, however, was due to viral pneumonia alone (HR, 3.94; P < 0.01), without significant impact from symptomatic viral infection (HR, 0.97; P = 0.94) nor from asymptomatic viral infection (HR, 0.99; P = 0.98). The risk of graft loss was not increased by asymptomatic CARV infection (HR, 0.74; P = 0.37) nor symptomatic CARV infection (HR, 1.39; P = 0.41). Viral pneumonia did, however, significantly increase the risk of graft loss (HR, 2.78; P < 0.01).ConclusionsWith respect to CARV, only viral pneumonia increased the risk of both CLAD and graft loss after lung transplantation. In the absence of pneumonia, respiratory viruses had no impact on measured outcomes.

Idiopathic pulmonary fibrosis is a fatal lung disease with a median survival of 2 to 5 years. A decade of studies has downplayed inflammation contributing to its pathogenesis. However, these studies preceded the discovery of regulatory T cells (Tregs) and all of their functions. On the basis of human studies demonstrating Tregs can decrease graft-versus-host disease and vasculitides, there is consideration of their use to treat idiopathic pulmonary fibrosis. We hypothesized that Treg therapy would attenuate the fibroplasia involved in a preclinical murine model of pulmonary fibrosis. IL-2 complex was used in vivo to expand CD4(+)CD25(hi)Foxp3(+) cells in the lung during intratracheal bleomycin challenge; however, this unexpectedly led to an increase in lung fibrosis. More important, this increase in fibrosis was a lymphocyte-dependent process. We corroborated these results using a CD4(+)CD25(hi)Foxp3(+) cellular-based therapy. Mechanistically, we demonstrated that CD4(+)CD25(hi)Foxp3(+) cells undergo alterations during bleomycin challenge and the IL-2 complex had no effect on profibrotic (eg, transforming growth factor-β) or type 17 immune response cytokines; however, there was a marked down-regulation of the type 1 and augmentation of the type 2 immune response cytokines from the lungs. Collectively, our animal studies show that a specific lung injury can induce Treg alterations, which can augment pulmonary fibrosis.

RationaleThe clinical course of chronic lung allograft dysfunction (CLAD) is heterogeneous. Forced vital capacity (FVC) loss at onset, which may suggest a restrictive phenotype, was associated with worse survival for bilateral lung transplant recipients in one previously published single-center study.ObjectivesWe sought to replicate the significance of FVC loss in an independent, retrospectively identified cohort of bilateral lung transplant recipients and to investigate extended application of this approach to single lung recipients.MethodsFVC loss and other potential predictors of survival after the onset of CLAD were assessed using Kaplan-Meier and Cox proportional hazards models.Measurements and main resultsFVC loss at the onset of CLAD was associated with higher mortality in an independent cohort of bilateral lung transplant recipients (hazard ratio [HR], 2.75; 95% confidence interval [CI], 2.02-3.73; P

BackgroundThe diagnosis of antibody-mediated rejection (AMR) in the lung transplant is still an area under investigation. We performed a blinded multicenter study to determine if any statistically significant histologic findings in transbronchial biopsy specimens from lung transplant patients correlate with the presence of donor-specific antibodies (DSAs).MethodsWe asked 9 pathologists with experience in lung transplantation to evaluate 161 lung transplant biopsy specimens for various histologic parameters. The findings were correlated with antibody status positive for DSAs, positive for non-DSAs, and no antibodies (NABs) present. The significance of each histologic variable was reviewed.ResultsWe found no statistically significant association with acute cellular rejection, airway inflammation, or bronchiolitis obliterans and the presence or absence of antibodies. However, biopsy specimens with DSAs had a statistically significant difference vs NABs in the setting of acute lung injury, with or without diffuse alveolar damage (p = 0.0008), in the presence of capillary neutrophilic inflammation (p = 0.0014), and in samples with endotheliitis (p = 0.0155). In samples with complement 4d staining, there was a trend but no statistically significant difference between specimens associated with DSAs and specimens with NABs.ConclusionsCapillary inflammation, acute lung injury, and endotheliitis significantly correlated with DSAs. The infrequently observed diffuse staining for complement 4d limits the usefulness of this stain.

RationaleThe mechanism by which acute allograft rejection leads to chronic rejection remains poorly understood despite its common occurrence. Exosomes, membrane vesicles released from cells within the lung allograft, contain a diverse array of biomolecules that closely reflect the biologic state of the cell and tissue from which they are released. Exosome transcriptomes may provide a better understanding of the rejection process. Furthermore, biomarkers originating from this transcriptome could provide timely and sensitive detection of acute cellular rejection (AR), reducing the incidence of severe AR and chronic lung allograft dysfunction and improving outcomes.ObjectivesTo provide an in-depth analysis of the bronchoalveolar lavage fluid exosomal shuttle RNA population after lung transplantation and evaluate for differential expression between acute AR and quiescence.MethodsSerial bronchoalveolar lavage specimens were ultracentrifuged to obtain the exosomal pellet for RNA extraction, on which RNA-Seq was performed.Measurements and main resultsAR demonstrates an intense inflammatory environment, skewed toward both innate and adaptive immune responses. Novel, potential upstream regulators identified offer potential therapeutic targets.ConclusionsOur findings validate bronchoalveolar lavage fluid exosomal shuttle RNA as a source for understanding the pathophysiology of AR and for biomarker discovery in lung transplantation.

Survival in patients with cystic fibrosis (CF) has improved dramatically over the past 30 to 40 years, with mean survival now approximately 40 years. Nonetheless, progressive respiratory insufficiency remains the major cause of mortality in CF patients, and lung transplantation (LT) is eventually required. Timing of listing for LT is critical, because up to 25 to 41% of CF patients have died while awaiting LT. Globally, approximately 16.4% of lung transplants are performed in adults with CF. Survival rates for LT recipients with CF are superior to other indications, yet LT is associated with substantial morbidity and mortality (∼50% at 5-year survival rates). Myriad complications of LT include allograft failure (acute or chronic), opportunistic infections, and complications of chronic immunosuppressive medications (including malignancy). Determining which patients are candidates for LT is difficult, and survival benefit remains uncertain. In this review, we discuss when LT should be considered, criteria for identifying candidates, contraindications to LT, results post-LT, and specific complications that may be associated with LT. Infectious complications that may complicate CF (particularly Burkholderia cepacia spp., opportunistic fungi, and nontuberculous mycobacteria) are discussed.

Type 2 alveolar epithelial cells (AEC2s) function as progenitor cells in the lung. We have shown previously that failure of AEC2 regeneration results in progressive lung fibrosis in mice and is a cardinal feature of idiopathic pulmonary fibrosis (IPF). In this study, we identified deficiency of a specific zinc transporter, SLC39A8 (ZIP8), in AEC2s from both IPF lungs and lungs of old mice. Loss of ZIP8 expression was associated with impaired renewal capacity of AEC2s and enhanced lung fibrosis. ZIP8 regulation of AEC2 progenitor function was dependent on SIRT1. Replenishment with exogenous zinc and SIRT1 activation promoted self-renewal and differentiation of AEC2s from lung tissues of IPF patients and old mice. Deletion of Zip8 in AEC2s in mice resulted in impaired AEC2 renewal, increased susceptibility to bleomycin injury, and development of spontaneous lung fibrosis. Therapeutic strategies to restore zinc metabolism and appropriate SIRT1 signaling could improve AEC2 progenitor function and mitigate ongoing fibrogenesis., Introduction Idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease (ILD), with most patients dying or requiring a lung transplant within 3-5 years of diagnosis (1). The [...]

RationaleAfter lung transplantation, insults to the allograft generally result in one of four histopathologic patterns of injury: (1) acute rejection, (2) lymphocytic bronchiolitis, (3) organizing pneumonia, and (4) diffuse alveolar damage (DAD). We hypothesized that DAD, the most severe form of acute lung injury, would lead to the highest risk of chronic lung allograft dysfunction (CLAD) and that a type I immune response would mediate this process.ObjectivesDetermine whether DAD is associated with CLAD and explore the potential role of CXCR3/ligand biology.MethodsTransbronchial biopsies from all lung transplant recipients were reviewed. The association between the four injury patterns and subsequent outcomes were evaluated using proportional hazards models with time-dependent covariates. Bronchoalveolar lavage (BAL) concentrations of the CXCR3 ligands (CXCL9/MIG, CXCL10/IP10, and CXCL11/ITAC) were compared between allograft injury patterns and "healthy" biopsies using linear mixed-effects models. The effect of these chemokine alterations on CLAD risk was assessed using Cox models with serial BAL measurements as time-dependent covariates.Measurements and main resultsThere were 1,585 biopsies from 441 recipients with 62 episodes of DAD. An episode of DAD was associated with increased risk of CLAD (hazard ratio, 3.0; 95% confidence interval, 1.9-4.7) and death (hazard ratio, 2.3; 95% confidence interval, 1.7-3.0). There were marked elevations in BAL CXCR3 ligand concentrations during DAD. Furthermore, prolonged elevation of these chemokines in serial BAL fluid measurements predicted the development of CLAD.ConclusionsDAD is associated with marked increases in the risk of CLAD and death after lung transplantation. This association may be mediated in part by an aberrant type I immune response involving CXCR3/ligands.

Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, yet markers for early detection and intervention are currently lacking. Given the role of regulatory T cells (Treg) in modulation of immunity, we hypothesized that frequencies of Treg in bronchoalveolar lavage fluid (BALF) after lung transplantation would predict subsequent development of BOS. Seventy BALF specimens obtained from 47 lung transplant recipients were analyzed for Treg lymphocyte subsets by flow cytometry, in parallel with ELISA measurements of chemokines. Allograft biopsy tissue was stained for chemokines of interest. Treg were essentially all CD45RA(-), and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4(+) and CD103(-) and neither of these subsets correlated to risk for BOS. In contrast, higher percentages of CCR7(+) Treg correlated to reduced risk of BOS. Additionally, the CCR7 ligand CCL21 correlated with CCR7(+) Treg frequency and inversely with BOS. Higher frequencies of CCR7(+) CD3(+)CD4(+)CD25(hi)Foxp3(+)CD45RA(-) lymphocytes in lung allografts is associated with protection against subsequent development of BOS, suggesting that this subset of putative Treg may down-modulate alloimmunity. CCL21 may be pivotal for the recruitment of this distinct subset to the lung allograft and thereby decrease the risk for chronic rejection.

Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, yet markers for early detection and intervention are currently lacking. Given the role of regulatory T cells (Treg) in modulation of immunity, we hypothesized that frequencies of Treg in bronchoalveolar lavage fluid (BALF) after lung transplantation would predict subsequent development of BOS. Seventy BALF specimens obtained from 47 lung transplant recipients were analyzed for Treg lymphocyte subsets by flow cytometry, in parallel with ELISA measurements of chemokines. Allograft biopsy tissue was stained for chemokines of interest. Treg were essentially all CD45RA(-), and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4(+) and CD103(-) and neither of these subsets correlated to risk for BOS. In contrast, higher percentages of CCR7(+) Treg correlated to reduced risk of BOS. Additionally, the CCR7 ligand CCL21 correlated with CCR7(+) Treg frequency and inversely with BOS. Higher frequencies of CCR7(+) CD3(+)CD4(+)CD25(hi)Foxp3(+)CD45RA(-) lymphocytes in lung allografts is associated with protection against subsequent development of BOS, suggesting that this subset of putative Treg may down-modulate alloimmunity. CCL21 may be pivotal for the recruitment of this distinct subset to the lung allograft and thereby decrease the risk for chronic rejection.

The prognosis of patients with hematopoietic stem cell transplants (HSCTs) who require admission to the intensive care unit (ICU) has been regarded as extremely poor. We sought to re-evaluate recent outcomes and predictive factors in a retrospective cohort study. Among the 605 adult patients that received an HSCT between 2001 and 2006, 154 required admission to the ICU. Of these, 47% were discharged from the ICU, 36% were discharged from the hospital, and 19% survived 6 months. Allogeneic transplant, mechanical ventilation, vasopressor-use, and neutropenia were each associated with increased mortality, and the mortality of patients with all four characteristics was 100%. Hemodialysis was also associated with increased mortality in a Kaplan-Meier analysis but did not appear important in a multivariate tree analysis. A final Cox model confirmed that allogeneic transplant, mechanical ventilation, and vasopressor-use were each independent risk factors for mortality in the 6 months following ICU admission.