Lung Allograft Epithelium DNA Methylation Age Is Associated With Graft Chronologic Age and Primary Graft Dysfunction.

Advanced donor age is a risk factor for poor survival following lung transplantation. However, recent work identifying epigenetic determinants of aging has shown that biologic age may not always reflect chronologic age and that stressors can accelerate biologic aging. We hypothesized that lung allografts that experienced primary graft dysfunction (PGD), characterized by poor oxygenation in the first three post-transplant days, would have increased biologic age. We cultured airway epithelial cells isolated by transbronchial brush at 1-year bronchoscopies from 13 subjects with severe PGD and 15 controls matched on age and transplant indication. We measured epigenetic age using the Horvath epigenetic clock. Linear models were used to determine the association of airway epigenetic age with chronologic ages and PGD status, adjusted for recipient PGD risk factors. Survival models assessed the association with chronic lung allograft dysfunction (CLAD) or death. Distributions of promoter methylation within pathways were compared between groups. DNA methyltransferase (DNMT) activity was quantified in airway epithelial cells under hypoxic or normoxic conditions. Airway epigenetic age appeared younger but was strongly associated with the age of the allograft (slope 0.38 per year, 95% CI 0.27-0.48). There was no correlation between epigenetic age and recipient age (P = 0.96). Epigenetic age was 6.5 years greater (95% CI 1.7-11.2) in subjects who had experienced PGD, and this effect remained significant after adjusting for donor and recipient characteristics (P = 0.03). Epigenetic age was not associated with CLAD-free survival risk (P = 0.11). Analysis of differential methylation of promoters of key biologic pathways revealed hypomethylation in regions related to hypoxia, inflammation, and metabolism-associated pathways. Accordingly, airway epithelial cells cultured in hypoxic conditions showed suppressed DNMT activity. While airway methylation age was primarily determined by donor chronologic age, early injury in the form of PGD was associated with increased allograft epigenetic age. These data show how PGD might suppress key promoter methylation resulting in long-term impacts on the allograft.

Chronic lung allograft dysfunction small airways reveal a lymphocytic inflammation gene signature.

Chronic lung allograft dysfunction (CLAD) is the major barrier to long-term survival following lung transplantation, and new mechanistic biomarkers are needed. Lymphocytic bronchitis (LB) precedes CLAD and has a defined molecular signature. We hypothesized that this LB molecular signature would be associated with CLAD in small airway brushings independent of infection. We quantified RNA expression from small airway brushings and transbronchial biopsies, using RNAseq and digital RNA counting, respectively, for 22 CLAD cases and 27 matched controls. LB metagene scores were compared across CLAD strata by Wilcoxon rank sum test. We performed unbiased host transcriptome pathway and microbial metagenome analysis in airway brushes and compared machine-learning classifiers between the two tissue types. This LB metagene score was increased in CLAD airway brushes (p = .002) and improved prediction of graft failure (p = .02). Gene expression classifiers based on airway brushes outperformed those using transbronchial biopsies. While infection was associated with decreased microbial alpha-diversity (p ≤ .04), neither infection nor alpha-diversity was associated with LB gene expression. In summary, CLAD was associated with small airway gene expression changes not apparent in transbronchial biopsies in this cohort. Molecular analysis of airway brushings for diagnosing CLAD merits further examination in multicenter cohorts.

Natural killer cells activated through NKG2D mediate lung ischemia-reperfusion injury.

Pulmonary ischemia-reperfusion injury (IRI) is a clinical syndrome of acute lung injury that occurs after lung transplantation or remote organ ischemia. IRI causes early mortality and has no effective therapies. While NK cells are innate lymphocytes capable of recognizing injured cells, their roles in acute lung injury are incompletely understood. Here, we demonstrated that NK cells were increased in frequency and cytotoxicity in 2 different IRI mouse models. We showed that NK cells trafficked to the lung tissue from peripheral reservoirs and were more mature within lung tissue. Acute lung ischemia-reperfusion injury was blunted in a NK cell-deficient mouse strain but restored with adoptive transfer of NK cells. Mechanistically, NK cell NKG2D receptor ligands were induced on lung endothelial and epithelial cells following IRI, and antibody-mediated NK cell depletion or NKG2D stress receptor blockade abrogated acute lung injury. In human lung tissue, NK cells were increased at sites of ischemia-reperfusion injury and activated NK cells were increased in prospectively collected human bronchoalveolar lavage in subjects with severe IRI. These data support a causal role for recipient peripheral NK cells in pulmonary IRI via NK cell NKG2D receptor ligation. Therapies targeting NK cells may hold promise in acute lung injury.

Type-1 immunity and endogenous immune regulators predominate in the airway transcriptome during chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) remains the major complication limiting long-term survival among lung transplant recipients (LTRs). Limited understanding of CLAD immunopathogenesis and a paucity of biomarkers remain substantial barriers for earlier detection and therapeutic interventions for CLAD. We hypothesized the airway transcriptome would reflect key immunologic changes in disease. We compared airway brush-derived transcriptomic signatures in CLAD (n = 24) versus non-CLAD (n = 21) LTRs. A targeted assessment of the proteome using concomitant bronchoalveolar lavage (BAL) fluid for 24 cytokines/chemokines and alloimmune T cell responses was performed to validate the airway transcriptome. We observed an airway transcriptomic signature of differential genes expressed (DGEs) in CLAD marked by Type-1 immunity and striking upregulation of two endogenous immune regulators: indoleamine 2, 3 dioxygenase 1 (IDO-1) and tumor necrosis factor receptor superfamily 6B (TNFRSF6B). Advanced CLAD staging was associated with a more intense airway transcriptome signature. In a validation cohort using the identified signature, we found an area under the curve (AUC) of 0.77 for CLAD LTRs. Targeted proteomic analyses revealed a predominant Type-1 profile with detection of IFN-γ, TNF-α, and IL-1ß as dominant CLAD cytokines, correlating with the airway transcriptome. The airway transcriptome provides novel insights into CLAD immunopathogenesis and biomarkers that may impact diagnosis of CLAD.

Cystic Fibrosis Lung Transplant Recipients Have Suppressed Airway Interferon Responses during Pseudomonas Infection.

Lung transplantation can be lifesaving in end-stage cystic fibrosis (CF), but long-term survival is limited by chronic lung allograft dysfunction (CLAD). Persistent upper airway Pseudomonas aeruginosa (PsA) colonization can seed the allograft. While de novo PsA infection is associated with CLAD in non-CF recipients, this association is less clear for CF recipients experiencing PsA recolonization. Here, we evaluate host and pathogen contributions to this phenomenon. In the context of PsA infection, brushings from the airways of CF recipients demonstrate type 1 interferon gene suppression. Airway epithelial cell (AEC) cultures demonstrate similar findings in the absence of pathogens or immune cells, contrasting with the pre-transplant CF AEC phenotype. Type 1 interferon promoters are relatively hypermethylated in CF AECs. CF subjects in this cohort have more mucoid PsA, while non-CF PsA subjects have decreased microbiome α diversity. Peri-transplant protocols may benefit from consideration of this host and microbiome equilibrium.

Dectin-1 genetic deficiency predicts chronic lung allograft dysfunction and death.

BACKGROUNDInnate immune activation impacts lung transplant outcomes. Dectin-1 is an innate receptor important for pathogen recognition. We hypothesized that genotypes reducing dectin-1 activity would be associated with infection, graft dysfunction, and death in lung transplant recipients.METHODSWe assessed the rs16910526 CLEC7A gene polymorphism Y238X, which results in dectin-1 truncation, in 321 lung allograft recipients at a single institution and in 1,129 lung allograft recipients in the multicenter Lung Transplant Outcomes Group (LTOG) cohort. Differences in dectin-1 mRNA, cytokines, protein levels, immunophenotypes, and clinical factors were assessed.RESULTSY238X carriers had decreased dectin-1 mRNA expression (P = 0.0001), decreased soluble dectin-1 protein concentrations in bronchoalveolar lavage (P = 0.008) and plasma (P = 0.04), and decreased monocyte surface dectin-1 (P = 0.01) compared with wild-type subjects. Y238X carriers had an increased risk of fungal pathogens (HR 1.17, CI 1.0-1.4), an increased risk of graft dysfunction or death (HR 1.6, CI 1.0-2.6), as well increased mortality in the UCSF cohort (HR 1.8, CI 1.1-3.8) and in the LTOG cohort (HR 1.3, CI 1.1-1.6), compared with wild-type CLEC7A subjects.CONCLUSIONIncreased rates of graft dysfunction and death associated with this dectin-1 polymorphism may be amplified by immunosuppression that drives higher fungal burden from compromised pathogen recognition.FUNDINGThe UCSF Nina Ireland Program for Lung Health Innovative Grant program, the Clinical Sciences Research & Development Service of the VA Office of Research and Development, and the Joel D. Cooper Career Development Award from the International Society for Heart and Lung Transplantation.

Attenuated Airway Epithelial Cell Interleukin-22R1 Expression in the Infant Nonhuman Primate Lung.

Respiratory tract infections are a leading cause of morbidity and mortality in children under 5 years of age. Increased susceptibility to infection is associated with deficiencies in immunity during early childhood. Airway epithelium represents the first line of mucosal defense against inhaled pathogens. However, little is known about epithelial immune mechanisms in the maturing lung. IL-22 and its receptor IL-22R1 are important in host defense and repair of epithelial barriers. The objective of this study was to determine whether a quantitative difference in IL-22R1 exists between infant and adult airways using the rhesus macaque monkey as a model of childhood lung development. Immunofluorescence staining of tracheal tissue revealed minimal expression of IL-22R1 in epithelium at 1 month of age, with a progressive increase in fluorescence-positive basal cells through 1 year of age. Western blot analysis of tracheal lysates confirmed significant age-dependent differences in IL-22R1 protein content. Further, primary tracheobronchial epithelial cell cultures established from infant and adult monkeys showed differential IL-22R1 mRNA and protein expression in vitro. To begin to assess the regulation of age-dependent IL-22R1 expression in airway epithelium, the effect of histone deacetylase and DNA methyltransferase inhibitors was evaluated. IL-22R1 mRNA in adult cultures was not altered by 5-aza-2'-deoxycytidine or trichostatin A. IL-22R1 mRNA in infant cultures showed no change with 5-aza-2'-deoxycytidine but was significantly increased after trichostatin A treatment; however, IL-22R1 protein did not increase concurrently. These data suggest that IL-22R1 in airway epithelium is regulated, in part, by epigenetic mechanisms that are dependent on chronologic age.

A comparison of the clinical utility of several published formulae for estimated osmolality of canine serum.

OBJECTIVE: To determine the normal osmole gap for 18 previously published formulae used to estimate serum osmolality in dogs. DESIGN: Prospective study. SETTING: University veterinary medical teaching hospital. ANIMALS: Two hundred and fifty client-owned dogs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serum samples were saved and frozen at -80°C after routine biochemical analysis as ordered by attending clinicians. An Advanced Micro Osmometer 3300 was used to measure serum osmolality. Eighteen distinct formulae previously reported in the medical literature were used to calculate the osmolality from the biochemical analysis results. The calculated osmolality was then subtracted from the measured osmolality to determine the osmole gap. Osmole gaps for azotemic and hyperglycemic dogs were compared to those of dogs without azotemia or hyperglycemia using each formula. The median measured osmolality for all dogs in the study was 302 mOsm/kg (interquartile range 297-307). The osmole gaps varied widely depending on the formula used to calculate osmolality and the presence or absence of hyperglycemia or azotemia. Eleven formulae led to calculated osmolality and osmole gaps that were not statistically different when hyperglycemia or azotemia was present. Four out of these 11 formulae resulted in osmole gaps near zero. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple formulae reported to calculate serum osmolality can be used in the clinical setting, but they result in significantly different normal osmole gaps. Clinicians should be aware of the specific reference interval for the formula being used. The authors recommend the formula 2(Na(+) ) + [glucose/18] + [BUN/2.8] because it is easy to use and is reliable even when hyperglycemia or azotemia are present.