Urinary eicosanoid levels in early life and risk of atopic disease in childhood.

BACKGROUND: Eicosanoids are lipid mediators including thromboxanes (TXs), prostaglandins (PGs), and leukotrienes with a pathophysiological role in established atopic disease. However, their role in the inception of disease is unclear. This study aimed to investigate the association between urinary eicosanoids in early life and development of atopic disease. METHODS: This study quantified the levels of 21 eicosanoids in urine from children from the COPSAC2010 (Copenhagen Prospective Studies on Asthma in Childhood 2010) (age 1 year, n = 450) and VDAART (Vitamin D Antenatal Asthma Reduction Trial) (age 3 years, n = 575) mother-child cohorts and analyzed the associations with development of wheeze/asthma, atopic dermatitis, and biomarkers of type-2 inflammation, applying false discovery rate of 5% (FDR5%) multiple testing correction. RESULTS: In both cohorts, analyses adjusted for environmental determinants showed that higher TXA2 eicosanoids in early life were associated with increased risk of developing atopic dermatitis (P < FDR5%) and type-2 inflammation (P < .05). In VDAART, lower PGE2 and PGI2 eicosanoids and higher isoprostanes were also associated with increased risk of atopic dermatitis (P < FDR5%). For wheeze/asthma, analyses in COPSAC2010 showed that lower isoprostanes and PGF2 eicosanoids and higher PGD2 eicosanoids at age 1 year associated with an increased risk at age 1-10 years (P < .05), whereas analyses in VDAART showed that lower PGE2 and higher TXA2 eicosanoids at age 3 years associated with an increased risk at 6 years (P < FDR5%). CONCLUSIONS: This study suggests that early life perturbations in the eicosanoid metabolism are present before the onset of atopic disease in childhood, which provides pathophysiological insight in the inception of atopic diseases.

Development of a Desorption Electrospray Ionization-Multiple-Reaction-Monitoring Mass Spectrometry (DESI-MRM) Workflow for Spatially Mapping Oxylipins in Pulmonary Tissue.

Oxylipins are a class of low-abundance lipids formed via oxygenation of fatty acids. These compounds include potent signaling molecules (e.g., octadecanoids, eicosanoids) that can exert essential functions in the pathophysiology of inflammatory diseases including asthma. While some oxylipin signaling cascades have been unraveled using LC-MS/MS-based methods, measurements in homogenate samples do not represent the spatial heterogeneity of lipid metabolism. Mass spectrometry imaging (MSI) directly detects analytes from a surface, which enables spatial mapping of oxylipin biosynthesis and migration within the tissue. MSI has lacked the sensitivity to routinely detect low-abundance oxylipins; however, new multiple-reaction-monitoring (MRM)-based MSI technologies show increased sensitivity. In this study, we developed a workflow to apply desorption electrospray ionization coupled to a triple quadrupole mass spectrometer (DESI-MRM) to spatially map oxylipins in pulmonary tissue. The targeted MSI workflow screened guinea pig lung extracts using LC-MS/MS to filter oxylipin targets based on their detectability by DESI-MRM. A panel of 5 oxylipins was then selected for DESI-MRM imaging derived from arachidonic acid (TXB2, 11-HETE, 12-HETE), linoleic acid (12,13-DiHOME), and α-linolenic acid (16-HOTrE). To parse this new data type, a custom-built R package (quantMSImageR) was developed with functionality to label regions of interest as well as quantify and analyze lipid distributions. The spatial distributions quantified by DESI-MRM were supported by LC-MS/MS analysis, with both indicating that 16-HOTrE and 12-HETE were associated with airways, while 12,13-DiHOME and arachidonic acid were mapped to parenchyma. This study realizes the potential of targeted MSI to routinely map low-abundance oxylipins with high specificity at scale.

Body mass index trajectories from birth to early adulthood and lung function development.

BACKGROUND: Limited studies have investigated the influence of body mass index (BMI) trajectories on lung function covering the entire growth period. METHODS: We conducted a prospective study utilizing data from the Swedish BAMSE birth cohort. Latent class mixture modelling was employed to examine the diversity in BMI z-scores from birth to 24 years of age. Participants with four or more BMI z-scores were included (n=3204, 78·4%). Pre-bronchodilator (BD) spirometry was tested at 8, 16, and 24 years, while post-BD spirometry, multiple-breath nitrogen washout (for lung clearance index, LCI), and urinary metabolomics data were assessed at 24 years. RESULTS: Six distinct BMI development groups were identified. Compared to the stable normal BMI group, the accelerated increasing BMI group exhibited reduced pre- and post-BD FEV1/FVC ratio z scores (ß=-0·26, 95% CI=[-0·44, -0·08], and -0·22, [ -0·39, -0·05], respectively), along with elevated LCI (0·30, [0·22, 0·42]) at 24 years. The persistent high BMI group demonstrated lower FEV1, and FVC z scores growth between 16 and 24 years (-0.24, [-0.42, -0.05], and -0.27, [-0.45, -0.01], respectively), and elevated LCI (0·20, [0·03, 0·39]) at 24 years. However, those impairments were not observed in the accelerated resolving BMI group. Conversely, the persistent low BMI group displayed persistently decreased FEV1, and FVC from 8 to 24 years, as well as decreased lung function growth. Additionally, histidine-related metabolites were associated with pre- and post-BD FEV1 (hypergeometric FDR=0.008 and <0.001, respectively). CONCLUSIONS: Early interventions aiming for normal BMI during childhood may contribute to improved lung health later in life.

Radiomultiomics: quantitative CT clusters of severe asthma associated with multi-omics.

RATIONALE: Lung quantitative computed tomographic (qCT) severe asthma clusters have been reported, but their replication and underlying disease mechanisms are unknown. We identified and replicated qCT clusters of severe asthma in two independent asthma cohorts and determined their association with molecular pathways. METHODS: We used consensus clustering on qCT measurements of airway and lung CT scans, performed in 105 severe asthmatic adults from the U-BIOPRED cohort. The same qCT measurements were used to replicate qCT clusters in a subsample of the ATLANTIS asthma cohort (n=97). We performed integrated enrichment analysis using blood, sputum, bronchial biopsies, bronchial brushings and nasal brushings transcriptomics and blood and sputum proteomics to characterize radiomultiomic-associated clusters (RACs). RESULTS: qCT clusters and clinical features in U-BIOPRED were replicated in the matched ATLANTIS cohort. In the U-BIOPRED cohort, RAC1 (n=30) was predominantly female with elevated BMI, mild airflow limitation, normal qCT parameters and upregulation of the complement pathway. RAC2 (n=34) subjects had a lower degree of airflow limitation, airway wall thickness and dilatation, with upregulation of proliferative pathways, including neurotrophic receptor tyrosine kinase 2/tyrosine kinase receptor B (NTRK2/TRKB), and down-regulation of semaphorin pathways. RAC3 (n=41) showed increased lung attenuation area and air trapping, severe airflow limitation, hyperinflation, and upregulation of cytokine signaling and signaling by interleukin pathways, and matrix metallopeptidase 1, 2 and 9. CONCLUSIONS: U-BIOPRED severe asthma qCT clusters were replicated in a matched independent asthmatic cohort and associated with specific molecular pathways. Radiomultiomics might represent anovel strategy to identify new molecular pathways in asthma pathobiology.

Workshop report - interdisciplinary metabolomic epidemiology: the pathway to clinical translation.

Metabolomic epidemiology studies are complex and require a broad array of domain expertise. Although many metabolite-phenotype associations have been identified; to date, few findings have been translated to the clinic. Bridging this gap requires understanding of both the underlying biology of these associations and their potential clinical implications, necessitating an interdisciplinary team approach. To address this need in metabolomic epidemiology, a workshop was held at Metabolomics 2023 in Niagara Falls, Ontario, Canada that highlighted the domain expertise needed to effectively conduct these studies -- biochemistry, clinical science, epidemiology, and assay development for biomarker validation -- and emphasized the role of interdisciplinary teams to move findings towards clinical translation.

Large airway T cells in adults with former bronchopulmonary dysplasia.

BACKGROUND: Bronchopulmonary Dysplasia (BPD) in infants born prematurely is a risk factor for chronic airway obstruction later in life. The distribution of T cell subtypes in the large airways is largely unknown. OBJECTIVE: To characterize cellular and T cell profiles in the large airways of young adults with a history of BPD. METHODS: Forty-three young adults born prematurely (preterm (n = 20), BPD (n = 23)) and 45 full-term-born (asthma (n = 23), healthy (n = 22)) underwent lung function measurements, and bronchoscopy with large airway bronchial wash (BW). T-cells subsets in BW were analyzed by immunocytochemistry. RESULTS: The proportions of both lymphocytes and CD8 + T cells in BW were significantly higher in BPD (median, 6.6%, and 78.0%) when compared with asthma (3.4% and 67.8%, p = 0.002 and p = 0.040) and healthy (3.8% and 40%, p < 0.001 and p < 0.001). In all adults born prematurely (preterm and BPD), lymphocyte proportion correlated negatively with forced vital capacity (r= -0.324, p = 0.036) and CD8 + T cells correlated with forced expiratory volume in one second, FEV1 (r=-0.448, p = 0.048). Correlation-based network analysis revealed that lung function cluster and BPD-birth cluster were associated with lymphocytes and/or CD4 + and CD8 + T cells. Multivariate regression analysis showed that lymphocyte proportions and BPD severity qualified as independent factors associated with FEV1. CONCLUSIONS: The increased cytotoxic T cells in the large airways in young adults with former BPD, suggest a similar T-cell subset pattern as in the small airways, resembling features of COPD. Our findings strengthen the hypothesis that mechanisms involving adaptive and innate immune responses are involved in the development of airway disease due to preterm birth.

Sphingolipid classes and the interrelationship with pediatric asthma and asthma risk factors.

BACKGROUND: While dysregulated sphingolipid metabolism has been associated with risk of childhood asthma, the specific sphingolipid classes and/or mechanisms driving this relationship remain unclear. We aimed to understand the multifaceted role between sphingolipids and other established asthma risk factors that complicate this relationship. METHODS: We performed targeted LC-MS/MS-based quantification of 77 sphingolipids in plasma from 997 children aged 6 years from two independent cohorts (VDAART and COPSAC2010 ). We examined associations of circulatory sphingolipids with childhood asthma, lung function, and three asthma risk factors: functional SNPs in ORMDL3, low vitamin D levels, and reduced gut microbial maturity. Given racial differences between these cohorts, association analyses were performed separately and then meta-analyzed together. RESULTS: We observed elevations in circulatory sphingolipids with asthma phenotypes and risk factors; however, there were differential associations of sphingolipid classes with clinical outcomes and/or risk factors. While elevations from metabolites involved in ceramide recycling and catabolic pathways were associated with asthma and worse lung function [meta p-value range: 1.863E-04 to 2.24E-3], increased ceramide levels were associated with asthma risk factors [meta p-value range: 7.75E-5 to .013], but not asthma. Further investigation identified that some ceramides acted as mediators while some interacted with risk factors in the associations with asthma outcomes. CONCLUSION: This study demonstrates the differential role that sphingolipid subclasses may play in asthma and its risk factors. While overall elevations in sphingolipids appeared to be deleterious overall; elevations in ceramides were uniquely associated with increases in asthma risk factors only; while elevations in asthma phenotypes were associated with recycling sphingolipids. Modification of asthma risk factors may play an important role in regulating sphingolipid homeostasis via ceramides to affect asthma. Further function work may validate the observed associations.

A Walnut-Enriched Diet for 2 Years Changes the Serum Oxylipin Profile in Healthy Older Persons.

BACKGROUND: Oxylipins are products derived from polyunsaturated fatty acids (PUFAs) that play a role in cardiovascular disease and aging. Fish oil-derived n-3 PUFAs promote the formation of anti-inflammatory and vasodilatory oxylipins; however, there are little data on oxylipins derived from α-linolenic acid (C18:3n-3), the primary plant-derived n-3 PUFA. Walnuts are a source of C18:3n-3. OBJECTIVES: To investigate the effect on serum oxylipins of a diet enriched with walnuts at 15% energy (30-60 g/d; 2.6-5.2 g C18:3n-3/d) for 2 y compared to a control diet (abstention from walnuts) in healthy older males and females (63-79 y). METHODS: The red blood cell proportion of α-linolenic acid was determined by gas chromatography as a measure of compliance. Ultra-performance liquid chromatography-tandem mass spectrometry was used to measure serum concentrations of 53 oxylipins in participants randomly assigned to receive the walnut diet (n = 64) or the control diet (n = 51). Two-year concentration changes (final minus baseline) were log-transformed (base log-10) and standardized (mean-centered and divided by the standard deviation of each variable). Volcano plots were then generated (fold change ≥1.5; false discovery rate ≤0.1). For each oxylipin delta surviving multiple testing, we further assessed between-intervention group differences by analysis of covariance adjusting for age, sex, BMI, and the baseline concentration of the oxylipin. RESULTS: The 2-y change in red blood cell C18:3n-3 in the walnut group was significantly higher than that in the control group (P < 0.001). Compared to the control diet, the walnut diet resulted in statistically significantly greater increases in 3 C18:3n-3-derived oxylipins (9-HOTrE, 13-HOTrE, and 12,13-EpODE) and in the C20:5n-3 derived 14,15-diHETE, and greater reductions of the C20:4n-6-derived 5-HETE, 19-HETE, and 5,6-diHETrE. CONCLUSIONS: Long-term walnut consumption changes the serum oxylipin profile in healthy older persons. Our results add novel mechanistic evidence on the cardioprotective effects of walnuts. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01634841.

A single extraction 96-well method for LC-MS/MS quantification of urinary eicosanoids, steroids and drugs.

Urinary eicosanoid concentrations reflect inflammatory processes in multiple diseases and have been used as biomarkers of disease as well as suggested for patient stratification in precision medicine. However, implementation of urinary eicosanoid profiling in large-scale analyses is restricted due to sample preparation limits. Here we demonstrate a single solid-phase extraction of 300 µL urine in 96-well-format for prostaglandins, thromboxanes, isoprostanes, cysteinyl-leukotriene E4 and the linoleic acid-derived dihydroxy-octadecenoic acids (9,10- and 12,13-DiHOME). A simultaneous screening protocol was also developed for cortisol/cortisone and 7 exogenous steroids as well as 3 cyclooxygenase inhibitors. Satisfactory performance for quantification of eicosanoids with an appropriate internal standard was demonstrated for intra-plate analyses (CV = 8.5-15.1%) as well as for inter-plate (n = 35) from multiple studies (CV = 22.1-34.9%). Storage stability was evaluated at - 20 °C, and polar tetranors evidenced a 50% decrease after 5 months, while the remaining eicosanoids evidenced no significant degradation. All eicosanoids were stable over 3.5-years in urine stored at - 80 °C. This method will facilitate the implementation of urinary eicosanoid quantification in large-scale screening.

Cohort-based strategies as an in-house tool to evaluate and improve phenotyping robustness of LC-MS/MS lipidomics platforms.

In recent years, instrumental improvements have enabled the spread of mass spectrometry-based lipidomics platforms in biomedical research. In mass spectrometry, the reliability of generated data varies for each compound, contingent on, among other factors, the availability of labeled internal standards. It is challenging to evaluate the data for lipids without specific labeled internal standards, especially when dozens to hundreds of lipids are measured simultaneously. Thus, evaluation of the performance of these platforms at the individual lipid level in interlaboratory studies is generally not feasible in a time-effective manner. Herein, using a focused subset of sphingolipids, we present an in-house validation methodology for individual lipid reliability assessment, tailored to the statistical analysis to be applied. Moreover, this approach enables the evaluation of various methodological aspects, including discerning coelutions sharing identical selected reaction monitoring transitions, pinpointing optimal labeled internal standards and their concentrations, and evaluating different extraction techniques. While the full validation according to analytical guidelines for all lipids included in a lipidomics method is currently not possible, this process shows areas to focus on for subsequent method development iterations as well as the robustness of data generated across diverse methodologies.

Maternal vitamin D-related metabolome and offspring risk of asthma outcomes.

BACKGROUND: Gestational vitamin D deficiency is implicated in development of respiratory diseases in offspring, but the mechanism underlying this relationship is unknown. OBJECTIVE: We sought to study the link between gestational vitamin D exposure and childhood asthma phenotypes using maternal blood metabolomics profiling. METHODS: Untargeted blood metabolic profiles were acquired using liquid chromatography-mass spectrometry at 1 week postpartum from 672 women in the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) mother-child cohort and at pregnancy weeks 32 to 38 from 779 women in the Vitamin D Antenatal Asthma Reduction Trial (VDAART) mother-child cohort. In COPSAC2010, we employed multivariate models and pathway enrichment analysis to identify metabolites and pathways associated with gestational vitamin D blood levels and investigated their relationship with development of asthma phenotypes in early childhood. The findings were validated in VDAART and in cellular models. RESULTS: In COPSAC2010, higher vitamin D blood levels at 1 week postpartum were associated with distinct maternal metabolome perturbations with significant enrichment of the sphingomyelin pathway (P < .01). This vitamin D-related maternal metabolic profile at 1 week postpartum containing 46 metabolites was associated with decreased risk of recurrent wheeze (hazard ratio [HR] = 0.92 [95% CI 0.86-0.98], P = .01) and wheeze exacerbations (HR = 0.90 [95% CI 0.84-0.97], P = .01) at ages 0 to 3 years. The same metabolic profile was similarly associated with decreased risk of asthma/wheeze at ages 0 to 3 in VDAART (odds ratio = 0.92 [95% CI 0.85-0.99], P = .04). Human bronchial epithelial cells treated with high-dose vitamin D3 showed an increased cytoplasmic sphingolipid level (P < .01). CONCLUSIONS: This exploratory metabolomics study in 2 independent birth cohorts demonstrates that the beneficial effect of higher gestational vitamin D exposure on offspring respiratory health is characterized by specific maternal metabolic alterations during pregnancy, which involves the sphingomyelin pathway.

Precision medicine in complex diseases-Molecular subgrouping for improved prediction and treatment stratification.

Complex diseases are caused by a combination of genetic, lifestyle, and environmental factors and comprise common noncommunicable diseases, including allergies, cardiovascular disease, and psychiatric and metabolic disorders. More than 25% of Europeans suffer from a complex disease, and together these diseases account for 70% of all deaths. The use of genomic, molecular, or imaging data to develop accurate diagnostic tools for treatment recommendations and preventive strategies, and for disease prognosis and prediction, is an important step toward precision medicine. However, for complex diseases, precision medicine is associated with several challenges. There is a significant heterogeneity between patients of a specific disease-both with regards to symptoms and underlying causal mechanisms-and the number of underlying genetic and nongenetic risk factors is often high. Here, we summarize precision medicine approaches for complex diseases and highlight the current breakthroughs as well as the challenges. We conclude that genomic-based precision medicine has been used mainly for patients with highly penetrant monogenic disease forms, such as cardiomyopathies. However, for most complex diseases-including psychiatric disorders and allergies-available polygenic risk scores are more probabilistic than deterministic and have not yet been validated for clinical utility. However, subclassifying patients of a specific disease into discrete homogenous subtypes based on molecular or phenotypic data is a promising strategy for improving diagnosis, prediction, treatment, prevention, and prognosis. The availability of high-throughput molecular technologies, together with large collections of health data and novel data-driven approaches, offers promise toward improved individual health through precision medicine.

Recognising the importance of chronic lung disease: a consensus statement from the Global Alliance for Chronic Diseases (Lung Diseases group).

BACKGROUND: Chronic respiratory diseases are disorders of the airways and other structures of the lung, and include chronic obstructive pulmonary disease (COPD), lung cancer, asthma, bronchiectasis, interstitial lung diseases, occupational lung diseases and pulmonary hypertension. Through this article we take a broad view of chronic lung disease while highlighting (1) the complex interactions of lung diseases with environmental factors (e.g. climate change, smoking and vaping) and multimorbidity and (2) proposed areas to strengthen for better global patient outcomes. CONCLUSION: We suggest new directions for the research agenda in high-priority populations and those experiencing health disparities. We call for lung disease to be made a research priority with greater funding allocation globally.

Metabo-Endotypes of Asthma Reveal Differences in Lung Function: Discovery and Validation in Two TOPMed Cohorts.

Rationale: Current guidelines do not sufficiently capture the heterogeneous nature of asthma; a more detailed molecular classification is needed. Metabolomics represents a novel and compelling approach to derive asthma endotypes (i.e., subtypes defined by functional and/or pathobiological mechanisms). Objectives: To validate metabolomic-driven endotypes of asthma and explore their underlying biology. Methods: In the Genetics of Asthma in Costa Rica Study (GACRS), untargeted metabolomic profiling, similarity network fusion, and spectral clustering was used to identify metabo-endotypes of asthma, and differences in asthma-relevant phenotypes across these metabo-endotypes were explored. The metabo-endotypes were recapitulated in the Childhood Asthma Management Program (CAMP), and clinical differences were determined. Metabolomic drivers of metabo-endotype membership were investigated by meta-analyzing findings from GACRS and CAMP. Measurements and Main Results: Five metabo-endotypes were identified in GACRS with significant differences in asthma-relevant phenotypes, including prebronchodilator (p-ANOVA = 8.3 × 10-5) and postbronchodilator (p-ANOVA = 1.8 × 10-5) FEV1/FVC. These differences were validated in the recapitulated metabo-endotypes in CAMP. Cholesterol esters, trigylcerides, and fatty acids were among the most important drivers of metabo-endotype membership. The findings suggest dysregulation of pulmonary surfactant homeostasis may play a role in asthma severity. Conclusions: Clinically meaningful endotypes may be derived and validated using metabolomic data. Interrogating the drivers of these metabo-endotypes has the potential to help understand their pathophysiology.

Association of Differential Mast Cell Activation with Granulocytic Inflammation in Severe Asthma.

Rationale: Mast cells (MCs) play a role in inflammation and both innate and adaptive immunity, but their involvement in severe asthma (SA) remains undefined. Objectives: We investigated the phenotypic characteristics of the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) asthma cohort by applying published MC activation signatures to the sputum cell transcriptome. Methods: Eighty-four participants with SA, 20 with mild/moderate asthma (MMA), and 16 healthy participants without asthma were studied. We calculated enrichment scores (ESs) for nine MC activation signatures by asthma severity, sputum granulocyte status, and three previously defined sputum molecular phenotypes or transcriptome-associated clusters (TACs) 1, 2, and 3 using gene set variation analysis. Measurements and Main Results: MC signatures except unstimulated, repeated FcεR1-stimulated and IFN-γ-stimulated signatures were enriched in SA. A FcεR1-IgE-stimulated and a single-cell signature from asthmatic bronchial biopsies were highly enriched in eosinophilic asthma and in the TAC1 molecular phenotype. Subjects with a high ES for these signatures had elevated sputum amounts of similar genes and pathways. IL-33- and LPS-stimulated MC signatures had greater ES in neutrophilic and mixed granulocytic asthma and in the TAC2 molecular phenotype. These subjects exhibited neutrophil, NF-κB (nuclear factor-κB), and IL-1ß/TNF-α (tumor necrosis factor-α) pathway activation. The IFN-γ-stimulated signature had the greatest ES in TAC2 and TAC3 that was associated with responses to viral infection. Similar results were obtained in an independent ADEPT (Airway Disease Endotyping for Personalized Therapeutics) asthma cohort. Conclusions: Gene signatures of MC activation allow the detection of SA phenotypes and indicate that MCs can be induced to take on distinct transcriptional phenotypes associated with specific clinical phenotypes. IL-33-stimulated MC signature was associated with severe neutrophilic asthma, whereas IgE-activated MC was associated with an eosinophilic phenotype.

Disturbed glucose and pyruvate metabolism in glaucoma with neuroprotection by pyruvate or rapamycin.

Intraocular pressure-sensitive retinal ganglion cell degeneration is a hallmark of glaucoma, the leading cause of irreversible blindness. Here, we used RNA-sequencing and metabolomics to examine early glaucoma in DBA/2J mice. We demonstrate gene expression changes that significantly impact pathways mediating the metabolism and transport of glucose and pyruvate. Subsequent metabolic studies characterized an intraocular pressure (IOP)-dependent decline in retinal pyruvate levels coupled to dysregulated glucose metabolism prior to detectable optic nerve degeneration. Remarkably, retinal glucose levels were elevated 50-fold, consistent with decreased glycolysis but possibly including glycogen mobilization and other metabolic changes. Oral supplementation of the glycolytic product pyruvate strongly protected from neurodegeneration in both rat and mouse models of glaucoma. Investigating further, we detected mTOR activation at the mechanistic nexus of neurodegeneration and metabolism. Rapamycin-induced inhibition of mTOR robustly prevented glaucomatous neurodegeneration, supporting a damaging role for IOP-induced mTOR activation in perturbing metabolism and promoting glaucoma. Together, these findings support the use of treatments that limit metabolic disturbances and provide bioenergetic support. Such treatments provide a readily translatable strategy that warrants investigation in clinical trials.

Targeting a scavenger receptor on tumor-associated macrophages activates tumor cell killing by natural killer cells.

Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.

Mapping atopic dermatitis and anti-IL-22 response signatures to type 2-low severe neutrophilic asthma.

BACKGROUND: Transcriptomic changes in patients who respond clinically to biological therapies may identify responses in other tissues or diseases. OBJECTIVE: We sought to determine whether a disease signature identified in atopic dermatitis (AD) is seen in adults with severe asthma and whether a transcriptomic signature for patients with AD who respond clinically to anti-IL-22 (fezakinumab [FZ]) is enriched in severe asthma. METHODS: An AD disease signature was obtained from analysis of differentially expressed genes between AD lesional and nonlesional skin biopsies. Differentially expressed genes from lesional skin from therapeutic superresponders before and after 12 weeks of FZ treatment defined the FZ-response signature. Gene set variation analysis was used to produce enrichment scores of AD and FZ-response signatures in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes asthma cohort. RESULTS: The AD disease signature (112 upregulated genes) encompassing inflammatory, T-cell, TH2, and TH17/TH22 pathways was enriched in the blood and sputum of patients with asthma with increasing severity. Patients with asthma with sputum neutrophilia and mixed granulocyte phenotypes were the most enriched (P < .05). The FZ-response signature (296 downregulated genes) was enriched in asthmatic blood (P < .05) and particularly in neutrophilic and mixed granulocytic sputum (P < .05). These data were confirmed in sputum of the Airway Disease Endotyping for Personalized Therapeutics cohort. IL-22 mRNA across tissues did not correlate with FZ-response enrichment scores, but this response signature correlated with TH22/IL-22 pathways. CONCLUSIONS: The FZ-response signature in AD identifies severe neutrophilic asthmatic patients as potential responders to FZ therapy. This approach will help identify patients for future asthma clinical trials of drugs used successfully in other chronic diseases.

Hyperglycemia Induces Trained Immunity in Macrophages and Their Precursors and Promotes Atherosclerosis.

BACKGROUND: Cardiovascular risk in diabetes remains elevated despite glucose-lowering therapies. We hypothesized that hyperglycemia induces trained immunity in macrophages, promoting persistent proatherogenic characteristics. METHODS: Bone marrow-derived macrophages from control mice and mice with diabetes were grown in physiological glucose (5 mmol/L) and subjected to RNA sequencing (n=6), assay for transposase accessible chromatin sequencing (n=6), and chromatin immunoprecipitation sequencing (n=6) for determination of hyperglycemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into (normoglycemic) Ldlr-/- mice was used to assess its functional significance in vivo. Evidence of hyperglycemia-induced trained immunity was sought in human peripheral blood mononuclear cells from patients with diabetes (n=8) compared with control subjects (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. RESULTS: In macrophages, high extracellular glucose promoted proinflammatory gene expression and proatherogenic functional characteristics through glycolysis-dependent mechanisms. Bone marrow-derived macrophages from diabetic mice retained these characteristics, even when cultured in physiological glucose, indicating hyperglycemia-induced trained immunity. Bone marrow transplantation from diabetic mice into (normoglycemic) Ldlr-/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated assay for transposase accessible chromatin, chromatin immunoprecipitation, and RNA sequencing analyses of hematopoietic stem cells and bone marrow-derived macrophages revealed a proinflammatory priming effect in diabetes. The pattern of open chromatin implicated transcription factor Runt-related transcription factor 1 (Runx1). Similarly, transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for Runx1 targets, consistent with a potential role in human disease. Pharmacological inhibition of Runx1 in vitro inhibited the trained phenotype. CONCLUSIONS: Hyperglycemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Development of a Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry Platform for the Quantitative Metabolic Profiling of Octadecanoid Oxylipins.

Octadecanoids are broadly defined as oxylipins (i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem mass spectrometry detection for quantification of a broad panel of octadecanoids. The platform includes 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in <13 min, while the achiral LC method separated 67 octadecanoids in 13.5 min. The LC method provided a robust complementary approach with greater sensitivity relative to the SFC method. Both methods were validated in solvent and surrogate matrix in terms of linearity, lower limits of quantification (LLOQ), recovery, accuracy, precision, and matrix effects. Instrumental linearity was good for both methods (R2 > 0.995) and LLOQ ranged from 0.03 to 6.00 ng/mL for SFC and 0.01 to 1.25 ng/mL for LC. The average accuracy in the solvent and surrogate matrix ranged from 89 to 109% in SFC and from 106 to 220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentrations) and 26% (at low concentrations). Validation in the surrogate matrix showed negligible matrix effects (<16% for all analytes), and average recoveries ranged from 71 to 83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little-studied compounds.