Multi-organ phenotypes in mice lacking latent TGFß binding protein 2 (LTBP2).

BACKGROUND: Latent TGFß binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFß, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS: Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.

Nanoparticle targeting of neutrophil glycolysis prevents lung ischemia-reperfusion injury.

Neutrophils exacerbate pulmonary ischemia-reperfusion injury (IRI) resulting in poor short and long-term outcomes for lung transplant recipients. Glycolysis powers neutrophil activation, but it remains unclear if neutrophil-specific targeting of this pathway will inhibit IRI. Lipid nanoparticles containing the glycolysis flux inhibitor 2-deoxyglucose (2-DG) were conjugated to neutrophil-specific Ly6G antibodies (NP-Ly6G[2-DG]). Intravenously administered NP-Ly6G(2-DG) to mice exhibited high specificity for circulating neutrophils. NP-Ly6G(2-DG)-treated neutrophils were unable to adapt to hypoglycemic conditions of the lung airspace environment as evident by the loss of demand-induced glycolysis, reductions in glycogen and ATP content, and an increased vulnerability to apoptosis. NP-Ly6G(2-DG) treatment inhibited pulmonary IRI following hilar occlusion and orthotopic lung transplantation. IRI protection was associated with less airspace neutrophil extracellular trap generation, reduced intragraft neutrophilia, and enhanced alveolar macrophage efferocytotic clearance of neutrophils. Collectively, our data show that pharmacologically targeting glycolysis in neutrophils inhibits their activation and survival leading to reduced pulmonary IRI.

Why Lungs Keep Time: Circadian Rhythms and Lung Immunity.

Circadian rhythms are daily cycles in biological function that are ubiquitous in nature. Understood as a means for organisms to anticipate daily environmental changes, circadian rhythms are also important for orchestrating complex biological processes such as immunity. Nowhere is this more evident than in the respiratory system, where circadian rhythms in inflammatory lung disease have been appreciated since ancient times. In this focused review we examine how emerging research on circadian rhythms is being applied to the study of fundamental lung biology and respiratory disease. We begin with a general introduction to circadian rhythms and the molecular circadian clock that underpins them. We then focus on emerging data tying circadian clock function to immunologic activities within the respiratory system. We conclude by considering outstanding questions about biological timing in the lung and how a better command of chronobiology could inform our understanding of complex lung diseases.

Establishment of the early cilia preassembly protein complex during motile ciliogenesis.

Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Förster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.

Long noncoding RNA FOXD3-AS1 regulates oxidative stress-induced apoptosis via sponging microRNA-150.

The function of most human long noncoding RNAs (lncRNAs) remains unclear. Our studies identified a highly up-regulated mammalian lncRNA, FOXD3-AS1, known as linc1623 in mice, in the setting of hyperoxia/reactive oxygen species (ROS)-induced lung injury. We found that ROS induced a robust expression of FOXD3-AS1 in mouse lung tissue. Functionally, FOXD3-AS1 promoted oxidative stress-induced lung epithelial cell death. In human lung epithelial cells, the microRNA-150 (miR-150) was identified to interact with FOXD3-AS1; this finding was confirmed using the luciferase reporter assays. Consistently, mutation on the miR-150 pairing sequence in FOXD3-AS1 abolished the interactions between FOXD3-AS1 and miR-150. Additionally, miR-150 mimics suppressed the level of FOXD3-AS1. The antisense oligos of FOXD3-AS1 significantly augmented the intracellular level of miR-150, supporting the theory of sponging effects of FOXD3-AS1 on miR-150. We further investigated the cellular function of miR-150 in our lung injury models. MiR-150 conferred a cytoprotective role in lung epithelial cells after oxidative stress, whereas FOXD3-AS1 promoted cell death. Taken together, our studies indicated that FOXD3-AS1 serves as a sponge or as a competing endogenous noncoding RNA for miR-150, restricting its capability to promote cell growth and thereby exaggerating hyperoxia-induced lung epithelial cell death.-Zhang, D., Lee, H., Haspel, J. A., Jin, Y. Long noncoding RNA FOXD3-AS1 regulates oxidative stress-induced apoptosis via sponging microRNA-150.

MiR-15a/16 Regulates Apoptosis of Lung Epithelial Cells after Oxidative Stress.

Lung epithelial cell apoptosis is an important feature of hyperoxia-induced lung injury. Death receptor-associated extrinsic pathway and mitochondria-associated intrinsic pathway both mediate the development of lung epithelial cell apoptosis. Despite decades of research, molecular mechanisms of hyperoxia-induced epithelial cell apoptosis remain incompletely understood. Here we report a novel regulatory paradigm in response to hyperoxia-associated oxidative stress. Hyperoxia markedly up-regulated miR-15a/16 levels in lung epithelial cells, broncho-alveolar lavage fluid (BALF) and lung tissue. This effect was mediated by hyperoxia-induced reactive oxygen species (ROS). Functionally, miR-15a/16 inhibitors induced caspase 3-mediated lung epithelial cell apoptosis, in the presence of hyperoxia. MiR-15a/16 inhibitors robustly enhanced FADD level and down-regulated Bcl-2 expression. Consistently, cleaved caspase 8 and 9 were highly induced in the miR-15a/16 deficient cells, after hyperoxia. Using airway epithelial cell specific, miR-15a/16-/- mice, we found that Bcl-2 significantly reduced in lung epithelial cells in vivo after hyperoxia. In contrast, caspase 3, 8 and Bcl-2 associated death promoter (BAD) were highly elevated in the miR-15a/16-/- epithelial cells in vivo. Interestingly, in lung epithelial malignant cells, rather than benign cells, deletion of miR-15a/16 prevented apoptosis. Furthermore, deletion of miR-15a/16 in macrophages also prohibited apoptosis, opposite to what we have found in normal lung epithelial cells. Taken together, our data suggested that miR-15a/16 may exert differential roles in different cell types. MiR-15a/16 deficiency result in lung epithelial cell apoptosis in response to hyperoxia, via modulating both intrinsic and extrinsic apoptosis pathways.

Autophagy in pulmonary diseases.

(Macro)autophagy provides a membrane-dependent mechanism for the sequestration, transport, and lysosomal turnover of subcellular components, including proteins and organelles. In this capacity, autophagy maintains basal cellular homeostasis and healthy organelle populations such as mitochondria. During starvation, autophagy prolongs cell survival by recycling metabolic precursors from intracellular macromolecules. Furthermore, autophagy represents an inducible response to chemical and physical cellular stress. Increasing evidence suggests that autophagy, and its regulatory proteins, may critically influence vital cellular processes such as programmed cell death, cell proliferation, inflammation, and innate immune functions and thereby may play a critical role in the pathogenesis of human disease. The function of autophagy in disease pathogenesis remains unclear and may involve either impaired or accelerated autophagic activity or imbalances in the activation of autophagic proteins. This review examines the roles of autophagy in the pathogenesis of pulmonary diseases, with emphasis on pulmonary vascular disease and acute and chronic lung diseases.

Syndecan-2 exerts antifibrotic effects by promoting caveolin-1-mediated transforming growth factor-ß receptor I internalization and inhibiting transforming growth factor-ß1 signaling.

RATIONALE: Alveolar transforming growth factor (TGF)-ß1 signaling and expression of TGF-ß1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-ß receptor TßRI inhibits TGF-ß signaling and could attenuate development of experimental lung fibrosis. OBJECTIVES: To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-ß1 signaling in alveolar epithelial cells. METHODS: Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2-dependent changes in epithelial cell TGF-ß1 signaling, TGF-ß1, and TßRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury. MEASUREMENTS AND MAIN RESULTS: We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-ß1 signaling and downstream expression of TGF-ß1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1-dependent internalization of TGF-ß1 and TßRI in alveolar epithelial cells, which inhibited TGF-ß1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice. CONCLUSIONS: Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1-dependent TGF-ß1 and TßRI internalization and inhibiting TGF-ß1 signaling in alveolar epithelial cells. Hence, molecules that facilitate TßRI degradation via endocytosis represent potential therapies for pulmonary fibrosis.

Circadian rhythms in solid organ transplantation.

Circadian rhythms are daily cycles in physiology that can affect medical interventions. This review considers how these rhythms may relate to solid organ transplantation. It begins by summarizing the mechanism for circadian rhythm generation known as the molecular clock, and basic research connecting the clock to biological activities germane to organ acceptance. Next follows a review of clinical evidence relating time of day to adverse transplantation outcomes. The concluding section discusses knowledge gaps and practical areas where applying circadian biology might improve transplantation success.

Circadian immunity from bench to bedside: a practical guide.

The immune system is built to counteract unpredictable threats, yet it relies on predictable cycles of activity to function properly. Daily rhythms in immune function are an expanding area of study, and many originate from a genetically based timekeeping mechanism known as the circadian clock. The challenge is how to harness these biological rhythms to improve medical interventions. Here, we review recent literature documenting how circadian clocks organize fundamental innate and adaptive immune activities, the immunologic consequences of circadian rhythm and sleep disruption, and persisting knowledge gaps in the field. We then consider the evidence linking circadian rhythms to vaccination, an important clinical realization of immune function. Finally, we discuss practical steps to translate circadian immunity to the patient's bedside.

Inflammasome-regulated cytokines are critical mediators of acute lung injury.

RATIONALE: Despite advances in clinical management, there are currently no reliable diagnostic and therapeutic targets for acute respiratory distress syndrome (ARDS). The inflammasome/caspase-1 pathway regulates the maturation and secretion of proinflammatory cytokines (e.g., IL-18). IL-18 is associated with injury in animal models of systemic inflammation. OBJECTIVES: We sought to determine the contribution of the inflammasome pathway in experimental acute lung injury and human ARDS. METHODS: We performed comprehensive gene expression profiling on peripheral blood from patients with critical illness. Gene expression changes were assessed using real-time polymerase chain reaction, and IL-18 levels were measured in the plasma of the critically ill patients. Wild-type mice or mice genetically deficient in IL-18 or caspase-1 were mechanically ventilated using moderate tidal volume (12 ml/kg). Lung injury parameters were assessed in lung tissue, serum, and bronchoalveolar lavage fluid. MEASUREMENTS AND MAIN RESULTS: In mice, mechanical ventilation enhanced IL-18 levels in the lung, serum, and bronchoalveolar lavage fluid. IL-18-neutralizing antibody treatment, or genetic deletion of IL-18 or caspase-1, reduced lung injury in response to mechanical ventilation. In human patients with ARDS, inflammasome-related mRNA transcripts (CASP1, IL1B, and IL18) were increased in peripheral blood. In samples from four clinical centers, IL-18 was elevated in the plasma of patients with ARDS (sepsis or trauma-induced ARDS) and served as a novel biomarker of intensive care unit morbidity and mortality. CONCLUSIONS: The inflammasome pathway and its downstream cytokines play critical roles in ARDS development.

Biological rhythms in COVID-19 vaccine effectiveness in an observational cohort study of 1.5 million patients.

BACKGROUNDCircadian rhythms are evident in basic immune processes, but it is unclear if rhythms exist in clinical endpoints like vaccine protection. Here, we examined associations between COVID-19 vaccination timing and effectiveness.METHODSWe retrospectively analyzed a large Israeli cohort with timestamped COVID-19 vaccinations (n = 1,515,754 patients over 12 years old, 99.2% receiving BNT162b2). Endpoints included COVID-19 breakthrough infection and COVID-19-associated emergency department visits and hospitalizations. Our main comparison was among patients vaccinated during morning (800-1159 hours), afternoon (1200-1559 hours), or evening hours (1600-1959 hours). We employed Cox regression to adjust for differences in age, sex, and comorbidities.RESULTSBreakthrough infections differed based on vaccination time, with lowest the rates associated with late morning to early afternoon and highest rates associated with evening vaccination. Vaccination timing remained significant after adjustment for patient age, sex, and comorbidities. Results were consistent in patients who received the basic 2-dose series and who received booster doses. The relationship between COVID-19 immunization time and breakthrough infections was sinusoidal, consistent with a biological rhythm that modifies vaccine effectiveness by 8.6%-25%. The benefits of daytime vaccination were concentrated in younger (<20 years old) and older patients (>50 years old). COVID-19-related hospitalizations varied significantly with the timing of the second booster dose, an intervention reserved for older and immunosuppressed patients (HR = 0.64, morning vs. evening; 95% CI, 0.43-0.97; P = 0.038).CONCLUSIONWe report a significant association between the time of COVID-19 vaccination and its effectiveness. This has implications for mass vaccination programs.FUNDINGNIH.

Autophagy: a core cellular process with emerging links to pulmonary disease.

Autophagy is a highly conserved homeostatic pathway by which cells transport damaged proteins and organelles to lysosomes for degradation. Dysregulation of autophagy contributes to the pathogenesis of clinically important disorders in a variety of organ systems but, until recently, little was known about its relationship to diseases of the lung. However, there is now growing evidence at the basic research level that autophagy is linked to the pathogenesis of important pulmonary disorders such as chronic obstructive pulmonary disease, cystic fibrosis, and tuberculosis. In this review, we provide an introduction to the field of autophagy research geared to clinical and research pulmonologists. We focus on the best-studied autophagic mechanism, macroautophagy, and summarize studies that link the regulation of this pathway to pulmonary disease. Last, we offer our perspective on how a better understanding of macroautophagy might be used for designing novel therapies for pulmonary disorders.

Perfect timing: circadian rhythms, sleep, and immunity - an NIH workshop summary.

Recent discoveries demonstrate a critical role for circadian rhythms and sleep in immune system homeostasis. Both innate and adaptive immune responses - ranging from leukocyte mobilization, trafficking, and chemotaxis to cytokine release and T cell differentiation -are mediated in a time of day-dependent manner. The National Institutes of Health (NIH) recently sponsored an interdisciplinary workshop, "Sleep Insufficiency, Circadian Misalignment, and the Immune Response," to highlight new research linking sleep and circadian biology to immune function and to identify areas of high translational potential. This Review summarizes topics discussed and highlights immediate opportunities for delineating clinically relevant connections among biological rhythms, sleep, and immune regulation.

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux.

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.

Adventures in spacetime: circadian rhythms and the dynamics of protein catabolism.

Circadian rhythms help cells to organize complex processes, but how they shape the kinetics of protein catabolism is unclear. In a recent paper, we employed proteomics to map daily biological rhythms in autophagic flux in mouse liver, and correlated these rhythms with proteasome activity. We also explored the effect of inflammation caused by endotoxin on autophagy dynamics. Our data provide insight into how circadian rhythms serve as a framework for connecting the spatial, temporal, and metabolic aspects of autophagy at a system-wide level. Our observations also have implications for how to optimize autophagy-directed therapies in patients.

Diurnal Rhythms Spatially and Temporally Organize Autophagy.

Circadian rhythms are a hallmark of physiology, but how such daily rhythms organize cellular catabolism is poorly understood. Here, we used proteomics to map daily oscillations in autophagic flux in mouse liver and related these rhythms to proteasome activity. We also explored how systemic inflammation affects the temporal structure of autophagy. Our data identified a globally harmonized rhythm for basal macroautophagy, chaperone-mediated autophagy, and proteasomal activity, which concentrates liver proteolysis during the daytime. Basal autophagy rhythms could be resolved into two antiphase clusters that were distinguished by the subcellular location of targeted proteins. Inflammation induced by lipopolysaccharide reprogrammed autophagic flux away from a temporal pattern that favors cytosolic targets and toward the turnover of mitochondrial targets. Our data detail how daily biological rhythms connect the temporal, spatial, and metabolic aspects of protein catabolism.

Sleep Loss and Circadian Rhythm Disruption in the Intensive Care Unit.

Critical illness is associated with profound sleep disruption. Causality is diverse and includes physiologic, psychological, and environmental factors. There are limited pharmacologic interventions available to treat sleep disturbances in critical illness; however, multidisciplinary strategies that alter the intensive care unit (ICU) environment and cluster care delivery have shown promise in sleep and circadian promotion and delirium reduction. With the appropriate administrative support and involvement of diverse ICU stakeholders, effective strategies could be created, implemented, and maintained to improve sleep disruption in critically ill patients.

Circadian rhythm reprogramming during lung inflammation.

Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian programme exhibits unique features, including a divergent group of rhythmic genes and metabolites compared with the basal state and a distinct periodicity and phase distribution. At the cellular level, endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex re-organization of cellular and molecular circadian rhythms that are relevant to early events in lung injury.

Metabolomic derangements are associated with mortality in critically ill adult patients.

OBJECTIVE: To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults. RATIONALE: Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible. METHODS: We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study. RESULTS: We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations). CONCLUSION: Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.