Circadian Clock and Hypoxia.

The timing of life on Earth is remarkable: between individuals of the same species, a highly similar temporal pattern is observed, with shared periods of activity and inactivity each day. At the individual level, this means that over the course of a single day, a person alternates between two states. They are either upright, active, and communicative or they lie down in a state of (un)consciousness called sleep where even the characteristic of neuronal signals in the brain shows distinctive properties. The circadian clock governs both of these time stamps-activity and (apparent) inactivity-making them come and go consistently at the same approximate time each day. This behavior thus represents the meeting of two pervasive systems: the circadian clock and metabolism. In this article, we will describe what is known about how the circadian clock anticipates daily changes in oxygen usage, how circadian clock regulation may relate to normal physiology, and to hypoxia and ischemia that can result from pathologies such as myocardial infarction and stroke.

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.

Age-Dependent Reduction in Asthmatic Pathology through Reprogramming of Postviral Inflammatory Responses.

Asthma is a chronic disease of childhood, but for unknown reasons, disease activity sometimes subsides as children mature. In this study, we present clinical and animal model evidence suggesting that the age dependency of childhood asthma stems from an evolving host response to respiratory viral infection. Using clinical data, we show that societal suppression of respiratory virus transmission during coronavirus disease 2019 lockdown disrupted the traditional age gradient in pediatric asthma exacerbations, connecting the phenomenon of asthma remission to virus exposure. In mice, we show that asthmatic lung pathology triggered by Sendai virus (SeV) or influenza A virus is highly age-sensitive: robust in juvenile mice (4-6 wk old) but attenuated in mature mice (>3 mo old). Interestingly, allergen induction of the same asthmatic traits was less dependent on chronological age than viruses. Age-specific responses to SeV included a juvenile bias toward type 2 airway inflammation that emerged early in infection, whereas mature mice exhibited a more restricted bronchiolar distribution of infection that produced a distinct type 2 low inflammatory cytokine profile. In the basal state, aging produced changes to lung leukocyte burden, including the number and transcriptional landscape of alveolar macrophages (AMs). Importantly, depleting AMs in mature mice restored post-SeV pathology to juvenile levels. Thus, aging influences chronic outcomes of respiratory viral infection through regulation of the AM compartment and type 2 inflammatory responses to viruses. Our data provide insight into how asthma remission might develop in children.

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.

Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.

Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1ß (IL-1ß) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1ß and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.

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.

Age-dependent rebound in asthma exacerbations after COVID-19 lockdown.

Background: Virus mitigation measures enacted early in the coronavirus infectious disease 2019 (COVID-19) pandemic suppressed common respiratory viruses and reduced the number of obstructive lung disease exacerbations. However, many localities began to ease these precautions in the year 2021, leading to a resurgence of non-COVID viruses. How asthma and chronic obstructive pulmonary disease (COPD) activity responded to this upswing in viral abundance is unclear. Objective: Our aim was to examine how viral resurgence during the relaxation of COVID-19 restrictions affected asthma and COPD exacerbations. Methods: We analyzed electronic medical records for emergency department (ED) respiratory virus positivity, asthma visits, and COPD visits. We compared the 52-week interval before the COVID-19 restrictions (the pre-lockdown period [March 22, 2019-March 19, 2020]), the 52-week period immediately following enactment of the restrictions (the lockdown period [March 20, 2020-March 18, 2021]), and the 52-week period thereafter (the post-lockdown period [March 19, 2021-March 18, 2022]). We used MetaCYCLE to analyze seasonal trends in our data. Results: The post-lockdown period was marked by a 400% increase in viral positivity compared with during the lockdown period. Asthma- and COPD-related ED visits each rose 37% compared with during the lockdown, with the rebound in asthma ED visits concentrated in individuals younger than 20 years. Interestingly, after the lockdown period, asthma ED visits overcorrected in children younger than 5 years, rising 81% compared with before the lockdown. Seasonal rhythms in asthma and COPD exacerbations were suppressed during the lockdown and recovered after the lockdown. Conclusions: COVID-19 precautions had the unexpected effect of magnifying early-childhood asthma activity once common respiratory viruses recurred. These results may have implications for the future use of virus mitigation strategies in young children.

Effect of the COVID-19 Lockdown on Asthma Biological Rhythms.

Asthma has a striking temporal character, in which time-of-day, patient age, and season each influence disease activity. The extent to which rhythms in asthma activity reflect exposure to specific disease triggers remains unclear. In this study, we examined how virus mitigation strategies enacted during the COVID-19 pandemic ("lockdown measures") affected rhythms in asthma clinical activity in children. To this end, we retrospectively analyzed asthma clinical presentations in children aged <18 years to our regional academic medical center, comparing 4 years of medical records prior to COVID-19 lockdown measures with the 12 months immediately after the institution of such measures. We correlated these data to positive viral test results, febrile seizures, and allergic clinical surrogates (allergic reaction visits and Emergency Department [ED] antihistamine prescriptions, respectively) over the same time frame. In the 12 months following the institution of the COVID-19 lockdown, positivity rates for common respiratory viruses dropped by 70.2% and ED visits for asthma among children dropped by 62% compared to pre-COVID years. Lockdown suppressed seasonal variation in positive viral tests and asthma ED visits, while diurnal rhythms in asthma visits were unchanged. Asthma seasonality correlated most strongly with rhinovirus positivity both before and after the institution of COVID lockdown measures. Altogether, our data support a causal role for viruses in driving seasonal variability in asthma exacerbations in children.

A Timely Call to Arms: COVID-19, the Circadian Clock, and Critical Care.

We currently find ourselves in the midst of a global coronavirus disease 2019 (COVID-19) pandemic, caused by the highly infectious novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we discuss aspects of SARS-CoV-2 biology and pathology and how these might interact with the circadian clock of the host. We further focus on the severe manifestation of the illness, leading to hospitalization in an intensive care unit. The most common severe complications of COVID-19 relate to clock-regulated human physiology. We speculate on how the pandemic might be used to gain insights on the circadian clock but, more importantly, on how knowledge of the circadian clock might be used to mitigate the disease expression and the clinical course of COVID-19.

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.