Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons.

The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism.

Chronic inflammatory arthritis drives systemic changes in circadian energy metabolism.

Chronic inflammation underpins many human diseases. Morbidity and mortality associated with chronic inflammation are often mediated through metabolic dysfunction. Inflammatory and metabolic processes vary through circadian time, suggesting an important temporal crosstalk between these systems. Using an established mouse model of rheumatoid arthritis, we show that chronic inflammatory arthritis results in rhythmic joint inflammation and drives major changes in muscle and liver energy metabolism and rhythmic gene expression. Transcriptional and phosphoproteomic analyses revealed alterations in lipid metabolism and mitochondrial function associated with increased EGFR-JAK-STAT3 signaling. Metabolomic analyses confirmed rhythmic metabolic rewiring with impaired ß-oxidation and lipid handling and revealed a pronounced shunt toward sphingolipid and ceramide accumulation. The arthritis-related production of ceramides was most pronounced during the day, which is the time of peak inflammation and increased reliance on fatty acid oxidation. Thus, our data demonstrate that localized joint inflammation drives a time-of-day­dependent build-up of bioactive lipid species driven by rhythmic inflammation and altered EGFR-STAT signaling.

Circadian clock function does not require the histone methyltransferase MLL3.

The circadian clock controls the physiological function of tissues through the regulation of thousands of genes in a cell-type-specific manner. The core cellular circadian clock is a transcription-translation negative feedback loop, which can recruit epigenetic regulators to facilitate temporal control of gene expression. Histone methyltransferase, mixed lineage leukemia gene 3 (MLL3) was reported to be required for the maintenance of circadian oscillations in cultured cells. Here, we test the role of MLL3 in circadian organization in whole animals. Using mice expressing catalytically inactive MLL3, we show that MLL3 methyltransferase activity is in fact not required for circadian oscillations in vitro in a range of tissues, nor for the maintenance of circadian behavioral rhythms in vivo. In contrast to a previous report, loss of MLL3-dependent methylation did not affect the global levels of H3K4 methylation in liver, indicating substantial compensation from other methyltransferases. Furthermore, we found little evidence of genomic repositioning of H3K4me3 marks. We did, however, observe repositioning of H3K4me1 from intronic regions to intergenic regions and gene promoters; however, there were no changes in H3K4me1 mark abundance around core circadian clock genes. Output functions of the circadian clock, such as control of inflammation, were largely intact in MLL3-methyltransferase-deficient mice, although some gene-specific changes were observed, with sexually dimorphic loss of circadian regulation of specific cytokines. Taken together, these observations indicate that MLL3-directed histone methylation is not essential for core circadian clock function; however, it may influence the inflammatory response.

Nuclear receptor REVERBα is a state-dependent regulator of liver energy metabolism.

The nuclear receptor REVERBα is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. Using antibody-independent ChIP-sequencing of REVERBα in mouse liver, we reveal a high-confidence cistrome and define direct target genes. REVERBα-binding sites are highly enriched for consensus RORE or RevDR2 motifs and overlap with corepressor complex binding. We find no evidence for transcription factor tethering and DNA-binding domain-independent action. Moreover, hepatocyte-specific deletion of Reverbα drives only modest physiological and transcriptional dysregulation, with derepressed target gene enrichment limited to circadian processes. Thus, contrary to previous reports, hepatic REVERBα does not repress lipogenesis under basal conditions. REVERBα control of a more extensive transcriptional program is only revealed under conditions of metabolic perturbation (including mistimed feeding, which is a feature of the global Reverbα-/- mouse). Repressive action of REVERBα in the liver therefore serves to buffer against metabolic challenge, rather than drive basal rhythmicity in metabolic activity.

Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator.

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.

Seasonal changes in NRF2 antioxidant pathway regulates winter depression-like behavior.

Seasonal changes in the environment lead to depression-like behaviors in humans and animals. The underlying mechanisms, however, are unknown. We observed decreased sociability and increased anxiety-like behavior in medaka fish exposed to winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes in 68 metabolites, including neurotransmitters and antioxidants associated with depression. Transcriptome analysis identified 3,306 differentially expressed transcripts, including inflammatory markers, melanopsins, and circadian clock genes. Further analyses revealed seasonal changes in multiple signaling pathways implicated in depression, including the nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway. A broad-spectrum chemical screen revealed that celastrol (a traditional Chinese medicine) uniquely reversed winter behavior. NRF2 is a celastrol target expressed in the habenula (HB), known to play a critical role in the pathophysiology of depression. Another NRF2 chemical activator phenocopied these effects, and an NRF2 mutant showed decreased sociability. Our study provides important insights into winter depression and offers potential therapeutic targets involving NRF2.

The clock gene Bmal1 inhibits macrophage motility, phagocytosis, and impairs defense against pneumonia.

The circadian clock regulates many aspects of immunity. Bacterial infections are affected by time of day, but the mechanisms involved remain undefined. Here we show that loss of the core clock protein BMAL1 in macrophages confers protection against pneumococcal pneumonia. Infected mice show both reduced weight loss and lower bacterial burden in circulating blood. In vivo studies of macrophage phagocytosis reveal increased bacterial ingestion following Bmal1 deletion, which was also seen in vitro. BMAL1-/- macrophages exhibited marked differences in actin cytoskeletal organization, a phosphoproteome enriched for cytoskeletal changes, with reduced phosphocofilin and increased active RhoA. Further analysis of the BMAL1-/- macrophages identified altered cell morphology and increased motility. Mechanistically, BMAL1 regulated a network of cell movement genes, 148 of which were within 100 kb of high-confidence BMAL1 binding sites. Links to RhoA function were identified, with 29 genes impacting RhoA expression or activation. RhoA inhibition restored the phagocytic phenotype to that seen in control macrophages. In summary, we identify a surprising gain of antibacterial function due to loss of BMAL1 in macrophages, associated with a RhoA-dependent cytoskeletal change, an increase in cell motility, and gain of phagocytic function.

The circadian clock protein REVERBα inhibits pulmonary fibrosis development.

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinß1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

The histone methyltransferase Ezh2 restrains macrophage inflammatory responses.

Robust inflammatory responses are critical to survival following respiratory infection, with current attention focused on the clinical consequences of the Coronavirus pandemic. Epigenetic factors are increasingly recognized as important determinants of immune responses, and EZH2 is a prominent target due to the availability of highly specific and efficacious antagonists. However, very little is known about the role of EZH2 in the myeloid lineage. Here, we show EZH2 acts in macrophages to limit inflammatory responses to activation, and in neutrophils for chemotaxis. Selective genetic deletion in macrophages results in a remarkable gain in protection from infection with the prevalent lung pathogen, pneumococcus. In contrast, neutrophils lacking EZH2 showed impaired mobility in response to chemotactic signals, and resulted in increased susceptibility to pneumococcus. In summary, EZH2 shows complex, and divergent roles in different myeloid lineages, likely contributing to the earlier conflicting reports. Compounds targeting EZH2 are likely to impair mucosal immunity; however, they may prove useful for conditions driven by pulmonary neutrophil influx, such as adult respiratory distress syndrome.

Night shift work is associated with an increased risk of asthma.

INTRODUCTION: Shift work causes misalignment between internal circadian time and the external light/dark cycle and is associated with metabolic disorders and cancer. Approximately 20% of the working population in industrialised countries work permanent or rotating night shifts, exposing this large population to the risk of circadian misalignment-driven disease. Analysis of the impact of shift work on chronic inflammatory diseases is lacking. We investigated the association between shift work and asthma. METHODS: We describe the cross-sectional relationship between shift work and prevalent asthma in >280000 UK Biobank participants, making adjustments for major confounding factors (smoking history, ethnicity, socioeconomic status, physical activity, body mass index). We also investigated chronotype. RESULTS: Compared with day workers, 'permanent' night shift workers had a higher likelihood of moderate-severe asthma (OR 1.36 (95% CI 1.03 to 1.8)) and all asthma (OR 1.23 (95% CI 1.03 to 1.46)). Individuals doing any type of shift work had higher adjusted odds of wheeze/whistling in the chest. Shift workers who never or rarely worked on nights and people working permanent nights had a higher adjusted likelihood of having reduced lung function (FEV1 <80% predicted). We found an increase in the risk of moderate-severe asthma in morning chronotypes working irregular shifts, including nights (OR 1.55 (95% CI 1.06 to 2.27)). CONCLUSIONS: The public health implications of these findings are far-reaching due to the high prevalence and co-occurrence of both asthma and shift work. Future longitudinal follow-up studies are needed to determine if modifying shift work schedules to take into account chronotype might present a public health measure to reduce the risk of developing inflammatory diseases such as asthma.

Circadian asthma airway responses are gated by REV-ERBα.

BACKGROUND: The circadian clock powerfully regulates inflammation and the clock protein REV-ERBα is known to play a key role as a repressor of the inflammatory response. Asthma is an inflammatory disease of the airways with a strong time of day rhythm. Airway hyper-responsiveness (AHR) is a dominant feature of asthma; however, it is not known if this is under clock control. OBJECTIVES: To determine if allergy-mediated AHR is gated by the clock protein REV-ERBα. METHODS: After exposure to the intra-nasal house dust mite (HDM) allergen challenge model at either dawn or dusk, AHR to methacholine was measured invasively in mice. MAIN RESULTS: Wild-type (WT) mice show markedly different time of day AHR responses (maximal at dusk/start of the active phase), both in vivo and ex vivo, in precision cut lung slices. Time of day effects on AHR were abolished in mice lacking the clock gene Rev-erbα, indicating that such effects on asthma response are likely to be mediated via the circadian clock. We suggest that muscarinic receptors one (Chrm 1) and three (Chrm 3) may play a role in this pathway. CONCLUSIONS: We identify a novel circuit regulating a core process in asthma, potentially involving circadian control of muscarinic receptor expression, in a REV-ERBα dependent fashion. CLINICAL IMPLICATION: These insights suggest the importance of considering the timing of drug administration in clinic trials and in clinical practice (chronotherapy).

Circadian variation in pulmonary inflammatory responses is independent of rhythmic glucocorticoid signaling in airway epithelial cells.

The circadian clock is a critical regulator of immune function. We recently highlighted a role for the circadian clock in a mouse model of pulmonary inflammation. The epithelial clock protein Bmal1 was required to regulate neutrophil recruitment in response to inflammatory challenge. Bmal1 regulated glucocorticoid receptor (GR) recruitment to the neutrophil chemokine, CXC chemokine ligand 5 (CXCL5), providing a candidate mechanism. We now show that clock control of pulmonary neutrophilia persists without rhythmic glucocorticoid availability. Epithelial GR-null mice had elevated expression of proinflammatory chemokines in the lung under homeostatic conditions. However, deletion of GR in the bronchial epithelium blocked rhythmic CXCL5 production, identifying GR as required to confer circadian control to CXCL5. Surprisingly, rhythmic pulmonary neutrophilia persisted, despite nonrhythmic CXCL5 responses, indicating additional circadian control mechanisms. Deletion of GR in myeloid cells alone did not prevent circadian variation in pulmonary neutrophilia and showed reduced neutrophilic inflammation in response to dexamethasone treatment. These new data show GR is required to confer circadian control to some inflammatory chemokines, but that this alone is insufficient to prevent circadian control of neutrophilic inflammation in response to inhaled LPS, with additional control mechanisms arising in the myeloid cell lineage.-Ince, L. M., Zhang, Z., Beesley, S., Vonslow, R. M., Saer, B. R., Matthews, L. C., Begley, N., Gibbs, J. E., Ray, D. W., Loudon, A. S. I. Circadian variation in pulmonary inflammatory responses is independent of rhythmic glucocorticoid signaling in airway epithelial cells.

Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion.

Pulmonary airway epithelial cells (AECs) form a critical interface between host and environment. We investigated the role of the circadian clock using mice bearing targeted deletion of the circadian gene brain and muscle ARNT-like 1 (Bmal1) in AECs. Pulmonary neutrophil infiltration, biomechanical function, and responses to influenza infection were all disrupted. A circadian time-series RNA sequencing study of laser-captured AECs revealed widespread disruption in genes of the core circadian clock and output pathways regulating cell metabolism (lipids and xenobiotics), extracellular matrix, and chemokine signaling, but strikingly also the gain of a novel rhythmic transcriptome in Bmal1-targeted cells. Many of the rhythmic components were replicated in primary AECs cultured in air-liquid interface, indicating significant cell autonomy for control of pulmonary circadian physiology. Finally, we found that metabolic cues dictate phasing of the pulmonary clock and circadian responses to immunologic challenges. Thus, the local circadian clock in AECs is vital in lung health by coordinating major cell processes such as metabolism and immunity.-Zhang, Z. Hunter, L., Wu, G., Maidstone, R., Mizoro, Y., Vonslow, R., Fife, M., Hopwood, T., Begley, N., Saer, B., Wang, P., Cunningham, P., Baxter, M., Durrington, H., Blaikley, J. F., Hussell, T., Rattray, M., Hogenesch, J. B., Gibbs, J., Ray, D. W., Loudon, A. S. I. Genome-wide effect of pulmonary airway epithelial cell-specific Bmal1 deletion.

Incidence of primary graft dysfunction after lung transplantation is altered by timing of allograft implantation.

The importance of circadian factors in managing patients is poorly understood. We present two retrospective cohort studies showing that lungs reperfused between 4 and 8 AM have a higher incidence (OR 1.12; 95% CI 1.03 to 1.21; p=0.01) of primary graft dysfunction (PGD) in the first 72 hours after transplantation. Cooling of the donor lung, occurring during organ preservation, shifts the donor circadian clock causing desynchrony with the recipient. The clock protein REV-ERBα directly regulates PGD biomarkers explaining this circadian regulation while also allowing them to be manipulated with synthetic REV-ERB ligands.

Natural selection against a circadian clock gene mutation in mice.

Circadian rhythms with an endogenous period close to or equal to the natural light-dark cycle are considered evolutionarily adaptive ("circadian resonance hypothesis"). Despite remarkable insight into the molecular mechanisms driving circadian cycles, this hypothesis has not been tested under natural conditions for any eukaryotic organism. We tested this hypothesis in mice bearing a short-period mutation in the enzyme casein kinase 1ε (tau mutation), which accelerates free-running circadian cycles. We compared daily activity (feeding) rhythms, survivorship, and reproduction in six replicate populations in outdoor experimental enclosures, established with wild-type, heterozygous, and homozygous mice in a Mendelian ratio. In the release cohort, survival was reduced in the homozygote mutant mice, revealing strong selection against short-period genotypes. Over the course of 14 mo, the relative frequency of the tau allele dropped from initial parity to 20%. Adult survival and recruitment of juveniles into the population contributed approximately equally to the selection for wild-type alleles. The expression of activity during daytime varied throughout the experiment and was significantly increased by the tau mutation. The strong selection against the short-period tau allele observed here contrasts with earlier studies showing absence of selection against a Period 2 (Per2) mutation, which disrupts internal clock function, but does not change period length. These findings are consistent with, and predicted by the theory that resonance of the circadian system plays an important role in individual fitness.

The circadian clock regulates inflammatory arthritis.

There is strong diurnal variation in the symptoms and severity of chronic inflammatory diseases, such as rheumatoid arthritis. In addition, disruption of the circadian clock is an aggravating factor associated with a range of human inflammatory diseases. To investigate mechanistic links between the biological clock and pathways underlying inflammatory arthritis, mice were administered collagen (or saline as a control) to induce arthritis. The treatment provoked an inflammatory response within the limbs, which showed robust daily variation in paw swelling and inflammatory cytokine expression. Inflammatory markers were significantly repressed during the dark phase. Further work demonstrated an active molecular clock within the inflamed limbs and highlighted the resident inflammatory cells, fibroblast-like synoviocytes (FLSs), as a potential source of the rhythmic inflammatory signal. Exposure of mice to constant light disrupted the clock in peripheral tissues, causing loss of the nighttime repression of local inflammation. Finally, the results show that the core clock proteins cryptochrome (CRY) 1 and 2 repressed inflammation within the FLSs, and provide novel evidence that a CRY activator has anti-inflammatory properties in human cells. We conclude that under chronic inflammatory conditions, the clock actively represses inflammatory pathways during the dark phase. This interaction has exciting potential as a therapeutic avenue for treatment of inflammatory disease.-Hand, L. E., Hopwood, T. W., Dickson, S. H., Walker, A. L., Loudon, A. S. I., Ray, D. W., Bechtold, D. A., Gibbs, J. E. The circadian clock regulates inflammatory arthritis.

Pin1 promotes GR transactivation by enhancing recruitment to target genes.

The glucocorticoid receptor (GR) is a ligand activated transcription factor, serving to regulate both energy metabolism and immune functions. Factors that influence cellular sensitivity to glucocorticoids (GC) are therefore of great interest. The N-terminal of the GR contains numerous potential proline-directed phosphorylation sites, some of which can regulate GR transactivation. Unrestricted proline isomerisation can be inhibited by adjacent serine phosphorylation and requires a prolyl isomerise, Pin1. Pin1 therefore determines the functional outcome of proline-directed kinases acting on the GR, as cis/trans isomers are distinct pools with different interacting proteins. We show that Pin1 mediates GR transactivation, but not GR trans-repression. Two N-terminal GR serines, S203 and S211, are targets for Pin1 potentiation of GR transactivation, establishing a direct link between Pin1 and the GR. We also demonstrate GC-activated co-recruitment of GR and Pin1 to the GILZ gene promoter. The Pin1 effect required both its WW and catalytic domains, and GR recruitment to its GRE was Pin1-dependent. Therefore, Pin1 is a selective regulator of GR transactivation, acting through N-terminal phospho-serine residues to regulate GR recruitment to its target sites in the genome. As Pin1 is dysregulated in disease states, this interaction may contribute to altered GC action in inflammatory conditions.

The nuclear receptor REV-ERBα mediates circadian regulation of innate immunity through selective regulation of inflammatory cytokines.

Diurnal variation in inflammatory and immune function is evident in the physiology and pathology of humans and animals, but molecular mechanisms and mediating cell types that provide this gating remain unknown. By screening cytokine responses in mice to endotoxin challenge at different times of day, we reveal that the magnitude of response exhibited pronounced temporal dependence, yet only within a subset of proinflammatory cytokines. Disruption of the circadian clockwork in macrophages (primary effector cells of the innate immune system) by conditional targeting of a key clock gene (bmal1) removed all temporal gating of endotoxin-induced cytokine response in cultured cells and in vivo. Loss of circadian gating was coincident with suppressed rev-erbα expression, implicating this nuclear receptor as a potential link between the clock and inflammatory pathways. This finding was confirmed in vivo and in vitro through genetic and pharmacological modulation of REV-ERBα activity. Circadian gating of endotoxin response was lost in rev-erbα(-/-) mice and in cultured macrophages from these animals, despite maintenance of circadian rhythmicity within these cells. Using human macrophages, which show circadian clock gene oscillations and rhythmic endotoxin responses, we demonstrate that administration of a synthetic REV-ERB ligand, or genetic knockdown of rev-erbα expression, is effective at modulating the production and release of the proinflammatory cytokine IL-6. This work demonstrates that the macrophage clockwork provides temporal gating of systemic responses to endotoxin, and identifies REV-ERBα as the key link between the clock and immune function. REV-ERBα may therefore represent a unique therapeutic target in human inflammatory disease.

The circadian clock and asthma.

It is characteristic of asthma that symptoms worsen overnight, particularly in the early hours of the morning. Nocturnal symptoms in asthma are common and are an important indicator for escalation of treatment. An extensive body of research has demonstrated that nocturnal symptoms of cough and dyspnea are accompanied by circadian variations in airway inflammation and physiologic variables, including airflow limitation and airways hyper-responsiveness. The molecular apparatus that underpins circadian variations, controlled by so called 'clock' genes, has recently been characterised. Clock genes control circadian rhythms both centrally, in the suprachiasmatic nucleus of the brain and peripherally, within every organ of the body. Here, we will discuss how clock genes regulate circadian rhythms. We will focus particularly on the peripheral lung clock and the peripheral immune clock and discuss how these might relate to both the pathogenesis and treatment of asthma.