Circadian regulation of pulmonary disease: the importance of timing.

Circadian regulation causes the activity of biological processes to vary over a 24-h cycle. The pathological effects of this variation are predominantly studied using two different approaches: pre-clinical models or observational clinical studies. Both these approaches have provided useful insights into how underlying circadian mechanisms operate and specifically which are regulated by the molecular oscillator, a key time-keeping mechanism in the body. This review compares and contrasts findings from these two approaches in the context of four common respiratory diseases (asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and respiratory infection). Potential methods used to identify and measure human circadian oscillations are also discussed as these will be useful outcome measures in future interventional human trials that target circadian mechanisms.

Effects of sleep disturbance on dyspnoea and impaired lung function following hospital admission due to COVID-19 in the UK: a prospective multicentre cohort study.

BACKGROUND: Sleep disturbance is common following hospital admission both for COVID-19 and other causes. The clinical associations of this for recovery after hospital admission are poorly understood despite sleep disturbance contributing to morbidity in other scenarios. We aimed to investigate the prevalence and nature of sleep disturbance after discharge following hospital admission for COVID-19 and to assess whether this was associated with dyspnoea. METHODS: CircCOVID was a prospective multicentre cohort substudy designed to investigate the effects of circadian disruption and sleep disturbance on recovery after COVID-19 in a cohort of participants aged 18 years or older, admitted to hospital for COVID-19 in the UK, and discharged between March, 2020, and October, 2021. Participants were recruited from the Post-hospitalisation COVID-19 study (PHOSP-COVID). Follow-up data were collected at two timepoints: an early time point 2-7 months after hospital discharge and a later time point 10-14 months after hospital discharge. Sleep quality was assessed subjectively using the Pittsburgh Sleep Quality Index questionnaire and a numerical rating scale. Sleep quality was also assessed with an accelerometer worn on the wrist (actigraphy) for 14 days. Participants were also clinically phenotyped, including assessment of symptoms (ie, anxiety [Generalised Anxiety Disorder 7-item scale questionnaire], muscle function [SARC-F questionnaire], dyspnoea [Dyspnoea-12 questionnaire] and measurement of lung function), at the early timepoint after discharge. Actigraphy results were also compared to a matched UK Biobank cohort (non-hospitalised individuals and recently hospitalised individuals). Multivariable linear regression was used to define associations of sleep disturbance with the primary outcome of breathlessness and the other clinical symptoms. PHOSP-COVID is registered on the ISRCTN Registry (ISRCTN10980107). FINDINGS: 2320 of 2468 participants in the PHOSP-COVID study attended an early timepoint research visit a median of 5 months (IQR 4-6) following discharge from 83 hospitals in the UK. Data for sleep quality were assessed by subjective measures (the Pittsburgh Sleep Quality Index questionnaire and the numerical rating scale) for 638 participants at the early time point. Sleep quality was also assessed using device-based measures (actigraphy) a median of 7 months (IQR 5-8 months) after discharge from hospital for 729 participants. After discharge from hospital, the majority (396 [62%] of 638) of participants who had been admitted to hospital for COVID-19 reported poor sleep quality in response to the Pittsburgh Sleep Quality Index questionnaire. A comparable proportion (338 [53%] of 638) of participants felt their sleep quality had deteriorated following discharge after COVID-19 admission, as assessed by the numerical rating scale. Device-based measurements were compared to an age-matched, sex-matched, BMI-matched, and time from discharge-matched UK Biobank cohort who had recently been admitted to hospital. Compared to the recently hospitalised matched UK Biobank cohort, participants in our study slept on average 65 min (95% CI 59 to 71) longer, had a lower sleep regularity index (-19%; 95% CI -20 to -16), and a lower sleep efficiency (3·83 percentage points; 95% CI 3·40 to 4·26). Similar results were obtained when comparisons were made with the non-hospitalised UK Biobank cohort. Overall sleep quality (unadjusted effect estimate 3·94; 95% CI 2·78 to 5·10), deterioration in sleep quality following hospital admission (3·00; 1·82 to 4·28), and sleep regularity (4·38; 2·10 to 6·65) were associated with higher dyspnoea scores. Poor sleep quality, deterioration in sleep quality, and sleep regularity were also associated with impaired lung function, as assessed by forced vital capacity. Depending on the sleep metric, anxiety mediated 18-39% of the effect of sleep disturbance on dyspnoea, while muscle weakness mediated 27-41% of this effect. INTERPRETATION: Sleep disturbance following hospital admission for COVID-19 is associated with dyspnoea, anxiety, and muscle weakness. Due to the association with multiple symptoms, targeting sleep disturbance might be beneficial in treating the post-COVID-19 condition. FUNDING: UK Research and Innovation, National Institute for Health Research, and Engineering and Physical Sciences Research Council.

ClinCirc identifies alterations of the circadian peripheral oscillator in critical care patients.

BackgroundAssessing circadian rhythmicity from infrequently sampled data is challenging; however, these types of data are often encountered when measuring circadian transcripts in hospitalized patients.MethodsWe present ClinCirc. This method combines 2 existing mathematical methods (Lomb-Scargle periodogram and cosinor) sequentially and is designed to measure circadian oscillations from infrequently sampled clinical data. The accuracy of this method was compared against 9 other methods using simulated and frequently sampled biological data. ClinCirc was then evaluated in 13 intensive care unit (ICU) patients as well as in a separate cohort of 29 kidney-transplant recipients. Finally, the consequences of circadian alterations were investigated in a retrospective cohort of 726 kidney-transplant recipients.ResultsClinCirc had comparable performance to existing methods for analyzing simulated data or clock transcript expression of healthy volunteers. It had improved accuracy compared with the cosinor method in evaluating circadian parameters in PER2:luc cell lines. In ICU patients, it was the only method investigated to suggest that loss of circadian oscillations in the peripheral oscillator was associated with inflammation, a feature widely reported in animal models. Additionally, ClinCirc was able to detect other circadian alterations, including a phase shift following kidney transplantation that was associated with the administration of glucocorticoids. This phase shift could explain why a significant complication of kidney transplantation (delayed graft dysfunction) oscillates according to the time of day kidney transplantation is performed.ConclusionClinCirc analysis of the peripheral oscillator reveals important clinical associations in hospitalized patients.FundingUK Research and Innovation (UKRI), National Institute of Health Research (NIHR), Engineering and Physical Sciences Research Council (EPSRC), National Institute on Academic Anaesthesia (NIAA), Asthma+Lung UK, Kidneys for Life.

Adipocyte NR1D1 dictates adipose tissue expansion during obesity.

The circadian clock component NR1D1 (REVERBα) is considered a dominant regulator of lipid metabolism, with global Nr1d1 deletion driving dysregulation of white adipose tissue (WAT) lipogenesis and obesity. However, a similar phenotype is not observed under adipocyte-selective deletion (Nr1d1Flox2-6:AdipoqCre), and transcriptional profiling demonstrates that, under basal conditions, direct targets of NR1D1 regulation are limited, and include the circadian clock and collagen dynamics. Under high-fat diet (HFD) feeding, Nr1d1Flox2-6:AdipoqCre mice do manifest profound obesity, yet without the accompanying WAT inflammation and fibrosis exhibited by controls. Integration of the WAT NR1D1 cistrome with differential gene expression reveals broad control of metabolic processes by NR1D1 which is unmasked in the obese state. Adipocyte NR1D1 does not drive an anticipatory daily rhythm in WAT lipogenesis, but rather modulates WAT activity in response to alterations in metabolic state. Importantly, NR1D1 action in adipocytes is critical to the development of obesity-related WAT pathology and insulin resistance.

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.

Timekeeping in the hindbrain: a multi-oscillatory circadian centre in the mouse dorsal vagal complex.

Metabolic and cardiovascular processes controlled by the hindbrain exhibit 24 h rhythms, but the extent to which the hindbrain possesses endogenous circadian timekeeping is unresolved. Here we provide compelling evidence that genetic, neuronal, and vascular activities of the brainstem's dorsal vagal complex are subject to intrinsic circadian control with a crucial role for the connection between its components in regulating their rhythmic properties. Robust 24 h variation in clock gene expression in vivo and neuronal firing ex vivo were observed in the area postrema (AP) and nucleus of the solitary tract (NTS), together with enhanced nocturnal responsiveness to metabolic cues. Unexpectedly, we also find functional and molecular evidence for increased penetration of blood borne molecules into the NTS at night. Our findings reveal that the hindbrain houses a local network complex of neuronal and non-neuronal autonomous circadian oscillators, with clear implications for understanding local temporal control of physiology in the brainstem.

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.

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.

Targeting of the circadian clock via CK1δ/ε to improve glucose homeostasis in obesity.

Growing evidence indicates that disruption of our internal timing system contributes to the incidence and severity of metabolic diseases, including obesity and type 2 diabetes. This is perhaps not surprising since components of the circadian clockwork are tightly coupled to metabolic processes across the body. In the current study, we assessed the impact of obesity on the circadian system in mice at a behavioural and molecular level, and determined whether pharmacological targeting of casein kinase 1δ and ε (CK1δ/ε), key regulators of the circadian clock, can confer metabolic benefit. We demonstrate that although behavioural rhythmicity was maintained in diet-induced obesity (DIO), gene expression profiling revealed tissue-specific alteration to the phase and amplitude of the molecular clockwork. Clock function was most significantly attenuated in visceral white adipose tissue (WAT) of DIO mice, and was coincident with elevated tissue inflammation, and dysregulation of clock-coupled metabolic regulators PPARα/γ. Further, we show that daily administration of a CK1δ/ε inhibitor (PF-5006739) improved glucose tolerance in both DIO and genetic (ob/ob) models of obesity. These data further implicate circadian clock disruption in obesity and associated metabolic disturbance, and suggest that targeting of the clock represents a therapeutic avenue for the treatment of metabolic disorders.

Adiponectin induces A20 expression in adipose tissue to confer metabolic benefit.

Obesity is a major risk factor for metabolic disease, with white adipose tissue (WAT) inflammation emerging as a key underlying pathology. We detail that mice lacking Reverbα exhibit enhanced fat storage without the predicted increased WAT inflammation or loss of insulin sensitivity. In contrast to most animal models of obesity and obese human patients, Reverbα(-/-) mice exhibit elevated serum adiponectin levels and increased adiponectin secretion from WAT explants in vitro, highlighting a potential anti-inflammatory role of this adipokine in hypertrophic WAT. Indeed, adiponectin was found to suppress primary macrophage responses to lipopolysaccharide and proinflammatory fatty acids, and this suppression depended on glycogen synthase kinase 3ß activation and induction of A20. Attenuated inflammatory responses in Reverbα(-/-) WAT depots were associated with tonic elevation of A20 protein and ex vivo shown to depend on A20. We also demonstrate that adipose A20 expression in obese human subjects exhibits a negative correlation with measures of insulin sensitivity. Furthermore, bariatric surgery-induced weight loss was accompanied by enhanced WAT A20 expression, which is positively correlated with increased serum adiponectin and improved metabolic and inflammatory markers, including C-reactive protein. The findings identify A20 as a mediator of adiponectin anti-inflammatory action in WAT and a potential target for mitigating obesity-related pathology.

A novel mechanism controlling resetting speed of the circadian clock to environmental stimuli.

Many aspects of mammalian physiology are driven through the coordinated action of internal circadian clocks. Clock speed (period) and phase (temporal alignment) are fundamental to an organism's ability to synchronize with its environment. In humans, lifestyles that disturb these clocks, such as shift work, increase the incidence of diseases such as cancer and diabetes. Casein kinases 1δ and ε are closely related clock components implicated in period determination. However, CK1δ is so dominant in this regard that it remains unclear what function CK1ε normally serves. Here, we reveal that CK1ε dictates how rapidly the clock is reset by environmental stimuli. Genetic disruption of CK1ε in mice enhances phase resetting of behavioral rhythms to acute light pulses and shifts in light cycle. This impact of CK1ε targeting is recapitulated in isolated brain suprachiasmatic nucleus and peripheral (lung) clocks during NMDA- or temperature-induced phase shift in association with altered PERIOD (PER) protein dynamics. Importantly, accelerated re-entrainment of the circadian system in vivo and in vitro can be achieved in wild-type animals through pharmacological inhibition of CK1ε. These studies therefore reveal a role for CK1ε in stabilizing the circadian clock against phase shift and highlight it as a novel target for minimizing physiological disturbance in shift workers.