The circadian system is as an important regulator of immune function. Human inflammatory lung diseases frequently show time-of-day variation in symptom severity and lung function, but the mechanisms and cell types that are underlying these effects remain unclear. We show that pulmonary antibacterial responses are modulated by a circadian clock within epithelial club (Clara) cells. These drive circadian neutrophil recruitment to the lung via the chemokine CXCL5. Genetic ablation of the clock gene Bmal1 (also called Arntl or MOP3) in bronchiolar cells disrupts rhythmic Cxcl5 expression, resulting in exaggerated inflammatory responses to lipopolysaccharide and bacterial infection. Adrenalectomy blocks rhythmic inflammatory responses and the circadian regulation of CXCL5, suggesting a key role for the adrenal axis in driving CXCL5 expression and pulmonary neutrophil recruitment. Glucocorticoid receptor occupancy at the Cxcl5 locus shows circadian oscillations, but this is disrupted in mice with bronchiole-specific ablation of Bmal1, leading to enhanced CXCL5 expression despite normal corticosteroid secretion. In clock-gene disrupted mice the synthetic glucocorticoid dexamethasone loses anti-inflammatory efficacy. We now define a regulatory mechanism that links the circadian clock and glucocorticoid hormones to control both time-of-day variation and also the magnitude of pulmonary inflammation and responses to bacterial infection.

Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.

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 glucocorticoid receptor (GR) is a major drug target in inflammatory disease. However, chronic glucocorticoid (GC) treatment leads to disordered energy metabolism, including increased weight gain, adiposity, and hepatosteatosis — all programs modulated by the circadian clock. We demonstrated that while antiinflammatory GC actions were maintained irrespective of dosing time, the liver was significantly more GC sensitive during the day. Temporal segregation of GC action was underpinned by a physical interaction of GR with the circadian transcription factor REVERBa and co-binding with liver-specific hepatocyte nuclear transcription factors (HNFs) on chromatin. REVERBa promoted efficient GR recruitment to chromatin during the day, acting in part by maintaining histone acetylation, with REVERBa-dependent GC responses providing segregation of carbohydrate and lipid metabolism. Importantly, deletion of Reverba inverted circadian liver GC sensitivity and protected mice from hepatosteatosis induced by chronic GC administration. Our results reveal a mechanism by which the circadian clock acts through REVERBa in liver on elements bound by HNF4A/HNF6 to direct GR action on energy metabolism.

Resistance to the intestinal parasitic helminth Trichuris muris requires T-helper 2 (TH2) cellular and associated IgG1 responses, with expulsion typically taking up to 4 weeks in mice. Here, we show that the time-of-day of the initial infection affects efficiency of worm expulsion, with strong TH2 bias and early expulsion in morning-infected mice. Conversely, mice infected at the start of the night show delayed resistance to infection, and this is associated with feeding-driven metabolic cues, such that feeding restriction to the day-time in normally nocturnal-feeding mice disrupts parasitic expulsion kinetics. We deleted the circadian regulator BMAL1 in antigen-presenting dendritic cells (DCs) in vivo and found a loss of time-of-day dependency of helminth expulsion. RNAseq analyses revealed that IL-12 responses to worm antigen by circadian-synchronised DCs were dependent on BMAL1. Therefore, we find that circadian machinery in DCs contributes to the TH1/TH2 balance, and that environmental, or genetic perturbation of the DC clock results in altered parasite expulsion kinetics.

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 integrinβ1 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 objective of this paper was to estimate the effect of the costs of mastitis on the profitability of Irish dairy farms as indicated by various ranges of bulk milk somatic cell count (BMSCC). Data were collected from 4 sources and included milk production losses, cases treated, and on-farm practices around mastitis management. The Moorepark Dairy Systems Model, which simulates dairying systems inside the farm gate, was used to carry out the analysis. The cost components of mastitis that affect farm profitability and that were included in the model were milk losses, culling, diagnostic testing, treatment, veterinary attention, discarded milk, and penalties. Farms were grouped by 5 BMSCC thresholds of ≤ 100,000, 100,001-200,000, 200,001-300,000, 300,001-400,000, and > 400,000 cells/mL. The ≤ 100,000 cells/mL threshold was taken as the baseline and the other 4 thresholds were compared relative to this baseline. For a 40-ha farm, the analysis found that as BMSCC increased, milk receipts decreased from €148,843 at a BMSCC <100,000 cells/mL to €138,573 at a BMSCC > 400,000 cells/mL. In addition, as BMSCC increased, livestock receipts increased by 17%, from €43,304 at a BMSCC <100,000 cells/mL to €50,519 at a BMSCC > 400,000 cells/mL. This reflected the higher replacement rates as BMSCC increased and the associated cull cow value. Total farm receipts decreased from €192,147 at the baseline (< 100,000 cells/mL) to €189,091 at a BMSCC > 400,000 cells/mL. Total farm costs increased as BMSCC increased, reflecting treatment, veterinary, diagnostic testing, and replacement heifer costs. At the baseline, total farm costs were €161,085, increasing to €177,343 at a BMSCC > 400,000 cells/mL. Net farm profit decreased as BMSCC increased, from €31,252/yr at the baseline to €11,748/yr at a BMSCC > 400,000 cells/mL. This analysis highlights the impact that mastitis has on the profitability of Irish dairy farms. The analysis presented here can be used to develop a "cost of mastitis" tool for use on Irish dairy farms to motivate farmers to acknowledge the scale of the problem, realize the value of improving mastitis control, and implement effective mastitis control practices.

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. FASEB J. 33, 6226–6238 (2019). http://www.fasebj.org