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.

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.

Parasites-The importance of time.

The special edition of Parasite Immunology 'Parasites-The importance of time' embraces the intersection between three established research disciplines-parasitology, immunology, and circadian biology. Each of these research areas has a longstanding history littered with landmark discoveries with the intersect between the three bringing exciting findings and new questions and perhaps even a greater sense of awe in terms of how parasites have evolved to interact and live with their hosts.

Circadian rhythms in immunity and host-parasite interactions.

The mammalian immune system adheres to a 24 h circadian schedule, exhibiting daily rhythmic patterns in homeostatic immune processes, such as immune cell trafficking, as well as the inflammatory response to infection. These diurnal rhythms are driven by endogenous molecular clocks within immune cells which are hierarchically coordinated by a light-entrained central clock in the suprachiasmatic nucleus of the hypothalamus and responsive to local rhythmic cues including temperature, hormones and feeding time. Circadian control of immunity may enable animals to anticipate daily pathogenic threat from parasites and gate the magnitude of the immune response, potentially enhancing fitness. However, parasites also strive for optimum fitness and some may have co-evolved to benefit from host circadian timing mechanisms, possibly via the parasites' own intrinsic molecular clocks. In this review, we summarize the current knowledge surrounding the influence of the circadian clock on the mammalian immune system and the host-parasitic interaction. We also discuss the potential for chronotherapeutic strategies in the treatment of parasitic diseases.

Circadian rhythms in adaptive immunity.

The circadian clock provides organisms with the ability to track time of day, allowing them to predict and respond to cyclical changes in the external environment. In mammals this clock consists of multiple auto-regulatory feedback loops generated by a network of circadian clock proteins. This network provides the fundamental basis for rhythms in behaviour and physiology. This clockwork machinery exists in most cells, including those of the immune system. In recent years evidence has emerged highlighting the important role of molecular clocks in dictating the response of immune pathways. While initial work highlighted the effect of the clock in the 'first line of defence', the innate immune system, it has become increasingly apparent that it also plays a role in the more tailored, later-stage adaptive immune response. This review provides an overview of the role of the circadian cycle in the adaptive immune response. We interrogate the depth of knowledge on cell intrinsic clocks within adaptive immune cells and how these cells may be temporally directed by extrinsic rhythmic signals. We discuss the role of the circadian clock in diseases associated with adaptive immunity such as multiple sclerosis, asthma and parasitic infection. We also discuss the current knowledge on timing of vaccination, and the implications this may have on how we can harness and modulate temporal gating of the adaptive immune response in a clinical setting.

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.

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.

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.

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.

Fibroblast-like synoviocytes orchestrate daily rhythmic inflammation in arthritis.

Rheumatoid arthritis is a chronic inflammatory disease that shows characteristic diurnal variation in symptom severity, where joint resident fibroblast-like synoviocytes (FLS) act as important mediators of arthritis pathology. We investigate the role of FLS circadian clock function in directing rhythmic joint inflammation in a murine model of inflammatory arthritis. We demonstrate FLS time-of-day-dependent gene expression is attenuated in arthritic joints, except for a subset of disease-modifying genes. The deletion of essential clock gene Bmal1 in FLS reduced susceptibility to collagen-induced arthritis but did not impact symptomatic severity in affected mice. Notably, FLS Bmal1 deletion resulted in loss of diurnal expression of disease-modulating genes across the joint, and elevated production of MMP3, a prognostic marker of joint damage in inflammatory arthritis. This work identifies the FLS circadian clock as an influential driver of daily oscillations in joint inflammation, and a potential regulator of destructive pathology in chronic inflammatory arthritis.

Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 (CK1) enzymes.

Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) epsilon or delta can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1epsilon and CK1delta using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1delta is the principal regulator of the clock period: pharmacological inhibition of CK1delta, but not CK1epsilon, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1delta inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1delta inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from Vipr2(-/-) mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the Vipr2(-/-) mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1delta offers an avenue for therapeutic modulation of perturbed circadian behavior.

Chronotype in Patients With Immune-Mediated Inflammatory Disease: A Systematic Review.

Immune-mediated inflammatory diseases (IMIDs) such as rheumatoid arthritis, inflammatory bowel disease, and asthma share common pathophysiological pathways characterized by chronic inflammation and subsequent tissue damage involving multiple body sites. Circadian rhythms are 24-h body cycles that regulate immune activity and control the magnitude of immune response based on time of day. Chronotype is a person's individual circadian phase preference, ranging from morningness to eveningness, which is known to influence the risk of cardiometabolic and mental health disease. We systematically reviewed the literature to assess the association of questionnaire-based chronotype and patients with IMID. A comprehensive search of MEDLINE and Embase identified 12 studies meeting the inclusion criteria, conducted in 7 countries and covering 4 IMIDs to include 15,625 IMID patients and 410,783 healthy controls. Results showed that later chronotype may be a risk factor for worse quality of life and increased symptom burden in patients with IMIDs. In addition, chronotype may be a risk factor for IMID incidence, but the direction and magnitude of this effect were not consistent across individual IMIDs. Chronotype assessment could contribute to risk stratification in patients with IMIDs. Cross-disciplinary collaboration to understand the role of circadian rhythms and chronotype in driving common inflammatory pathways could help to improve outcomes for patients with IMIDs.

Adaptive immunity, chronic inflammation and the clock.

The adaptive arm of the immune system facilitates recognition of specific foreign pathogens and, via the action of T and B lymphocytes, induces a fine-tuned response to target the pathogen and develop immunological memory. The functionality of the adaptive immune system exhibits daily 24-h variation both in homeostatic processes (such as lymphocyte trafficking and development of T lymphocyte subsets) and in responses to challenge. Here, we discuss how the circadian clock exerts influence over the function of the adaptive immune system, considering the roles of cell intrinsic clockwork machinery and cell extrinsic rhythmic signals. Inappropriate or misguided actions of the adaptive immune system can lead to development of autoimmune diseases such as rheumatoid arthritis, ulcerative colitis and multiple sclerosis. Growing evidence indicates that disturbance of the circadian clock has negative impact on development and progression of these chronic inflammatory diseases and we examine current understanding of clock-immune interactions in the setting of these inflammatory conditions. A greater appreciation of circadian control of adaptive immunity will facilitate further understanding of mechanisms driving daily variation in disease states and drive improvements in the diagnosis and treatment of chronic inflammatory diseases.

Rhythmicity of intestinal IgA responses confers oscillatory commensal microbiota mutualism.

Interactions between the mammalian host and commensal microbiota are enforced through a range of immune responses that confer metabolic benefits and promote tissue health and homeostasis. Immunoglobulin A (IgA) responses directly determine the composition of commensal species that colonize the intestinal tract but require substantial metabolic resources to fuel antibody production by tissue-resident plasma cells. Here, we demonstrate that IgA responses are subject to diurnal regulation over the course of a circadian day. Specifically, the magnitude of IgA secretion, as well as the transcriptome of intestinal IgA+ plasma cells, was found to exhibit rhythmicity. Oscillatory IgA responses were found to be entrained by time of feeding and were also found to be in part coordinated by the plasma cell-intrinsic circadian clock via deletion of the master clock gene Arntl. Moreover, reciprocal interactions between the host and microbiota dictated oscillatory dynamics among the commensal microbial community and its associated transcriptional and metabolic activity in an IgA-dependent manner. Together, our findings suggest that circadian networks comprising intestinal IgA, diet, and the microbiota converge to align circadian biology in the intestinal tract and to ensure host-microbial mutualism.

Circadian Host-Microbiome Interactions in Immunity.

The gut microbiome plays a critical role in regulating host immunity and can no longer be regarded as a bystander in human health and disease. In recent years, circadian (24 h) oscillations have been identified in the composition of the microbiota, its biophysical localization within the intestinal tract and its metabolic outputs. The gut microbiome and its key metabolic outputs, such as short chain fatty acids and tryptophan metabolites contribute to maintenance of intestinal immunity by promoting barrier function, regulating the host mucosal immune system and maintaining the function of gut-associated immune cell populations. Loss of rhythmic host-microbiome interactions disrupts host immunity and increases risk of inflammation and metabolic complications. Here we review factors that drive circadian variation in the microbiome, including meal timing, dietary composition and host circadian clocks. We also consider how host-microbiome interactions impact the core molecular clock and its rhythmic outputs in addition to the potential impact of this relationship on circadian control of immunity.

The circadian regulator Bmal1 in joint mesenchymal cells regulates both joint development and inflammatory arthritis.

BACKGROUND: The circadian clock plays a crucial role in regulating physiology and is important for maintaining immune homeostasis and responses to inflammatory stimuli. Inflammatory arthritis often shows diurnal variation in disease symptoms and disease markers, and it is now established that cellular clocks regulate joint inflammation. The clock gene Bmal1 is critical for maintenance of 24-h rhythms and plays a key role in regulating immune responses, as well as in aging-related processes. Fibroblast-like synoviocytes (FLS) are circadian rhythmic joint mesenchymal cells which are important for maintenance of joint health and play a crucial role in the development of inflammatory arthritis. The aim of this study was to investigate the importance of the joint mesenchymal cell circadian clock in health and disease. METHODS: Mice were generated which lack Bmal1 in Col6a1-expressing cells, targeting mesenchymal cells in the ankle joints. Joints of these animals were assessed by X-ray imaging, whole-mount staining and histology, and the composition of the synovium was assessed by flow cytometry. Arthritis was induced using collagen antibodies. RESULTS: Bmal1 deletion in joint mesenchymal cells rendered the FLS and articular cartilage cells arrhythmic. Targeted mice exhibited significant changes in the architecture of the joints, including chondroid metaplasia (suggesting a switch of connective tissue stem cells towards a chondroid phenotype), reductions in resident synovial macrophages and changes in the basal pro-inflammatory activity of FLS. Loss of Bmal1 in FLS rendered these resident immune cells more pro-inflammatory in response to challenge, leading to increased paw swelling, localised infiltration of mononuclear cells and enhanced cytokine production in a model of arthritis. CONCLUSIONS: This study demonstrates the importance of Bmal1 in joint mesenchymal cells in regulating FLS and chondrocyte development. Additionally, we have identified a role for this core clock component for restraining local responses to inflammation and highlight a role for the circadian clock in regulating inflammatory arthritis.

The circadian regulator BMAL1 programmes responses to parasitic worm infection via a dendritic cell clock.

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.