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.

Impact of Fluid Flow Shear Stress on Osteoblast Differentiation and Cross-Talk with Articular Chondrocytes.

Bone cells, in particular osteoblasts, are capable of communication with each other during bone growth and homeostasis. More recently it has become clear that they also communicate with other cell-types; including chondrocytes in articular cartilage. One way that this process is facilitated is by interstitial fluid movement within the pericellular and extracellular matrices. This stimulus is also an important mechanical signal in skeletal tissues, and is known to generate shear stresses at the micron-scale (known as fluid flow shear stresses (FFSS)). The primary aim of this study was to develop and characterize an in vitro bone-cartilage crosstalk system, to examine the effect of FFSS on these cell types. Specifically, we evaluated the response of osteoblasts and chondrocytes to FFSS and the effect of FFSS-induced soluble factors from the former, on the latter. This system will ultimately be used to help us understand the role of subchondral bone damage in articular cartilage degeneration. We also carried out a comparison of responses between cell lines and primary murine cells in this work. Our findings demonstrate that primary cells produce a more reliable and reproducible response to FFSS. Furthermore we found that at lower magnitudes , direct FFSS produces anabolic responses in both chondrocytes and osteoblasts, whereas higher levels produce more catabolic responses. Finally we show that exposure to osteoblast-derived factors in conditioned media experiments produced similarly catabolic changes in primary chondrocytes.

Circadian clock protein BMAL1 regulates IL-1ß in macrophages via NRF2.

A variety of innate immune responses and functions are dependent on time of day, and many inflammatory conditions are associated with dysfunctional molecular clocks within immune cells. However, the functional importance of these innate immune clocks has yet to be fully characterized. NRF2 plays a critical role in the innate immune system, limiting inflammation via reactive oxygen species (ROS) suppression and direct repression of the proinflammatory cytokines, IL-1ß and IL-6. Here we reveal that the core molecular clock protein, BMAL1, controls the mRNA expression of Nrf2 via direct E-box binding to its promoter to regulate its activity. Deletion of Bmal1 decreased the response of NRF2 to LPS challenge, resulting in a blunted antioxidant response and reduced synthesis of glutathione. ROS accumulation was increased in Bmal1-/- macrophages, facilitating accumulation of the hypoxic response protein, HIF-1α. Increased ROS and HIF-1α levels, as well as decreased activity of NRF2 in cells lacking BMAL1, resulted in increased production of the proinflammatory cytokine, IL-1ß. The excessive prooxidant and proinflammatory phenotype of Bmal1-/- macrophages was rescued by genetic and pharmacological activation of NRF2, or through addition of antioxidants. Our findings uncover a clear role for the molecular clock in regulating NRF2 in innate immune cells to control the inflammatory response. These findings provide insights into the pathology of inflammatory conditions, in which the molecular clock, oxidative stress, and IL-1ß are known to play a role.

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.

Immunometabolism: Is it under the eye of the clock?

Molecular clocks allow an organism to track time of day, providing the means to anticipate and respond to the daily changes within the environment. In mammals the molecular clock consists of a network of proteins that form auto-regulatory feedback loops that drive rhythms in physiology and behavior. In recent times the extent to which the molecular clock controls key metabolic and immune pathways has begun to emerge. For example, the main clock protein BMAL1 has been linked to mitochondrial metabolism, mitochondrial dynamics and various host defense pathways. The molecular clock may function to integrate daily metabolic changes driven by feeding-fasting to immune function and output. Understanding how the clock intersects with metabolic pathways within immune cells to affect immune phenotypes will have broad implications for the management of metabolic, inflammatory and infectious diseases.

Circadian control of innate immunity in macrophages by miR-155 targeting Bmal1.

The response to an innate immune challenge is conditioned by the time of day, but the molecular basis for this remains unclear. In myeloid cells, there is a temporal regulation to induction by lipopolysaccharide (LPS) of the proinflammatory microRNA miR-155 that correlates inversely with levels of BMAL1. BMAL1 in the myeloid lineage inhibits activation of NF-κB and miR-155 induction and protects mice from LPS-induced sepsis. Bmal1 has two miR-155-binding sites in its 3'-UTR, and, in response to LPS, miR-155 binds to these two target sites, leading to suppression of Bmal1 mRNA and protein in mice and humans. miR-155 deletion perturbs circadian function, gives rise to a shorter circadian day, and ablates the circadian effect on cytokine responses to LPS. Thus, the molecular clock controls miR-155 induction that can repress BMAL1 directly. This leads to an innate immune response that is variably responsive to challenges across the circadian day.

The circadian protein BMAL1 in myeloid cells is a negative regulator of allergic asthma.

Our body clock drives rhythms in the expression of genes that have a 24-h periodicity. The transcription factor BMAL1 is a crucial component of the molecular clock. A number of physiological processes, including immune function, are modulated by the circadian clock. Asthma, a disease with very strong clinical evidence demonstrating regulation by circadian variation, is of particular relevance to circadian control of immunity. Airway hypersensitivity and asthma attacks are more common at night in humans. The molecular basis for this is unknown, and there is no model of asthma in animals with genetic distortion of the molecular clock. We used mice lacking BMAL1 in myeloid cells (BMAL1-LysM-/-) to determine the role of BMAL1 in allergic asthma. Using the ovalbumin model of allergic asthma, we demonstrated markedly increased asthma features, such as increased lung inflammation, demonstrated by drastically higher numbers of eosinophils and increased IL-5 levels in the lung and serum, in BMAL1-LysM-/- mice. In vitro studies demonstrated increased proinflammatory chemokine and mannose receptor expression in IL-4- as well as LPS-treated macrophages from BMAL1-LysM-/- mice compared with wild-type controls. This suggests that Bmal1 is a potent negative regulator in myeloid cells in the context of allergic asthma. Our findings might explain the increase in asthma incidents during the night, when BMAL1 expression is low.

The circadian clock influences T cell responses to vaccination by regulating dendritic cell antigen processing.

Dendritic cells play a key role in processing and presenting antigens to naïve T cells to prime adaptive immunity. Circadian rhythms are known to regulate many aspects of immunity; however, the role of circadian rhythms in dendritic cell function is still unclear. Here, we show greater T cell responses when mice are immunised in the middle of their rest versus their active phase. We find a circadian rhythm in antigen processing that correlates with rhythms in both mitochondrial morphology and metabolism, dependent on the molecular clock gene, Bmal1. Using Mdivi-1, a compound that promotes mitochondrial fusion, we are able to rescue the circadian deficit in antigen processing and mechanistically link mitochondrial morphology and antigen processing. Furthermore, we find that circadian changes in mitochondrial Ca2+ are central to the circadian regulation of antigen processing. Our results indicate that rhythmic changes in mitochondrial calcium, which are associated with changes in mitochondrial morphology, regulate antigen processing.

Innate Rhythms: Clocks at the Center of Monocyte and Macrophage Function.

The circadian cycle allows organisms to track external time of day and predict/respond to changes in the external environment. In higher order organisms, circadian rhythmicity is a central feature of innate and adaptive immunity. We focus on the role of the molecular clock and circadian rhythmicity specifically in monocytes and macrophages of the innate immune system. These cells display rhythmicity in their internal functions, such as metabolism and inflammatory mediator production as well as their external functions in pathogen sensing, phagocytosis, and migration. These inflammatory mediators are of clinical interest as many are therapeutic targets in inflammatory disease such as cardiovascular disease, diabetes, and rheumatoid arthritis. Moreover, circadian rhythm disruption is closely linked with increased prevalence of these conditions. Therefore, understanding the mechanisms by which circadian disruption affects monocyte/macrophage function will provide insights into novel therapeutic opportunities for these chronic inflammatory diseases.

Loss of the molecular clock in myeloid cells exacerbates T cell-mediated CNS autoimmune disease.

The transcription factor BMAL1 is a core component of the molecular clock, regulating biological pathways that drive 24 h (circadian) rhythms in behaviour and physiology. The molecular clock has a profound influence on innate immune function, and circadian disruption is linked with increased incidence of multiple sclerosis (MS). However, the mechanisms underlying this association are unknown. Here we show that BMAL1 and time-of-day regulate the accumulation and activation of various immune cells in a CNS autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). In myeloid cells, BMAL1 maintains anti-inflammatory responses and reduces T cell polarization. Loss of myeloid BMAL1 or midday immunizations to induce EAE create an inflammatory environment in the CNS through expansion and infiltration of IL-1ß-secreting CD11b+Ly6Chi monocytes, resulting in increased pathogenic IL-17+/IFN-γ+ T cells. These findings demonstrate the importance of the molecular clock in modulating innate and adaptive immune crosstalk under autoimmune conditions.