Detecting and exploiting the circadian clock in rheumatoid arthritis

Over the past four decades, research on 24‐h rhythms has yielded numerous remarkable findings, revealing their genetic, molecular, and physiological significance for immunity and various diseases. Thus, circadian rhythms are of fundamental importance to mammals, as their disruption and misalignment have been associated with many diseases and the abnormal functioning of many physiological processes. In this article, we provide a brief overview of the molecular regulation of 24‐h rhythms, their importance for immunity, the deleterious effects of misalignment, the link between such pathological rhythms and rheumatoid arthritis (RA), and the potential exploitation of chronobiological rhythms for the chronotherapy of inflammatory autoimmune diseases, using RA as an example.

cellular clocks through nerve connections, humoral signals, changes in core body temperature, and sleepwake behavior. 1,2The core cellular circadian pacemaker in mammals oscillates with a period of approximately 24 h.4][5][6][7][8][9] These molecules are tightly regulated by different kinases. 10,11Notably, adenosine monophosphate-dependent protein kinase, which increases its activity in response to cellular ATP deprivation, not only controls cellular metabolic functions but also has been implicated in the degradational process of PER and CRY proteins-linking metabolism to the molecular clock. 12,13The degradation of PER and CRY terminates the negative feedback and restarts the cycle of the circadian network.5][16] Other regulators within this circuitry include the transcription factors albumin D site-binding protein (DBP) and E4 promoter-binding protein 4 (E4BP4 or NFIL3). 17he core regulatory components of the molecular clock, such as BMAL1, CLOCK, PER, CRY, NR1D1, and ROR, keep the cellular clock ticking.Furthermore, they are also involved in regulating the physiological outputs of the circadian clock, including the sleep-wake cycle, heart rate and blood pressure, body temperature and, crucially important, hormone secretion, and immune responses. 18,191][22] Briefly, E4BP4 suppresses Th17 cell development by directly binding and repressing the Rorγt promoter, thus inhibiting the expression of RORγt, the lineage-specific transcription factor for Th17 cells. 20In addition, REV-ERBα binds to ROR response elements, antagonizes RORγt function in Th17 cells and The dynamic synchronization of the circadian clock in mammals and the molecular machinery of the central and peripheral clock.The central clock in the suprachiasmatic nucleus (SCN) acts as a master synchronizer to orchestrate the rhythms of ubiquitous peripheral clocks, thereby ensuring coherent and functional systemic rhythms.The central clock processes photic and non-photic cues and relays them to the other parts of the brain and peripheral clocks via neuronal and humoral signals, core body temperature, and behavior to synchronize their rhythms.The clockwork machinery in almost every cell is conceptualized as an autoregulatory, time-delayed transcription-translation feedback loop.The core loop consists of the BMAL1:CLOCK heterodimer that binds to the e-box element of clockcontrolled genes (CCGs), including PER and CRY genes, to induce their transcription.PER and CRY build heterodimers in the cytoplasm and translocate back into the nucleus to inhibit their transcription by interaction with BMAL1:CLOCK.In the secondary loop, REV-ERB and ROR proteins compete to bind RORE to modulate the expression of CCGs, including BMAL1, which regulates the expression of REV-ERBs together with CLOCK.DBP, whose expression is controlled by BMAL1:CLOCK, can induce ROR proteins and also the expression of PER and REV-ERBA through the D-Box element (not shown).In addition, the clock genes modulate the expression of CCGs in regulating various physiological processes.Created with BioRe nder.com.
inhibits the expression of RORγt-dependent genes, including Il17a and Il17f. 21,22Deletion of REV-ERBα enhanced Th17-mediated pro-inflammatory cytokine expression, exacerbating experimental autoimmune encephalomyelitis (EAE), and colitis. 21Moreover, treatment with a synthetic REV-ERB agonist suppressed Th17 cell development, was effective in colitis intervention studies, and significantly delayed the onset and impeded the progression of EAE. 21,22he circadian clock influences immune responses under homeostatic conditions and during inflammation. 23everal innate immune cells, including monocytes, 24 macrophages, [25][26][27] mast cells, 28,29 neutrophils, 30 eosinophils, 28 and natural killer (NK) cells 31 and also cells of the adaptive immune system, such as T cells [32][33][34] and B cells, 26 possess intrinsic clocks and exhibit daily rhythms in activity and function.In addition, professional antigen-presenting dendritic cells (DCs), bridging innate and adaptive immunity, show oscillations in core clock components. 26A consequence of the existence of intrinsic clocks in almost all immune cells is the temporal control of immune responses regarding their magnitude and characteristics as a function of circadian rhythmicity (as reviewed in Scheiermann et al. 23 ).Disruption or disarrangement of the circadian clock, whether by genetically targeting the central clock components or by imposing phase shifts in the light-dark cycle, has significant deleterious effects on the immune response, often leading to exacerbating inflammation and has been associated with the development of autoimmunity. 23owever, mechanisms that condition the association between circadian misalignment and disease are unclear.Furthermore, the understanding of rhythmic variations in clinical manifestations of inflammatory diseases is scarce.
The following section provides an overview of current knowledge about the effects of circadian disruption on human health, including the association with RA as an example.

RHYTHMS
There is evidence that circadian misalignment due to artificial light at night, shift work, night-time eating, sleep disorders, and jet lag is common in modern life and contributes to many human diseases (Figure 2).Along this F I G U R E 2 Circadian misalignment due to artificial light, night-time-eating, social jet lag, sleep disorders, shift work, and jet lag is common in modern life and contributes to many human diseases affecting all organs.Created with BioRe nder.com.6][37][38][39][40][41][42][43] Conflicting photic information received by the SCN with non-photic information, such as food intake or exercise can be hypothesized as a major reason for the desynchronization or mismatch of circadian rhythms.Shift workers are regularly exposed to artificial light at night, which leads to circadian misalignment of various biological rhythms, including melatonin and cortisol. 44Melatonin secretion is regulated by light and peaks during the rest phase in humans but during the active phase in mice. 45,46Melatonin can have contradictory effects on the immune system (as reviewed by Carrillo-Vico et al. 46 ).It can act as an immunostimulant proinflammatory substance, for example, by promoting the stimulation of NK cell activity and chemotaxis of neutrophils from healthy subjects but also has antiinflammatory effects by neutralizing the exacerbation of proinflammatory cytokines, in various in vivo models of inflammation as reviewed by Carrillo-Vico et al. 46 Therefore, a disruption of melatonin rhythms may affect the immune system's normal function.Even though our bodies are designed to adapt to environmental changes, a metaanalysis of 6-sulphatoxymelatonin (excreted metabolite of melatonin) rhythms showed that only a tiny percentage (3%) of long-term night workers could completely adjust their biological rhythms to night work schedules (as shown by the low levels of melatonin during the course of the night shift), while a larger number of participants showed only partial adjustment. 47ecently, Aslam et al. reported that shift work also affects expression of molecular components of the clock.Circadian clock genes CLOCK, PER1, PER3, and REV-ERBα expression in blood was higher in shift workers than in regular workers, while BMAL1 and CRY1 were lower. 48Expression of BMAL1 and PER1 was found in antiphase to each other in peripheral blood mononuclear leukocytes (PBMCs) of day workers, while BMAL1 expression was found arrhythmic in night workers. 491][52][53][54][55] BMAL1 was reported as a key metabolic sensor in macrophages, and its deficiency leads to a heightened proinflammatory state. 50BMAL1 controls the immune response by regulating the epigenetic states of macrophages. 51Mice lacking the Per1 gene are shown to be more susceptible to lipopolysaccharide (LPS)/ d-galactosamine-induced macrophage-dependent acute live failure compared with wild-type mice. 54Deletion of Per1/2 in murine myeloid cells exacerbates diet-induced inflammation and insulin resistance. 55In contrast, PER1 was upregulated in peripheral blood from 12 patients with multiple sclerosis compared with 15 unaffected controls. 56ollectively, the current data indicate a possible role of misaligned and disturbed circadian rhythm in clock genes such as BMAL1 (expressed low and misaligned) and PER1/2 (expressed high but misaligned), in the establishment and perpetuation of inflammation in shift workers.The altered circadian rhythms may increase the risk for the development of chronic inflammatory diseases such as RA. 43ince the human central circadian pacemaker can shift by only about 1 h per day, rapid air travel across multiple time zones results in a misalignment between the destination environment and the internal clock, also known as jet lag. 57Persistent misalignment of the clock, for example, due to a prolonged change in the light-dark cycle, is closely associated with the development of severe chronic inflammation.In mice, ongoing experimental jet lag altered the amplitude and period of Per2 in a tissue-specific manner.It exacerbated LPS-induced inflammation leading to persistent hypothermia, increased mortality, and severe chronic inflammatory symptoms, including elevated expression of pro-inflammatory cytokines, particularly IL-1β, IL-12, IL-13, and granulocyte-macrophage colonystimulating factor (GM-CSF). 58Minami et al. also reported the deleterious effects of chronic jet lag, leading to splenomegaly and an increased number of myeloid cells in the bone marrow, culminating in early death. 59Of note, these deleterious effects were observed during a lightdark cycle with a chronic phase advance (8-h advance per every 4 days) which induced un-entrainable circadian behavioral rhythm.In contrast, a light-dark cycle with chronic phase delay (8-h delay per every 7 days) showed a behavioral pattern that was entrained and synchronized within 7 days.
Furthermore, under a chronic jet lag condition, NK cell rhythms were disrupted, and the cytotoxicity activity was substantially diminished. 60Consequently, tumor incidence and frequency upon injection with MADB106 tumor cells were significantly increased.In addition, chronic jet lag promotes NK cell aging. 61he effects of circadian disruption and sleep deprivation on the immune system are similar.Thus, it is hard to disentangle them from each other.Sleep disorder or inconsistent sleep time between work days and days off, termed social jet lag, can be further exacerbated by exposure to phase-delaying blue light in the evening from electronic devices or another artificial lighting. 57However, sleep loss might disturb rhythms in hormones and immune parameters, thus contributing to the deleterious health consequences of circadian disruption, which is comprehensively reviewed elsewhere. 62,63Collectively, the available data presented here confirm the importance of a well-timed circadian clock for maintaining immunity and health.Disrupted or disturbed circadian rhythms by chronic jet lag, sleep deprivation, and shift work are closely related to the development of inflammatory diseases such as rheumatoid arthritis (RA).

| Rheumatoid arthritis: When lifestyle changes and time matters
Rheumatoid arthritis is one of the most common systemic autoimmune diseases, and the most common progressive and chronic inflammatory joint disease first recognized more than 20 centuries ago. 64,65The average prevalence of RA is 0.5%-1.0% in the population worldwide, displaying ethnic and demographic differences. 64lthough a progressive decline in the incidence of RA was detected from 1955 to 1995, suggesting that an environmental factor or genetic drift/a population change may play a role in the etiology of RA, the survival rate in RA patients was significantly lower than the expected rate in the general population with no improvement over time. 66,67In recent decades, the lifestyle of our modern society has undergone major changes, which to a certain extent has led to an increase in comfort, but also contributes to circadian disruption, which leads to many human diseases due to artificial light, shift work, night meals, and jet lag.A more recent systematic analysis of the Global Burden of Disease Study 2017 modeling the burden of RA for 195 countries from 1990 to 2017 now reported that the age-standardized prevalence and incidence rates are increasing. 68A pathogenesis is characterized by chronic inflammation of the joints due to immune cell infiltration into the synovial membrane and the joint cavity and the formation of hyperplastic and invasive synovium.Both results in progressive cartilage destruction and subchondral bone erosion in the late stages of the disease if not treated.Along with the joints, RA can affect many of the body's organs, including the heart, eyes, skin, intestine, kidney, lungs, brain, and skeleton. 69,70Affected patients experience a significant loss of quality of life.The cost and effort of health care are substantial, resulting in a considerable economic and social burden on society. 71RA is a multifactorial disease associated with numerous environmental and genetic factors, including lifestyle, sleep loss, shift work, age, obesity, smoking, sex, and major histocompatibility complex regions encoding human leukocyte antigen (HLA) proteins that are suggested to be involved in the initiation of the disease. 57,62,63,69,72Of note, patients with RA show an earlier circadian rhythm (i.e., serum melatonin peaks earlier during the night, indicating an earlier timing of the internal circadian pacemaker) and an earlier chronotype. 73Autoantibodies against modified self-proteins can be detected years before the first clinical symptoms of RA appear, enabling an early determination of the onset of the disease. 69,70

| The pathogenesis of RA: Circadian disruption and chronic inflammation
The pathogenesis of RA involves several different stages.As a prerequisite, a certain level of RA susceptibility and risk factors are part of the preclinical phase of RA, leading to synovial inflammation, which, if not resolved, ultimately leads to the development of RA. 64,69,72 Notably, the manifestation of the RA symptoms, such as pain, joint swelling, and stiffness, follows a clear diurnal pattern, with the highest severity observed, in most cases, in the early morning (Figure 3). 74-773.2.1 | The breach of tolerance alters the spatial and temporal arrangement of cells in the inflamed joint In the preclinical stage, numerous nuclear proteins (e.g., histones) and cellular proteins (e.g., collagen) are posttranslationally modified, including the process of protein citrullination, in which the amino acid arginine is converted into citrulline. 69,70Citrullination can be triggered by smoking, the microbiota, and an exaggerated presence of neutrophils expressing peptidylarginine deiminases. 64,69,72These events are suggested to contribute to the breach of tolerance because altered, modified selfantigens are generated and engage antigen-presenting cells of the synovial lining, including synovial tissue macrophages and DCs. 78,79The tissue-resident macrophages of the synovial lining usually form a protective barrier that maintains lining layer integrity and limits the development of sterile and pathogen-induced inflammation by phagocytic and immunoregulatory activity.They inhibit effector T-cell activation while facilitating the expansion of regulatory T cells (Tregs) by promoting their lipid metabolism. 78,79Tregs, well known to mediate peripheral tolerance, displayed diurnal variation with an acrophase observed during the active phase in the inflamed joints in collagen-induced arthritis (CIA), an animal model of RA, coinciding with the decreased inflammation. 80In the preclinical phase of arthritis, the altered modified selfproteins are regularly recognized as foreign and induce a "normal" immune response via the help of T cells which are engaged preferably in the efferent lymph nodes, get expanded and stimulate B cells to produce a wide range of (auto)antibodies recognizing self-proteins such as rheumatoid factor and anti-citrullinated protein antibodies.
In developing arthritis, macrophages in the lining layer appear reduced 81 and are replaced by M1-like macrophages from the peripheral blood 78,79 not supporting Treg expansion.These rapid changes in spatial and temporal cell composition supports the pathogenic behavior of immune cells. 82This includes inflammation-mediated disruption of the macrophage lining barrier that permits the invasion of activated fibroblasts and infiltrating immune cells (peripheral Th cell infiltration and differentiation into Tfh, Th1, and Th17 cells) and the expansion of pathogenic fibroblasts and macrophages in the sublining layer that results in a proinflammatory microenvironment and supports the infiltration and survival of inflammatory cells and the perpetuation of inflammation. 82,833.2.25][86][87][88][89][90][91][92][93] For instance, the response of TLR-4 expressing macrophages following stimulation with LPS shows diurnal variations. 27It is, therefore, not unlikely that a misaligned and disturbed circadian rhythm exacerbates the disturbance of tolerance in the development of arthritis and also affects the perpetuation of inflammation in chronic inflammatory autoimmunity such as RA.As a major sign circadian disruption, altered circulating leukocyte rhythms have been reported in RA patients. 94pecifically, compared to healthy controls, the rhythms of effector CD8 + T cells, effector CD4 + T cells, and IL6R+ CD8 + T cells were abolished in RA patients, whereas IL8R+ monocytes, CD20 + CD27 + memory B cells, and CD20 + HLA-DR+ activated B cells, that were not rhythmic in healthy individuals displayed rhythmic circulation in the peripheral blood of RA patients. 94mportantly, Clock, Bmal1, Rev-Erbα, and Cry are required for the rhythmic expression of macrophage-derived cytokines in mice. 25,95,96Indeed, deletion of some but not all of these clock genes in macrophages abrogates either the rhythmic expression of inflammatory cytokines or increases their basal expression, thereby amplifying inflammatory responses.Mice lacking Cry1 and Cry2 develop a more aggressive form of collagen antibody-induced arthritis, characterized by significantly higher serum levels of IL-1β, TNF, IL-6, and MMP-3. 97The absence of Cry genes induces the up-regulation of protein kinase A signaling, which leads to increased phosphorylation of p65, one of the five components of the nuclear factor kappa-lightchain-enhancer activated B cells (NF-κB) resulting in elevated TNF and IL-6 levels. 95NF-κB regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory responses. 98eregulated NF-κB activation contributes to the pathogenic processes of various inflammatory diseases including RA. Deregulated activation of NF-κB also contributes to aberrant survival of self-reactive B cells and production of auto-antibodies that contribute to the pathogenesis of RA. 99 Thus, Cry1 and Cry2 are indispensable for normal F I G U R E 3 Manifestation of the rheumatoid arthritis (RA) symptoms, such as pain, joint swelling, and stiffness, follows a clear diurnal pattern, with the highest severity observed, in most cases, in the early morning and lowest in the late afternoon.The numbers around the clock face indicate hours of the 24-h day.Melatonin secretion initiated/stops refer to detectable circulating levels.Created with BioRe nder.com.
B-cell development and function. 100Furthermore, Mice lacking Cry1 and Cry2 in the bone marrow only (after transplanting BM cells into lethally irradiated WT B6 or Rag1 KO mice) also spontaneously develop an autoimmune phenotype, showing elevated serum immunoglobulin G (IgG) concentrations, the presence of serum antinuclear antibodies, and the precipitation of IgG, IgM, and complement component C3 in their glomeruli similar to the double knock out mice. 100 In addition, synovial fibroblasts of RA patients show an impaired circadian clock gene expression, particularly a loss of BMAL1 and PER1 rhythmic expression, indicating a possible dampening of clocks at the site of inflammation. 101,102Deletion of Bmal1 in FLS rendered these resident joint cells more proinflammatory in the CIA model, leading to increased paw swelling, synovial immune cell infiltration, and enhanced cytokine production. 103Moreover, REV-ERBα is decreased in RA fibroblastlike synoviocytes (FLS) stimulated with IL-1β in vitro but increased in synovial tissues from patients with RA. 104 Increased REV-ERBα in the RA-FLS seems to be a sign of affected circadian regulation impacting inflammation, because REV-ERBα activation using the agonist SR9009 decreased the expression of proinflammatory cytokines and matrix metalloproteinases, reduced reactive oxygen species generation, blocked mitogen-activated protein kinase and NF-κB pathways, inhibited M1 macrophage polarization, and suppressed osteoclastogenesis and osteoclastrelated genes expression. 104Moreover, treatment with REV-ERBα agonist SR9009 significantly suppressed synovial hyperplasia, inflammatory cell infiltration and cartilage and bone destruction in CIA. 104These findings may indicate that a disturbed cellular clock associated with RA contributes to the severity of joint inflammation.

| RA symptoms vary depending on the time of day
The diurnal variation of clinical symptoms in RA is mainly attributed to the elevated levels of the critical proinflammatory cytokines, IL-6 and TNF, which mostly peak before the increased severity of the clinical signs. 105Cortisol is the most potent anti-inflammatory hormone in the body.6][107] Despite the potent anti-inflammatory property of cortisol, its rise in the early morning somehow fails to prevent the manifestation of RA symptoms.This is possibly due to the peak of the proinflammatory messenger IL-6 occurring earlier than the peak of cortisol. 105n CIA, joint swelling and inflammatory markers like serum cytokines IL-1β, IL-10, IFN-γ, and TNF exhibited a diurnal variation peaking during the rest period, and the rhythmic expression of circadian clock genes within the joint was disrupted. 108As mentioned before, Tregs peak within inflamed joints during the active phase, representing the NADIR of inflammation. 108Interestingly, naïve Tregs do not display an intrinsic clock.Therefore, the group concluded that the rhythmicity in joint swelling in CIA mice might be due to cell-extrinsic rhythms.One of these cell-external cues might be glucocorticoids (GCs), since dexamethasone administration induces the expression of the chemokine receptor CXCR4 in vivo on Tregs harvested from lymph nodes and spleen.CXCR4 conveys T-cell homing to the bone marrow, lymph nodes, and spleen in a circadian manner and in response to its ligand stromal-cell-derived factor-1α.As a result, endogenous GC rhythmicity might enhance the numbers of Tregs in joints, reducing joint inflammation during the active phase.
An increase in Th1-type responses at night was suggested many years ago by Petrovsky et al. 109 In the study, they measured LPS-stimulated production of IFN-γ, IL-10, plasma cortisol, and melatonin in humans and found that the IFN-y/IL-10 ratio is high at night.However, they did not distinguish between the contribution of T cells and monocytes to IL-10 production in their study.The study also mentioned the potential contribution of melatonin in regulating the diurnal rhythmicity of Th1/Th2 balance, since the high ratio of IFN-γ/IL-10 coincides with the peak of plasma melatonin.Indeed, melatonin was reported to enhance the production of some cytokines, including IFN-γ. 110Certainly, melatonin is significantly higher in patients with RA than in controls and more elevated in patients with RA from northern Europe than in those from further south. 111In addition, decreased sleep quality due to severe pain is prevalent in RA patients. 112,113leep deprivation disrupts the rhythmic functions of immune cells, increases inflammatory responses, impairs the proper function of immune cells, and exacerbates circadian disruption, thus potentially feeding into a vicious circle. 112,1133.2.4| Circadian disruption alters the associated metabolic programmes Besides melatonin, altered metabolic programs are associated with rhythmic joint inflammation. 114,115RA is associated with an increased incidence of diabetes and cardiovascular disease.Using transcriptome and proteome analyses on PBMCs and lipidomics on serum samples from patients with RA, Poolman et al. found an amplification of circadian rhythmicity, both in immune cells and system-wide, involving circadian coupling of ceramide synthesis and joint inflammation. 115In addition, transcriptional and phosphoproteomic analyses identified alterations in lipid metabolism and mitochondrial function associated with increased EGFR-JAK-STAT3 signaling. 114β-Oxidation and lipid handling were impaired, leading to a pronounced shunt towards sphingolipid and ceramide accumulation.Arthritis-related production of ceramides was most evident during the day, the time when inflammation was highest and fatty acid oxidation was most engaged.The data demonstrate a link between rhythmic gene expression within the inflamed joint and widespread changes to rhythmic transcriptional, phosphoproteomic, and metabolomic profiles in the liver and muscle, leading to system-wide damaging ceramide accumulation. 114s the course of RA may differ among individuals regarding symptoms, pathogenesis, and subtypes of the disease, the ultimate goal must be a personalized and targeted medicine at the right time to achieve successful remission.

| Re-establishing the circadian rhythms of immunity
A timed drug treatment, known as chronotherapy, was applied decades ago to improve outcomes of RA as detailed in the following.The first study implementing the concept of chronotherapy in RA was reported in 1964, involving 56 RA patients.In this double-blinded study, administering low-dose GC in RA patients at night proved more effective than in the morning. 116In 1976, a study reported that an evening dose of indomethacin was more effective in controlling the morning symptoms of inflammatory arthropathies than a morning dose. 117This result is supported by two other studies from the 1980s showing that the same regimen with either indomethacin or flurbiprofen improved the morning symptoms better than a morning or day dose in osteoarthritis and RA, respectively. 118,119One of the most convincing pieces of evidence for the successful application of chronotherapy was demonstrated through the administration of modifiedrelease prednisone (a GC) before bedtime (10 p.m.) in RA patients. 74,120IL-6 shows a rhythmic expression that peaks at night between 2 and 6 a.m. in RA. 75 Ideally, antiinflammatory drugs, such as GCs, should be taken before the rise of inflammatory molecules to maximize the suppression effect.However, it is inconvenient for the patients to wake up after midnight to take their medication.When taken at 10 p.m., the modified-release prednisone will release the active compound GC 4 h later at 2 a.m., just in time to interfere with the increase of IL-6 finally altering the IL-6 profile back to almost normal.Administration of modified-release prednisone was shown to be significantly more effective in reducing joint stiffness in the morning compared to the application of conventional GCs. 120n a subsequent study, the modified-release prednisone showed a superior effect compared to the placebo, indicated by increased treatment response rates and physical function and reduced morning stiffness, severity of RA, and fatigue. 121esides modified-release prednisone, chronotherapy revealed beneficial effects for disease-modifying antirheumatic drugs (DMARDs).An important DMARD in the treatment of RA is methotrexate (MTX).However, not all patients benefit from MTX due to inefficacy and adverse effects. 122,1235][126] In these mice, which develop an autoimmune disease resembling systemic lupus erythematosus (SLE), administration of MTX before the surge of TNF levels resulted in a significantly lower RA score compared to the control group. 125imilarly, timing MTX medication in synchronization with the 24-h rhythms of IL-6 were shown to improve the therapeutic index in CIA rats. 126CIA rats that received MTX before the increase of IL-6 showed lower RA scores and lower expression of plasma TNF, IL-6, and CRP in comparison to the group that received MTX when IL-6 level was the highest and the group that was treated traditionally with MTX (once a week same dose of MTX by gastric perfusion).In humans, the same study demonstrated the use of chronotherapy in 17 Japanese RA patients.In Japan, MTX is regularly administered three times a week.On the first day, MTX is administered after breakfast and supper, and on the second day, once after breakfast.Patients receiving treatment with this regimen were switched to a regimen in which MTX is administered once a day before bedtime in the same dose and maintained the same number of administrations.Throughout the 3 months of the study period, disease activity improved under chronotherapy.No severe adverse events were reported during the study period. 124nother DMARD approved for RA treatment is baricitinib, a janus kinase inhibitor.In CIA models, the administration of baricitinib at the beginning of the rest phase is more beneficial than administration at the beginning of the active phase.The mRNA expression of IL-6, IFNγ, TNF, and GM-CSF was significantly reduced compared to the control group that did not receive any treatment.This effect was not observed when baricitinib was administered during the active phase. 127xperimental drugs that have been used in mouse models of arthritis such as the REV-ERBα ligand SR9009 reducing CIA symptoms 104 or the CRY activator KL001 which has been demonstrated to have anti-inflammatory effects in FLS 108 point to the importance of targeting the clock and the effectiveness in really drugging the clock.It should be noted, however, that constant activation of otherwise temporally regulated molecules may not exclusively produce anti-inflammatory effects and can have undesirable side effects, at least in other tissues and organs.

| A step towards personalized medicine
Personalized medicine also with regard to chronotherapy matters, because individuals display a diurnal and interpersonal variability in their biological rhythms, their hormone secretion, number of circulating leucocytes, and immune reactions.The phase, period, and amplitude of the circadian rhythms are influenced by various factors, for example, genetic predisposition, the environment, daily habits of sleeping, eating, and other activities.Accordingly, patients suffering from the same disease could manifest specific clinical symptoms at different times of the day.For example, although the rhythm of IL-6 in most RA patients peaks at night before the worsening of the clinical symptoms (as reviewed in Buttgereit et al. 74 ), some patients exhibit aberrant peaks that emerge in the morning or during the day, 137 which might be a result of different chronotypes or the temporal association with flare-up of symptoms.
Our unique biological rhythms are accountable for the outcome of diagnostic tests and the pharmacokinetics, pharmacodynamics, tolerance, and toxicity of various medications.Pharmacokinetics describes drug liberation, absorption, distribution, metabolism, and elimination (LADME).The LADME processes have been reported to show diurnal variation and are also regulated by the circadian clock. 138They are influenced by several processes in our body showing diurnal rhythms, including liver metabolism, gastric acid secretion, pH, mobility of digestive tubes, membrane permeability, blood flow, plasma protein, bile volume, salt excretion, renal glomerular filtration rate, tubular reabsorption rate, and all other rhythmic physiology. 136,139The pharmacodynamics of the drug-the effect of the drug on the body-is affected by the diurnal availability and sensitivity of the target and its activated pathways.It is, therefore, reasonable to assume that not only chronotherapy but also chronodiagnostics should be implemented in future research.In this line, Wen et al. identified four circadian rhythm genes (EGR1, FOSL2, GADD45B, and NFIL3/E4BP4) differentially expressed between RA and normal control samples with a high specificity and sensitivity for RA diagnosis. 140Moreover, Kaneshiro et al. identified clock gene expression in leukocytes that could be useful as biomarkers predicting disease activities and therapeutic efficacies for biological DMARDs in RA treatments analyzing 15 RA patients treated with TNF inhibitor (5), IL-6 inhibitor (5), or CTLA4-IgG (5). 141Finally, assays that very easily and accurately estimate the internal circadian time in humans as described in the following section are needed to identify the right time for diagnosis and therapy.
Using high-resolution multiorgan expression data (RNA-seq and DNA arrays) to quantify the transcriptomes of 12 mouse organs over time Zhang et al. characterized the role of the circadian clock in mouse physiology and behavior showing that nearly half of all genes in the mouse genome oscillate with circadian rhythm in one or more tissues within the body.They found a majority of the 100 best-selling drugs in the United States target circadian gene products but have relatively short half-lives and would therefore benefit from timed dosing.Although proven in mice, this approach is a prime example of the importance of chronotherapeutic personalized holistic medicine to treat chronobiologically related diseases effectively. 142or these reasons, it is only sensible to personalize medication and disease treatment based on the patient's biological rhythm.Through this, the maximum efficacy of the treatment could be achieved while lowering unwanted adverse effects and toxicity.

| Identifying the right time
Identifying time-telling markers to determine individual circadian rhythms might offer many advantages in tackling circadian rhythm-related diseases.A first step towards time-telling markers is the identification of differences in clock gene expression that are associated with disease and which might act as potential biomarkers.Recently, in breast cancer, a machine learning tool was developed to distinguish healthy from malignant-tissue based on the detection of circadian clock dysfunction. 143ollowing this line, Wu et al. established an algorithm to report the circadian phase within 3 h of analysis from a human skin sample. 144Once biomarkers for circadian rhythms are identified, a strategy to identify the circadian rhythms of individual patients (chronodiagnosis) would pave the way for an individual, personalized therapeutic approach.Wittenbrink and co-workers followed a 3-stage biomarker development strategy to accurately estimate the internal circadian time in humans from a single blood sample. 145Moreover, Hughey et al. developed a method termed 'ZeitZeiger' based on a genome-wide gene expression data set to develop and validate a multiorgan predictor of circadian time in mice. 146In the end, Wittenbrink and co-workers was able to establish a cost-effective, low-complexity, and accurate 'BodyTime' assay using ≤13 genes that can predict the body's time.Among the reported genes are circadian clock genes (PER1, PER2, PER3, NR1D1, NR1D2, CRY1, CRY2) and three genes loosely associated with the clock. 145ery recently, a robust method based on two existing mathematical methods (Lomb-Scargle periodogram and cosinor) was established that permits characterization of circadian changes from infrequently sampled clinical blood specimens collected over a 24-48 h period in hospitalized patients termed ClinCirc. 147ClinCirc revealed from ICU (intensive care unit) patients that inflammation is associated with loss of circadian rhythmicity on ICU.Second, this new method revealed that a kidney transplant recipients' clock is entrained to the time of operation and that this shift in phase potentially explains the circadian regulation of delayed graft function.This study offers new insights into the implementation of circadian mechanisms and a new way to determine the right time to act.

| CONCLUSION AND PERSPECTIVES
The genetic, biochemical, and physiological relevances of circadian rhythms in a number of disorders have been shown during the past 40 years.With respect to the immune system, understanding of some underlying mechanisms and the importance of 24-h rhythms in immune populations, especially in humans, is still scarce.The molecular players that underlie the effect of disrupted circadian rhythms have also not been entirely uncovered.However, circadian disruption or misalignment due to modern life style (e.g., artificial light at night, jet lag) appear to be implicated in the pathomechanism of RA by altering the timing of clock genes leading to a pathologically altered immune cell migration and function, and to exacerbate synovial inflammation during the development and maintenance of RA.Thus, it is not surprising that the application of chronotherapy has been shown to exceed the benefit of conventional therapy in many clinical studies, including RA.Some chronotherapy has even reached clinical study phase III with promising results, although long-acting therapeutic approaches in the treatment of chronic inflammatory disease are available including bDMARDs that are not suitable for timed administration.For other rheumatic diseases, the benefit of chronotherapy has not yet been tested.One of the reasons might be the absence of disease-related circadian markers.Personalized chronotherapy is also a very promising approach.Tailored timing of medication according to individual biological rhythms might yield even better outcomes than general chronotherapy, since almost all diseases are heterogeneous and the biological rhythms of each person are unique.The challenge lies in developing practical and cost-effective methods to identify the individual circadian rhythms of the patients.However, in the past few years, remarkable findings on circadian biomarkers suggest that individual circadian rhythm measurement is possible.How well the current ways to find personal rhythms work in a clinical setting still needs to be answered.Overall, further research is required to gain understanding of circadian rhythms.Thus, a systematic approach could be established to identify individual biological rhythms that can be used in the clinic to improve diagnosis, existing treatments, find new treatment targets, and even stop the progression of diseases that are linked to circadian rhythms.