Circadian Clock: A Regulator of Immunity in Autoimmune Diseases

The immune system is distributed throughout the body, including two branches: innate and adaptive immunity. Innate immunity is the frontline response against exogenous and endogenous danger signals in a nonspecific manner. In this process, classical innate immune cells, such as neutrophils, macrophages, dendritic cells, and innate lymphoid cells, are activated and some pro‐inflammatory factors are released [44]. Other than innate immunity, there is also infiltration of adaptive immune cells (T cells and B cells) in tissue inflammation. T cells receive signals from antigens and then differentiate into effector T cells, which produce pro‐inflammatory or anti‐inflammatory cytokines while B cells differentiate into plasma cells and produce specific antibodies upon stimulation [45].

Melatonin and cortisol participate in the modulation of the circadian clock. Since melatonin and cortisol both have immunomodulatory effects [43, 46, 47], the immune activities exhibit circadian rhythmicity. Virtually all levels of immune function have circadian rhythms, including leukocyte development, leukocyte trafficking, and specific cell effects. We list the findings about circadian rhythms in immune activities by cell type (Table 1) and the regulation of circadian clock on immune systems (Figure 1).

The rhythmic characteristic of the immune circuit is the result of the coordinated action of neurons of the SCN, structural cells in tissues, and immune cells [84]. Circadian misalignment in various stages of rhythmic immune regulation might lead to immune disorders, and disease progression and exacerbation. A study focused on response patterns of immune rhythms in night shifters and pointed out that the altered pattern expressions of immune cells might increase susceptibility to infections and decrease the efficiency of vaccination in night workers [85]. In addition, it was indicated that in the case of chronic jet leg, the circadian clocks in immune cells were altered and thus the pace for cell growth was dysregulated, accompanied by a deficient or compromised immune system against tumor generation, leading to an increased risk of tumor development or progression [86, 87, 88]. It was also found that circadian disruption induced by light/dark cycle reversal promoted immune disorder, exacerbated renal fibrosis, and accelerated renal dysfunction, facilitating renal injury in chronic kidney disease [89]. There was also research highlighting the immunological effects of circadian dysregulation in chronic inflammation. Cheng et al. revealed that circadian disruption could help exacerbate allergenic reactions in the nasal mucosa and lung tissues by increasing Th2‐like immune response [90]. Moreover, intestinal inflammation was also reported to be associated with circadian clock dysfunction. It was found that light/dark phase shifts contributed to abnormal intestinal barrier function, leading to low‐grade intestinal inflammation [91]. Another research found that mice with colitis that had a lost circadian rhythm exhibited no daily rhythm in the infiltration of immune cells and worsened colitis severity [92]. Swanson et al. also suggested that circadian dysregulation was related to increased intestinal permeability, elevated blood pro‐inflammatory factors, and elevated intestinal inflammation in inflammatory bowel diseases [93]. In addition to inflammatory bowel disease, the role of circadian dysregulation in immunity in a lot of other autoimmune diseases has raised much interest, and we will discuss these in the following text.

Another important impact of circadian rhythm dysregulation on immunity lies in vaccine administration. The antituberculosis vaccine bacillus Calmette‐Guerin (BCG), influenza, and hepatitis A vaccine all showed a higher antibody response in the morning compared with immunization in the afternoon [94, 95, 96]. However, several studies showed different results that vaccination in the afternoon had higher responsiveness or the timing of vaccination did not affect the immune responsiveness [97, 98, 99]. Liu et al. indicated that age might influence the effects of the timing of vaccination and vaccination in the morning might enhance the immune responsiveness in adults over 65 years old [100]. Although there is currently no consensus on the best timing of vaccination, some studies have demonstrated the biological rhythm regulation involved in vaccine immunization. It was indicated that the circadian clock modulated the expression of Toll‐like receptor (TLR) 9. Therefore, using TLR‐9 as an adjuvant, immunization at the time when TLR‐9 had the highest activity might present an improved immune response [101]. Antigen processing also had circadian rhythms, and it was reported that oscillations in mitochondrial morphology and the associated metabolic output influence the antigen presentation process [102]. Ince et al. found a time‐of‐day‐dependent manner of the dendritic cell migration from the skin to the draining lymph node, which ensured the interaction between dendritic cells and T cells, and rhythmic expression of tumor necrosis factor and intercellular cell adhesion molecule‐1 (ICAM‐1), contributing to lymphocyte infiltration and lymph node expansion. It was suggested that lymph node cellularity governed the immune activities to vaccinations [103]. Nobis et al. used a vaccination model of antigen‐presenting cells loaded with an antigen to focus on T cell‐intrinsic functions and revealed the role of the CD8 T cell clock in the T cell response to vaccination [82]. Since there has been no definitive conclusion on the best timing of vaccination in clinical practice, further investigation on the mechanism and the layer of clinical setting is needed.

Autoimmune diseases are characterized as immune responses to self‐antigens, which contribute to damage to self‐organs or tissues. The etiologies of autoimmune diseases are complicated, and the widely accepted point is that the onset of autoimmune diseases is the result of the combined action of genetic predispositions, infection, and environment [104]. Previous studies have revealed higher incidences of rheumatoid arthritis (RA) [105], type 1 diabetes (T1D) [106], multiple sclerosis (MS) [107] in those who have a disturbance of circadian genes. Due to the circadian modulation in the immune system, circadian clocks have a role in the pathogenesis of autoimmune diseases. Considering the underlying links between autoimmune diseases and circadian clocks, we summarize the association between the circadian clocks and the immune changes in autoimmune diseases, prospecting for a better understanding of the pathogenesis of autoimmune diseases and potential synchronization therapy.

RA is a chronic autoimmune disease, impairing many organs and tissues, and affecting polyarthritis (including large, medium, and small) systematically. The main characteristic of RA is synovitis and the inflammation in the synovial tissues contributes to the irreversible destruction of the joints [108]. Patients with RA exhibit diurnal variations in their symptoms, and the most representative manifestation is morning stiffness, which is recognized to be associated with increased pro‐inflammatory factors such as interleukin (IL)‐6 in the morning [109, 110].

Studies have also revealed several circadian genes involved in the modulation of rhythmic variations in RA. It was demonstrated that the Cry proteins had an inhibitory effect on the modulation of pro‐inflammatory tumor necrosis factor‐α (TNF‐α), as the deletion of Cry1 and Cry2 contributed to an increased production of TNF‐α [111]. Moreover, in Cry1^(‐/‐)Cry2^(‐/‐) mouse models, exacerbation of joint swelling and elevated inflammatory mediators such as TNF‐α and IL‐6 were observed [111]. In mouse models of anti‐type II collagen antibody‐induced arthritis, it was observed that the use of melatonin contributed to increased infiltration of inflammatory cells, exacerbation of synovial hyperplasia, and worsened articular cartilage destruction while markedly decreased expression of Cry1, suggesting that protein Cry1 might be involved in the melatonin‐mediated aggravation of joint inflammation and destruction [55]. Du et al. found that NFIL3 also contributed to RA pathogenesis as they observed elevated expression of NFIL3 in the peripheral blood and synovial tissues of RA patients, and an association between an increased NFIL3 level and abnormal inflammatory responses [112]. In turn, the inflammatory pathways could also directly affect the expression of clock genes. It was reported that TNF‐α induced the expression of Bmal1 through calcium‐dependent pathways in synovial cells of RA [113]. Cavadini et al. found that TNF‐α and IL‐1β had inhibitory effects on the expression of Per1/2/3, Dbp, and Tef while promoted slightly the expression of Clock and Rev‐erbα [114]. Moreover, in vitro studies using RA‐FLS cells, it was observed that TNF‐α could inhibit the expression of Per2 and promote the expression of Clock, Bmal1, and Cry1 [115].

Systematic changes in circadian energy metabolism were also observed in RA. In chronic inflammatory diseases, immune activities require large energy consumption, which is crucial for energy regulation. In healthy individuals, energy allocation to immune activities is time‐dependent, as many aspects of immune activities (including antigen presentation, clonal expansion, and antibody production) are inhibited in the morning due to the immunosuppressive effects of cortisol and noradrenaline, and they are promoted in the midnight following low activities of the hypothalamic‐pituitary‐adrenal axis and the sympathetic nervous system [116, 117]. In parallel with waking up and increased levels of cortisol and noradrenaline in the morning, circulating cytokines are increased and lipolysis, β‐oxidation of fatty acids, glycogenolysis, and gluconeogenesis are stimulated [118]. However, in patients with RA, rhythmic metabolic rewiring was observed as β‐oxidation and lipid handling were impaired while sphingolipid and ceramide were accumulated [119]. It was demonstrated that the arthritis‐related production of ceramides was most pronounced and it was at the time of peak inflammation [119]. Since the circadian symptoms of RA arise during the night when energy consumption is allocated for increased immune activities and ceramide accumulation accompanied by peak inflammation, energy redistribution might be a promising treatment target for patients with RA and it deserves further investigation.

Modified prednisone releases were indicated in patients with active RA: plasma cortisol levels started to rise at 23:00–02:00, earlier than healthy controls, and had a higher peak [120]. This earlier rise of cortisol was preceded by a rise in IL‐6, which was tightly associated with the pathogenesis of RA including the clinical manifestations such as joint swelling and stiffness [43, 120]. IL‐6 also played a role in stimulating the cortisol secretion [43]. Based on this biorhythm in RA, a study compared the treatment effects of using prednisone at 2:00 a.m. with that at 7:30 a.m. and found improved morning stiffness, pain, and disease activity when using prednisone at 2:00 am [121]. Another study used a novel modified‐release prednisone tablet at 10:00 p.m. which released prednisone at 2:00 a.m. and found significant improvement in clinical manifestations [122]. Moreover, Buttgereit et al. assessed the efficacy and safety of the modified‐release prednisone compared with standard prednisone and found that patients receiving the modified‐release prednisone achieved a mean reduction of 44 min in the duration of morning stiffness, and the absolute difference between the two groups was 29.2 min, indicating a better efficacy of the modified‐release prednisone (p = 0.072) [123]. Since morning stiffness is one of the early symptoms recommended for diagnosis of RA and is associated with abnormal immune rhythms, it is of great importance to carry out multi‐center large‐scale studies to investigate the efficacy and safety of treatment strategies that consider the timing of inflammatory activities.

T1D derives from a progressive loss of pancreatic β‐cell function and requires daily insulin treatment. T1D is mediated by a cell‐mediated immune response, which is driven by aberrant T cells, causing the loss of self‐recognition of T cells and targeting the antigens of pancreatic β cells [124]. Previous studies have revealed that circadian oscillators play a role in the pathogenesis of T1D. Hofmann et al. investigated the rhythmic expression of clock genes and clock‐controlled genes in rat models with T1D including streptozotocin‐treated rats, spontaneous T1D LEW.1AR1‐iddm (lddm) rats and lddm rats used insulin for 10 days, and found that the clock genes all exhibited a rhythmic expression pattern while the expression of Bmal1, Per2, and Clock was altered in lddm and insulin‐treated lddm rats [125]. Furthermore, using insulin helped normalize the rhythm in the expression of Dbp, Rev‐erbα, and E4bp4 [125]. Marcheva et al. also observed oscillations of the expression of Clock and Bmal1 in pancreatic islets, and they found that conditional knockout of the pancreatic clock genes (including Clock and Bmal1) contributed to insulin deficiency, glucose tolerance impairment, and the onset of diabetes [126]. In addition, the aryl hydrocarbon receptor (AHR), a member of the family of the homologous genes of the transcriptional‐translational feedback loops, was demonstrated to be associated with the pathogenesis of T1D [127]. It was suggested that AHR had a role in the development of Treg and Th17 [128, 129]. Moreover, the use of AHR ligand 2,3,7,8‐Tecrachlorodibenzo‐p‐dioxin (TCDD) in Nonobese diabetic (NOD) mice could reduce the pancreatic islet insulitis with an expansion of Treg in the pancreatic lymph nodes, which indicated a suppression in the development of T1D [127]. Aryl hydrocarbon receptor nuclear translocator‐like 2 (ARNTL2), also known as Bmal2, was suggested to contribute to variation in the regulation of circadian rhythm [130, 131]. In NOD mice, Arntl2 was indicated to be a candidate gene for T1D as the expression of Arntl2 was downregulated in NOD mice compared with that in nondiabetic mice [132, 133]. It was also demonstrated that Arntl2 could bind to the promotor of the Il‐21 gene to control the expression of Il‐21 without interaction with other oscillators such as Bmal1 or Clock [134]. IL‐21 could control the proliferation of immune cells, and the gene Il‐21 is located at the T1D locus Idd3 [135, 136]. It was shown that IL‐21 was associated with the pathogenesis of T1D [137], and it was shown to have an increase in the number of IL‐21‐secreting effector T cells in patients with T1D [138]. However, there is no data about the circadian changes of ARNTL2 or IL‐21 in T1D. Another study revealed the circadian dysrhythmia of immune cells of T1D, as patients with T1D had significant phase shifts in the time of peak occurrence of B cells ( + 4.8 h), CD4 and CD8 T cells ( + 5 h), and their naive and effector memory subsets ( + 3.3 to +4.5 h), and regulatory T cells ( + 4.1 h) [139]. Further investigations are needed to find the diurnal changes in the interaction between circadian oscillators and immunity in T1D.

MS, an immunological disease that is characterized by chronic inflammation and acute inflammatory lesions in the central nervous system (CNS), could lead to neural demyelination and neurodegeneration [140, 141]. It has been generally accepted that autoimmunity plays a major role in the pathogenesis of MS, and specific T cells and B cells are both involved in mediating the disease [142, 143]. MS includes three subtypes: relapsing‐remitting subtype of MS (RRMS) which is characterized by recurrent episodes of neurological dysfunction caused by acute inflammatory demyelination, primary progressive MS (PPMS), and secondary progressive MS (SPMS) that is defined as progressive neurodegeneration leading to neurological disability [144]. Previous studies supported a significant association between shift work and increased MS risk [145, 146]. Moreover, it was revealed that insufficient sleep and low sleep quality might increase the risk of developing MS [147, 148]. These findings suggested that MS might be associated with a dysregulation of circadian rhythms.

Several clock genes have been reported to be associated with MS. The polymorphism of genes ARNTL, CLOCK, and PER3 were all reported to be associated with MS risk or MS exacerbation [149, 150]. Six genes (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) encoded for circadian entrainment components were shown to be associated with MS [151]. Buenafe also found that the diurnal rhythm of Per2 expression was altered in mice with EAE [152]. Moreover, Sutton et al. used a mouse model of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and found that Bmal1 regulated the accumulation and activation of immune cells in EAE, and the loss of myeloid Bmal1 exacerbated inflammation in the CNS through the expansion of IL‐1β secreting monocytes, which increased pathogenic IL‐17⁺/IFN‐γ⁺ T cells [107]. Although data on the association between circadian clock genes and immunity are limited, the modulation of Bmal1 on inflammation in CNS of EAE indicated that circadian dysfunction might result in immunity disruption and malfunction in MS. More studies are needed to investigate the link between circadian disruption and immune cell dysfunction in MS.

It was indicated that the level of melatonin was decreased in patients with MS [153]. Kern et al. observed that lower melatonin levels within the first hour after awakening were indicative of longer MS duration. They also found that melatonin levels correlated with neurological disability, fatigue, and health‐related quality of life [154]. The findings of Damasceno et al. also supported that the disruption of melatonin circadian rhythm production was associated with disability and fatigue [155]. Several studies also investigated the circadian rhythm of cortisol in MS. An increase in the cortisol awakening response and an increase in cortisol within approximately 30 min after awakening were found, and this change in the cortisol level was found to be associated with neurological disability worsening and fatigue [156, 157, 158]. However, the association between immunity and the changes in melatonin and cortisol levels in MS has not been studied yet.

MS symptoms also showed diurnal variations. It was observed that fatigue and pain increase over the day while muscle strength, objective, and subjective cognitive performance decrease over the day [159, 160]. The reason why MS symptoms have circadian rhythmicity has not been investigated yet. Davis et al. indicated that circadian temperature variations might be associated with the fluctuation of motor function [161]. More studies are needed to investigate the underlying circadian mechanisms.

Systemic lupus erythematosus (SLE) is a chronic connective tissue disease, characterized by loss of immune tolerance to self‐antigens, and the formation of immune complexes [162]. Dan et al. performed a case‐control study and found that PER2 gene single‐nucleotide polymorphisms were related to the SLE pathogenesis [163]. Moreover, IL‐17A was suggested to be involved in the development and progression of SLE, and it was found that the level of IL‐17A was increased in patients with SLE [164, 165]. The expression of IL‐17A required RORγt [166], a receptor activates transcription and participates in circadian rhythms [167]. However, there is no research concerning the role of clock genes, especially the ROR family in SLE. Existing data on the association between circadian modulation in immunity and the pathogenesis of SLE are also scarce and more research is warranted.

Helvaci et al. provided the first evidence for an association between autoimmune thyroid disease and clock gene PER3, indicating the possible relevance of PER3 gene polymorphisms in the pathogenesis of autoimmune thyroid disease [168]. Ando et al. also established a link between psoriasis and the circadian clock, as they found that Clock could regulate psoriasis‐like skin inflammation in mice models via modulation of the expression of IL‐23R in γ/δ + T cells [169]. Lizuka et al. revealed the crucial role of RORγt in the development of Sjogren's syndrome [170]. However, data on circadian modulation in the pathogenesis of these diseases are limited.

Recent studies linked the association between circadian disruption and intestinal dysbiosis in the pathogenesis of inflammatory bowel disease (IBD). Genetic deficiencies in various circadian clock components (PER1/2, BMAL1, CLOCK, RORα, and REV‐ERBα) have been linked to an increased susceptibility of IBD mainly via influencing colonic permeability by regulating the integrity of the intestinal barrier [56, 83, 92, 171, 172, 173]. Cao et al. also revealed the role of the modulation of Cry proteins in B cell development and the BCR‐signaling pathway in ulcerative colitis [78]. In addition, mice with induced colitis and patients with IBD also exhibit disruptions in clock gene expression (including genes encoding BMAL1, CLOCK, PER1/2, CRY1/2, NPAS2, REV‐ERBα, RORα, etc.) [56, 174, 175]. Moreover, there are also studies investigating the association between circadian disruption and intestinal dysbiosis in the pathogenesis of extra‐autoimmune diseases, which mainly focused on RA. It was suggested that barrier permeability was affected by arthritis and increased barrier permeability during disease initiation is reported to be accompanied by increased frequencies of Th1 and Th17 cells in the small intestines, which have the potential to migrate to the joints [176]. Moreover, temporal remodeling not only in the microbial composition of the gut [Alistipes and Odoribacter (both Bacteroidetes) and Lactobacillus (a Firmicute)] but also metabolic outputs (indolelactate and methyl indole‐3‐acetate) was observed in collagen‐induced arthritis [177]. However, the mechanisms underlying crosstalk between the inflamed joint, the colon and its constituents are unknown and need further investigation. These findings might also indicate new directions for the study of the association of circadian clock and intestinal homeostasis in other extra‐intestinal autoimmune diseases since intestinal dysbiosis was reported to be related with many autoimmune diseases including MS, T1D, and SLE [178].

Immune thrombocytopenia (ITP) is an autoimmune bleeding disease characterized by decreased platelet counts and an increased bleeding risk [179]. The pathogenesis of ITP includes decreased platelet production by megakaryocytes in the bone marrow and increased platelet destruction [179]. A previous study demonstrated that the Per2 gene was required for platelet formation [180]. Moreover, it was indicated that the total platelet numbers and plasma thrombopoietin (TPO) concentration were in circadian rhythms and entrained by the light‐dark cycle [181]. Tracey et al. observed that CLOCK regulated the diurnal expression of the TPO gene in human cell lines [182]. The above studies provided evidence that platelet production was in circadian modulation, and further studies are needed to investigate whether there are circadian changes in ITP, which might provide new treatment targets for ITP.

Remarkable progress has been made in the understanding of the circadian clock in innate and adaptive immunity, and autoimmune diseases in the last few years. Therefore, this evidence might support the idea of modulating or maintaining circadian rhythms for treating diseases [183].

Many clock genes have been demonstrated to influence immunity and the pathogenesis of autoimmune diseases, among which RORs might be the most promising clock genes to act as therapeutic targets. Previous studies have demonstrated that RORs are responsible for regulating Th17 differentiation and regulating the IL‐17 pathway [184, 185, 186]. RORγt, a subtype of RORγ, is not only a key circadian rhythm regulator but also a crucial transcription factor of immune cells. Recently, RORγt has attracted much interest as a potential therapeutic target in autoimmune diseases. Digoxin, SR1001, SR1555, TMP778, and TMP920 were all proposed to display effects on RORγt and further regulated immunity [187, 188, 189, 190]. Moreover, another important circadian modulator, Rev‐erbα, is suggested to act as an antagonist of RORγt and regulate the differentiation of Th17 [191]. Therefore, REV‐ERBα might also serve as a potential treatment target for autoimmune diseases. There have been some studies to investigate the agonist of REV‐ERBα in autoimmune diseases, and the involved agents include GS2667, GSK5072, GSK2945, SR9009, and so on [192, 193]. However, these studies have all focused on the effects of the agents on the differentiation of Th17 cells and cytokine secretions. No research has focused on the circadian regulation of the immune system under the condition of autoimmune diseases by these agents, which deserves more research attention.

Factors reported to have an impact on the circadian system include light, food, exercise, and hormones (including melatonin and cortisol), et al., which have been investigated as treatment approaches to reset or bolster the circadian rhythms in many diseases [194, 195, 196, 197]. In autoimmune diseases, there have also been some reports. In a study using collagen‐induced arthritis mice models, chronotherapy with Baricitinib could target the secretion of cytokines and was demonstrated to be effective in the treatment of arthritis [198]. Dietary approaches, such as intermittent fasting, which means alternate day fasting involving a combination of no‐eating days with eating days, have also been investigated and were indicated to impact circadian rhythms and improve inflammatory parameters in MS [199]. However, there are also negative results. De Carvalho et al. summarized the efficacy of melatonin supplementation in rheumatological disease and found that melatonin supplementation was not effective in RA and SLE [200]. A series of clinical trials are currently being conducted to explore the effects of zeitgeber regulation on circadian rhythms and disease progression itself in autoimmune diseases. For example, a study focused on the effects of light therapy in progressive MS (NCT 06261528). Another study aimed to determine the effects of intermittent fasting and a Mediterranean diet in patients with MS (NCT 06546033). Furthermore, melatonin and cortisol are both vital hormones that are involved in circadian regulation. Considering the changes in the two hormones observed in autoimmune diseases, melatonin and corticosteroid treatment for autoimmune diseases are proposed. Indeed, corticosteroids are the first‐line or recognized effective treatment of many autoimmune diseases, including RA, and SLE. However, due to the changes in the circadian rhythmicity of corticosteroids in disease conditions [120], further studies on the best timing of treatment are needed. Melatonin supplementation is also explored in autoimmune diseases, such as MS, and is indicated to improve sleep in patients with MS [201]. However, there are still not many related studies available. Taken together, there have been limited data on the effects of zeitgeber modulation on immunity and autoimmune diseases, and more studies are needed to find new treatment targets.