Circadian Rhythm Dysregulation in Inflammatory Bowel Disease: Mechanisms and Chronotherapeutic Approaches

The core of the molecular mechanism of circadian rhythms is the transcription-translation feedback loop of clock genes [7,8], and the genes involved in this circuit are called “core clock genes” [9].

Specifically, the aryl hydrocarbon receptor nuclear translocator (ARNTL, also known as BMAL1), circadian locomotor output cycle kaput (CLOCK), periods (PER), and cryptochromes (CRY) form the core loop. In addition, there are several subloops that couple to the core loop, including a stabilizing loop that mainly consists of the nuclear receptor reverse erythroblastosis virus-α (REV-ERBα) and the retinoic acid-related orphan receptor (ROR)-response element (RORE) sequence, as well as a D-box loop in which D-box binding protein (DBP), E4 promoter binding protein 4 (E4BP4, also known as NFIL3), hepatic leukemia factor (HLF), and thyrotroph embryonic factor (TEF) bind to the D-box and function as output clock genes.

The heterodimer of CLOCK-BMAL1, a set of the core clock gene products, binds to the promoters of the PER and CRY clock genes, thereby activating both PER and CRY transcription [10]. The translated PER and CRY suppress CLOCK-BMAL1 transcription through a negative feedback mechanism, and this loop cycles once every 24 h to generate the circadian rhythm [11]. In addition, nuclear receptors such as REV-ERBα and RORα regulate transcription through the ROR/REV-ERB response element (RRE) present in transcriptional control regions such as the BMAL1 gene, thereby reinforcing this feedback loop [12].

Core clock genes, such as BMAL1 and CLOCK, generate circadian rhythms by regulating a group of genes with E-box sequences that provide a rhythm underlying cellular functions. The output clock genes include DBP, TEF, HLF, and E4BP4, and the CLOCK- BMAL1 complex binds to the E-box in the promoter region of the DBP gene. The promoter region of the DBP gene is a CLOCK/BMAL1 binding region, known as a cis-regulatory element, referred to as the E-Box. The translated DBP binds to DNA recognition sites called the D-box and activates the transcription of downstream genes. The E4BP4 promoter has an RRE region to which ROR and REV-ERB bind, and translated E4BP4 binds to the same D-Box as DBP [13] (Figure 1). Although DBP and E4BP4 are not considered core clock genes for circadian rhythms, they may play a role in fine-tuning the core network, since the cycle length of circadian rhythms varies depending on the expression levels of DBP and E4BP4 [14]. Multiple regulatory subloops back up the oscillations of the core loop, generating peaks in gene transcriptional activity at different times of day [15].

The SCN functions as the master circadian clock. The circadian rhythm is modulated by environmental signals such as light, temperature, social interactions, and feeding patterns. The most important signal is light, which reaches the circadian system through retinal ganglion cells that contain the light-sensitive melanopsin [16,17]. In addition to light exposure, exogenous melatonin administration and physical activity can induce circadian phase shifts [16]. Rhythms in clock gene and protein expression have been observed in cells and tissues throughout the mammalian body, and these rhythms persist in culture, indicating that non-SCN cells also contain endogenous circadian oscillators [17]. The SCN acts as a “standard time regulator”, synchronizing peripheral tissue clocks [18]. For instance, in a study, when SCN-ablated hamsters received SCN transplants, behavioral rhythms were restored, and gene expression rhythms led to recovery in some peripheral tissues such as the liver and kidneys [19]. The central clock is primarily controlled by the brain’s SCN, which controls the circadian rhythms of several physiological and behavioral events through central nervous system neurons, including the paraventricular nucleus of the hypothalamus, which is important for neuroendocrine, homeostatic, and autonomic function integration [18].

However, circadian rhythms can also be mediated by “peripheral clocks” located in multiple tissues, including the intestines, liver, pancreas, and adipose tissue [20]. Feeding time serves as the main factor regulating peripheral clocks. When feeding is restricted to limited times in the light–dark cycle, peripheral clocks become disconnected from the brain’s master clock and synchronize according to feeding times and their autonomy [20]. Time-restricted feeding (TRF) can at least partially restore transcriptional and behavioral rhythms in mice with genetically disrupted circadian clocks [20]. Liver-specific deletion of the circadian clock gene (Bmal1) in mice attenuates glucose-regulated gene expression and impairs whole-body glucose homeostasis [21]. This demonstrates that peripheral clocks are essential for gene expression within tissues and overall tissue function. Peripheral clocks are synchronized to the SCN through neural and hormonal pathways [17]. The endocrine system is central in synchronizing the SCN and peripheral clocks [22]. In summary, central and peripheral clocks influence each other through various factors and are synchronized and regulated by external factors.

Immune dysfunction is among the most important pathogenic features of many chronic diseases, including IBD. Immune cell numbers and functions exhibit diurnal variations, resulting in time-dependent changes in immune response efficiency. Neutrophil mobilization from the bone marrow shows marked circadian rhythms, with relatively low neutrophil numbers in the blood during the day, significantly increasing at the beginning of the night activity phase, reaching peak numbers [23,24]. This rhythmic mobilization of neutrophils from the bone marrow into the blood is regulated by adrenergic neural transmission and circadian expression changes in the bone marrow stromal cell chemokine CXCL12 [24]. Similarly, lymphocytes exhibit significant circadian rhythms in their flow and quantity variations within lymph nodes; during the daytime, lymph nodes contain relatively fewer lymphocytes, but when the nighttime active phase begins, a large number of lymphocytes rapidly enter the lymph nodes, causing rapid accumulation [23,25]. Interestingly, immunizations or exposure to vaccines during the time window when lymphocyte numbers peak daily can induce stronger immune responses [25,26]. Innate immune recognition pathways also exhibit distinct circadian rhythm patterns. For instance, Toll-like receptor 9 (TLR9) gene expression and the production of mRNA and protein peak in the early active period, resulting in rhythmic changes in its ability to recognize pathogens due to the receptor’s own daily fluctuations in expression [27].

The immune system is largely controlled by circadian rhythms, with immune cell activity, gene expression, and cytokine production showing circadian variations throughout the day [28]. NFIL3 (E4BP4) is necessary for innate lymphoid cell (ILC) development, including natural killer cells; similarly, B cell development depends on BMAL1 [29]. Clock genes are important in regulating immune cell function and inflammatory responses, contributing to maintaining immune system homeostasis in the body [28]. Other research has shown that clock proteins control fundamental aspects of immune responses [30]. For example, the CLOCK-BMAL1 heterodimer directly controls the expression of TLR9, a pattern recognition receptor that recognizes bacterial and viral DNA, and suppresses inflammatory monocyte chemokine ligand (CCL2) expression. In addition, REV-ERBα suppresses IL-6 induction [30]. The molecular clock functions as a “controller” of immune cells, regulating immune responses depending on the integration of various inputs, including neural, hormonal, and local factors, and circadian rhythms in cellular gene expression [30].

The SCN clock harmonizes peripheral clocks through the hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system and their respective hormones, glucocorticoids, and catecholamines (epinephrine and norepinephrine), functioning as synchronization messengers or “zeitgebers” for peripheral clocks [30]. Other hormones, such as glucocorticoids, catecholamines, prolactin, melatonin, and growth hormones, affect the immune system and peak at specific times of the day. This SCN control over autonomic and endocrine outputs enables the synchronization of peripheral clocks, including immune cells, allowing the coordination of temporal physiological programs across many tissues at specific times of day [30]. The central clock in the SCN and its rhythmic control over autonomic and endocrine systems are important determinants of immune regulation. Circadian rhythms divide the immune system into two states: one where animals prepare for activity and heightened alertness when infection and injury risks are high, requiring increased white blood cell counts and immune cell sensitivity, and another state occurring during rest periods, providing opportunities for inflammation suppression and tissue repair [30]. Circadian rhythms influence innate immunity and adaptive immune responses. These are regulated through complex mechanisms, and circadian rhythm disruption is involved in autoimmune diseases, cancer, and inflammatory diseases [28].

IBD symptoms vary depending on inflammation severity and location. Symptoms range from mild to severe and alternate between active and remission periods. Common symptoms in both UC and CD include abdominal pain and/or diarrhea (often with blood or mucus). These symptoms can fluctuate and may persist for weeks to months, unlike infectious gastroenteritis symptoms, which typically last only days [35]. Unlike functional gastrointestinal disorders where symptoms appear only during the day, IBD symptoms may continue at night. Additional symptoms may include loss of appetite, weight loss, extreme fatigue, and fever [35].

UC consistently shows mucosal inflammation causing edema, ulceration, bleeding, and electrolyte leakage. UC inflammation typically begins in the rectum and extends continuously to the proximal colon [3]. CD features skip lesions and can affect any part of the digestive tract, potentially causing strictures, inflammation, and fistulas. While UC’s continuous inflammation is limited to the mucosa and submucosa, CD affects the entire thickness of the digestive tract mucosa [3].

Common complications of IBD include colon cancer, intestinal perforation, fistulas, pelvic or perirectal abscesses, toxic megacolon, and extraintestinal manifestations, such as osteoporosis, deep vein thrombosis, anemia, gallstones, primary sclerosing cholangitis, arthritis, iritis, and skin lesions. Additionally, psychiatric illness rates are higher, including depression, suicidal tendencies, and anxiety [3].

IBD diagnosis involves clinical findings, blood tests, stool tests, imaging (CT scans or MRI), and endoscopic biopsies [35]. The Crohn’s Disease Activity Index (CDAI) is the most commonly used scale for disease activity assessment for CD. This index scores factors including abdominal pain frequency, diarrhea episodes, weight changes, and general condition. The Harvey Bradshaw Index (HBI) is also used for CD. Similarly, UC uses measures such as the Mayo Score/DAI [3].

IBD treatment aims to induce and maintain remission [3,35]. Drug therapy is used to improve symptoms and control mucosal inflammation. Early remission induction is desirable to minimize intestinal damage and should be achieved within three months [35].

UC treatment is tailored based on severity, extent, patient age, comorbidities, drug safety and efficacy, patient preferences, administration route, urgency, cost, extraintestinal manifestations (EIMs), and specific factors such as pregnancy, surgery, and pediatric populations [36].

Various treatment options are available for mild to moderate UC, including 5-aminosalicylates, corticosteroids, and immunosuppressants such as thiopurines and methotrexate [37]. For moderate to severe UC, treatment approaches shift to advanced biologics, such as anti-TNFα, anti-α4β integrin biological drugs, anti-interleukin 12/23, selective IL-23 inhibitors, and small molecule drugs like Janus kinase (JAK) inhibitors and sphingosine-1-phosphate (S1P) modulators [37]. Surgical treatment, such as total colectomy, may be necessary for treatment-resistant cases [3].

CD treatment varies depending on the affected digestive tract portions, degree of fistulas or strictures, and extraintestinal complications [3]. Mild ileocecal disease typically starts with mesalamine treatment and is intensified with budesonide, a steroid that undergoes first-pass metabolism in the liver to prevent systemic side effects. More extensive cases require systemic steroid therapy with prednisone. The goal is steroid withdrawal within six weeks; patients unable to achieve this may need additional immunomodulators such as 6-mercaptopurine, azathioprine, or low-dose methotrexate [3]. Moderate to severe cases may need anti-TNFα agents. Patients with severe fistulas may require surgical treatment.

Both diseases require attention to nutritional status, mental health, bone health, vaccinations against preventable diseases, and cancer screening [3]. Patients with IBD should have their iron, vitamin B12, folate, and vitamin D levels measured every six months and take recommended supplements in case of deficiencies. IBD symptoms significantly impact psychological well-being and quality of life. Anxiety and depression are common in patients with IBD compared to the general population, making screening and psychiatric referrals important [35]. Patients with IBD have approximately 1.5–5 times higher mortality rates compared to the general population. Major causes of death include infections, disease progression, surgical complications, and multi-organ involvement. Importantly, due to higher colorectal cancer rates, colonoscopy screening is recommended every 1–2 years [3].

The intestinal tissue has its own peripheral clock, which controls the circadian rhythm of various physiological functions, including immune response, intestinal barrier function, and nutrient absorption [6]. This peripheral clock system is a dynamic system that is entrained by neural and humoral signals from the central clock and influenced by changes in meal timing and gut microbiota [6]. Of particular interest, the gut microbiota itself is regulated by gender and the host circadian clock, suggesting that there is a bidirectional interaction between the gut microbiota and the host’s clock system [38]. Analysis of the fecal microbiota of mice in a previous study showed circadian rhythms in the absolute amount of fecal bacteria and the abundance of bacteroides, which were more pronounced in female mice [27]. When the host circadian clock was disrupted by the deletion of Bmal1, the rhythmicity in the fecal microbiota composition disappeared in both genders, and interaction was observed [38]. Furthermore, changes in the gut microbiota induced by a high-fat diet drive the reprogramming of liver circadian rhythms through PPARγ-mediated transcriptional activation in mice, suggesting that the intestinal peripheral clock also plays an important role in regulating the body’s metabolism [39].

The expression of clock genes in intestinal tissue has been confirmed in various cell types. The intestinal epithelial cells either continue to proliferate or cease dividing and differentiate depending on cell cycle regulation. Crosstalk may occur between circadian rhythms and molecules that control cell cycle progress, and several reports have linked cell proliferation in the gastrointestinal epithelium to the circadian clock [40]. Although the detailed mechanism explaining the temporal regulation is unclear, one possible mechanism is the direct regulation of key cell cycle regulators through transcriptional regulation via the circadian core clock loop. Another possible mechanism is the regulation of the proliferation rhythm by extrinsic luminal signals, neural output signals, gut intestinal hormones, and growth factors [40].

The innate immunity in the gut utilizes a host of antimicrobial polypeptides known as defensins to combat ingested bacteria [41]. The mouse intestinal defensins (cryptdins) are constitutively produced and secreted but are overexpressed during infection and inflammation. Under healthy conditions, the expression of cryptdin in the mouse intestine shows a circadian rhythm, suggesting that defensin expression in humans also peaks during the day [41].

Tight junction proteins, occludin and claudin-1, are important in regulating intestinal epithelial permeability. In mice, the expression of occludin and claudin-1 shows circadian oscillation opposite to that of the Per2 mRNA level, and CLOCK and BMAL1 directly bind to the E-box of occludin and claudin-1 promoters and increase the transcriptional response [42]. Mice with a mutated Per2 gene(mPer2^m/m) showed constitutively high levels of occludin and claudin-1 proteins and were more resistant to dextran sulfate sodium (DSS)-induced colitis, whereas Clock^Δ19/Δ19 mice showed decreased levels of these tight junction proteins and were more susceptible to DSS-induced colitis [42]. These results suggest that the expression of the tight junction proteins occludin and claudin-1 is under the control of circadian rhythms and may affect the diurnal variation of intestinal permeability and the susceptibility to colitis.

The peripheral clock is important for nutrient absorption and metabolism. The expression and activity of H(+)/peptide cotransporter 1 (PEPT1) in intestinal epithelial cells is important for the transport of nutrients and drugs. Its expression and activity show circadian rhythms, which regulate the efficiency of nutrient absorption in a time-dependent manner [43]. In animal models, the expression of DBP was synchronized with PEPT1, and DBP directly induced the transcriptional activity of PEPT1, indicating that DBP plays a crucial role in the circadian oscillation of PEPT1 [43]. Interestingly, some aspects of circadian rhythmicity in small intestinal function, such as nutrient transport and enzyme activities, persist even during intravenous feeding that bypasses the intestine, suggesting regulation by neuroendocrine mechanisms rather than direct nutrient sensing [44].

These findings indicate that the peripheral clock system in the intestinal tract regulates three major functions in an integrated manner: immune response, barrier function, and nutrient absorption. More importantly, these functions are closely interrelated, and the disruption of one function may affect the others. The integrated breakdown of these functions may play a role in the pathogenesis of IBD.

Alterations in clock gene expression in patients with IBD have been reported by several research groups. IBD is a multifactorial disease that causes an abnormal immune response in genetically susceptible individuals and results from a complex interrelationship between environmental/microbial factors and the intestinal immune system. In CD and UC patients, genetic polymorphisms in the clock gene PER3 were evaluated, and the rs2797685 variant was significantly increased in both CD (p = 1.6 × 10⁻⁴, odds ratio [OR] = 1.38, 95% confidence interval [CI]: 1.17–1.63) and UC (p = 0.12, OR = 1.25, 95% CI: 1.05–1.48) patient groups compared to healthy controls [45]. A study by Liu et al. showed that the expression rhythms of key clock genes, including BMAL1, CLOCK, PER1, PER2, PER3, CRY1, and CRY2, were significantly disrupted in the colon mucosa of patients with active IBD. In particular, the expression of BMAL1 and PER2 was significantly decreased by up to one third, and these changes correlated with increases in inflammatory cytokines [5]. Furthermore, the expression of BMAL1, CRY1/2, and REV-ERBα decreased by up to one half in patients with IBD and sleep disorders, and sleep quality was correlated with disease severity in these patients [46].

Another study analyzed clock gene expression in endoscopic mucosal biopsies from IBD patients and found that ARNTL2 and RORα were elevated in IBD patients, whereas CSNK2B, NPAS2, PER1, and PER3 were decreased in CD patients [47]. On the other hand, ARNTL2, CRY1, CSNK1E, and TIPIN were elevated in UC patients, whereas NR1D2 and PER3 were decreased, indicating differences in clock gene expression between CD and UC [47].

Clock gene expression is decreased in patients with active UC. However, recent studies have demonstrated that the expression of clock genes, such as BMAL1, CLOCK, PER1, and CRY1, is significantly increased in the colonic mucosa of children with active UC, whereas the expression of PER2 is decreased [48]. Although this report differs from previous reports, patients with active IBD show great variability in gene expression [5,49]. Interestingly, a uniform pattern of gene expression was found in healthy controls compared to the highly variable expression pattern in patients with UC. Among the healthy controls, inflammatory genes (TNFα, IL10, IL6, NFκB) were positively correlated with clock genes (BMAL1, CLOCK, PER1, PER2, CRY1, CRY2) [48].

Clock genes crucially regulate the intestinal immune system. Recent studies have demonstrated that PER2 negatively regulates CD4+ T cell IFN-γ production in UC [50]. PER2 reduces the binding activity of ADAMS12, a T cell costimulatory molecule that contributes to tissue inflammation, and suppresses IFN-γ production by CD4+ T cells. In patients with IBD, decreased expression of PER2 in the intestinal mucosa may promote intestinal inflammation through enhanced IFN-γ production [50]. Interestingly, this pathogenic role of decreased PER2 in human IBD appears to contrast with findings in mouse models [42], where mice with a mutated Per2 gene showed increased expression of tight junction proteins (occludin and claudin-1) and decreased susceptibility to DSS-induced colitis. These seemingly contradictory findings suggest that PER2 may have distinct roles in different aspects of intestinal homeostasis: immune regulation and barrier function.

When comparing clock protein expression in the intestinal mucosal epithelial cells of 24 patients with CD and 26 patients with UC and controls, the expressions of BMAL1, PER1, PER3, TIMELESS, and NPAS2 decreased in patients with IBD, while no significant differences were observed in BMAL2, CLOCK, and PER2 expression [51]. Although the findings regarding PER2 expression levels appear inconsistent with its functional roles discussed above, PER2 remains a potential therapeutic target for IBD that merits further investigation.

In contrast, the role of BMAL1 in intestinal homeostasis is more clearly defined. Decreased expression of Bmal1 due to disruption of the circadian rhythm leads to decreased levels of tight junction proteins and increased apoptosis of intestinal epithelial cells, thereby impairing intestinal barrier function. Studies using DSS-induced colitis models in mice have demonstrated that decreased Bmal1 expression exacerbates inflammation through impaired intestinal barrier function [42,52].

Supplementation therapy with butyrate, a short-chain fatty acid, may contribute to the normalization of clock gene expression in patients with IBD. A double-blind randomized controlled trial showed that butyrate supplementation increased the expression of CRY1/2, PER1, and BMAL1 in patients with active UC while simultaneously reducing the biomarkers of inflammation and improving sleep quality and quality of life [53].

These findings suggest that abnormalities in clock gene expression play an important role in the pathophysiology of IBD and that correcting these abnormalities may be a promising new therapeutic strategy. In particular, the importance of the control of the intestinal immune system through the regulation of clock gene expression, as well as sleep and circadian rhythms, is emphasized.

Output system clock genes are a group of downstream genes controlled by core clock genes. Several reports have described the relationships between output clock genes and metabolic regulation [13], but reports on their relationship with immune function are scarce. E4BP4 is essential for the development of NK cells and dendritic cells and is also involved in macrophage activation and the polarization of CD4+ T cell responses [54,55]. E4BP4 is widely distributed in immune cells and is an important transcription factor in maintaining intestinal immune homeostasis. Dysfunctional E4BP4 is involved in the pathogenesis of IBD [55].

In cultured macrophages and in vivo models, E4BP4 expression is increased upon exposure to microorganisms. The expression of E4BP4 is reduced in CD14-positive lamina propria mononuclear cells from patients with CD and UC compared with healthy controls. The cytokines IL-12 and IL-23, which are important in the pathogenesis of IBD, are mainly produced by macrophages and dendritic cells [55]. The two cytokines form heterodimeric proteins that share a common subunit, p40, and E4BP4 is involved in the development of IBD by negatively regulating the secretion of IL-12p40 in the intestine. Indeed, E4BP4 knockout mice spontaneously develop colitis and rectal prolapse, which are associated with the overproduction of IL-12p40 in the serum and colon [56]. E4BP4 is induced by IL-10 and serves as an IL-12p40 inhibitor in macrophages upon encounter with bacterial products [56]. E4BP4 and IL-10 double-knockout mice develop severe early-onset colitis and show upregulated IL-12p40 expression [55]. Genetic deletion of Il12b in E4BP4 knockout mice completely prevents the development of colitis. These results suggest that E4BP4 and IL-10 are involved in intestinal mucosal homeostasis and dysregulation of IL-12b is involved in the development of colitis [55]. Furthermore, E4BP4 knockout mice are more susceptible to colitis when housed in conventional environments, indicating that the colitis development in E4BP4-deficient mice is dependent on the gut microbiota [56]. E4BP4 in macrophages induces an anti-inflammatory phenotype that ameliorates the severity of DSS-induced colitis [57]. E4BP4 can bind to the Il4rα gene, which promotes anti-inflammatory properties, and may contribute to increased anti-inflammatory gene expression. Not only the core clock genes but also E4BP4, one of the output clock genes, may be potentially important factors regulating gut immunity. E4BP4 is a promising new therapeutic target for IBD.

This is a review article and does not contain any new data. All information discussed is based on previously published studies that are cited within the text.