Aryl hydrocarbon receptor (AhR) impairs circadian regulation: Impact on the aging process

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Introduction
The aryl hydrocarbon receptor (AhR) is an evolutionarily conserved defence sensor against diverse environmental toxins. The AhR gene evolved over 600 million years ago, i.e., before the Cambrian Period (Hahn et al., 2017). However, the AhR protein is not only an environmental sensor but it has also a significant role in the early development of the organism and the functions of both the innate and adaptive immunity (Nebert, 2017;Trikha and Lee, 2020). There is clear evidence that AhR signaling displays antagonistic pleiotropy in its regulation of organismal development and the ageing process (Salminen, 2022a). The concept of antagonistic pleiotropy postulates that a certain gene may control many functional traits that are beneficial during embryogenesis but which have harmful effects later in life, e.g., promoting the aging process (Austad and Hoffman, 2018). For instance, while AhR signaling has an important role in the growth and differentiation of the embryo, later its properties can disturb cellular homeostasis during the ageing process (Section 4.). An activation of AhR signaling is also associated with a wide variety of chronic diseases, especially inflammatory diseases (Neavin et al., 2018). Interestingly, there is robust evidence that although the expression of the AhR protein is under circadian regulation, AhR signaling can impair the function of circadian clocks and thus promote the aging process (Section 3.3.).
Life on planet earth is synchronized by the solar cycle with a periodicity of 24 h. The circadian oscillation in the activities of metabolism and many other cellular functions have been found even in cyanobacteria and Archaea (Bhadra et al., 2017). For instance, in humans, circadian regulation has been organized around the central pacemaker in the hypothalamus which synchronizes the rhythms of circadian clocks in peripheral tissues (Mohawk et al., 2012). Circadian rhythmicity can be observed in many physiological functions and cellular activities, e.g., the immune system is subjected to a circadian regulation (Scheiermann et al., 2018;Hergenhan, 2020). Circadian rhythms are evident in several crucial functions, such as energy metabolism, redox balance, autophagy, cellular senescence, and immune responses (Section 3.1.). These are functions which gradually become disturbed in the aging process. Interestingly, AhR signaling not only regulates the functions of core clocks but it also is involved in the appearance of many of the hallmarks of the aging process (Salminen, 2022a). Next, I will examine in detail the crosstalk between AhR signaling and circadian clocks in an attempt to E-mail address: antero.salminen@uef.fi. understand their role in the aging process.

AhR signaling pathways
The Ah receptor is a ligand-regulated transcription factor which is a major environmental sensor for a wide variety of environmental toxins, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbon (PAH) compounds (Nebert, 2017). The AhR gene is an evolutionarily conserved member of the ancient basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) domain family (Vazquez-Rivera et al., 2021). In the cytoplasm, the Ah receptor is inactivated via the binding of three chaperones; HSP90, p23, and XAP2 proteins. The binding of a specific agonist to the Ah receptor triggers the release of the chaperones from the complex and the AhR protein becomes translocated into the nucleus where it forms a heterodimer with another PAS domain protein, i.e., the AhR nuclear translocator (ARNT) protein (Nebert, 2017;Larigot et al., 2022). The complex of AhR/ARNT factors induces the transcription of target genes by binding to the dioxin/xenobiotic response elements (DRE/XRE). In the AhR/ARNT heterodimer, the ARNT protein contains the DNA-binding motif, whereas the AhR protein carries the transactivation domain. The Ah receptor can also form non-canonical complexes with other PAS domain transcription factors (Section 3.3.) and with many non-PAS transcription factors, such as RelB, a member of the NF-κB family, and E2F1, a tumor suppressor protein (Vogel et al., 2007;Marlowe et al., 2008). The non-PAS AhR complexes mostly inhibit the transcription driven by the interacting partner but the non-canonical heterodimers can also bind to novel sites at gene promoters, e.g., the AhR/RelB complex binds to the specific RelBAhRE site and mediates the transcription of many chemokines, such as IL-8 and CCL20, in an AhR ligand-dependent manner (Vogel et al., 2007;Ishihara et al., 2019). Interestingly, the ligand-activated AhR signaling can also stimulate non-genomic responses by activating protein kinases, such as the FAK/RhoA and FAK/Src pathways (Chang et al., 2009;Tomkiewicz et al., 2013). Currently, the role of the FAK/RhoA and FAK/Src signaling in the regulation of circadian rhythms needs to be clarified.
In addition to the environmental xenobiotics, such as TCDD and PAH, there is a broad diversity of endogenous ligands which can activate AhR signaling. It seems that during evolution the AhR protein has adapted many novel properties beyond the detoxification of environmental toxins. In particular, the catabolism of L-tryptophan generates several endogenous AhR ligands, both as a result of inflammatory responses and due to metabolic activity of the gut microbiota and dietary supply of tryptophan amino acids (Mezrich et al., 2010;Ma et al., 2020;Larigot et al., 2022) (Fig. 1). A chronic low-grade inflammation, a phenomenon called inflammaging (Franceschi et al., 2000), stimulates the expression of indoleamine 2,3-dioxygenase 1 (IDO1) which triggers the kynurenine (KYN) pathway, generating a number of KYN metabolites which are potent endogenous agonists for the Ah receptor (Mezrich et al., 2010;Ma et al., 2020;Salminen, 2022b) (Fig. 1). For instance, it is known that KYN, kynurenic acid (KYNA), anthranilic acid, cinnabarinic acid, and xanthurenic acid are able to activate AhR signaling. Interestingly, there exists a positive feedback loop since the activation of AhR signaling stimulates the expression of IDO1 and thus augments the function of the IDO1/KYN/AhR pathway under inflammatory conditions (Vogel et al., 2008). The activation of the NF-κB factor, a master gene of inflammatory responses, can also transactivate the human AhR gene (Vogel et al., 2014) (Fig. 1).
There are several other sources of tryptophan catabolites which can activate AhR signaling. For example, the exposure of the skin to UV radiation triggers the production of the 6-formylindolol[3,2-b]carbazole (FICZ) derivative from L-tryptophan (Fritsche et al., 2007). FICZ stimulates AhR signaling which promotes the generation of photoaging in the skin (Salminen et al., 2022). Many bacteria present in gut microbiota possess a tryptophanase enzyme which can convert L-tryptophan into indole 3-pyruvic acid (Ma et al., 2020). Subsequently, this compound can be metabolized into several other indole compounds, such as indole 3-aldehyde and indoxyl 3-sulfate, which are effective agonists for AhR signaling. There are some other catabolic pathways which are able to produce potent AhR agonists, e.g., certain derivatives of arachidonic acid, such as prostaglandin G2 (PgG2), lipoxin A4 (LXA4), and 12-hydroxyeicosatetraenoic acid (12-HETE), as well as bilirubin and biliverdin, catabolic products from the heme protein (Nebert, 2017) (Fig. 1). Finally, many dietary compounds, such as phytochemicals, can modify not only the activity of the gut microbiota but also affect the function of AhR in immune cells and peripheral tissues.
The AhR protein is a multifunctional transcription factor which is able to cooperate with many signaling pathways (see above). In addition to the detoxification of xenobiotics, AhR signaling has an important role in microbial defence as well as in the regulation of inflammatory responses and the immunity of the organism (Gutierrez-Vazquez and Quintana, 2018;Rothhammer and Quintana, 2019;Trikha and Lee, 2020). For instance, AhR signaling stimulates the expression of the NADPH oxidase subunits, i.e., the p40phox in mouse liver (Wada et al., 2013) and the p47phox in human macrophages (Pinel-Marie et al., 2009). NADPH oxidase has a crucial role in microbial defence. Moreover, AhR signaling stimulates the expression of the FoxP3 protein, a master regulator of immunosuppressive regulatory T cells (Treg) (Mezrich et al., 2010). AhR signaling also suppresses the function of NLRP3 inflammasomes (Huai et al., 2014) as well as stimulating the expression of the suppressor of cytokine signaling 3 (SOCS3) protein (Wada et al.,

Fig. 1.
AhR signaling impairs circadian regulation. Several factors stimulate the activation of AhR signaling, e.g., (i) inflammaging activates AhR signaling via the IDO1 and NF-κB pathways, (ii) metabolites of heme and arachidonic acid catabolism, (iii) dietary compounds and metabolites generated by microbiota, and (iv) environmental insults induced by UVB radiation and xenobiotics. The activation of AhR signaling impairs the function of circadian clocks leading to disturbances in circadian rhythms. The disruption of circadian rhythms impairs the maintenance of physiological and cellular homeostasis promoting cellular senescence and premature aging process. Abbreviations: AA, arachidonic acid; AhR, aryl hydrocarbon receptor; BMAL1, brain and muscle ARNT-Like 1; 12-HETE, 12-hydroxyeicosatetraenoic acid; IDO1, indoleamine 2,3-dioxygenase 1; LXA4, lipoxin A4; NF-κB, nuclear factor-κB; PgG2, prostaglandin G2; UVB, ultraviolet B radiation. 2016). For instance, Tapinarof, a natural agonist of AhR signaling, has been used as a therapy for atopic dermatitis and psoriasis (Furue et al., 2019). Recently, Trikha and Lee (2020) reviewed in detail the function of AhR signaling in immune cell development and in the responses of the innate and adaptive immunity.
There is convincing evidence that epigenetic mechanisms control the transcription of the AhR gene (Wajda et al., 2020;Akhtar et al., 2022;Habano et al., 2022). The promoter sequences of the mouse and human AhR gene contain GC-rich regions but not a TATA or CCAAT box (Schmidt et al., 1993;Eguchi et al., 1994). Mulero-Navarro et al. (2006) revealed that the CpG sites were hypermethylated and the transcription of the AhR gene down-regulated in several human tumor cells. Interestingly, Ko et al. (2014) demonstrated in mouse embryonal stem cells (ES) that the distal promoter of the AhR gene contained two clusters of the binding sites for pluripotency factors, i.e., NANOG, OCT3/4, and SOX2 proteins. The binding of these factors to the promoter sequences repressed the transcription of the AhR gene, whereas the binding of Sp1/Sp3 factors and the removal of pluripotency factors stimulated the differentiation of mouse ES cells. They also observed that the differentiation of ES cells was associated with increased histone acetylation, especially at the H3K9 site, as well as histone methylation at the H3K4 and H3K27 sites in the proximal promoter of the AhR gene. Short-chained fatty acids (SCFA), especially butyrate produced by gut microbiome, robustly increased the transcription of the AhR gene since SCFAs are potent inhibitors of histone deacetylases (HDAC) and thus they enhanced the level of histone acetylation (Jourova et al., 2022). It is now evident that epigenetic mechanisms regulate not only the expression of the AhR gene but they also control the AhR-mediated transcription of target genes. For instance, there are studies indicating that the Ah receptor could act as a reader of DNA methylation sites recognizing unmethylated CpG sites in the DRE/XRE elements and subsequently this stimulated the transcription of target genes (Habano et al., 2022). Moreover, the activation of AhR signaling induced a robust DNA demethylation at the promoter of the CYP1a1 gene and subsequently stimulated its expression in mouse liver (Amenya et al., 2016). On the other hand, Papoutsis et al. (2012) reported that TCDD exposure induced an AhR-dependent hypermethylation of the CpG island in the proximal promoter of the BRCA-1 gene, which repressed the transcription of this gene in human MCF-7 breast cancer cells. Recently, Akhtar et al. (2022) reviewed in detail the AhR-driven epigenetic regulation in cancer stem cells. The AhR protein can also cooperate with many transposons, e.g., with the Alu retrotransposon regulating the transcription of the NANOG gene which controls cell differentiation during development (Gonzalez-Rico et al., 2020). Mulero-Navarro and Fernandez-Salguero (2016) have clarified the role of the AhR receptor in the expression of its target genes through the cooperation with many retrotransposons. Finally, AhR signaling is also able to regulate gene expression via microRNAs (Disner et al., 2021). It is known that epigenetic regulation controls the function of circadian clocks and vice versa many epigenetic mechanisms are subjected to circadian rhythmicity (Du et al., 2019;Pacheco-Bernal et al., 2019). Currently, it is not known whether there are epigenetic mechanisms involved in the AhR-induced disturbances in circadian regulation.

AhR signaling promotes the age-related degeneration of tissues
According to antagonistic pleiotropy, AhR signaling has a crucial role in development but it can promote tissue degeneration with aging (Salminen, 2022a). However, it needs to be clarified whether AhR signaling directly controls the aging process and lifespan in mammals although there are clear evidences that AhR signaling is involved in many of the degenerative changes appearing in tissues during the aging process (Eckers et al., 2016;Vogeley et al., 2019;Brinkmann et al., 2020;Ojo and Tischkau, 2021;Salminen, 2022a). It is known that AhR signaling can enhance diverse cellular stresses in aged tissues, e.g., oxidative stress, sphingolipid accumulation, and a depletion of NAD + , a crucial coenzyme in metabolism. AhR signaling activates certain target genes which generate ROS/NO compounds, such as (i) CYP1A1 which enhances superoxide production in mitochondria and (ii) the p40phox component of NADPH oxidase present in neutrophils and endothelial cells (Dalton et al., 2002;Wada et al., 2013). For instance, oxidative stress activates NLRP3 inflammasomes which are able to enhance the inflammaging process . Accordingly, the activation of AhR signaling was shown to increase the synthesis of sphingolipids, especially ceramides, by increasing the expression of serine palmitoyltransferase small subunit A (SPTSSA), whereas that of sphingosine 1-phosphate lyase (S1PL) was reduced causing an inhibition of ceramide degradation Majumder et al., 2020). An increase in the amounts of ceramides, i.e., ceramide stress, has been associated with cellular senescence and the aging process (Trayssac et al., 2018). Moreover, it is known that AhR signaling stimulated the expression of TCDD-induced PARP (TiPARP/PARP7) which significantly decreased the intracellular concentration of NAD + (Ma, 2002;Diani-Moore et al., 2017). The cellular level of NAD + significantly declines with aging in several rat tissues, thus disturbing energy metabolism in aged tissues (Braidy et al., 2011). Interestingly, there is robust evidence that the AhR-induced stresses, i.e., an increase in the levels of ROS and ceramides as well as a deficiency of NAD + , can impair the function of circadian clocks and disturb diurnal rhythmicity with aging (Section 3.2.).
The activation of AhR signaling is associated with the appearance of common hallmarks of the aging process although currently it is not known whether the outcomes are either ligand-related or tissue-specific responses. For example, AhR signaling can (i) inhibit autophagy in different conditions (Kim et al., 2020;Kondrikov et al., 2020), (ii), arrest cell proliferation and promote cellular senescence Koizumi et al., 2014;Wan et al., 2014), (iii) augment extracellular matrix (ECM) degeneration (Hillegass et al., 2006;Kung et al., 2009), provoke osteoporosis, tissue fibrosis, and vascular inflammation (Ito et al., 2016;Eisa et al., 2020;Yu et al., 2022), and (iv) induce a remodeling of the immune system, e.g., enhancing thymic atrophy, generating either pro-or anti-inflammatory responses, and activating the cells of the immunosuppressive network (Quintana et al., 2008;Mezrich et al., 2010;Wang et al., 2015;Gutierrez-Vazquez and Quintana, 2018). Given that inflammation is a potent inducer of AhR signaling ( Fig. 1), it seems that AhR signaling is one of the down-stream effectors of the inflammaging-driven tissue degeneration. Currently, there is clear evidence that the disruption of circadian rhythms enhances inflammatory responses (Wang and Li, 2021) and thus it could augment AhR signaling with aging. Moreover, there are studies indicating that AhR signaling is increased in many age-related diseases where it seems to promote pathogenesis (Neavin et al., 2018;Yi et al., 2018).
AhR knockout mice displayed a sick phenotype and experienced a shorter lifespan (Fernandez-Salguero et al., 1995;Schmidt et al., 1996;Bravo-Ferrer et al., 2019). For instance, the knockout mice suffered from cardiovascular lesions, hepatic fibrosis, impairments of the immune system, and increased tumorigenesis. Moreover, the AhR-null mice exhibited features of premature aging, such as increased inflammation and cellular senescence (Bravo-Ferrer et al., 2019;Nacarino-Palma et al., 2022). Nacarino-Palma et al. (2022) reported that with aging the AhR knockout mice established a very high incidence of hepatic tumors. They also reported that the number of senescent cells was robustly expanded in the livers of the knockout mice at the age of 15 months. This seems to indicate that a lack of the AhR protein could accelerate the premature aging process. Recently, Serna et al. (2020) also proposed that the AhR protein could possess anti-aging survival properties since the loss of AhR signaling induced cellular senescence and promoted the premature aging. However, it seems that cellular senescence in the tumorous liver of AhR knockout mice is a defence mechanism against cancer expansion rather than a sign of premature aging. Over twenty years ago, Campisi (2001) proposed that cellular senescence represents a tumor-suppressor mechanism which prevents the expansion of cancers. Interestingly, Zhang et al. (2016) revealed that the AhR-null mice displayed a robust decline in the number of liver-resident natural killer (NK) cells. In addition, Shin et al. (2013) reported that the NK cells from the AhR knockout mice exhibited a clear defect in their tumoricidal activity. It is known that NK cells are involved in the immunosurveillance of senescent cells (Sagiv et al., 2013) as well as cancer cells (Waldhauer and Steinle, 2008), thus it seems that the accumulation of both tumor cells and senescent cells in the livers of AhR-null mice could be attributed to a defect in the immune surveillance capabilities of these mice.

Circadian clockwork
Circadian clocks maintain an internal sleep-wake periodicity of 24 h synchronized by the solar cycle. Early studies indicated that circadian oscillations might have evolved about 2.5 billion years ago after the Great Oxidation Event as a detoxification mechanism for reactive oxygen species (ROS) (Edgar et al., 2012;Loudon, 2012). For instance, Edgar et al. (2012) reported that the oxidation-reduction cycle of peroxiredoxin proteins displays circadian rhythms in all domains of life. However, subsequent studies have revealed that cellular redox oscillations seem to undergo a crosstalk with the circadian clocks which are based on the transcription-translation feedback loops. Currently, the molecular mechanisms of circadian outputs through the transcription-translation repressive loops have been revealed in great detail and described in many review articles (Takahashi, 2017;Honma, 2018;Patke et al., 2020). In humans, the suprachiasmatic nuclei (SCN) in the hypothalamus represent the central circadian pacemaker which synchronizes the oscillations of peripheral clocks via neuronal and humoral signals (Mohawk et al., 2012). For instance, the central clock of the SCN has a crucial role in the control of metabolic rhythms in mouse (Petrus et al., 2022b). Melatonin and cortisol are the major chemical messengers controlling diurnal rhythmicity in the human body. Several external stimuli, e.g., the timing of light/dark and feeding/fasting phases, can reset circadian rhythms meaning that the circadian clocks are entrainable in the organism. The brain and muscle ARNT-like 1 (BMAL1) and the clock circadian regulator (CLOCK) proteins are the drivers of the clockwork in both the SCA and peripheral tissues (Takahashi, 2017;Wu and Rastinejad, 2017). These proteins are transcription factors belonging to the bHLH/PAS family, i.e., they can heterodimerize with each other through their PAS domain. Interestingly, given that the AhR protein also is a PAS-domain protein, the BMAL1/CLOCK proteins can form heterodimers with the AhR protein (Section 3.3.). The PAS domain proteins include many sensor proteins of oxygen level, redox balance, and light sensing (Taylor and Zhulin, 1999). The PAS domains can recruit coactivators which affect the oligomerization and the activity of the PAS complexes (Partch and Gardner, 2010). The canonical BMAL1/CLOCK heterodimer can transactivate hundreds of target genes through the E-boxes in their promoters in a cell-type dependent manner. Given that the activity of circadian clocks oscillates, there exist repressive mechanisms which can suppress the activity of the BMAL1/CLOCK complex (Takahashi, 2017;Honma, 2018). For instance, the BMAL1/CLOCK complex stimulates the transcription of the period (PER) and the cryptochrome circadian regulator (CRY) genes. The PER and CRY proteins form heterodimers which repress the activity of the BMAL1/CLOCK complex, i.e., these proteins represent a negative feedback loop which suppresses the activity of the BMAL1/CLOCK complexes. Subsequently, the PER and CRY proteins have been degraded via proteasomes thus permitting the circadian rhythmicity. Moreover, the REV-ERBα and RORα transcription factors compete for binding to the RORE/RevRE in the promoter sequences of the BMAL1 gene (Preitner et al., 2002;Akashi and Takumi, 2005;Guillaumond et al., 2005). Accordingly, the BMAL1/CLOCK complex stimulates the expression of the REV-ERBα protein which subsequently represses the transcription of the BMAL1 gene by interacting with the RORE/RevRE sites. In contrast, the binding of the RORα protein to the RORE/RevRE site stimulates the expression of the BMAL1 protein. The RORα protein, a retinoic acid-related orphan protein, has many effects on lipid metabolism and inflammation, e.g., it was reported to regulate anti-inflammatory functions in human macrophages (Nejati Moharrami et al., 2018). The circadian rhythmicity is based on the oscillation between the activation of transcriptional enhancers and their translational repressors in both the central oscillator and the peripheral tissues.
In addition to the major feedback regulation, there are several other signaling mechanisms which affect both the core clocks and their feedback systems. Disturbances in these rheostat mechanisms can impair the homeostasis of the circadian rhythmicity and evoke pathological processes, even promoting the aging process. There are certain metabolites, such as NAD + , AMP, and ketone bodies, that can affect the circadian rhythms of both the central and peripheral clocks. For instance, there are diurnal changes in the level of NAD + concentration which modify the function of circadian clocks (Imai, 2010). NAD + is a crucial activator of sirtuins (SIRT) and it is known that SIRT1 can deacetylate and trigger the degradation of the PER2 protein, an inhibitor of the BMAL1/CLOCK complex, and in this way NAD + can stimulate the function of circadian clocks (Asher et al., 2008) (Fig. 2). The activation of the SIRT1 enzyme deacetylates and induces the degradation of histone-lysine N-methyltransferase 2 A (KMT2A), thus suppressing the transcription of the H3K4-dependent genes (Aguilar-Arnal et al., 2015). It is known that the concentration of NAD + clearly declines with aging in several tissues (Gomes et al., 2013) inhibiting the activity of SIRT1, a well-known longevity factor (Salminen and Kaarniranta, 2009). The rhythmic acetylation of the H3 histones is accompanied with the function of the Cry and Per genes in mouse liver (Etchegaray et al., 2003). Currently, there is substantial evidence that many epigenetic mechanisms, not only histone acetylation, regulate circadian rhythms (Pacheco-Bernal et al., 2019) and in this manner, the age-related epigenetic changes might control circadian rhythms. In addition to protein acetylation, protein phosphorylation is another important regulator of circadian rhythms (Narasimamurthy and Virshup, 2021). For instance, AMP-activated protein kinase (AMPK) phosphorylates the CRY and PER proteins inducing their degradation, thus allowing AMPK to enhance the activity of the BMAL1/CLOCK complex and increase the expression of circadian genes (Lee and Kim, 2013) (Fig. 2). It is known that AMPK signaling has an important role in the regulation of the aging process via an integrated Fig. 2. AhR signaling inhibits the activation of the BMAL1/CLOCK complex induced by longevity factors, such as AMPK, SIRT1, and FOXO3. Dietary restriction and pharmacological interventions by metformin and rapamycin enhance the activity of the BMAL1/CLOCK complex. The activity of the BMAL1/CLOCK complex supports healthy aging of the organism. Abbreviations: AhR, aryl hydrocarbon receptor; AMPK, AMP-activated protein kinase; BMAL1, brain and muscle ARNT-Like 1; FOXO3, Forkhead box O3; SIRT1, sirtuin 1. signaling network . Moreover, Chaves et al. (2014) revealed that Forkhead box O3 (FoxO3), a well-known longevity factor (Morris et al., 2015), was responsible for the maintenance of circadian rhythms in mouse liver (Fig. 2). Insulin-FoxO3 signaling stimulated the transcription of the Clock gene via the binding of FoxO3 factor to the DBE element in the promoter of the Clock gene. The knockout of the FoxO3 gene dampened the oscillation of the major clock genes and the IGF1 receptor. These studies indicate that several longevity factors, such as AMPK, SIRT1, and FoxO3, regulate the function of circadian clocks (Fig. 2).
The circadian clockwork has a crucial role in the regulation of both systemic and peripheral physiological processes and disturbances in the diurnal rhythms are encountered in many diseases (Richards and Gumz, 2013;Dibner and Schibler, 2015;Poggiogalle et al., 2018;Patke et al., 2020). Zhang et al. (2014) demonstrated that about 43% of all protein-coding genes in mouse genome were under circadian regulation. In addition, non-coding RNAs also control circadian rhythmicity (Mehta and Cheng, 2013). Circadian clocks regulate systemic and cellular metabolism, immune responses, and the functions of the endocrine, reproductive, renal, and cardiovascular systems. With respect to the aging process, probably all of those are modified but here it is better to focus on metabolism and immunity since both processes are robustly affected by aging and AhR signaling. It is known that the expression of metabolic enzymes oscillates and thus circadian clocks are able to control metabolic rates in tissues. For example, the REV-ERBα and RORα proteins have an important role in the regulation of lipid metabolism (Solt et al., 2011) and a deficiency of the REV-ERB protein aggravated steatosis in mouse liver (Bugge et al., 2012). Moreover, the peroxisome proliferator-activated receptor γ (PPARγ) protein integrates circadian clocks and energy metabolism in adipocytes, i.e., a disruption of the natural circadian rhythm can promote obesity (Wang et al., 2022b). In addition, AMPK and SIRT1, important circadian regulators (see above), are well-known guardians of cellular energy status (Chang and Guarente, 2014;Lin and Hardie, 2018). Given that inflammaging is associated with metabolic disorders enhancing chronic inflammatory state (Franceschi et al., 2018a), it seems that disruptions in circadian rhythms via AhR signaling could trigger metabolic disturbances and thus promote the aging process and age-related metabolic diseases.
There is convincing evidence that circadian clocks regulate the diurnal expression of immune genes and control inflammatory conditions but on the other hand, inflammatory pathways, especially NF-κB signaling, can reciprocally manipulate the function of circadian clocks and thus disturb diurnal rhythmicity (Cermakian et al., 2013;Scheiermann et al., 2018;Hergenhan et al., 2020;Wang et al., 2022a). Several studies have revealed that the RelA and RelB proteins, the transactivating components of NF-κB complex, can physically interact with the BMAL1 protein in the presence of the CLOCK protein in the E-boxes of circadian target genes (Bellet et al., 2012;Shen et al., 2021). The interaction of the RelA or RelB protein to the transactivation domain of the BMAL1 protein represses the transcription of circadian-driven genes and impairs diurnal rhythmicity. Interestingly, the RelB protein can also bind to the AhR protein and thus it was able to control the expression of IL-22 and CCL20 induced by AhR ligands in mouse bone marrow-derived macrophages and the thymus (Vogel et al., 2007;Ishihara et al., 2019). There is abundant evidence that the administration of pro-inflammatory mediators, such as TNF-α and LPS, suppressed the expression of clock genes and their target genes, both in the SCN and peripheral tissues (Cavadini et al., 2007;Okada et al., 2008;Cermakian et al., 2013). It is known that inflammatory conditions can disturb circadian rhythmicity, e.g., during microbial attacks (Scheiermann et al., 2018). On the other hand, it has been revealed that circadian clocks can control the functions of the immune system, e.g., regulating the intensity of inflammatory responses (Scheiermann et al., 2018;Hergenhan et al., 2020;Timmons et al., 2020;Wang et al., 2022a). For instance, disturbances in the expression of clock genes appear in the early phase of many inflammatory diseases, such as inflammatory bowel disease (IBD) (Weintraub et al., 2020). Currently, chronotherapeutic strategies are popular in drug discovery programs for the treatment of chronic inflammatory diseases (Jacob et al., 2020). There seems to exist different mechanisms through which circadian clocks can suppress inflammatory responses. For instance, Wang et al. (2018) demonstrated that the REV-ERBα protein repressed the transcription of the Nlrp3 gene in mice via the binding to the RevRE site in the promoter of the Nlrp3 gene. The expression of Nlrp3 mRNA reached its peak in both mouse liver and colon during the dark time, whereas a low level of expression existed during the light period . Given that the NLRP3 inflammasomes act as a crucial pro-inflammatory mechanism in the innate immune system, this explains why the intensity of inflammatory responses also oscillates in inflammatory diseases (Cermakian et al., 2013). Interestingly, it is known that melatonin alleviates the function of NLRP3 inflammasomes and thus it seems to have therapeutic activity in relieving the symptoms of many inflammatory diseases (Arioz et al., 2021).

Circadian rhythms are impaired in the aging process
A deterioration of circadian rhythms is one of the hallmarks of the aging process and there are many review articles examining in detail the age-related decline in the function of circadian clocks in both the SCN and peripheral tissues (Weinert, 2000;Kondratov, 2007;Froy, 2011;Hood and Amir, 2017;Welz and Benitah, 2020). Currently, it is not known whether this circadian disruption is a promoting cause or a consequence of the degenerative aging process. It is evident that aging disturbs circadian regulation at multiple levels, i.e., the central and peripheral clocks, behavioral and physiological rhythms, as well as via cellular, molecular, and epigenetic mechanisms. The common markers that link circadian disruption with aging include a significant decline in the amplitude of oscillating rhythms, a diminished synchronization of the rhythms, and an impaired phase-shifting times to external stimuli. There are major structural and functional changes that take place in the SCN, such as a decline in the numbers of some specific neurons and a reduced coupling of neurons within the SCN and other hypothalamic nuclei, thus impairing the stability of the diurnal rhythms (Weinert, 2000;Nakamura et al., 2011;Hood and Amir, 2017). For instance, the number of neurons expressing arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) was clearly reduced in the human SCN with aging. Chang and Guarente (2013) demonstrated that SIRT1 stimulated the expression of the BMAL1 and CLOCK in mouse SCN amplifying their circadian oscillations involving also those rhythms occurring in SIRT1 expression. They observed that the SCN of aged mice contained a clearly decreased level of the SIRT1 protein as well as the expression of the BMAL1 and PER2 proteins was significantly reduced as compared to that present in young mice. Accordingly, circadian rhythms of the aged mice were disrupted and were unable to adapt to changes in light entrainment. They also reported that young mice with the knockout of SIRT1 displayed the circadian disturbances reminiscent of those in the SCN of aged mice. These results indicated that the SIRT1 protein has a crucial role in the control of the central pacemaker and the decline of SIRT1 in the SCN with aging might also disturb the functions of peripheral clocks. Aging also resets the hormonal and neural outputs from the SCN which directly or indirectly control the peripheral rhythms, e.g., sleep-wake cycle and metabolic oscillations (Weinert, 2000;Froy, 2011).
Interestingly, it is known that caloric restriction and different feeding regimens, e.g., intermittent fasting and time-restricted feeding (TRF), can control the circadian clocks in the SCN and peripheral tissues (Froy et al., 2008;Froy and Miskin, 2010;Mendoza et al., 2012;Franceschi et al., 2018b). There is clear evidence emerging from rodent models that feeding entrainment can enhance circadian rhythmicity of gene transcription. Recently, Deota et al. (2023) demonstrated that TRF treatment affected the transcription of nearly 80% of the genes in the samples taken from 22 mouse organs and brain regions. TRF enhanced the rhythmicity of global gene expression in major metabolic tissues although many changes were tissue-and pathway-specific responses. For instance, the TRF protocol reduced the expression of the genes associated with inflammatory signaling and glycerolipid metabolism, whereas it increased the expression of many genes involved in the maintenance of tissue homeostasis, such as RNA processing, protein folding, and autophagy. Wei et al. (2022) also reported that there seems to exist tissue-specific oscillation signatures of gene expression in the tissues of mice subjected to an intermittent fasting protocol. They revealed that proteasomes coordinate the transcriptional switch during intermittent fasting, especially in the liver. It seems that the increased longevity associated with caloric restriction and intermittent fasting could be attributed to the feeding-induced entrainment in circadian regulation. Given that repeated low-level stresses induce hormetic adaptation, it seems that dietary interventions could act as hormetic modulators and thus enhance healthspan and even lifespan (Santoro et al., 2020).
The molecular mechanisms underpinning the decline in the function of circadian clocks with aging still need to be clarified. There are several transcriptome profiling studies investigating the age-related alterations in the activity of circadian clocks in different tissues (Kolker et al., 2003;Chen et al., 2016;Sato et al., 2017;Barth et al., 2021;Blacher et al., 2022). However, the expression studies have not revealed any consistent age-related changes in circadian rhythms and moreover, there seem to be robust, tissue-specific differences in the diurnal expression of clock proteins. This might indicate that the maintenance of circadian rhythms is unrelated to protein expression levels of the core clocks but rather it is under the control of other mechanisms, e.g., heterodimerization of PAS-containing proteins (Section 3.3.) or functions of co-activators and repressors in the canonical core complex. However, in the rodent SCN, the expression of the Bmal1 gene seems to be consistently downregulated with aging (Kolker et al., 2003;Chang and Guarente, 2013;Bonaconsa et al., 2014). It is known that the expression of the Bmal1 gene is epigenetically regulated via DNA methylation in cultured human cells (Satou et al., 2013). Given that both the circadian clocks and the aging process are under epigenetic regulation (Sen et al., 2016;Cronin et al., 2017;Samoilova et al., 2021), it could be argued that the CpG sites at the regulatory sequences of the Bmal1 gene have a crucial role in the age-related regulation of circadian clocks.
The knockout mouse models have provided an interesting approach to elucidate the role of clock genes in the regulation of the aging process. Kondratov et al. (2006) demonstrated that the knockout of the mouse Bmal1 gene resulting in a phenotype reminiscent of the premature aging process associated with many age-related diseases. The Bmal1-null mice displayed a disruption of circadian rhythms and a clearly reduced lifespan. The premature aging syndrome involved sarcopenia, osteoporosis, cataracts, and a reduction in the amount of subcutaneous adipose tissue. They also detected an increased level of ROS compounds in many tissues although it probably was not the primary cause of tissue degeneration. Moreover, Ali et al. (2015) reported that the depletion of the Bmal1 gene induced an accelerated age-dependent decline in the neurogenesis of adult mice. Subsequently, Yang et al. (2016) used a conditional Bmal1 knockout technology to compare the results between conventional and conditional knockout models. They reported that the conditional knockout of the Bmal1 gene in adult mice inhibited the circadian oscillations but only minor changes appeared in their phenotype. However, Barca-Mayo et al. (2020) demonstrated that the conditional knockout of the Bmal1 gene in mouse astrocytes impaired the activities of the hypothalamic clocks, induced age-dependent astrogliosis and astrocyte apoptosis, disturbed glucose metabolism, and reduced the animals' lifespan. The cardiomyocyte-specific deletion of the mouse Bmal1 gene developed age-related cardiac hypertrophy associated with fibrosis, accelerated degeneration of the extracellular matrix, and impaired the resolution of inflammation (Ingle et al., 2015). Huo et al. (2017) revealed that the deletion of the Bmal1 gene in mouse myeloid cells increased the proinflammatory M1 phenotype of macrophages. Accordingly, the Bmal1 deficiency promoted atherogenesis by increasing the recruitment of monocytes into atherosclerotic lesions. There are also studies indicating that the lack of the Bmal1 gene in myeloid cells enhanced proinflammatory responses in chronic inflammatory diseases, such as in mouse sepsis (Curtis et al., 2015;Early et al., 2018). Given that the activation of NF-κB signaling inhibits the function of the Bmal1 protein (Section 3.1.), it seems that the increased activity of NF-κB signaling with aging (Salminen et al., 2008) can inhibit the Bmal1-driven transcription and thus promote the age-related inflammatory state. These studies indicate that a deficiency of the Bmal1 gene can impair circadian regulation and promote a premature aging process although it still needs to be clarified whether the circadian-independent, i.e., the so-called moonlighting, functions of the Bmal1 protein, e.g., the stabilization of telomeric heterochromatin, affect the aging process (Park et al., 2019). Dubrovsky et al. (2010) demonstrated that the mice with a knockout of the Clock gene did not develop a similar premature aging phenotype as the deficiency of the Bmal1 gene although these two factors form the transactivating heterodimeric complex. However, the Clock-null mice displayed a shorter lifespan than their wild-type counterparts and the deficiency of the Clock gene increased the incidence of cataract and dermatitis with aging. Another model, the Clock mutant mice (lack of exon 19) displayed an age-dependent cardiac hypertrophy (Alibhai et al., 2017) as well as displaying an accelerated aging phenotype if these mutant animals were exposed to a low-dose irradiation (Antoch et al., 2008). There are also knockout and mutant models of the Per genes which have revealed that a deficiency of the Per1 and Per2 genes enhanced the intensity of pro-inflammatory responses to inflammatory stimuli, e.g., LPS exposure (Wang et al., 2016). In the mouse brain, the ablation of the Per1 gene increased presenilin immunoreactivity, activated microglia, and induced an accumulation of β-amyloid and lipofuscin (Börner et al., 2021). Bacalini et al. (2022) demonstrated in an Italian population that there was a significant association between the rs3027178 polymorphism in the human PER1 gene with both Alzheimer's disease (AD) and longevity. Interestingly, the G allele decreased the risk for AD but concurrently it reduced the expectancy of an extended lifespan. Recently, Zhu et al. (2022) reviewed the currently identified polymorphic sites in the clock genes which were associated with the hallmarks of aging and age-related diseases. Many of the polymorphic sites were associated with cognitive impairment, sleep disorders, and the risk for AD and Parkinson's disease.
There is robust evidence that circadian rhythms are disturbed during the aging process. However, it still needs to be clarified how these processes are connected with each other, i.e., it is unclear whether a decline in the function of circadian clocks promotes the aging process or whether the aging process impairs the circadian regulation. It seems probable that it is not a one-way street but involves feedback or even feed-forward systems. There exists a close reciprocal regulation between circadian clocks and the activity of the mammalian target of rapamycin (mTOR) (Cao, 2018;Ramanathan et al., 2018;Wu et al., 2019). This is an interesting connection since mTOR signaling is a crucial modifier of the aging process and age-related diseases (Johnson et al., 2013). There is substantial evidence that the circadian clock proteins can control the activity of mTOR signaling and thus regulate the aging process (Khapre et al., 2014;Cao, 2018;Wu et al., 2019). For instance, Khapre et al. (2014) demonstrated that the Bmal1 protein inhibited the activity of mTOR complex 1 (mTORC1) in mouse fibroblasts. They also revealed that a deficiency of the Bmal1 protein increased the activity of mTORC1 and elevated protein synthesis in cultured cells and mouse liver. The increase in the activity of mTORC1 significantly accelerated the aging process in the Bmal1 knockout mice. It is known that rapamycin, an inhibitor of mTORC1 signaling, increases the lifespan of mice (Blagosklonny, 2019). Wu et al. (2019) revealed another mechanism, a Per2-dependent signaling, which suppressed the activity of mTORC1 in the cytoplasm of mouse hepatocytes. They reported that the Per2 protein acted as a scaffold protein which tethered the tuberous sclerosis complex 1 (Tsc1), Raptor, and mTOR together in the cytoplasm. This complex suppressed the activity of mTORC1 thus inhibiting protein synthesis while enhancing autophagy. On the other hand, there are studies indicating that mTORC1 signaling can regulate the function of the central and peripheral clocks (Cao, 2018;Ramanathan et al., 2018). Ramanathan et al. (2018) revealed that the inhibition of mTOR signaling reduced the amplitude and lengthened the wave periods of circadian rhythms in mouse hepatocytes, whereas the activation of mTOR augmented the amplitudes and shortened the wave lengths. A constitutive activation of mTOR signaling increased the expression of the Bmal1 and Clock genes in mouse hepatocytes. It is known that an activation of mTOR can enhance circadian rhythmicity in both the SCA and peripheral clocks (Cao, 2018). There are several other age-related signaling pathways which are known to regulate the circadian clocks and the aging process, such as inflammation-related signaling and SIRT1 activation (Section 3.1.).

AhR signaling impairs circadian regulation
Circadian clocks and the AhR protein are environmental sensors which evolved in microorganisms early during evolution. Currently, it is known that these factors have many mutual interactions, e.g., the expression of the AhR protein is under circadian regulation and thus also the daily defence against xenobiotics and some endogenous insults shows diurnal oscillation (Richardson et al., 1998;Huang et al., 2002;Claudel et al., 2007;Tanimura et al., 2011) (Fig. 1). There is convincing evidence that the activation of AhR signaling inhibits the expression of circadian clock genes and accordingly impair diurnal rhythms in different experimental models (Mukai et al., 2008;Shimba and Watabe, 2009;Xu et al., 2013;Fader et al., 2019). For instance, Fader et al. (2019) demonstrated that TCDD exposure reduced the amplitude of the daily oscillations of lipid and glucose metabolism in mouse liver. They reported that this was attributable to a decrease in the expression level and rhythmicity of several clock genes, e.g., the Bmal1, Per1, Per2, and Cry1 genes. Accordingly, Xu et al. (2013) reported that the exposure to β-naphthoflavone (BNF), a potent inducer of AhR signaling, inhibited the light-induced transcription of the Per1 gene in both mouse SCN and liver. Jaeger et al. (2017) demonstrated that a deficiency of the Ah receptor even enhanced the amplitudes of circadian rhythms by increasing the expression of the core clock genes in mouse liver. These studies clearly indicated that the AhR protein could act as a suppressor of circadian rhythms in both central and peripheral clocks. Petrus et al. (2022a) demonstrated that certain tryptophan metabolites regulated the rhythmicity of circadian clocks in mouse SCN and liver. It is known that these tryptophan metabolites are potent agonists of AhR signaling through the IDO1/KYN/AhR pathway (Fig. 1). There is substantial evidence that inflammatory conditions disrupt diurnal rhythmicity (Anderson et al., 2013;Xu et al., 2021). Interestingly, the inflammaging state is associated with an activation of IDO1 which subsequently stimulates the KYN pathway (Salminen, 2022b), probably impairing circadian rhythmicity with aging. The gut microbiota is another source which generates different agonists of AhR signaling (Section 2.1.). There is robust evidence that the gut microbiome is able to modulate host diurnal rhythms and thus it can affect human health (Parkar et al., 2019).
Currently, it is known that AhR signaling disturbs circadian rhythmicity and vice versa circadian clocks control the expression of the AhR protein. Interestingly, the AhR and ARNT proteins and circadian core clocks, i.e., the BMAL1 and CLOCK proteins, are members of the conserved bHLH/PAS domain family. Although the heterodimeric AhR/ ARNT and CLOCK/BMAL1 complexes are canonical complexes, there are several studies indicating that the AhR protein can bind to the BMAL1 protein via their PAS domains (Xu et al., 2010;Jaeger and Tischkau, 2016;Tischkau, 2020). These complexes seem to be inhibitory since they suppress the formation and activity of the canonical complexes. Xu et al. (2010) demonstrated that treatment of mouse hepatoma cells with BNF induced the formation of the AhR/BMAL1 complexes but concurrently this treatment decreased the level of the canonical CLOCK/BMAL1 complexes. Correspondingly, the activation of AhR signaling repressed the CLOCK/BMAL1-induced transactivation of the Per1 promoter. The transfection of hepatoma cells with the AhR gene also repressed the CLOCK/BMAL1-induced activation of the Per1 promoter. The activation of AhR signaling displaced the CLOCK protein from the promoter of the Per1 gene and thus inhibited the transcription of the Per1 gene. Tischkau et al. (2011) reported that administration of TCDD inhibited the expression of the Bmal1 transcripts in mouse ovary. They also revealed that the AhR protein interacted with the BMAL1 protein and the inhibitory interaction was enhanced after TCDD treatment. Recently, Khazaal et al. (2023) demonstrated that AhR signaling inhibited the transcription of many lipolytic genes as a result of the binding of the AhR protein to the circadian-driven E-boxes at their promoter sequences. It is known that circadian clocks control lipolysis and thus AhR signaling seems to inhibit lipolysis. The role of AhR signaling in the control of lipid metabolism is underlined by the fact that the AhR knockout mice are slim and protected against a diet-induced obesity as well as being resistant to hepatic steatosis and insulin resistance (Xu et al., 2015). These studies indicate that the activation of AhR signaling impairs circadian rhythms by disturbing the oligomerization of the core clock proteins and thus inhibiting their transactivation functions.
Interestingly, the promoter sequences of the circadian core clock genes contain several putative binding sites for the AhR protein, especially the regulatory regions of the Clock, Rorα, Per2, Cry1, and Cry2 genes (Fader et al., 2019). However, their role in the transcription of circadian genes needs to be clarified. Fader et al. (2019) examined the changes of transcriptome profiles in the liver of the TCDD-treated mice. They observed that TCDD exposure affected the diurnal expression of over 5000 genes. For instance, the transcription levels of Bmal1, Clock, Per1, and Cry1 genes were clearly diminished and the rhythmic expression of the clock-controlled hepatic genes was mostly abolished. Concurrently, the rhythms of oscillating metabolites were lost following TCDD treatment. Fader et al. (2019) also demonstrated that there was an enrichment in the binding of the AhR protein to the DRE sites at the promoter sequences of seven clock genes out of 15 genes. The binding of the AhR protein probably induced a silencing of these clock genes through epigenetic regulation. It is known that the TCDD-activated AhR signaling can stimulate the methylation of the CpG islands at the promoter of the BRCA-1 gene, thus silencing its transcription (Papoutsis et al., 2012). It is known that the transcription of many clock genes is controlled through epigenetic mechanisms (Papazyan et al., 2016;Du et al., 2019). For instance, Cronin et al. (2017) demonstrated that the rhythmic DNA methylation correlated with the expression of the Bmal1 gene in human fibroblasts and brain tissue. Interestingly, they reported that the early stages of Alzheimer's disease were associated with an aberrant methylation pattern in the promoter of the Bmal1 gene. It seems that the Ah receptor can inhibit the expression of some clock genes via epigenetic regulation and thus disturb circadian rhythms.
There are several other mechanisms through which AhR signaling can reduce the activity of clock genes and thus lead to disturbances in circadian rhythms. For instance, it is known that AhR signaling induces the expression of microRNAs which are able to affect circadian clocks. Recently, Kinoshita et al. (2020) compiled a list of all miRs known to control circadian regulation and induce disruption of circadian rhythms. Accordingly, Disner et al. (2021) enumerated a number of miRs produced by AhR signaling and their primary targets were specified. For instance, AhR signaling stimulated the expression of miR-132/212, especially in neuronal and immune compartments (Wanet et al., 2012;Nakahama et al., 2013). The induction was not present in the AhR knockout mice. The expression of miR-132/212 displayed signs of circadian oscillation and targeted several mRNAs (Wanet et al., 2012). It seems that the effects of miR-132 on the circadian clocks are mediated by targeting the mRNAs of the methyl CpG binding protein 2 (MeCP2) and Sirt1 genes disturbing neuronal plasticity and cognition in mice (Aten et al., 2018). Alvarez-Saavedra et al. (2011) demonstrated that light exposure induced the expression of miR-132 in mouse SCN and attenuated the resetting of circadian clocks. They reported that miR-132 reduced the binding of MeCP2 and Jarid1a proteins to the promoters of mPer1 and mPer2 genes. The light-induced expression of mir-132 attenuated the level of the mPer1 and mPer2 proteins in the mouse SCN and thus an excessive expression of miR-132 by AhR signaling would be able to disrupt circadian regulation. The light-stimulated generation of FICZ is a potential inducer of AhR signaling in the SCN (Mukai and Tischkau, 2007). The expression of miR-132 is a possible enhancer of sleep disorders caused by a disruption of the circadian clocks (Davis et al., 2011). The activation of AhR signaling can also downregulate the expression of SIRT1 via the miR-132 axis (Aten et al., 2018) and thus inhibit the degradation of the PER2 protein and consequently disturb diurnal rhythms (Asher et al., 2008). Moreover, Levine et al. (2020) demonstrated that the age-related decrease in the level of NAD + reduced the activity of SIRT1 inducing an accumulation of acetylated PER2 protein which evoked a prolonged PER2-induced repression of the CLOCK/BMAL1 complex and dampened circadian oscillations. It seems that an excessive AhR signaling can disturb circadian regulation via different signaling connections, probably in a tissue-dependent manner.

Does AhR signaling promote the ageing process by impairing circadian regulation?
As described earlier, AhR signaling impairs the function of circadian clocks as well as promoting the aging process, however, it is not known whether or not it enhances the aging process, e.g., via disturbing diurnal autophagy or immune regulation driven by circadian clocks. For instance, circadian clocks control autophagy, a process which is known to be impaired by AhR signaling and which is significantly declined with aging. The control of autophagy is an important tool exploited by circadian clocks in the diurnal regulation of cellular metabolism Wang et al., 2020;Juste et al., 2021). The circadian control of autophagy involves several signaling mechanisms which upregulate the expression of the genes stimulating autophagy. Ma et al. (2011) demonstrated that the CCAAT/enhancer-binding protein-β (C/EBPβ) increased the expression of the Ulk1, LC3B, and Bnip3 genes, all of which are important autophagy genes, in mouse liver. The C/EBPβ factor transactivated the expression of these genes and subsequently increased the protein degradation in mouse liver. Ma et al. (2011) also revealed that the oscillation of autophagic flux was dependent on the circadian and nutritional regulation of the Bmal1 gene. Mammucari et al. (2007) reported that the FoxO3 factor, an inducer of the Clock gene, also stimulated the transcription of many autophagy-related genes, e.g., the LC3 and Bnip3 genes, and increased lysosomal protein degradation in mouse skeletal muscle during atrophy. Several other signaling pathways are known to be involved in the circadian regulation of autophagic activities, e.g., the AMPK and TFEB/TFE3 pathways (Pastore et al., 2019;Wang et al., 2020). On the other hand, there are observations indicating that the chaperone-mediated autophagy (CMA) is able to undertake the rhythmic removal of selective clock proteins, a process called selective chronophagy (Toledo et al., 2018;Juste et al., 2021). Selective chronophagy is thought to target several clock proteins, e.g., the Bmal1, Clock, Cry1, and Rev-Erbα proteins, in mouse tissues. The selective degradation of clock proteins affected the cellular proteome inducing a CMA-dependent circadian sub-proteome (Juste et al., 2021). However, the activity of circadian CMA was under the regulation of the BMAL1 factor in mouse liver, an indication of a mutual regulation between the circadian clocks and CMA. Ulgherait et al. (2021) demonstrated in Drosophila that the circadian control of autophagy could promote the increase in longevity mediated by the intermittent time-restricted feeding. Interestingly, the night-specific induction of autophagy extended lifespan, whereas the day-specific induction did not increase the lifespan of Drosophila. Intermittent and periodic fasting protocols are also known to increase healthspan and lifespan in mammals (Longo et al., 2021).
There are several studies indicating that AhR signaling can suppresses autophagic activity in many experimental models (Tsai et al., 2017;Kim et al., 2020;Kondrikov et al., 2020;Kim et al., 2022). For instance, Kim et al. (2020) demonstrated that the activation of the Ah receptor by exposure to TCDD increased the expression of the AhR and CYP1A1 proteins, whereas TCDD significantly decreased the expression of many autophagy-related factors, such as ATG5, Beclin 1, and LC3, and augmented the accumulation of autophagosomes into human keratinocytes. Accordingly, the knockdown of AhR expression prevented the TCDD-induced down-regulation of the autophagy-related factors and the deposition of autophagosomes indicating that an increased AhR signaling inhibited autophagy in human keratinocytes. Tsai et al. (2017) revealed that AhR signaling promoted the ubiquitination of BNIP3 protein, an enhancer of mitophagy (Ney, 2015), and subsequently induced its proteosomal degradation in human A549 cells. Recently, Kondrikov et al. (2020) reported that KYN exposure disrupted autophagic flux via the activation of AhR signaling in bone marrow mesenchymal stem cells (BMSC) obtained from aged mice. The administration of KYN at the physiological level repressed autophagy which was induced by a serum-deficiency in BMSCs. This is an interesting observation because inflammation stimulates AhR signaling via the IDO1-KYN pathway (Fig. 1). The mechanisms underpinning the AhR-induced suppression of autophagy still needs to be clarified although the ubiquitination of BNIP3 clearly seems to be one of these mechanisms. In addition, Polager et al. (2008) reported that the E2F1 transcription factor induced autophagy by stimulating the expression of the ATG1, ATG5, LC3, and DRAM genes in human osteosarcoma cells. There are studies indicating that AhR signaling is a potent inhibitor of the E2F1-mediated signaling (Marlowe et al., 2008). On the other hand, many studies have revealed that the inhibition of autophagy robustly increased the expression of the AhR and CYP1A1 proteins. It is known that the AhR protein is degraded via selective autophagy in diverse human cell lines (Yang and Chan, 2020). This clearly indicates that AhR signaling and autophagy establish a rheostatic balance, similarly as performed by circadian clocks and autophagic activity (Wang et al., 2020). Currently, it is known that the expression of AhR mRNA peaks at noon, whereas that of BMAL1 is reduced during daytime and achieves its highest-level during the night (Richardson et al., 1998;Shimba and Watabe, 2009;Shi et al., 2016). However, there exist tissue-specific differences in oscillation times. Moreover, feeding times affect the diurnal rhythms of autophagy, e.g., in the liver. It seems that the regulation of AhR activity and circadian entrainment are crucial regulators of autophagy affecting cellular proteostasis in the aging process and many age-related diseases.
It is known that the knockout of core clock genes induces a premature aging phenotype and reduces the lifespan in mouse models (Section 3.2.). Inokawa et al. (2020) also demonstrated that the chronic circadian misalignment of mice, i.e., a chronic jet-lag protocol, desynchronized the circadian rhythms in the SCN and decreased the lifespan of mice. The mRNA expression analysis revealed a suppression of the genes controlling the circadian feedback loops in mouse liver and kidney. In addition, a systemic enhancement of inflammatory responses and the appearance of immune senescence were observed in the liver and kidney of diurnally misaligned mice. Khapre et al. (2011) demonstrated that the depletion of the Bmal1 protein increased the accumulation of senescent cells within lungs, liver, and spleen of mice. The Bmal1-deficiency increased the sensitivity of mouse fibroblasts to oxidative stress and also impaired circadian oscillations in cultured fibroblasts. On the other hand, there are studies indicating that cellular senescence can impair the function of circadian clocks in both in vitro and in vivo models (Kunieda et al., 2006;Ahmed et al., 2019;Ahmed et al., 2022). For instance, senescent human TIG-3 cells displayed circadian rhythms with decreased amplitudes and a delayed peak-time of BMAL1, PER1, and PER2 mRNAs (Ahmed et al., 2019). Given that the aging process increases the accumulation of senescent cells within tissues, it seems that this process could impair the function of clock genes both in the SCN and peripheral tissues. It is known that AhR signaling stimulates the cellular senescence in different experimental models (Koizumi et al., 2014;Wan et al., 2014;Kondrikov et al., 2020). Interestingly, an increased AhR signaling has been associated with disturbances in circadian clocks in several age-related diseases, such as cardiovascular diseases (Thosar et al., 2018;Yi et al., 2018) and neurodegenerative diseases (Ojo and Tischkau, 2021;Nassan and Videnovic, 2022).
There is robust evidence that both AhR signaling and circadian clocks regulate the function of immune system, especially the intensity of inflammatory responses. Circadian clocks can display different antiinflammatory effects, e.g., the REV-ERBα protein is able to inhibit NF-κB signaling as well as repressing the function of NLRP3 inflammasomes . Interestingly, melatonin, a synchronizer of circadian oscillations, can control the expression of many circadian genes (Zeman and Herichova, 2013) but it also inhibits the activity of the NLRP3 inflammasomes (Arioz et al., 2021). The function of immune cells is known to be subjected to circadian rhythms and circadian clocks control the activity of many immune cells, e.g., macrophages, NK cells, and T cells (Cermakian et al., 2013;Hand et al., 2020;Zeng et al., 2020;Blacher et al., 2022). For instance, Zeng et al. (2020) demonstrated that chronic shift-lag cycles disturbed the circadian rhythmicity in mouse NK cells and subsequently impaired their immunosurveillance activity and promoted the aging phenotype of NK cells. This is an important observation since NK cells are driving the immunosurveillance of senescent and cancer cells (Waldhauer and Steinle, 2008;Sagiv et al., 2013). Ovadya et al. (2018) revealed that an impairment in the activity of NK cells increased the accumulation of senescent cells in several mouse tissues. Accordingly, Logan et al. (2012) reported that a loss of circadian rhythms reduced the cytolytic activity of NK cells and impaired the immunosurveillance against the development of rat lung tumors. Several studies have revealed that a disruption of circadian rhythms can stimulate the function of NLRP3 inflammasomes and promote inflammatory responses (Cermakian et al., 2014;Fernandez-Ortiz et al., 2022). For instance, the inflammaging state is associated with the activation of NLRP3 inflammasomes . On the other hand, there is substantial evidence that cytokines and other inflammatory mediators disrupt the rhythms of the circadian clocks (Cavadini et al., 2007;Cermakian et al., 2014). For instance, Cavadini et al. (2007) reported that TNF-α exposure down-regulated the expression of the E-box-driven clock genes, such as the Per1-3 genes, in mouse liver, whereas the expression of the Bmal1 gene, an E-box-independent gene, was unaffected. There is robust evidence that the inflammaging process is associated with disturbances in the diurnal rhythms and the circadian-driven functions in immune cells, e.g., the cytotoxicity of lymphocytes, which might impair the maintenance of homeostasis with aging (Mazzoccoli et al., 2010;Blacher et al., 2022;Colombini et al., 2022).
As discussed in the Section 3.3., AhR signaling impairs the function of circadian clocks inducing the disruption of circadian rhythmicity. By disrupting circadian regulation, AhR signaling exposes tissues, especially aged tissues, to pro-inflammatory responses. Blacher et al. (2022) demonstrated that macrophages from aged mice had undergone a clear decrease in the expression of circadian clock genes and a loss of circadian rhythmicity. Disruption of circadian regulation was associated with increased inflammatory responses, impaired phagocytosis, and enhanced vulnerability to infections. There is convincing evidence that inflammatory mediators, such as cytokines and PGE2, activate the expression and signaling of the AhR protein (Vogel et al., 2014;Salminen, 2022b). Age-related chronic inflammation is associated with a positive feedback loop where the inflammatory state, the origin of which is largely unknown, stimulates AhR signaling which through disturbing circadian rhythms maintains and even promotes an inflammatory state. However, AhR signaling can also trigger a negative feedback loop inducing an immunosuppressive state which counteracts inflammatory insults. AhR signaling exploits different mechanisms to enhance anti-inflammatory responses. For instance, it is known that the Ah receptor transactivates the FoxP3 gene and induces the differentiation of immunosuppressive Tregs (Quintana et al., 2008). Moreover, AhR signaling can trigger the differentiation of regulatory B cells (Tousif et al., 2021) and tolerogenic dendritic cells (Barroso et al., 2021) as well as promoting the polarization of macrophages towards the anti-inflammatory M2 phenotype . In addition, AhR signaling can transactivate the Socs3 gene and thus prevent the appearance of hepatic steatosis induced by the feeding a high-fat diet to mice (Wada et al., 2016). Given that AhR signaling displays antagonistic pleiotropy with aging, it seems that a chronic low-grade inflammation enhances the expression of the Ah receptors which promote the aging process by disturbing circadian regulation and enhancing an immunosuppressive state. The increased immunosuppression with aging remodels the function of immune system and promotes cellular aging process (Salminen, 2020;Salminen, 2021).

Remarks for future research
The circadian clockwork has not only a crucial role in the regulation of daily metabolism but it also controls the diurnal activity of the immune system. Many age-related diseases are associated with a disruption of circadian rhythms although it is not known whether the impairment is a cause or only a consequence of pathological processes. Currently, there are many observations indicating that the core clocks have many moonlighting activities which might promote the aging process. For instance, Lipton et al. (2015) demonstrated that the BMAL1 protein can act as a translation factor in the cytoplasm of mouse fibroblasts and thus the BMAL1 protein can control the circadian rhythm of protein synthesis in a transcription-independent manner. Moreover, the expression of the Bmal1 gene is epigenetically regulated but on the other hand, the BMAL1 protein can stabilize heterochromatin in a non-canonical manner (Liang et al., 2022). For instance, Liang et al. (2022) reported that the BMAL1 protein could bind to and inactivate the human LINE1 retrotransposon which is able to promote senescence in primate cells. It is still unclear whether or not these BMAL1-dependent functions are involved in the age-related loss of heterochromatin and the down-regulation of protein synthesis. It is known that the AhR protein interacts with the BMAL1 and the CLOCK proteins via their PAS domains. However, these interactions should be specified by exploiting the novel techniques of structural biology. It seems that the expression of the AhR protein is controlled by circadian clocks but the oscillation mechanisms will need to be better unraveled to understand whether AhR signaling is a repressive component in the circadian feedback loops and thus a potential age-related inhibitor of circadian regulation. Given that inflammation is a potent activator of AhR signaling, it seems that arachidonic acid and KYN metabolites have a crucial role in the impairment of circadian regulation associated with aging and inflammatory diseases.

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