Ageing is related to the gradual waning of the efficiency and aptitude of cells/tissues/organs to signal reparative and regenerative processes in response to internal and external stress, thereby impeding the progress of age-related diseases. This complex process is strictly linked to oxidative damage and involves immunosenescence and tissue impairment triggering inflammation and inflammasome formation. Essentially, ageing is hallmarked by low-level, systemic, chronic inflammation acknowledged as “inflammaging” ^[1]. Other age-related processes include augmented levels of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR) [2,3]^[2][3] with disruption in Ca²⁺ balance and triggering of IRE1α, PERK, and ATF6 downstream signaling pathways, with inflammation and inflammasome formation ^[4]. The ER plays a key role in cell homeostasis as the principal cell compartment implicated in protein synthesis, folding, modification, and secretion [2,3]^[2][3]. The stress response prompted by disproportionate misfolded or unfolded proteins amassing in the ER lumen is termed UPR. UPR activates various signaling pathways and transcriptional processes to block protein translation, degrade misfolded proteins, and augment the assembly of chaperones required for protein folding, ultimately re-establishing ER homeostasis to avoid cell apoptosis [2,3]^[2][3]. A main protein turnover pathway through which cellular constituents are transported into the lysosomes for degradation and reprocessing is autophagy. This intracellular process preserves cellular homeostasis under stress conditions and its derangement could initiate physiological alterations. The activity of autophagic processes declines during ageing, resulting in the build-up of damaged macromolecules and organelles and aggravation of ageing-associated diseases ^[5].

The molecular processes involved in ageing are regulated by oscillatory patterns controlled by the circadian clock circuitry and mainly encompass derangement of inflammation and autophagy with immunesenescence. The complex functioning of the immune system is rhythmically ordered on different timescales, with prevalence of roughly 24-h periodicity termed circadian (from the Latin circa, approximately, and dies, within a day), showing a well-arranged time-qualified organization of the levels of humoral factors and cellular effectors with simultaneous or opposing phases of phagocytic, complement, lysozyme and peroxidase activity in innate immunity, and antibody and cytokine production, leukocyte trafficking, proliferation and apoptosis in adaptive immunity [6,7,8,9]^[6][7][8][9]. In the peripheral blood of healthy humans, lymphocyte subsets show 24-h rhythmic fluctuations impacting the amplitude and type of immune response, with the predominance of cytotoxic T cells during daytime and T helper cells at nighttime ^[10]. The temporal patterns originate from circadian variations of bone marrow production, turnover and redistribution of blood cells, as well as cell mobilization and migration to lymphatic system and peripheral tissues, implicating cyto/chemokines, hormones (cortisol, prolactin, growth hormone, thyroid stimulating hormone), sympathetic nerve fibers, and biogenic amine neurotransmitters (epinephrine, melatonin) [11,12,13,14,15]^{[11][12][13][14][15]}.

Studies performed in innate immune system cells and peripheral blood mononuclear cells showed the presence of biological clocks in inflammatory and immune competent cells [16,17]^[16][17]. Furthermore, circadian rhythmicity regulates the transcriptional processes controlling the expression of genes enriching the signaling pathways involved in inflammatory processes, such as the nuclear factor κB (NF-kB) and the NLR3P3 inflammasome pathway ^[18]. These multifaceted interactions represent a promising target for valuable interventions to thwart the progression of physiological changes leading to a decline in the organism’s adaptive capacity and weakening of biological functions.

2. The Circadian Clock Circuitry

Physiology and behavior of living beings, scheduled in keeping with sleep/wake, rest/activity, and fasting/feeding cycles, show nychthemeral variations driven by the circadian timing system (CTS), a hierarchical network of biological oscillators comprising a master pacemaker in the suprachiasmatic nuclei (SCN) of anterior hypothalamus ^[19]. The CTS drives extra-SCN cerebral clocks and self-sustained oscillators in the peripheral tissues through humoral (cortisol, melatonin) or neural (autonomic nervous system fibers) outputs ^[19]. The SCN are entrained by environmental cues, mainly photic stimuli transmitted by the retino-hypothalamic tract and signaling environmental light-darkness alternation due to Earth’s rotation on its axis to tissues, organs, and organ systems ^[19]. Biological rhythmicity is generated at the molecular level by way of a group of intertwining genes with their encoded proteins, generating a transcriptional-translational feedback loop (TTFL) revolving with a frequency of 1 cycle in 24 ± 4 h ^[19]. The TTFL is operated by an activation branch, hard-wired by the Period-Arnt-Single-minded and basic helix-loop-helix (PAS-bHLH) transcription activators CLOCK (circadian locomotor output cycles kaput), and its paralog NPAS2 (neuronal PAS domain protein 2), and BMAL1-2/ARNTL-2 (brain and muscle aryl-hydrocarbon receptor nuclear translocator-like/aryl-hydrocarbon receptor nuclear translocator-like), which heterodimerize and rhythmically bind to E-box (5′-CACGTG-3′) cis-regulatory enhancer sequences of Period (Per1–3) and Cryptochrome (Cry 1–2) genes driving waves of epigenetic modification to promote gene transcription [20,21]^[20][21]. These genes encode PER and CRY proteins, responsible for the inhibitory branch of the feedback loop, accumulate and heterodimerize in the cytoplasm, translocate back to the nucleus, and impede CLOCK/BMAL1-2 transcriptional activity [20,21]^[20][21]. The circadian proteins go through various types of post-translational modifications (PTMs). The key regulators of the clock machinery, the serine/threonine protein kinases casein kinase (CK) 1 δ/ε and CK2, encoded by CSNK1D, CSNK1E, and CSNK2 genes, bind to and phosphorylate multiple circadian substrates [20,21]^[20][21]. Robustness and amplitude of the core TTFL is increased by the nuclear receptors (NRs) REV-ERBs and RORs, whose expression is rhythmically driven by CLOCK/BMAL1 heterodimer. In turn, REV-ERBs and RORs regulate BMAL1 transcription in competition with binding specific ROR response elements (RORE) in BMAL1 promoter and eliciting transcription inhibition and activation, respectively [20,21]^[20][21]. CLOCK/BMAL1 heterodimers bind E-boxes in the second intron of the PPARA gene and activate transcription of PPARA ^[22], which upon ligand binding, assists BMAL1 expression through a PPARα response element located in the BMAL1 promoter ^[23]. Furthermore, CLOCK/BMAL1 heterodimers drive the rhythmic expression of first order clock-controlled genes encoding proline and acidic amino acid-rich domain basic leucine zipper (PAR-bZIP) transcription factors, which control the rhythmic expression of thousands of downstream genes ^[24]. These PAR-bZIP transcription factors comprise DBP (albumin gene D-site binding protein), TEF (thyrotroph embryonic factor), and HLF (hepatic leukemia factor), and the bHLH transcription factors differentially expressed in chondrocytes protein (DEC)1 and DEC2. In addition, the interaction between the opposite oscillatory phase of DBP and REV-ERBs with RORs on RORE at the promoter of Nfil3/E4bp4 gene regulates the rhythmic expression of the nuclear factor interleukin 3 regulated protein (NFIL3, also known as adenoviral E4 protein-binding protein, E4BP4), which plays a key role in immune competent cell development and commitment [24,25]^[24][25].

3. The Role of Biological Clock Derangement in the Ageing Process

During the ageing process, progressive remodeling and degeneration of tissues also occur at the level of the cellular elements of brain structures. At the level of the SCN, there is a progressive reduction in the number of neurons expressing vasoactive intestinal peptide and arginine-vasopressin, with weakening of the connectivity in the functional network that ensures the robustness of the oscillation of neuronal and astrocytic cells. With the process of neuronal degeneration associated with aging, the network inside the SCN loses its links and progressively disintegrates, with a reduction in the amplitude of the oscillatory signal and a tendency to shorten the period of oscillation ^[26]. An important element affected by the ageing process is the pineal gland, which is part of the circadian timing system and whose secretion of melatonin decreases during ageing ^[27]. Another factor is cortisol, whose secretion by the adrenal glands is not reduced in amplitude but changes in its oscillatory pattern during aging. These alterations determine progressive derangement in the context of the circadian clock circuitry, with alteration of the harmonization of cellular processes and tissue functions in various organs and organ systems ^[28]. Senescent cells accumulate with aging, modulate their microenvironment through a particular secretory pattern (senescence associated secretory phenotype, SASP), and produce molecules with pro-inflammatory, proapoptotic, and pro-fibrotic activity, such as growth factors, cytokines/chemokines, and extracellular proteases, which not only have an autocrine effect, but also act on neighboring cells (paracrine effect). This bioactive secretome can impact the cell fate by triggering a senescence program and prompting cell-cycle arrest, preventing transmission of DNA damage to daughter cells, and thus preventing potential malignant transformation or, conversely, promoting proliferation, as induced by pro-inflammatory cytokines ^[29]. SASP is brought on by different signaling pathways, comprising the DNA damage response, stress kinases, inflammation and inflammasome activation, metabolic sensors/pathways, cell survival-associated transcription factors, autophagy, and chromatin remodeling. All these cell processes, primarily genetic modifications based on p16INK4a-based or p21CIP1/WAF1, are rhythmically driven by the molecular clockwork [30,31]^[30][31]. Lingering senescent cells, which harbor a failing biological clock and drive age-related disorders, pave the way for preventive/therapeutic strategies (senotherapy) that specifically aim to remove senescent cells with senolytics to curb ageing [32,33]^[32][33]. All the processes entailed in ageing are enhanced by the signaling pathways sustaining anabolism, such as growth hormone and insulin/insulin-like growth factor 1 signaling pathways, which activate PI3K-Akt-mTOR signaling pathway enhancing the aging-related processes ^[34]. Anti-ageing action is carried out by SIRT1-related signaling pathways, acting in part through PGC1alpha, and by AMPK, an important nutrient sensor. AMPK plays an antagonistic role with respect to mTOR signaling and directly modulates the molecular clockwork through phosphorylation of cryptochorme proteins, tagging them for proteasomal degradation [35,36]^[35][36]. Another important player in ageing is mitochondrial dysfunction. Aged and dysfunctional mitochondria must be destroyed and then renewed with processes of mitochondrial biogenesis, for which PGC1alfa and PGC1beta are fundamental. The biological clock is involved in mitochondrial reactive oxygen species (ROS) production and detoxification through the control of nutrient flux, uncoupling mechanism, redox regulation, antioxidant defense, and mitochondrial dynamics. An important mechanism is the excess of oxidizing radicals and decrease in antioxidant mechanisms ^[37]. The biological clock controls the fundamental processes that counteract damage from oxidizing radicals. Additionally, the role played by the hormone secreted by the pineal gland, melatonin, one of the most powerful molecules with an antioxidant effect is significant ^[38]. The derangement of the circadian clock circuitry causes a gradual decline in the production of antioxidant molecules in the face of a continuous increase in oxidative damage, as seen in the mouse model with loss of functionality of BMAL1, characterized by increased oxidative stress, impaired expression of several redox defense genes, increased neuronal susceptibility to oxidative damage, synaptic damage, and increased gliosis ^[39]. When the biological clock is altered, this results in a reduction in life span ^[40]. Gene mutations, especially in the core circadian genes, have an important impact on the duration of life. In experimental animal models, the knockout of BMAL1 gene results in accelerated ageing processes, increased oxidative damage, reduced body weight, sarcopenia, and altered leukocyte formula ^[34]. On the other hand, silencing of CLOCK results in reduced lifespan and increased incidence of cataracts ^[41].

4. The Biological Clock and the Innate Immune System

Inflammation, an adaptive host response also involved in tissue repair, alerts the immune system to sites of infection and tissue damage. Under normal conditions, at the molecular level, it is crucial to rigorously control the expression of genes enriching the signaling pathways that manage inflammatory responses by maintaining repression when external or internal stimuli are absent, and promptly activating their expression in the case of infection or tissue injury. The transcriptional control of a large part of these genes relies on signal-dependent transcription factors comprising members of the NF-κB, activator protein 1 (AP-1, a heterodimer composed of c-Fos/c-Jun family members), and interferon regulatory factor (IRF) families of transcription factors. After activation, NF-κB, AP-1, and IRF elicit the expression of numerous genes that turn on and boost inflammatory processes and support the progression of acquired immunity in different immune competent and inflammatory cells. Many receptor systems for molecules derived from pathogens (PAMPs), endogenous damage-associated molecular patterns (DAMPs), and receptors for cell-derived inducers of inflammation, such as IL1β, TNFα, interferons, can prompt the expression of these transcription factors ^[42]. The key of the circadian pathways in managing innate immune response was recognized by studying animal models with molecular clockwork changes. A pathophysiological connection between disruption of the molecular clockwork and increased susceptibility to chronic inflammatory diseases was suggested by studies performed in fibroblasts with double knock-out of cryptochrome genes (Cry1^−/−Cry2^−/−). These studies showed that in a cell-autonomous manner, dual silencing of the cryptochrome (CRY1 and CRY2) proteins remove the inhibitory effect on cyclic adenosine monophosphate (cAMP) production, increase intracellular cAMP levels, and enhance protein kinase A (PKA) signaling, with augmented phosphorylation of p65 at S276 residue and NF-κB signaling activation leading to unremitting increase in the proinflammatory cytokines IL-6 and TNF-α, and considerably augmented expression of inducible nitric oxide (NO) synthase (iNOS) ^[43]. On the other hand, homozygous Clock mutant mice showed reduced expression of immune-related genes and dampened oscillations of leukocyte number in the peripheral blood ^[6]. In Per2^−/− mice, serum levels of the proinflammatory cytokines interferon IL-1β and (IFN)-γ were significantly reduced after lipopolysaccharide (LPS) challenge, while TNFα, IL-6, and IL-10 production was preserved. Per2^−/− mice were more resilient to LPS-induced endotoxic shock with respect to wild-type mice, proposing mPer2 as an essential regulator of natural killer (NK) cell function ^[44]. Macrophages are key controllers of innate immunity and are responsible for time-based gating of proinflammatory cytokine secretion and systemic immune responses. Mouse macrophages enclose autonomous molecular clockworks driving 24-h rhythmicity in more than 8% of the transcriptome, controlling numerous essential regulators of pathogen recognition and secretion of cytokines, including TNF-alpha and IL-6 upon challenge with bacterial endotoxin at diverse daily intervals ^[16]. Studies performed on mutant mice and human macrophages showed that REV-ERBα blocks innate immune response to endotoxin driving genes implicated in innate immunity. This circadian gating on inflammatory pathways is lost in REV-ERBα^−/− mice ^[45]. TNF-α interferes with the functioning of the molecular clockwork, mainly thwarting the expression of PER3, DBP, TEF, and HLF, possibly triggering the so-called ‘‘inflammatory clock gene response’’ responsible for weakness in innate immunity activation and inflammatory diseases [46,47]^[46][47]. Innate immune response includes the acute phase response (APR), consisting of prompt gene expression reprogramming and metabolism adjustments upon inflammatory cytokine secretion and acute phase protein (APP) production in the liver, with increased APP levels characteristic of metabolic disorders. APR, along with lipid sensing nuclear receptors (NR) and signal-dependent activation of proinflammatory transcription factors, such as NF-kB, signal transducers, and activators of transcription (STATs) and AP-1 family members backs the interplay among metabolic and inflammatory pathways, sustaining the so-called “metaflammation” process and upholding atherogenesis ^[48]. Numerous NR involved in metabolic pathways, such as liver X receptor (LXR, binding oxysterols), peroxisome proliferator-activated receptor (PPAR, binding fatty acids), and farnesoid X receptor (FXR, binding bile acids) transcriptionally interfere with the pro-inflammatory signal-dependent initiating transcription factors NF-kB [homo- or heterodimer composed of p50 and p65 (RelA)], signal transducers, and activators of transcription (STATs) and AP-1 family members ^[49]. Upon ligand binding, these NR suppress inflammatory gene expression with a process called transrepression, which consists in NR–proinflammatory transcription factor complexes exclusion from genomic binding sites ^[49]. Close interactions among NR and the molecular clockwork influence its entrainment in diverse tissues: PER2 inhibits PPARγ expression and prevents its recruitment to promoters of target genes. Conversely, PPARγ activates BMAL1 transcription. There is a mutual activation of BMAL1 expression by PPARα and PPARα expression by BMAL1, while CLOCK cooperatively activates the 24-h rhythmic expression of PPARα ^[50]. During the inflammatory response, PPARγ expression can be activated by IL-4 and other immunoregulatory molecules, while it is suppressed by IFN-γ and LPS ^[51]. In addition, REV-ERBα increases the expression of IL-6 and cyclooxygenase-2 in primary human macrophages in blood vessel wall ^[52]. REV-ERBα expression is prompted by ligands binding to LXRs, which bind to a specific response element in the human Rev-erba promoter and regulate cholesterol turn-over in macrophages and their role in inflammation and immune response. Moreover, LXRα induces transcriptional expression of TLR4, while REV-ERBα binds as a monomer to a RE overlying the LXR RE in the TLR4 promoter and rhythmically inhibits LXR transactivation of TLR4 expression ^[53]. Furthermore, PER2 drives the rhythmic expression of the TLR9, which recognizes deoxyribonucleic acid (DNA) leading to circadian fluctuation of cellular activation and cytokine production influencing the immune response ^[54].