Circadian rhythms in immunity

Abstract

Purpose of review

This review is focused on the existing evidence for circadian control of innate and adaptive immune responses to provide a framework for evaluating the contributions of diurnal rhythms to control of infections and pathogenesis of disease.

Recent findings

Circadian rhythms driven by cell-autonomous biological clocks are central to innate and adaptive immune responses against microbial pathogens. Research during the past few years has uncovered circadian circuits governing leukocyte migration between tissues, the magnitude of mucosal inflammation, the types of cytokines produced, and the severity of immune diseases. Other studies revealed how disruption of the circadian clock impairs immune function or how microbial products alter clock machinery.

Summary

Revelations concerning the widespread impact of the circadian clock on immunity and homeostasis highlight how the timing of inflammatory challenges can dictate pathological outcomes and how the timing of therapeutic interventions likely determines clinical efficacy. An improved understanding of circadian circuits controlling immune function will facilitate advances in circadian immunotherapy

Introduction

In order to anticipate and respond to daily environmental changes, virtually every living organism has evolved circadian clocks that drive daily oscillations in behavior and physiology. The 24-hour light-dark cycle along with other stimuli (e.g. feeding/fasting, oxygen, etc.) entrain cell-autonomous circadian clocks that regulate sleep, body temperature, digestion, locomotion, and metabolism [1]. Importantly, the responsiveness, localization, and activity of immune cells exhibit robust circadian rhythmicity. Mechanistically, this likely evolved to maximize immune defense during the greatest time of pathogen exposure and to facilitate tissue repair during rest [2, ●3].

This review summarizes recent advances in our understanding of diurnal rhythms and circadian clock circuitry that impact the magnitude and character of immune responses. Intricate changes in expression of migration factors and localization of immune cells are discussed in the context of immune surveillance, tissue repair, and disease pathogenesis. The impact of timing of vaccination or inflammatory challenge is described as a potent modulator of immune cell function and pathogen control. In addition, this review reveals the mechanism of microbe-mediated or disease-associated disruption of the circadian clock in immune cells as a driver of disease. In light of these factors, attention is devoted to the growing field of circadian medicine as a method for maximizing therapeutic efficacy via optimization of treatment timing or manipulation of the circadian clock.

Organism-level synchronization of the clock

The coordination of clocks within individual cells of the body is regulated by a master oscillator (Figure 1) in the hypothalamus called the suprachiasmatic nucleus (SCN) [4]. The SCN is entrained by light-dark cycles via innervation from the retina. In turn, the SCN clock transmits behavioral and rhythmic cues to peripheral oscillators via both neuropeptides and autonomic innervation [5]. Through SCN networking, the central clock also encodes day length and thereby drive seasonal responses [4]. Recent data revealing the astrocyte clocks can drive circadian rhythms of SCN [6], reveal evolutionary redundancy and importance of maintaining a master timekeeper.

Figure 1. Organization of central and cell-intrinsic peripheral oscillators.

The central oscillator is comprised of the SCN in the hypothalamus, which receives light signals and transmits rhythmic cues to the peripheral oscillators via hormone and neural pathways that are further modulated by timing of food intake and other environmental cues. The peripheral clock machinery is comprised of three interlocking feedback loops. BMAL1 and CLOCK binding to E-box elements drives transcription of multiple clock-controlled genes. As PER and CRY accumulate, they translocate back into the nucleus to repress BMAL1:CLOCK driven gene transcription, including that of PER and CRY. REV-ERBα and RORα alternatively regulate expression of RORE element associated genes, including BMAL1 and NFIL3. NFIL3 and DBP in turn alternatively regulate expression of genes like PER via D-box elements. Each element modulates expression of clock-controlled genes that may either contribute to circadian expression of other genes or have a circadian expression profile without feeding back into the molecular clock machinery.

Anatomy of the molecular clock

The 2017 Nobel Prize in Physiology or Medicine was awarded for the discovery of evolutionarily conserved molecular mechanisms governing the circadian clock [7]. Mammalian circadian rhythms are a consequence of a transcriptional- translational feedback loop controlled by self-sustained cell-autonomous molecular clocks (Figure 1). These oscillating transcription-translation feedback loops are driven by a set of dedicated clock proteins [8]. The transcription factors brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) heterodimerize and bind E-box regulatory sequence motifs throughout the genome to drive gene expression [9, 10]. Importantly, CLOCK-BMAL1 regulate expression of their own repressors, period (PER) proteins and cryptochromes (CRY), which increase in concentration over time, oligomerize and enter the nucleus to suppress CLOCK-BMAL1 activity [8]. This feedback loop operates in conjunction with two additional circuits to establish a 24-h rhythm. First, the timing and amplitude of BMAL1 expression is controlled by competitive binding of REV-ERBs (NR1D1 and NR1D2) and retinoic acid-related orphan receptor alpha (RORα) to the BMAL1 promoter, respectively, resulting in either repression or activation of BMAL1 transcription [11, 12]. Second, albumin D-box binding protein (DBP) transcriptional activator and its repressor, nuclear factor interleukin 3 regulated (NFIL3; also known as E4BP4), synergistically regulate expression of PER and other D-box genes [13]. The translation, trafficking, and degradation of clock proteins generates oscillations in gene expression that collectively establish the 24-h period of circadian timing (Figure 1).

Role of the clock in immune cell development

The majority of immune cell lineages possess intrinsic clocks that provide temporal control of development, differentiation, migration, and function of these cells [2, ●3]. In some cases, the circadian clock machinery contributes to development and differentiation of immune cell lineages. A primary example is NFIL3, which is required for the development of innate lymphoid cells (ILCs), including natural killer (NK) cells [14–18]. In the case of interferon-gamma (IFN-γ) producing ILC1 and NK cells, NFIL3 is essential both for development of these cells from a common ILC progenitor [15, 16, 19, 20], as well as, for the homeostatic maintenance of these lineages within intestines and lymphoid tissues [17, 18]. In contrast, group 3 ILC only require NFIL3 at the level of the common ILC progenitor [15].

Similar to the role of NFIL3 in ILC, the development of B cells depends on BMAL1. Bmal1−/− mice exhibit marked deficits in peripheral B cell numbers and immunoglobulin (IgG) titers [21]. However, cell transfer and conditional gene deletion experiments revealed a B cell-extrinsic role of BMAL1 in modulating the bone marrow microenvironment in which B cells develop [21, 22].

The circadian clock machinery is also implicated in the development of specific subsets of CD4⁺ T cells. Neither global nor T-cell-specific ablation of Bmal1 appreciably affected overall or subset-specific frequencies of T cells [21, 22]. However, loss of NFIL3 in T cells resulted in increased frequencies of T helper 17 (Th17) cells [23], whereas mice expressing a dominant-negative CLOCK protein or lacking either REV-ERBα or RORα exhibited reduced Th17 frequencies [23, 24]. Given the vital role of Th17 cells in intestinal health and immunity, the role of clock genes in Th17 homeostasis likely serves to balance the activity of the cells for optimal protection against pathogens with minimal tissue damage.

Clocks control leukocyte trafficking

During homeostasis, inflammatory cells undergo daily flux between the bone marrow, blood, and tissues. As a result, the number of circulating leukocytes in both mice and humans changes dynamically over the day [25, 26]. The rhythmic trafficking of immune cells during homeostasis is largely controlled via cell-extrinsic mechanisms [27]. Central clock cues from adrenergic nerves regulate expression of C-X-C motif chemokine ligand 12 (CXCL12) by bone marrow stromal cells. CXCL12 is a vital bone marrow retention signal and this mechanism controls the rhythmic egress of cells into the periphery.

Similar cell-extrinsic mechanisms control temporal variation in leukocyte numbers in tissues [28]. During acute inflammation, the rhythmic release of CXCL5 from bronchiolar epithelial Club cells in the lung promotes increased recruitment of neutrophils during the resting phase (daytime in mice) [29]. Elevated endogenous glucocorticoids dampen CXCL5 and neutrophil recruitment during the active phase (nighttime in mice), and BMAL1 deletion in Club cells abrogates this circadian circuit, resulting in a non-rhythmic pro-inflammatory influx of neutrophils into the lung.

Of note, diurnal expression of glucocorticoids also regulates IL-7 receptor (IL-7R) and CXCR4 in T cells [●30]. Circulating T cell frequencies in mouse blood peak during the resting phase when IL-7R expression is low, whereas elevations in IL-7R expression during the active phase enhanced T cell survival and accumulation in spleen and lymph nodes. As a result of active phase accumulation of T cells in secondary lymphoid tissues, antigen-specific T cell responses and humoral immunity induced by systemic bacterial infections and soluble antigen during this active phase were enhanced [●30].

A recent organism-wide analysis revealed that both the microenvironment and leukocyte-autonomous oscillations in migratory factors control the time-of-day-dependent homing of particular cell subsets to specific organs [●●31]. In multiple lymphoid and nonlymphoid tissues, pro-migratory molecules like intercellular adhesion molecule 1 (ICAM1), ICAM2, and vascular cell adhesion molecule 1 (VCAM1) undergo rhythmic oscillations. In contrast, addressins and selectins display tissue-restricted oscillations in the liver (E-selectin), lymph node (mucosal vascular addressin cell adhesion molecule 1, MAdCAM-1), gut (CD44 and peripheral lymph node addressin, PNAd), and skin (CD44). Correspondingly, some chemokine receptors (CXCR4) and adhesion molecules (P-selectin glycoprotein ligand-1, PSGL-1) exhibit circadian oscillations in nearly every leukocyte subset analyzed, including lymphocytes, myeloid cells, and granulocytes [●●31]. Genetic ablation of the clock in either the endothelium or in leukocyte ablates these time-of-day differences. Similarly, paired phasing of CC-chemokine receptor 7 (CCR7) on T cells and high endothelial venules CCL21 favored accumulation of mouse T cells in lymph nodes during the active phase [●32]. The authors conclude that an extensive circadian trafficking zip code system guides homeostatic leukocyte migration between circulation and organs.

Cell-intrinsic clocks also coordinate immune defense during inflammatory insult. Trafficking of inflammatory monocytes from blood to infected tissues is constrained by BMAL1 regulation of CXCR4 and CCL2 expression [25]. Likewise, BMAL1 regulation of CXCL2 expression modulates the migratory properties of circulating neutrophils, which is antagonized by CXCR4 [●33]. This process favors the egress of neutrophils from blood vessels of mice during the active phase, thereby enhancing anti-microbial activity within tissues. Disruption of this internal timer resulted in intravascular accumulation of neutrophils that predisposed mice to fatal vascular injury [●33]. Collectively, these studies reveal that leukocyte localization within the body is under strict environmental and cell-intrinsic circadian control that is vital to limit harmful pathology and promote optimal immune defense. In addition, these adaptations presumably optimize energy expenditure while pairing appropriate immune responses with the likely timing of pathogen encounter.

Rhythms of immune responses and function

In addition to controlling the location of leukocytes, circadian clocks impact the nature and amplitude of inflammatory responses induced by both pathogens and vaccines [●3]. The expression of pattern recognition receptors, secretion of complement or coagulation factors, the release of histamine, production of cytokines, and phagocytic activity of macrophages all exhibit circadian oscillations [2, 25, 26, 34–36]. For example, oscillations in NK cell capacity to make IFN-γ or kill target cells are dampened in the absence of PER1 [37]. Likewise, circadian control of IL-5 and IL-13 production by ILC2 determines blood eosinophilia and recruitment of eosinophils to tissue [38]. In the case of macrophages, REV-ERBs suppress CX3CR1, CCL2, CCL5, IL-12, and IL-6 expression to limit inflammation in mice during the resting phase [39, 40]. Desynchronization of the molecular clock among a population of macrophages promotes heterogeneity in inflammatory responses, with NFIL3 and DBP determining IL-12 production in response to lipopolysaccharide [●41].

Recent studies expanded our knowledge of circadian circuits on antimicrobial immunity to numerous bacteria, viruses, and parasites. Macrophage-associated BMAL1 is critical for control of Leishmania infection [42]. Dendritic cell (DC) BMAL1 dictates the Th1/Th2 balance via regulated production of IL-12, thereby contributing to strong Th2 bias and robust worm expulsion in morning-infected mice [43]. BMAL1 also controls IL-1 expression in myeloid cells [44].

In a similar fashion, BMAL1 determines innate immune control of infections with Sendai, respiratory syncytial, parainfluenza, hepatitis C, dengue, Zika, and influenza A virus at the level of both lung fibroblasts in vitro and intact mice [45–●47]. This likely drives changes in time-of-day susceptibility to influenza A and herpesvirus infections [48], while the absence of BMAL1-driven rhythms contributes to marked asthma-like airway changes [●47]. Importantly, oscillating leukocyte frequencies in tissues and circadian control of inflammatory gene expression critically affect harmful lung inflammation during influenza A virus infection [●●49]. As a result, infection just before the onset of the active phase resulted in more weight loss, increased clinical scores, and greater mortality than observed in mice infected just prior to the resting phase. Survival was independent of viral control but associated with the circadian influx of NK and NKT cells into the lung [●●49].

Microbial control of circadian immunity

Critical interactions between the microbiota and intestinal epithelium are orchestrated by the circadian clock. REV-ERBα and RORα control rhythmic gene expression of toll-like receptors (TLR) in intestinal epithelial cells (IEC) [50]. Moreover, intestinal bacteria trigger DC release of IL-23 during active phase in mice, thereby activating ILC3 to make IL-22 that in turn inhibits REV-ERBα in IEC [●51]. The resulting rhythmic expression of NFIL3 in IEC crucially regulates both Th17 differentiation and lipid metabolism. Intriguing new evidence points to synergistic contributions of light-dark cycles, food intake, and microbial cues via both the central clock and ILC3-intrinsic BMAL1 that shape intestinal health and lipid metabolism [●●52].

In fact, metabolism is synchronized withsleep-wake cycles and food intake. Recent work reveals an epigenetic mechanism by which gut microbiota contribute to daily metabolic cycles [●●53]. In small intestine but not colon IEC, microbiota drive oscillation in expression of metabolic genes involved in nutrient transport and lipid metabolism via rhythmic changes in histone deacetylase 3 (HDAC3) activity. Thus, mice lacking gut microbiota exhibit disrupted oscillations in metabolism and become obese on high-fat chow, providing a potential explanation for human obesity-associated with antibiotic damage to the microbiota.

Circadian disruption and clock contributions to disease

Circadian control of T and B cell responses affects the amplitude of pathogen-specific, allergen reactive and potentially harmful self-reactive lymphocytes. CLOCK promotes rhythmic proliferation responses of these cells by modulating timing of T-cell interaction with antigen-presenting cells [54]. Induction of sterile inflammation coincident with circadian timing of high cell counts in lymphoid tissues results in strong T [●●55, ●56] and B cell responses [●32, ●57]. This observation potentially explains stronger vaccine responses in humans immunized in the morning (early active phase) relative to the afternoon (late active phase) [58]. BMAL1 in DCs or myeloid cells was essential for oscillations in CD8 T cell responses [●●55, ●56]. Importantly, loss of myeloid BMAL1 increases inflammation in CNS with pathogenic expansions of Th1 and Th17 cells that exacerbate experimental autoimmune encephalomyelitis (EAE) [●●55]. Likewise, Cry1^−/−/Cry2^−/− mice exhibit marked elevations in IgG and autoantibody titers [●59], suggesting multiple circadian mechanisms exist to limit autoimmune disease.

The circadian clock has also been implicated in tumorigenesis [60]. In addition to the formation of tumors, some cancers (adenocarcinoma) mediate circadian remodeling in the liver via inflammatory mediators, where this process promotes a metabolic profile favorable to tumor growth [61]. In addition, therapies that disrupt circadian clock processes (REV-ERB agonists) selectively undermine the survival of tumor cells [●62], highlighting the importance of circadian rhythms in cancer. Intriguingly, BMAL1 was also implicated in suppression of programmed death-ligand 1 (PD-L1) expression on myeloid cells [63], which likely has implications for checkpoint blockade therapy targeting programmed cell death protein 1 (PD-1) on tumor-specific T cells.

Of note, asthma and allergic disease present robust circadian rhythms [●64]. There are marked day-night differences in clinical presentation of the disease, with symptoms usually worsening overnight (resting phase). Circadian variations in IgE and mast cells are likely important drivers of these disease oscillations [34]. As was the case with T cells [●●55], BMAL1 in myeloid cells constrains allergic asthma [65]. Likewise, CLOCK modulates the IL-33-mast cell axis through both temporal variation in IL-33-induced cytokine expression and oscillations in mast cell expression of the IL-33 receptor, ST2 [●66].

Therapeutic potential of clock modifiers

A large number of drugs used in the treatment of inflammatory and metabolic diseases directly target genes that exhibit circadian oscillations [●3, 67]. For example, targeting REV-ERB is beneficial in both atherosclerosis [68] and cancer [●62], whereas CRY-stabilizing drugs are anti-inflammatory [69]. The short in vivo half-life of many drugs and clock-dependent changes in drug metabolism genes provide further rationale for considerations of revisiting the time-of-day administration of these therapies. Some examples include the timing of vaccination [58], bone marrow transplantation [70], chemotherapy [71], and anti-inflammatory medication administration in arthritis, asthma, and multiple sclerosis [●3]. These promising results portend a future with carefully rationalized application of medical interventions and multiple new tools for modulation of the molecular clock to curtail disease.

Conclusions

Homeostasis and inflammatory responses of the immune system exhibit marked time-of-day variations. These oscillations are a product of cell-intrinsic molecular clocks and environmental cues- light, food, or microbiota. As a result, the number and function of leukocytes in specific tissues are highly dependent on circadian rhythms that determine the amplitude and nature of immune responses to immunological challenges. Circadian disruption of the rhythms is often observed in inflammatory diseases, including autoimmunity, atherosclerosis, and asthma. An improved understanding of the complex interplay between cell-intrinsic clocks in different immune cells and the central clock will be necessary to advance new circadian medicine opportunities.