Adaptive immunity, chronic inflammation and the clock

The immune system incorporates two functionally distinct, yet intertwining, arms of defence against pathogenic invasion or insult. The initial arm involves innate defence and includes maintaining barrier sites and non-specific killing of pathogens. Cells of the innate immune system recognise foreign bodies via their pathogen-associated molecular patterns (PAMPs), allowing an element of discrimination between self and non-self. Although crucial and effective, the first line of defence is not without limitations. The adaptive defence is far more fine-tuned, allowing specific recognition of foreign particles and the selective expansion of cells prepared to target specific pathogens and develop immunological memory. T and B lymphocytes are key component cells for adaptive immune responses with dendritic cells (DCs), which survey for foreign antigens, linking the innate and adaptive responses. Exogenous antigen is processed within DCs and displayed on their surface (via major histocompatibility complexes (MHC)). DCs then travel to lymph nodes where they interact with naïve T cells. Once in close proximity, the presented antigen joins with a corresponding T cell receptor (TCR) on the T cell surface; in the presence of a co-stimulatory signal, this triggers T cell activation and proliferation. If a B cell has encountered the same antigen, T cell-dependent B cell activation may occur (via CD40-CD40 ligand interaction) driving transformation of B cells to plasma cells, which secrete immunoglobulins.

Circadian rhythms are generated by the molecular clockwork machinery—a transcriptional translational feedback loop involving a small handful of genes and proteins (reviewed in [1]). In brief, the mammalian molecular clock involves two proteins CLOCK and BMAL1 which dimerise and bind to Ebox elements on the promoters of Period (Per1-3) and Cryptochrome (Cry1 and Cry2) to enhance their transcription. The transcribed proteins PER1/2 and CRY1/2 dimerise and in turn act to inhibit the action of CLOCK/BMAL1 on their own promoters to limit transcription. This cycle takes approximately 24 h to complete. Additionally, CLOCK/BMAL1 enhance the transcription of two nuclear receptors called Rev-erb (α and β) and Ror (α, β and γ) which initiate (ROR) or inhibit (REV-ERB) the transcription of Bmal1 respectively via a retinoic acid–related orphan receptor response element (RORE) within the promoter. The rhythmicity of the core clock is transferred onto physiological outputs via interaction with clock-controlled genes.

It has been well documented that cells of the innate immune system have this operational clockwork machinery which drives rhythms in cellular functions [2–4]. There is no doubt that cellular components of the adaptive immune system exhibit daily oscillations in behaviour, the most well-established example being 24-h rhythms in numbers of circulating lymphocytes [5, 6]. However, the evidence in favour or against functionally relevant clockwork machinery within adaptive immune cells is less abundant. Here, we discuss work in the field exploring whether adaptive immune cells possess functional intrinsic clocks.

At the highest level, T cells are divided by expression of surface proteins into CD4 + and CD8 + . CD8 + T cells are cytotoxic and can directly kill compromised cells and cancer cells. In contrast, CD4 + ‘helper’ T cells indirectly contribute to cellular destruction. Upon activation, CD4 + T cells differentiate into subsets, depending on the nature of the antigen and cytokines present within the local environment. Subsets identified to date include T helper1 (Th1), Th2, Th9, Th17, Th22, follicular helper T (Tfh) and regulatory T cells (Tregs). T helper subsets contribute to pro-inflammatory responses directed at eliminating specific microbial pathogens and activating other immune cell types. Tfh cells promote the survival and proliferation of germinal centre B cells. Tregs specialise in maintaining immune homeostasis and self-tolerance, dampening inflammation and preventing the development of autoimmune disease. Additionally, long-lived memory T cells can be derived from CD4 + and CD8 + T cells after antigen encounter. Work to date has demonstrated that some T cell subsets exhibit active rhythmic transcription of components of the core molecular clock. However, this has been limited to major T cell subsets, with scope remaining to further understand clock function within more specialised subsets.

Studies examining cellular clocks within B cells are lacking in number. Hemmers and Rudensky observed minor fluctuations in Per1 reporter (Per1^venus) expression over 24 h in murine B cells, but did not detect any abnormality in B cells lacking BMAL, leading them to conclude that functional clockwork machinery, if present, is not essential for normal B cell development [8].

DCs sit at the interface between the innate and adaptive immune system and as antigen presenting cells (APCs) prime the adaptive immune response. Early work in the field established oscillations in core clock genes within splenic DCs harvested from mice over a 24-h cycle [13]. More recently, Hopwood et al. demonstrated robust rhythmic PER2::luciferase bioluminescence from murine cultured bone marrow–derived DCs [14], confirming intrinsic rhythmicity.

Despite limited evidence for multiple cellular components of the adaptive immune system possessing functional cell intrinsic clocks, this defence system exhibits daily variation in cellular composition, cell trafficking and responses to insult. Here, we explore our current understanding of rhythmicity within the adaptive immune system, considering circadian control of homeostatic processes (such as maintenance of immune cell populations) and responses to challenge.

The circadian clock is important for the lineage specification and maintenance of intestinal Th17 cells. These cells make an important contribution to defence against extracellular bacteria, but also play a key role in the development of autoimmune diseases. Lineage specificity of Th17 cells is regulated by the transcription factor RORγt, which is a short isoform of RORγ. Expression of RORγt is limited to lymphoid cells and neutrophils (in contrast RORγ is not expressed in haematopoietic cells, but found more widely in peripheral tissues) [15]. Whilst expression of Rorγ is regulated by CLOCK/BMAL, this heterodimer cannot activate transcription of the RORγt promoter [16]. However, within activated murine CD4 + T cells, Rorγt expression exhibits diurnal variation [17], and this is reported to be due to diurnal variation in binding of nuclear factor interleukin 3 regulated (NFIL3) to the promoter (see below).

Studies in mice have demonstrated that REV-ERBα negatively regulates Th17 cell development both directly and indirectly. REV-ERBα can itself compete with RORγt to modulate Th17 signature genes [18], but also regulates expression of NFIL3, a transcription factor which directly binds and represses the RORγt promoter [17]. A consequence of these interactions is diurnal variation in numbers of Th17 cells within the lamina propria. Yu and colleagues demonstrated higher frequencies of intestinal Th17 in the day versus night [17]. Additional circadian mechanisms have been recognised to regulate enteric Th17 cell numbers through regulation of cell trafficking. Th17-specific IL17A secretion induces CCL20 production from the small intestine, which facilitate Th17 migration back to this site [19]. CCL20 expression exhibits circadian rhythms and likely contributes to diurnal variation in Th17 cell frequency within the small intestine [20].

T and B cells traffic through the lymph nodes (LN) in a circadian manner. In mice, lymphocyte accumulation in LNs peaks at the onset of night (Zeitgeber Time (ZT)12–13) and egress occurs during the day [10, 21]. Multiple mechanisms have been shown to facilitate this daily rhythm, with stress-associated hormones being at the forefront. The first is via adrenergic control [22]. Neural inputs to lymphocyte β₂-adrenergic receptors inhibit their egress from LNs at night, promoting retention (discussed in more depth below). In addition, daily rhythms in circulating glucocorticoids contribute to diurnal oscillations in T cell distribution [21, 23]. Activation of the glucocorticoid receptor (GR) induces IL-7R expression on T cells and upregulates expression of the chemokine receptor CXCR4 which binds CXCL12 and promotes homing to lymph nodes [21].

In addition to these rhythmic cell extrinsic signals, there is clear evidence for a role for cell intrinsic clocks directing rhythmic lymphocyte trafficking. Druzd and colleagues demonstrated diurnal variation in expression of two trafficking receptors on lymphocytes—CCR7 and sphingosine-1-phosphate receptor 1 (S1P1) [10]. CCR7 binds CCL21 to promote lymphocyte homing to lymph nodes. In contrast, S1P1 binds sphingosine-1-phosphate, which guides lymphocytes out of LNs and into efferent lymphatics. Critically, cell-specific ablation of Bmal1 in T cells results in loss of diurnal rhythms in expression of CCR7 and S1pr1 (which encodes S1P1) and loss of rhythms of lymphocyte trafficking [10]. Together, this suggests concerted action between cell intrinsic and extrinsic circadian signalling driving rhythmicity in a homeostatic process which is critical for immune surveillance and responses to antigen presentation. Of course, the role of extrinsic rhythmic signalling extends beyond the direct influence on chemokine signalling pathways and trafficking receptors to include influence on the core clock itself (reviewed in [24]). One example being the influence of glucocorticoids on Per1/2 expression and Rev-erbα [9, 25–27], which may then influence clock dependent trafficking mechanisms.

A second important aspect of lymphocyte trafficking which likely contributes to the daily variation in circulating numbers is movement between the bone marrow (BM) and blood. The BM contains mature T cells (CD4 + CD8 + and Tregs) and these make up approximately 3–8% of total nucleated BM cells [28]. Trafficking between compartments is well established [29] but the influence of the circadian clock is not yet fully understood. Given the influence of stress mediators (catecholamines and glucocorticoids) on trafficking of haematopoietic stem cells between BM and blood (reviewed in [30]), it seems likely that these rhythmic signals may also influence T cell trafficking from bone marrow. Indeed, work across humans and mouse models demonstrates a role for the glucocorticoid-regulated chemokine receptor CXCR4 [21] in retention of Tregs within the bone marrow (which expresses the ligand CXCL12) [31].

Vaccinations utilise the adaptive immune system to generate antibodies towards a target antigen [32]. Broadly, a small amount of the foreign antigen is introduced to the body, provoking APCs to traffic to lymph nodes, where they present the antigen to T cells. Activated T cells drive the development of B cells and concurrently, B cells are stimulated (via the B cell receptor) by soluble antigen. B cells provide an antibody response. Memory B cells are generated, which mediate long-term immune memory, along with CD8 + memory T cells which are primed to proliferate rapidly when they next encounter a pathogen. Evidence is emerging from clinical studies highlighting that time of day influences vaccine responses. Studies suggest that subjects exhibit enhanced immunogenicity after influenza, hepatitis A and bacillus Calmette-Guerin (BCG) vaccination in the morning compared to the evening [33–35]. Studies in animal models (utilising immunisation with an autoantigen) similarly show that responses by the adaptive immune system to immunisation follow a circadian rhythm [10, 12, 36]. In part, this is due to oscillations in numbers of CD4 + T cells in lymph nodes at the time the animal encounters an antigen, which is influenced by multiple rhythmic signals (as discussed above). Work by two independent groups has determined that in a model of experimental autoimmune encephalitis (EAE; whereby mice are immunised with a MOG_35-55 peptide in an adjuvant to induce an autoimmune response resulting in inflammation of the CNS), reactions to this immunisation are heightened if the antigen is encountered during the day (ZT6-ZT8) versus the night. This timing is perhaps counterintuitive in light of data from human studies, when encountering antigen during the early active phase provokes a greater response.

The circadian clock intrinsic to CD8 + T cells plays a role in regulating the magnitude of their response to antigen presentation [12]. Using a model whereby antigen is presented to T cells by bone marrow–derived DCs loaded with OVA_257-264 peptide, Nobis and colleagues show that CD8 + T cell expansion in responses to vaccination is heightened at circadian time (CT)6 (mid-rest phase) compared to CT18 (mid-active phase). Further studies whereby vaccinated mice were challenged with a lethal dose of OVA-expressing Listeria monocytogenes showed enhanced protection after vaccination at CT6. CD8 + T cell-specific deletion of Bmal1 abolishes circadian variation in CD8 + T cell expansion. Transcriptomics revealed circadian regulation of the TCR-dependent signalling pathway, thereby imparting temporal gating onto cell activation, proliferation and acquisition of effector function.

Murine responses to parasitic infection vary dependent on time of encounter (Fig. 1). This has been demonstrated using an infection model with Trichuris muris (a ceacal dwelling parasite) [14]. Here, DC presentation of antigen stimulates T cells to polarise towards a Th1 or Th2 response (dependent on factors including cytokine signal and antigen dose) with a Th2 response required for efficient worm expulsion. Hopwood et al. demonstrated a difference in worm expulsion rates 21 days post infection dependent on time of infection. Those animals infected at ZT0 (lights on) showed a strong Th2 bias and faster worm expulsion than those infected at ZT12 (lights off). In mice lacking Bmal1 specifically in CD11c + cells (a cell subset that is largely composed of DCs), this difference in worm burden was lost, favouring development of a protective Th2 response across the day. RNA sequencing showed down-regulation of IL12 pathways as a consequence of Bmal1 deletion in DCs. IL12 is critical for promoting Th1 polarisation, which could help explain the altered parasite expulsion ability. The study demonstrates clock influence over adaptive immune responses to parasite infection, and highlights the importance of clocks within DCs for tuning downstream T cell responses.

Circadian disruption can be brought about experimentally through genetic targeting or environmental manipulation. Genetic targeting takes the form of deletion or mutation of one of the core clock genes, often Bmal1 deletion or Clock mutation. Global targeting of these genes leads to varying effects on rhythms in rest-activity cycles but often impacts on the adaptive immune system. These effects on the immune system can be a consequence of direct action of that clock gene on immune processes integral to maintain homeostasis. An example here is the influence of Rev-erb α and β on DC development and maturation [37]. Deletion of Rev-erb α/β enhances expression of maturation markers and pro-inflammatory cytokines even under naïve conditions. Multiple studies have explored consequences of circadian disruption on development of chronic inflammatory diseases both in mouse and humans, and these are discussed below.

Many chronic inflammatory diseases are sensitive to perturbations in circadian rhythms, and recent advances are beginning to unravel how the clockwork machinery within individual cell types is important for balancing inflammatory responses and restraining inflammation. Of note, there is a growing field of literature investigating the role of the clock in the development and pathogenesis of inflammatory bowel disease, inflammatory arthritis, asthma and multiple sclerosis.

Common across these chronic inflammatory diseases is that the core clockwork machinery becomes dampened at the site of inflammation. For example, studies in human tissue have shown perturbations of clock genes and proteins within the synovial membrane [71] and specifically FLS [72] in RA patients. A mouse model of inflammatory arthritis additional shows clear evidence of down-regulation of core clock genes in inflammatory cells recruited to the joints, including macrophages and neutrophils [11]. Similarly, there is evidence for dampened clock gene expression within both the site of inflammation [41, 73, 74] and within the periphery [74, 75] in colitis and within the spinal cord in EAE [36]. In asthma patients, there is evidence for altered clock gene expression patterns in the respiratory system. Ehlers and colleagues report a decreased expression of 6 core clock genes (but increased Clock expression) in bronchial brushings from adult asthma patients compared to time matched controls [59]. A study of peripheral blood mononuclear cells in patients with bronchial asthma revealed down-regulation of eight circadian clock genes compared to healthy individuals [76]. Again, suggesting any effects on the molecular clock may extend from the site of local inflammation into the periphery.

It is hard to establish whether altered clock gene expression is cause or consequence of chronic inflammation, but it is clear that acute inflammation impacts on the clock machinery both at a transcriptional and post-transcriptional level [11, 77]. The transcription factor NFκB has been highlighted as a key mediator of inflammation-induced circadian disruption [78]. Activation of NFκB disrupts circadian transcription via inhibition of the negative arm of the clock (Per, Cry and Rev-erb). ChIP sequencing of p65 in the mouse liver after lipopolysaccharide stimulation revealed co-localisation of NFκB to sites occupied by CLOCK/BMAL, leading to circadian disruption [78]. Further work in human omental adipocytes has shown binding of p65 to the PER2 promoter, which prevent BMAL1 binding, thus inhibiting Per2 transcription and impairing circadian clock function [79]. In addition, clock proteins are sensitive to inflammation, with loss of REV-ERBα protein in lung tissue 2- to 4-h post-lipopolysaccharide stimulation [77]. This rapid degradation of REV-ERBα is promoted by the 26S proteasome. It remains to be seen whether these mechanisms underlie clock disruption in the setting of chronic inflammatory disease. Given the importance of the clock in regulating immune cell function, disruption is likely to have downstream impact on disease progression and potentially contribute to development of co-morbidities.

There is mounting evidence that circadian disruption is associated with chronic inflammatory disease and in the setting of chronic inflammation, cell intrinsic clocks become dampened. Yet, perhaps paradoxically, these diseases often present as circadian rhythmic, with daily oscillations in disease symptoms and markers. The best examples here are asthma and RA.

There is clear evidence that clockwork machinery within key effector cells, resident at the site of inflammation, plays an important role in repressing inflammation in the setting of chronic inflammatory disease. However often these are not adaptive immune cells themselves, but specialised tissue-resident cells such as IECs or FLS. However, we must also consider the importance of cell extrinsic rhythmic signals on the function of immune cells in the context of adaptive immunity and chronic inflammation. These rhythmic, non-photic time-cues (or ‘zeitgebers’) may take many forms including steroid hormones and adrenergic signals (Fig. 4).

Chronic inflammatory diseases exhibit rhythmicity in their symptoms, pathology and biomarkers. There is potential to utilise this knowledge to improve both diagnosis and treatment in the form of ‘chronotherapy’.

Studies in asthma patients demonstrate best how using knowledge of disease rhythmicity could both standardise and improve the diagnosis and treatment of chronic inflammatory conditions. Daily variation in asthma biomarkers, including exhaled biomarkers and cellular infiltrates within sputum, has been mapped. Wilkinson et al. identified a number of 24-h rhythmic exhaled volatile organic compounds (VOCs) in asthma patients (including acetone and isoprene) and noted rhythms in exhaled nitric oxide fraction (F_eNO) which are absent in healthy volunteers. Critically, F_eNO is utilised in diagnostic asthma algorithms; hence, time of sampling is critical to ensure stringent diagnosis [110]. Quantification of sputum eosinophils in severe asthma patients reveals higher eosinophil percentages in patients sampled during the morning clinic compared to those sampled in the afternoon clinic. Given that increased sputum eosinophil counts are an indicator for treatment escalation, time of sampling should be considered when making clinical decisions [87].

Chronotherapy can be described as altering the timing of administration of existing therapeutics to improve efficacy and reduce adverse effects. The best example of application of chronotherapy to treat chronic inflammatory diseases comes from RA. The Circadian Administration of Prednisone in Rheumatoid Arthritis (CAPRA) studies probe benefits of delayed release (4-h delay) prednisone in RA patients [111]. The rationale behind these studies was delivering the corticosteroid to coincide with the early morning rise in inflammatory cytokines. Trials, whereby delayed release prednisolone (or placebo) was administered alongside a disease-modifying antirheumatic drug, suggest that this approach has beneficial effects on morning joint stiffness [112] and fatigue [113].

Given established links between the molecular clock and processes underlying adaptive immunity, there is clear potential to target the clock for therapeutic gain. The last decade has seen the development of novel ligands which act on components of the clock including REV-ERB [114], ROR [115] and CRY [116, 117]. In vitro studies have demonstrated the anti-inflammatory potential of clock targeted compounds, such as the CRY activator KL001 [55] and REV-ERB agonists [37]. Furthermore, the effectiveness of these molecules in models of autoimmune diseases has been shown. For example, the REV-ERB activators SR9009 and SR12418 have a protective effect in DSS-induced colitis [18, 41], EAE [18, 118] and CIA [119]. To date, these interventions have been limited to pre-clinical models, but evidence is mounting to support the notion that the clock is a bona fide target for the treatment of autoimmune disease. As might be predicted, some of the beneficial effects of REV-ERB targeting on autoimmune diseases are due to the influence of this nuclear receptor on Th17 cells. In EAE, the therapeutic effects of SR9009 are in part due to modulation of Th17 activity. SR9009 treatment of mice undergoing EAE resulted in reduced numbers of IL17A producing CD4 + T cells in the CNS and reduced disease scores [18, 118]. In contrast, the effects of SR9009 in CIA are proposed to be mediated by anti-inflammatory effects on innate immune cells (including macrophages) and joint resident synovial fibroblasts [119] and within the colon in colitis [41].

The authors declare no competing interests.

Not applicable.

Not applicable.