REVIEWS
­Clocking in to immunity
Circadian
A free-​running rhythm with a
period of approximately 24 h
that persists in the absence of
external entrainment, such as
in constant darkness.
Suprachiasmatic nuclei
(SCN). A bilateral structure in
the anterior hypothalamus,
home to the central pacemaker,
which processes light input and
conveys timing information to
the rest of the body.
1
Walter Brendel Centre of
Experimental Medicine,
University Hospital, Ludwig-​
Maximilians–University
Munich, Biomedical Centre,
Planegg, Martinsried,
Germany.
2
DZHK (German Centre for
Cardiovascular Research),
partner site Munich Heart
Alliance, Munich, Germany.
3
School of Medical Sciences,
Faculty of Biology, Medicine
and Health, University of
Manchester, Manchester, UK.
*e-​mail: christoph.
scheiermann@
med.uni-​muenchen.de;
andrew.loudon@
manchester.ac.uk
https://doi.org/10.1038/
s41577-018-0008-4
Nature Reviews | Immunology
Christoph Scheiermann1,2*, Julie Gibbs3, Louise Ince   1 and Andrew Loudon3*
Abstract | Circadian rhythms are a ubiquitous feature of virtually all living organisms, regulating a
wide diversity of physiological systems. It has long been established that the circadian clockwork
plays a key role in innate immune responses, and recent studies reveal that several aspects of
adaptive immunity are also under circadian control. We discuss the latest insights into the genetic
and biochemical mechanisms linking immunity to the core circadian clock of the cell and
hypothesize as to why the immune system is so tightly controlled by circadian oscillations. Finally ,
we consider implications for human health, including vaccination strategies and the emerging
field of chrono-​immunotherapy.
The regular 24 h environmental cycles generated by secretion of hormones (such as glucocorticoids)7 and
the planet’s rotation have led to the evolution of daily metabolic cues8, which are driven by rhythmic feeding
circadian rhythms in almost all life forms on Earth. behaviour9,10. Although light is the key entrainment
Circadian rhythms are driven by cell-​autonomous bio- factor for the SCN, feeding-​regulated metabolic cues
logical clocks (Box 1), which allow organisms to adapt are now recognized as being pivotal to the regulation of
to and anticipate temporal changes in the environment. many peripheral clocks9,10.
These biological clocks probably evolved a few billion Phase coherence of the organism to the normally
years ago, perhaps in response to the need for single-​ occurring environmental light–dark cycle is known to
celled eukaryotes to avoid the damaging effects of ioniz- maintain organismal fitness, as animal studies show
ing radiation and oxidative stress during processes such that its disruption by abnormal lighting or feeding
as cell division in which single-​stranded nucleic acids schedules, or due to genetic mutation of key circa-
become exposed1,2. In higher organisms such as mam- dian timing genes, induces pathological changes11. In
mals, virtually all aspects of physiology are regulated by support of this, human lifestyles that disrupt inherent
internal circadian clocks, including sleep–wake cycles, timing systems (for example, exposure to abnormal
behaviour and locomotor activity, body temperature lighting schedules in chronic shift work) are associated
cycles, cardiovascular and digestive processes, endocrine with an increased risk of cancer, metabolic disorders
systems and metabolic and immune functions. and cardiovascular and cerebrovascular disease12–15. In
In the mammalian circadian system, peripheral addition, many human inflammatory diseases exhibit
clocks are normally maintained in phase coherence with rhythmicity in their pathology, including myocardial
the environment by entrainment from daily exposure infarction, asthma and rheumatoid arthritis, as we
to the light–dark cycle. This photic entrainment pathway discuss in detail later9,16,17.
operates via retinal innervation of a specialized neural Diurnal host responses to lethal infection and endo-
circadian pacemaker of around 20,000 neurons located toxins were first demonstrated almost 60 years ago18, but
in a pair of hypothalamic suprachiasmatic nuclei (SCN)3. it is only recently that studies have uncovered multiple
The mammalian SCN pacemaker has two unique prop- widespread aspects of immune functions that are under
erties: first, it is the only mammalian circadian struc- the control of the circadian clockwork. These include
ture that is directly entrainable to light. Second, the the trafficking of immune cells, host–pathogen interac-
SCN neurons remain coupled in ectopic culture con- tions and activation of innate and adaptive immunity,
ditions, persistently generating robust rhythmic circa- which have been discussed in recent reviews19–22. It is
dian cycles of electrical activity and gene expression for now apparent that circadian signals operate as a gate to
many months outside the body4,5. By contrast, periph- control the magnitude of immune responses in a pow-
eral circadian oscillators lose c­ir­ca­dian synchrony in erful time-​of-day manner. In this Review, we describe
culture because individual cells are unable to maintain the mechanisms that place the immune system under
coupled phase coherence. A­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­u­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­t­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­o­­­­­­n­­­­­­o­­m­ous c­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­i­­­­­­­­­­­­­­­­­­­­r­­­­­­­­­­­­­­­­­­­­c­­­a­­­­dian c­­­­l­­o­­­cks clock control. We address what is known of the mole­
h­­a­v­e b­­e­e­n d­­e­t­­ec­­ted i­­n a­­l­l major organs and tissues and cular elements that couple the core circadian clockwork
within many cell types of the body. Normal circadian to specific output arms of the immune system and
synchrony of peripheral cells and tissues is maintained how these new understandings may lead to improved
by a complex network involving neuronal signalling6, chronotherapeutic options for treatment of disease.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews

Fig. 1 | operation and disruption of the molecular clock ◀

Box 1 | How the clock works
the mammalian circadian core clockwork consists of a conserved set of transcription
factors operating as interlocked rhythmic transcription–translation feedback loops.
Central to this are the key regulatory transcriptional activators circadian locomoter
output cycles protein kaput (CLOCK), brain and muscle arNt-​like 1 (BMaL1) and
neuronal Pas domain-​containing protein 2 (neuronal Pas2), which form the
heterodimeric complexes CLOCK–BMaL1 and neuronal Pas2–BMaL1 and act on e-​box
elements on target genes. these target genes include the circadian transcription
factors period circadian protein homologue 1 (PER1) and PER2, cryptochrome 1 (CRY1)
and CRY2, rev-​erBα (NR1D1) and rev-​erBβ (NR1D2) and differentially expressed in
chondrocytes protein 1 (DEC1) and DEC2 — all of which are repressors of the core
clockwork. rhythmic negative feedback by Per and CrY proteins on e-​box sites leads
to the transcriptional repression of CLOCK–BMaL1 complexes on target genes,
including PER and CRY genes. this repression is lifted late at night owing to decay of the
Per–CrY complex and structural changes in the BMaL1 protein117. the CLOCK–BMaL1
complex can then reactivate transcription, leading to the generation of an oscillation.
Clock pace is strongly regulated by post-​translational control of feedback elements.
Genetic mutations altering the phosphorylation status, mobility or degradation of Per
and CrY proteins can have a profound effect on clock speed118,119. two additional
circuits cooperate with the core feedback loop to establish robust 24 h rhythms. First,
the retinoid-​related orphan receptor (rOr) nuclear receptors drive expression of
BMaL1 in a feedforward loop. rOrα shares a DNa-​binding site with rev-​erBs, and
rhythmic competitive repression by rev-​erBs generates an oscillation in BMAL1
expression. second, BMaL1–CLOCK heterodimers act on a family of Par domain basic
leucine zipper transcription factors — namely, albumin D box-​binding protein (DBP),
hepatic leukaemia factor (HLF) and thyrotroph embryonic factor (teF). these act on
D-​box elements in target genes, including those of the core feedback loop; for example,
DBP positively regulates PER1 expression120. Consequently, the interlocked loops of the
core circadian clock act via rhythmic control of e-​box, rOr or D-​box sites to generate
stable oscillations in target gene expression121. thus, the formation, trafficking and
degradation of different clock protein complexes throughout this transcriptional cycle
generate the intrinsic nature and stability of the clock.
in immune cells. a | The core molecular clockwork (centre)
consists of interlocked transcription– translation feedback
loops within each cell (Box 1). Rhythmic expression of
these clock genes (and other clock-​controlled genes) has
been described in cells of both the innate and adaptive
immune system, including macrophages24,25 and
microglia129, monocytes23, mast cells26,27, dendritic cells
(DCs)25, CD4+ T cells54,56, B cells25, natural killer (NK) cells29,
neutrophils28 and eosinophils27 as indicated by the clock
symbol. Other cell types such as CD8+ T cells54 and group 2
innate lymphoid cells (ILC2s)46 exhibit rhythmic function,
but clock gene expression has not been formally
demonstrated. In addition, circadian oscillations in gene
expression have also been reported in stromal cells such as
synoviocytes116, epithelial cells43,80 and blood vessels130,
which are known to modify immune function. b | Targeted
disruption of the clock in specific cell types has been
achieved largely by using the Cre-​loxP system. Crossing
Bmal1flox/flox mice with cell-​specific Cre lines abolishes
the molecular clockwork in those cells23,31,43,54,55,78,79,131. The
effects of such a disruption are varied, encompassing both
pro-​inflammatory and anti-​inflammatory responses, and
may depend upon the timing of the immune challenge.
BMAL1, brain and muscle ARNT-​like 1; CLOCK , circadian
locomoter output cycles protein kaput; CNS, central
nervous system; CRY, cryptochrome; CT0, circadian time 0;
CXCL5, CXC-​chemokine ligand 5; EAE, experimental
autoimmune encephalomyelitis; IEC, intestinal epithelial
cell; LPS, lipopolysaccharide; neuronal PAS2, neuronal PAS
domain-​containing protein 2; PER , period circadian protein
homologue; ROR , retinoid-​related orphan receptor ; TNF,
tumour necrosis factor.

Diurnal
A pattern that occurs over the
course of a day in which
external entrainment (such as
light–dark cycles) is used; the
onset of the light cycle is
defined as Zeitgeber time
0 (ZT0).
Period circadian protein
homologue 1
(PER1). PER1, PER2 and PER3
are PAS (PER–ARNT–SIM)
domain-​containing proteins
that associate with CRY
proteins to inhibit BMAL1–
CLOCK-​mediated gene
expression.
REV-​ERB
REV-​ERBα (encoded by
NR1D1) and REV-​ERBβ
(encoded by NR1D2) are
transcriptional repressors that
bind to ROR response element
(RORE) motifs in the BMAL1
promoter to regulate the
rhythmic expression of
BMAL1.
Cryptochromes
(CRYs). CRY1 and CRY2 are
transcriptional repressors that
associate with PER proteins to
inhibit BMAL1–CLOCK-​
mediated gene transcription.
Core clockwork in innate immunity
Multiple types of innate immune cells have been
demonstrated to possess intrinsic clocks, including
monocytes23, macrophages24,25, mast cells26,27, neutro-
phils28, eosinophils27 and natural killer (NK) cells29
(Fig. 1a). These intrinsic timers affect the function of
these cells, driving temporal gating of cell-​type-specific
processes (Fig. 1b). This temporal gating includes the
phagocytic activity of macrophages30 and their cytokine
release24,31, as well as histamine release and allergic reac-
tions of mast cells32,33. Evidence is emerging to suggest
that specific clock genes mediate different elements of
immune cell physiology. Logan and colleagues showed
that period circadian protein homologue 1 (PER1) is
necessary for efficient function of splenic NK cells34.
In the absence of Per1, these cells maintain rhyth-
micity but show altered rhythmic expression of IFNγ
and of the cytotoxic factors perforin and granzyme B.
Furthermore, rhythmicity in these factors and asso-
ciated cytotoxicity can be suppressed by chronic jet
lag35. In macrophages, the REV-​ERB nuclear receptors
are a critical nodal point between the clock and immu-
nity. For instance, REV-​ERBα (encoded by NR1D1)
and REV-​ERBβ (encoded by NR1D2) suppress gene
expression (for example, matrix metalloproteinase 9
(Mmp9) and CX3C-​chemokine receptor 1 (Cx3cr1))
in macrophages by repressing transcription from dis-
tal enhancers that are selected by macrophage-​lineage
determining factors36. Furthermore, REV-​ERBα modu­
lates the inflammatory functions of macrophages
(including adhesion, migration and integrin activation)
via direct regulation of CC-​chemokine ligand 2 (Ccl2)
through a REV-​ERB binding motif in the promoter37
(Fig.  2a) . Although not cell-​s pecific, cryptochromes
(CRYs) also directly affect inflammatory pathways
through action on adenylyl cyclase38. In the absence of
Cry1, basal levels of cAMP rise, leading to increased
protein kinase A (PKA) activation. In turn, this induces
phosphorylation of the p65 subunit of nuclear factor-​κB
(NF-​κ B) at S276, leading to NF-​κ B activation.
Consequently, basal levels of several pro-​inflammatory
cytokines (including IL-6, tumour necrosis factor (TNF)
and CXC-​chemokine ligand 1 (CXCL1)) are increased
in fibroblasts and bone-​marrow-derived macrophages
from Cry1−/−Cry2−/− mice38. Thus, molecular effectors
of pro-​inflammatory pathways are directly coupled
to clock genes.
Circadian control of innate immune cell movement.
The circadian clock is a key regulator of the temporal
abundance of innate immune cells around the body.
Studies by several laboratories have highlighted the
importance of a functional clock in regulating the daily
flux of inflammatory cells between bone marrow, blood
and immune organs under homeostatic conditions. For
instance, leukocyte numbers in blood show time-​of-day-​
dependent variation in humans39, mice40 and hamsters41.
This can be abolished by deletion of the core clock gene
brain and muscle ARNT-​like 1 (BMAL1; also known as
ARNTL)40 or by inducing chronic SCN arrhythmia41.

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 Reviews

Resident Mast cell DC CD4+ T cell Circulatory and/or migratory

Macrophage
CD8+ T cell

B cell

Microglia
PERs CRYs

Monocyte
PERs CRYs
PERs
Club cell
(bronchiolar CLOCK–
CRYs
exocrine cell) Neuronal
BMAL1 PAS2
NK cell
RORs
REV-ERBs RORs
Bmal1 REV-
ERBs

IEC
Neutrophil

Blood vessel Eosinophil

Fibroblast-like
synoviocyte
ILC

b
Cell group Cell type Driver Effect Refs
Innate Macrophage Lyz2–Cre 31

and/or (also known

monocyte as LysM–Cre) Increased monocyte recruitment and atherosclerosis lesion size; 79

polarization towards an M1-like phenotype (on ApoE–/– background)

Loss of diurnal variation in Ly6Chi monocyte numbers in spleen, blood 23

and bone marrow; reduced survival and higher inflammatory cytokine

production in Listeria monocytogenes infection; increased weight gain,
adiposity, insulin resistance and metabolic dysfunction on high-fat diet
78

LPS stimulation
Significantly enhanced serum IL-12–p40 under basal conditions, loss of 76
time-of-day susceptibility to EAE and exacerbated EAE pathology,
including increased CNS infiltration of inflammatory monocytes with
increased IL-1β production

Microglia Cd11b–Cre Reduced Il6 upregulation and attenuated neuronal 131

Adaptive T cell Cd4–Cre Differentiation and function unaffected, no impact on EAE severity and 55

reduced percentages of IFNγ+, IL-2+ and TNF+ T cells in L. monocytogenes

infection
Loss of oscillations in T cell numbers in lymph nodes and lymph; loss of 54

oscillations in homing capacity and Ccr7 and S1p1 expression

Lck–Cre Loss of time-of-day variation in susceptibility to EAE and reduced pathology 54

B cell Mb1–Cre Differentiation and function unaffected 55

Cd19–Cre Loss of oscillations in B cell numbers in lymph nodes and lymph 54

Stromal Club cell Ccsp–iCre Increased CXCL5 production and neutrophil influx 43

Nature Reviews | Immunology

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Reviews

Healthy Clock-disrupted
a
Day Night

REV–ERB Il6, ↑ β1 integrin

REV–ERB Il6, ↑ IL-6, IL-12–p40, Ccl2 ↑ Adhesion to
Ccl2 or RORα VCAM1
CCL5, CXCL1
and CCL2 RORα ↑ Chemotaxis
↑ IL-6 and CCL2
Overexpression

TLR9

b LPS LPS
Day Night Day Night
Corticosterone Bmal1-/- Corticosterone
GR Club cells GR

↑ CXCL5 ↓ CXCL5 ↑↑ CXCL5 ↑↑ CXCL5

Fig. 2 | Regulation of innate immunity in the macrophage and the lung. Clock regulation of innate immunity occurs in
an immune cell-​intrinsic and cell-​extrinsic manner. a | In macrophages, for example, the intrinsic clock regulates immune
function at multiple levels. Oscillations in the expression of the pattern-​recognition receptor Toll-​like receptor 9 (TLR9)
occur, along with temporal gating of cytokine release. In this way , the clock regulates not only the ability to sense
pathogens but also the extent of the cell’s response to such stimuli. Clock disruption in the macrophage yields a
pro-​inflammatory phenotype, as a subset of cytokines (for example, IL-6 and CC-​chemokine ligand 2 (CCL2)) loses the
rhythmic repression generated by REV-​ERB signalling. Overexpression of the transcriptional activator retinoid-​related
orphan receptor-​α (RORα), which binds to the same locus as REV-​ERB, generates a similar phenotype. b | Recruitment of
innate immune cells to tissues is also under clock control. In healthy murine lung exposed to lipopolysaccharide (LPS),
enhanced production of the neutrophil chemoattractant CXC-​chemokine ligand 5 (CXCL5) and increased neutrophil
recruitment are observed during the day. At night, endogenous glucocorticoids bind the glucocorticoid receptor (GR),
inhibiting Cxcl5 transcription and reducing neutrophil influx. Targeted ablation of Bmal1 in Club cells (Ccsp–Bmal1−/−)
has a clear pro-​inflammatory effect in this model. Lack of a functional oscillator in these cells greatly increased CXCL5
production and enhanced neutrophil recruitment after LPS at both time points. Although corticosterone still binds the
GR at night, the subsequent repression of Cxcl5 no longer occurs and neutrophilia increases. VCAM1, vascular cell
adhesion protein 1.

Brain and muscle
ARNT-like 1
(BMAL1). A basic helix–loop–
helix PER–ARNT–SIM (bHLH–
PAS) domain transcription
factor that dimerizes with
CLOCK to bind E-​boxes in gene
promoters to induce circadian
gene expression.
Leukocyte numbers also exhibit natural temporal var-
iation in the lung42. This persists under inflammatory
conditions but becomes less regular, with granulocytes
rather than lymphocytes dominating as the oscillating
cell type42. Gibbs and colleagues showed that neutro-
phil recruitment to the lung after acute inflammatory
challenge is strongly circadian gated, driven by the
rhythmic release of the chemokine CXCL5 from non-​
ciliated bronchiolar epithelial Club cells43 (Fig. 2b). Also,
neutrophil recruitment to sites infected with Leishmania
parasites has been shown to be circadian regulated44.
Neutrophils are the most abundant leukocytes in
human blood but appear to possess a weaker circadian
oscillator — perhaps modified as the neutrophil develops
from the myeloid progenitor28. Turnover of neutrophils
is known to be under circadian control, with rhythmic
clearance of aged neutrophils from the blood occurring
in the bone marrow (where they are engulfed by bone-​
marrow-resident macrophages)45. This clearance, which
occurs towards the end of the daily resting phase in mice,
leads to a subsequent egress of haematopoietic precursor
cells from the bone marrow in a liver X receptor (LXR)-
dependent manner. Ella et al. described rhythmic daily
changes in superoxide production by human neutrophils

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CLOCK
(Circadian locomoter output
cycles kaput). A basic helix–
loop–helix PER–ARNT–SIM
(bHLH–PAS) domain
transcription factor that can
dimerize with BMAL1 to
regulate circadian gene
expression.
Zeitgeber time
Zeitgeber, literally ‘time giver’,
is a cue (such as light) that
entrains the circadian clock.
Zeitgeber time (ZT) is the time
after light onset; for example,
lights on is ZT0 and lights
off is ZT12 in a 12 h light-12 h
dark cycle.
Nature Reviews | Immunology
and in their ability to engulf bacteria, and this was
attributed to time-​of-day variations in the composition
of the peripheral neutrophil pool, which in turn is a
result of circadian changes in circulating chemokine lev-
els (specifically in levels of CXCL12 and/or CXCL2)28.
Thus, both trafficking and localization of neutro-
phils and their function are time-​of-day-​dependent.
Similarly, IL-5 and IL-13 cytokine production by a sub-
set of group 2 innate lymphoid cells (ILC2s) in response
to hormonal cues induced by feeding influences the
number of blood eosinophils and their recruitment to
peripheral tissues46.
Under homeostatic conditions, the extravasation
of innate immune cells from postcapillary venules to
tissues is under clock control and is regulated by the
rhythmic expression of pro-​migratory factors (such
as intercellular adhesion molecule 1 (ICAM1) and
CCL2) by endothelial cells. This is dependent on local
delivery of sympathetic tone and signalling through
β-​adrenoreceptors40. Rhythmic extravasation persists
under inflammatory challenge, and in a model of septic
shock in mice, neutrophil recruitment to the liver exhib-
ited strong circadian oscillations. This influences mouse
survival following administration of lipopolysaccharide
(LPS; a component of the cellular wall of Gram-​negative
bacteria), with LPS delivery in the evening resulting in
dramatically increased lethality compared with in the
early morning40.
The examples discussed above describe how innate
immune cell trafficking can be regulated by both
intrinsic pathways and circadian signals from other
cells. The trafficking of Ly6Chi inflammatory mono-
cytes from blood to bone marrow and from blood to
tissue sites of infection is under circadian control23,47.
CXC-​chemokine receptor 4 (CXCR4) mediates the
rhythmic fluctuations in overall numbers of circu-
lating Ly6Chi monocytes and their homing to tissue
reservoirs 47. During peritoneal infection with the
bacterial pathogen Listeria monocytogenes, traffick-
ing of Ly6Chi monocytes to the peritoneum is regu-
lated by the rhythmic recruitment of the polycomb
repressor complex 2 (PRC2) to the Ccl2 promoter by
BMAL1–CLOCK heterodimers23. These studies show that
both cell-​autonomous as well as cell-​extrinsic micro­
environmental oscillations can govern the distribution
of distinct leukocyte populations within the body.
Innate immune cell clocks in pathogen responses. At
the level of the whole animal, circadian timers clearly
have an impact on how the host innate immune system
responds to pathogens. In part, this occurs through
the regulation of inflammatory cell recruitment, where
immune cell clocks are critical for maintaining effec-
tive host responses to disease23,43. Further control is
exerted through the regulation of tissue-​resident cells
at sites of inflammation. Ingestion of the bacterial
pathogen Salmonella enterica subsp. enterica serovar
Typhimurium (S. Typhimurium) causes intestinal
inflammation. Microarray analysis of the caecum of
mice 72 h post-​infection with this pathogen revealed
that genes encoding antimicrobial peptides show
strong circadian oscillations during infection that affect
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Reviews
patho­gen colonization of the colon48. This leads to ele-
vated pathogen levels following infection in the morning
(Zeitgeber time 4 (ZT4) (Box 2)) as compared with infec-
tion in the mid-​dark phase (ZT16)48. This response is
dependent on the presence of a functional copy of the
circadian-​regulating CLOCK (circadian locomoter
output cycles protein kaput) protein as global disrup-
tion of CLOCK alters time-​dependent responses to
S. Typhimurium. In a Leishmania infection model,
Kiessling and colleagues were able to show that para-
site burden was circadian in nature. In addition, bone
marrow chimaeras of Rag2–Bmal1−/− mice demonstrated
that Bmal1 in non-​lymphocyte immune cells (macro­
phages) was responsible for modulating the magnitude
of Leishmania infection44. Parasites themselves can
also exhibit circadian behaviour. For example, 10% of
the genes of Trypanosoma brucei (parasites responsi-
ble for inducing sleeping sickness) are expressed with
a circadian rhythm49. Furthermore, this circadian tran-
scriptome affects sensitivity to treatment. The circadian
clockwork also regulates cellular innate immunity against
viruses. Immortalized lung fibroblasts lacking BMAL1
show altered susceptibility to viruses50. Respiratory syn-
cytial virus (RSV) and parainfluenza virus type 3 (PIV3)
(which are both non-​segmented negative-​strand RNA
viruses) replicate at higher frequencies in the absence of
BMAL1 (ref.50). Bmal1−/− mice intranasally infected with
RSV had higher viral titres and increased weight loss
than wild-​type controls50. Along the same lines, the time
of day of infection affects the host response to herpes­
virus or influenza A virus, with enhanced viral titres seen
when levels of Bmal1 are low or in the absence of this
gene51. Therefore, the circadian clockwork can regulate
cellular immunity against bacteria, parasites and viruses.
Clock control of adaptive immunity
Recently, molecular evidence has emerged to show that
the adaptive immune system is also under circadian
control. This evidence is perhaps surprising given that
adaptive immune responses are mounted over weeks
and thus were not thought to be heavily dependent
on the initial time of day of challenge. However, this is
in line with early reports that had initially uncovered
T cell responses to be rhythmic52,53. In a similar manner
to innate immune cells, circadian oscillations have been
described in cells of the adaptive immune system, such
as T cells54–56 and B cells25. In addition, dendritic cells
(DCs), which are professional antigen-​presenting
cells (APCs) located at the interface between innate
and adaptive immunity, show oscillations in core clock
components25. Generally speaking, clock gene ampli-
tudes appear to be weaker in immune cells than in
other cells, such as hepatocytes55. Whether this is due to
a weaker oscillating system per se, a reduced coupling
capacity or increased heterogeneity in immune cell
populations is currently unknown and would need to
be addressed using single-​cell analyses.
Role of clock genes in lymphocyte development.
Circadian clock genes have been associated with the
development of lymphocytes, but the extent of their
involvement is not yet fully established. Deficiency
Reviews
Box 2 | Circadian terminologies
a circadian rhythm is an oscillation with a period (time from peak to peak) of
approximately 24 h that is able to be entrained by external factors and persists in
constant conditions (see the Figure). amplitudes (difference between peak and mean
values) of these oscillations vary, as does acrophase (the timing of the peak value), such
that some parameters may peak during the day (for example, leukocyte numbers in
blood in mice) and some during the night (for example, leukocyte numbers in lymph
node in mice). experiments assessing rhythmicity are often performed under one (or
both) of two conditions: light–dark or constant darkness. in the light–dark condition,
abbreviated as LD, animals or participants are exposed to controlled cycles of light and
dark (usually 12 h light:12 h dark) to mimic a day–night cycle. in this situation, the timing
nomenclature used is the ‘Zeitgeber time’ (Zt). a Zeitgeber (German for ‘time giver’) is a
stimulus that provides a timing signal, such as light onset, and Zt0 is simply the timing
of this stimulus. all other times are measured in a 24 h cycle relative to this stimulus. For
example, in a cycle with 12 h light:12 h dark, Zt6 is the middle of the light phase, Zt12 is
then the onset of the dark phase, Zt18 is the middle of the dark phase and Zt24/0 is the
onset of the light phase once more. an oscillation under these conditions would be
termed diurnal. in constant darkness, however, no such light stimulus exists. in this case,
times are noted as ‘circadian time’ (Ct) and better reflect the animal and/or
participant’s subjective time in the absence of external cues. thus, in dark–dark (DD)
experiments, Ct0 is the start of the subjective day (that is, when the lights would have
come on), Ct6 the middle of the subjective day, Ct12 the start of the subjective night
(that is, when the lights would have gone off), Ct18 the middle of subjective night and
Ct24/0 the start of the next subjective cycle. an oscillation under these conditions
would be termed circadian.
Period
Amplitude
Mean value
Acrophase
ZT LD
CT DD
0 12 24/0 12 24/0
of BMAL1 negatively affects mouse B cell develop-
ment, with Bmal1−/− animals showing significantly
reduced B cell numbers in blood and spleen57. Bmal1-
deficient B cells also showed a specific deficit in pro-
duction of IgG1 (but not in IgG2a, IgG2b, IgG3 or IgM)
antibodies after immunization, but they exhibited no
functional difference in response to LPS in vitro, suggest-
ing that those cells that do develop are still largely func-
tional. Adoptive transfer experiments revealed that it is
not the B cell intrinsic clock but rather loss of Bmal1 in the
bone marrow microenvironment that hampers B cell dif-
ferentiation57. This finding is also consistent with the data
of Hemmers and Rudensky, who showed that mice with
a B cell-​specific Bmal1 deficiency do not show altered
B cell differentiation or function55. These experiments
suggest that the influence of clock genes in the genera-
tion of B cells is not cell-​autonomous but dependent on
Circadian time
(CT). A measure of subjective the microenvironment.
time used when organisms are With respect to the influence of the circadian clock
isolated from Zeitgebers (for on the development of T cells, the overall T cell number
example, constant darkness). appears to be unaffected by global or cell-​specific Bmal1
CT0 represents the start of
subjective day and CT12
ablation55,57. However, Yu and colleagues reported a role
represents the start of for the clock in regulating the differentiation of T helper 17
subjective night. (TH17) cells58. TH17 cell development has been shown to
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
be suppressed by the circadian-​regulated basic leucine
zipper transcription factor nuclear factor IL-3-regulated
protein (NFIL3) in a cell-​intrinsic manner, and Nfil3−/−
mice exhibit increased TH17 cell frequencies in spleen,
small intestine and colon58. NFIL3 is crucial for the
development of ILCs59 and is a major regulator of TH17
cell frequency via rhythmic suppression of Rorc, which
encodes RORγt, a key transcription factor necessary for
TH17 cell differentiation58. Loss of ILC3s in the Nfil3−/−
mice would already skew towards a higher propensity
for TH17 cell differentiation as ILC3s are a significant
source of IL-22, which inhibits TH17 cell differentiation.
Depletion of ILCs thus enhances TH17 cell responses60.
Within CD4+ T cells themselves, Nfil3 and Rorγt are
rhythmically expressed in an anti-​phase manner, with
Nfil3 expression peaking during the dark phase (night)
and Rorγt expression peaking during the light phase
(day). T cells isolated during the day are therefore more
prone to differentiate into TH17 cells upon stimulation
than those isolated at night58. This propensity may addi-
tionally be due to the fact that during the day levels of the
night-​signalling hormone melatonin are low, because
melatonin treatment of human T cells has been shown
to inhibit TH17 cell differentiation via the NFIL3–RORγt
pathway61. Instead, melatonin promotes the develop-
ment of IL-10-producing type 1 regulatory (TR1) cells61.
The circadian clock protein REV-​ERBα directly inhibits
transcription of Nfil3, and Nfil3 expression is ele-
vated in both REV-​E RBα −/− T  cells and in T  cells
from ClockΔ19/Δ19 mice, which exhibit a lengthened
circadian period and arrhythmicity in complete dark-
ness 58. ClockΔ19/Δ19 mice also displayed reduced
frequencies of TH1 cells in the intestines, implying
additional pathways by which the circadian clock can
affect T cell polarization. The circadian regulation of
T cell development may be particularly important for
maintenance of an appropriate balance of cell types
by time of day, as chronic circadian disruption via
simulated jet lag resulted in increased proportions
of TH17 cells, rendering animals more susceptible to
experimental colitis58.
In addition, the circadian-​regulated proteins differ-
entially expressed in chondrocytes protein 1 (DEC1;
also known as BHLHE40) and DEC2 (also known as
BHLHE41) are driven by E-​box mediated BMAL1–
CLOCK binding. DEC1 is involved in essential CD4+
T cell effector functions, including in the regulation
of a large group of CD28-dependent genes62. Antigen-​
specific DEC1-deficient CD4+ T cells have cell-​intrinsic
defects in survival and proliferation62. Meanwhile, DEC2
is critical for the development of B-1a cells, and DEC2-
deficient B-1a cells show an abnormal B cell receptor
(BCR) repertoire63. Taken together, the proteins of
the circadian clock clearly play an important role in the
function of lymphocytes.
Lymphocyte trafficking rhythms. Similarly to innate
immune cells, T cells and B cells exhibit strong cir-
cadian oscillations in the blood, with peak numbers
during the respective behavioural rest phase of the
organism (that is, during the day in mice and at night
in humans)39,54,64–66. These rhythms have been linked to
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 Reviews

Day Night

Blood Blood
↓ CCR7 ↑ CCR7
(leukocytes) (leukocytes)

↑ NA

↓ Sympathetic tone ↑ Sympathetic tone

↓ Leukocyte content ↑ Leukocyte content

↑ S1p1 ↓ S1p1
(leukocytes) HEV (leukocytes)

Efferent Efferent
lymph lymph

B cell CD4+ T cell CD8+ T cell

Fig. 3 | Regulation of adaptive immunity in the lymph node. A coordinated oscillation in pro-​migratory factors drives
leukocyte oscillations in blood, lymph node and lymph.  During the active phase, sympathetic tone (noradrenaline (NA))
and concentrations of CC-​chemokine ligand 21 (CCL21) are high in the lymph node. In addition, expression of the
receptor for CCL21, CC-​chemokine receptor 7 (CCR7), is at its peak on T and B cells. The combination of high receptor
expression on the cells and high chemokine content in the lymph node provides a strong attraction signal, leading to
increased lymph node cellularity during this phase. During the rest phase, CCL21 and CCR7 expression decrease and
retention signals are reduced. At the same time, leukocytes begin to upregulate expression of the sphingosine
1-phosphate receptor 1 (S1P1), a critical mediator of cell egress. Thus, the reduction in sympathetic tone and homing
drive coupled with an increased egress potential lead to an emptying of the lymph node during the day. HEV, high
endothelial venule.

oscillations in CXCR4 and CX3CR1 expression, regulated
by glucocorticoids and catecholamines64,65, as well as
hypoxia-​inducible factor 1α (HIF1α) signalling67. Recent
papers found that under steady-​state conditions, lympho­
cytes within the lymph node also exhibit circadian
oscillations in mice, with the highest numbers present
at the beginning of the active phase (that is, at night in
mice)54,66,68 (Fig. 3). These higher numbers were not due
to local intranodal proliferation but depended on both
the rhythmic homing of cells from blood into lymph
nodes and the rhythmic egress of cells from lymph nodes
into efferent lymph. A key regulatory element is the
CC-​chemokine receptor 7 (CCR7), which exhibited diur-
nal oscillations in T cells and B cells, with its expression
peaking in phase with CCL21 levels on high endothelial
venules in lymph nodes54. This receptor–ligand pair is
critical for control of T cell migration into lymph nodes,
whereas B cells also rely on additional receptors such as
CXCR4 and CXCR5 (refs69,70). Peak expression levels
of both CCR7 and CCL21 were observed around night
onset, when the highest recruitment of CD4+ T cells,
CD8+ T cells and B cells occurred54. Both the microen-
vironment and lymphocytes themselves contribute to
this process, but T cell-​specific genetic deletion of the
circadian clock regulator BMAL1 was sufficient to ablate
the time-​dependent difference in lymph node homing
and cellularity54.
Interestingly, the egress of cells into efferent lym-
phatics was also found to be rhythmic, with lymphocyte
counts highest in lymph around ZT9 (refs54,66). This
finding was dependent on rhythmic expression of
sphingosine 1-phosphate receptor 1 (S1P1)54, which
promotes lymphocyte egress from lymph nodes
in response to the chemotactic lipid sphingosine
1-phosphate 71. Cells that migrated into the lymph
node at night also stayed longer within this tissue than
the (fewer) cells that had immigrated during the day.
Suzuki and colleagues demonstrated that this is depend-
ent on lymphocyte expression of β2-adrenergic recep-
tors72 and rhythmic environmental sympathetic tone66.
β2-Adrenergic-​receptor-deficient lymphocytes transited
through lymph nodes more quickly and failed to show a
diurnal pattern66. This finding indicates that enhanced
sympathetic tone and engagement of the β2-adren-
ergic receptor at night helps maintain cells in lymph
node compartments, likely by boosting lymphocyte
expression of the retention factors CCR7 and CXCR4
(ref. 72) . This longer lymph node dwell time plays an
important role in the generation of adaptive immune
responses54,66. Thus, time of day both dictates expression

Nature Reviews | Immunology

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews
of critical pro-​migratory molecules on lymphocytes and
endothelial cells and governs the trafficking behaviour
of lymphocytes from blood through lymph nodes and
into efferent lymphatics.
Rhythmic adaptive immune responses. Initial obser-
vations by Esquifino and colleagues showed that the
adaptive immune system exhibits functional rhythmi­
city. They demonstrated that proliferative responses of
rat lymph node cells following treatment with LPS or
concanavalin A were strongly rhythmic, with signifi-
cantly stronger proliferation occurring at midday than at
midnight73. Subsequent studies by Fortier and colleagues
confirmed these observations and further demonstrated
that this was dependent on the circadian clock. Mice
bearing the circadian mutation ClockΔ19/Δ19 exhib-
ited strongly blunted rhythms of proliferative responses
of T cells after direct stimulation of the T cell receptor
(TCR)74. Furthermore, they found that intravenous
injections of ovalbumin (OVA)-loaded DCs into recip-
ient mice at ZT6 (daytime), as opposed to at ZT18
(night-​time), generated higher numbers of OVA-​specific
T cells within the spleen. These data provide evidence
for the importance of circadian timing in the interaction
between T cells and APCs.
For an adaptive immune response to be generated,
APCs must functionally interact with lymphocytes.
Silver and colleagues were the first to investigate a
rhythmic link between the adaptive and innate immune
system. They demonstrated that the expression of Toll-​
like receptor 9 (TLR9) — which recognizes bacterial
CpG DNA — but not of other TLRs by macrophages,
DCs and B cells, exhibited oscillations that were con-
trolled by the circadian clock gene Per2 (ref.75). In line
with this, mice treated with OVA coupled to CpG at
the time of enhanced TLR9 expression (ZT19 versus
ZT7) showed elevated OVA-​specific adaptive immune
responses several weeks later75. Lymph node cultures
from these mice showed higher levels of OVA-​induced
lymphocyte proliferation and production of IFNγ at
ZT19. In addition, lymphocytes from mice lacking a
functional PER2 protein showed a stronger immune
response than wild-​type cells75.
These data demonstrate that circadian differences
in the expression of pattern-​recognition receptors
(PRRs) by APCs can drive rhythmic adaptive immune
responses and are thus under the control of the cir-
cadian clock. However, it is currently unclear how
the observed differences in the time of day of the
acrophase of the response arise. Whereas Silver and
Suzuki described stronger immune responses at night
(ZT17 and ZT19, respectively)66,75, Fortier and Druzd
observed peak responses during the day (ZT6 and ZT8,
respectively)54,74. An explanation for these discrepan-
cies might lie in the type of response elicited: injec-
tion sites (subcutaneous, intraperitoneal, intravenous
or intradermal), readout tissue (lymph node versus
spleen), lighting conditions, responding cell type and
antigen choice all contribute to such variations, par-
ticularly whether the antigen needs to be taken up by
APCs to be transported to lymph nodes or can move
there directly in a soluble form.
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Recent vaccination studies also showed diur-
nal rhythmicity in adaptive immune responses.
Responses to nitrophenyl (NP), a soluble antigen that
is directly transported to lymph nodes, or to myelin-​
oligodendrocyte glycoprotein (MOG), which is an
antigenic target in certain models of experimental
autoimmune encephalomyelitis (EAE), were higher
at the times when more cells were present in lymph
nodes 54,66,76. Stimulation at high cellularity times
resulted in a stronger humoral response, as demon-
strated by higher NP-​specific antibody titres, as well as
more NP-​specific IgM+ germinal centre B cells 7 days
after immunization66. In addition, in the EAE model a
stronger cellular immune response was observed with
more IL-17-producing TH17 cells being present, more
IL-2 being made, higher leukocyte infiltration into the
central nervous system and ultimately a higher dis-
ease score 14 days after inoculation54. Importantly, the
humoral immune response to NP became arrhythmic in
mice lacking the β2-adrenergic receptor66, and animals
with a myeloid-​cell-specific or T cell-​specific deficiency
in BMAL1 became arrhythmic in the EAE model of
autoimmunity54,76. Rhythmicity was not restricted solely
to sterile immunization responses but was also observed
following infection with pathogens, such as influenza A
virus and the bacterium Helicobacter pylori54.
Current research efforts now aim to take these find-
ings into the clinic and establish novel ways to enhance
responses to vaccines. A randomized trial of different
times of day of vaccination of three strains of influenza
virus yielded promising results. When 276 patients (over
65 years of age) were vaccinated in the morning they had
greater antibody titres 1 month later than patients vacci-
nated in the afternoon77. These results are encouraging,
but they will need to be validated in other preclinical
laboratory models and clinical trials using larger patient
cohorts. Taken together, this points to a strong role of
the circadian clock in the polarization of the adaptive
immune response, which is strongly dependent on the
initial time of day of antigen challenge.
Innate versus adaptive immune clocks. Whereas
myeloid-​specific ablation of the circadian gene Bmal1
leads to a general pro-​inflammatory phenotype in mice
that is characterized by higher levels of TNF, IL-6, IL-1β
and CCL2 (refs23,31,78,79), the phenotype of T cell-​specific
Bmal1 deficiency appears to be more time-​of-day-​
dependent54,55. Whether this is because the currently
employed genetic targeting strategies for lymphocytes
(CD4–Cre for T cells54,55) are more specific than for mye-
loid cells (that is, where Lyz2–Cre is currently used, thus
targeting most myeloid cell populations23) is unclear. To
better compare this, future studies will need to use pro-
moters that are specific for neutrophils, monocytes or
macrophages for Bmal1 ablation. Infection of mice with
L. monocytogenes resulted in a lower percentage of IL-2+,
IFNγ+ and TNF+ T cells in mice with a T cell-​specific
deficiency in Bmal1 than in control animals55. In addi-
tion, in models of EAE, mice with a T cell-​specific Bmal1
deficiency had lower disease scores than controls54
whereas mice with myeloid cell-​specific loss of Bmal1
showed exacerbated pathology76. This finding indicates
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RORα
The nuclear receptors RORα,
RORβ and RORγ are
transcriptional activators that
bind to ROR response element
(RORE) sites in target gene
promoters.
Nature Reviews | Immunology
Reviews
that the outcome of Bmal1 deletion on inflammation is Many bacterial genera within the gut microflora of
lineage dependent. mice and humans exhibit circadian oscillations them-
selves. For example, Lactobacillaceae show strong oscil-
Microbial regulation of circadian immunity lations in general abundance over the course of the day,
Recent evidence demonstrates that symbiosis between peaking at the onset of the mouse active phase85,86. This
microbiota and intestinal epithelial cells is critical in rhythmicity is driven by feeding rhythms and can be
maintaining immune homeostasis in the gut. Intriguingly, ablated by jet-​lag protocols (repeated shifts in timing of
this interaction is orchestrated by the circadian clock light–dark cycles) or antibiotic treatment85,86. Chronic
and cues from the microbiota that activate intestinal jet lag in mice leads to obesity, whereas in humans jet-​
TLRs. Mukherji and colleagues showed that the anti-​ lag protocols result in a type 2 diabetic phenotype86.
phasic binding of RORα and REV-​ERBα to TLR genes This finding indicated that aberrant circadian rhythms
acts as an activator and repressor, respectively, to medi- in humans can lead to metabolic disturbances. Gut
ate the circadian expression of TLR1–TLR5 and TLR9 bacteria exhibit circadian rhythmicity in terms of their
(but not TLR6 and TLR7)80. This oscillatory promoter numbers and activity, and their rhythmic function is
occupancy drives rhythmic activation of the key pro-​ also important for the systemic regulation of host tis-
inflammatory transcription factors activator protein 1 sues8. This is achieved by a specific rhythmic signature
(AP-1) and NF-​κB to exert their functions at diurnal of metabolites present in blood and is important in
times ZT20–ZT4 (which is the active phase in mice). driving epigenetic changes in host tissues distant from
Antibiotic-​mediated ablation of the microbiota disrupts the gut, such as the liver8. Thus, rhythmic release of
this rhythmicity and induces a pre-​diabetic syndrome in metabolites and LPS from the microbiota may be key
mice, characterized by hyperglycaemia and hypoinsuli- in directly driving important rhythmic immune-​related
naemia80. Intriguingly, rhythmicity could be restored by phenotypes, such as neutrophil ageing and associated
administration of LPS. Additional recent findings pro- activation45,82. In addition, rhythms in commensal bac-
vide a link between rhythmic Nfil3 expression in intesti- teria appear to help synchronize the body to the external
nal epithelial cells, microbiota and host metabolism that environment and thus indirectly influence a rhythmic
is mediated by immune cells81. The induction of TLR– immune system.
MYD88 signalling by bacteria stimulates DCs to secrete
IL-23 and activate ILC3s. This activates signal transducer Synchronization of the immune system. Microbial
and activator of transcription 3 (STAT3), leading to an products and feeding rhythms can synchronize periph-
inhibition of REV-​ERBα, a negative regulator of NFIL3. eral clocks in tissues87 but it is yet unclear how immune
Epithelial NFIL3 regulates lipid uptake and export in cells — most of which are migratory — are synchro-
intestinal epithelial cells so that mice with an intestinal-​ nized in  vivo to the external environment. Initial
specific deletion of Nfil3 are resistant to obesity induced studies focusing on circulating hormones were able
by a high-​fat diet81. These data indicate that the micro­ to demonstrate a correlative link between glucocorti-
biota is a previously unrecognized and important general coids and adrenaline and numbers of circulating T cells
regulator of immune rhythmicity in the host (Fig. 4). in blood by modulating surface CXCR4 and CX3CR1
levels65. It has now been shown using cell-​specific dis-
Microbial products shape circadian immunity. LPS ruption of glucocorticoid signalling that rhythmic T cell
also appears to be a critical factor in shaping the acti- trafficking is regulated via glucocorticoid receptor
vation status of neutrophils that are present in blood. (GR)-dependent induction of the IL-7 receptor (IL-7R)
Although blood neutrophils have long been thought to and CXCR4 (ref.68). Dexamethasone88 and high tran-
be a relatively homogeneous population, heterogeneity sient serum concentrations (a so-​called serum shock)87
arises owing to differences in their age and activation are used to synchronize cells in culture, indicating that
level. Neutrophils are short-​lived and — after having both glucocorticoids and feeding rhythms are impor-
been mobilized into the blood in the morning45 — age tant for the synchronization of migratory cells in vivo.
in blood over the course of the day45,82. Aged neutro- With respect to the sympathetic nervous system, lack
phils exhibit higher levels of CXCR4 on their surface of β2-adrenergic or β3-adrenergic receptors resulted
and lower levels of L-​selectin45,83,84 and are present at in an ablation of the rhythmic trafficking behaviour
higher concentrations during the day in mice45. This is of leuko­c ytes to tissues40,66. However, it is currently
associated with an enhanced activation status83, includ- unclear whether the clockwork in immune cells and/or
ing an increased capacity to form neutrophil extra- stromal cells was also affected in this case. Furthermore,
cellular traps82. Microbiota-​derived LPS drives this a recent surprising study has found that the cell-​intrinsic
ageing process82. Mechanistically, this is modulated by clock is sufficient for the rhythmic migration behaviour
TLR2- and TLR4-expressing haematopoietic cells, as of leuko­cytes67. Zhao and colleagues showed that in
well as by MYD88-expressing myeloid cells. Ablating humanized mice, human and mouse leukocyte num-
the microbiota dramatically reduced the number of bers within the same organism exhibited inverted oscil-
aged neutrophils and was also effective in alleviating lations in blood, reproducing the trafficking pattern
the pathogenesis of sickle cell disease and endotoxin-​ previously observed in both species. This cyclic pattern
induced septic shock82. Thus, rhythmically recurring of trafficking correlated with rhythmic CXCR4 expres-
cycles of neutrophil activation in the circulation can have sion, which was regulated by a HIF1α–BMAL1 hetero­
dramatic consequences on the time-​of-day-​dependent dimer. p38 mitogen-​activated protein kinases (MAPKs)
worsening of inflammatory disorders. and MAPK kinase activated protein kinase 2 (MK2)
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews

Outer layers Epithelium

Day Muscularis Night

mucosae
DC
Lamina propria
IL-23
MYD88
ILC3

IL-22 IEC
REV–ERBα
Lumen CD4+
T cell
CD4 T cell
+
Neutrophil Microbiome
REV–ERBα NFIL3
Blood MYD88 Bacteria
vessel
Metabolites
↑ CXCR4 ↓ CXCR4

NFIL3
RORγ t

RORγ t

↓ TH17 cell
↑ TH17 cell
in vitro in vitro

Fig. 4 | Interactions of the clock , microbiome and immune function. Circadian oscillations occur not only in host cells
but also in the composition of microbiota.  The relative abundance of microorganisms fluctuates across a day , as does the
quantity and variety of metabolites released from the gut into the bloodstream, which can, in turn, signal to peripheral
tissues or circulating cells (for example, via Toll-​like receptors (TLRs)). Expression of a subset of TLRs is rhythmic in intestinal
epithelial cells (IECs) and drives increased pro-​inflammatory responses via activator protein 1 (AP-1) and nuclear factor-​κB
(NF-​κB) at the light onset (Zeitgeber times ZT20–ZT4). Activation of the MYD88 pathway , a downstream cascade common
to many TLRs, was recently shown to regulate neutrophil ageing. This process occurs more prominently during the rest
phase, with aged neutrophils increasing CXC-​chemokine receptor 4 (CXCR4) expression and preferentially homing to
bone marrow. Homing of such senescent neutrophils triggers mobilization of haematopoietic progenitor cells, thereby
driving rhythmic modulation of this niche. Cell differentiation may also be modulated rhythmically , as in the case of T
helper 17 (TH17) cells and their key differentiation factor retinoid-​related orphan receptor γt (RORγt). In cells isolated
during the night, RORγt is suppressed by nuclear factor IL-3-regulated protein (NFIL3) and polarization towards TH17 cell
differentiation is reduced. However, in cells isolated during the day , the clock protein REV-​ERBα inhibits this suppression,
allowing RORγt levels to increase along with increased TH17 cell differentiation. In this way , T cell populations cross this
developmental checkpoint in a coordinated manner. Furthermore, MYD88-dependent activation of dendritic cells (DCs)
can trigger IL-22 release by type 3 innate lymphoid cells (ILC3s), which in turn promotes signal transducer and activator of
transcription 3 (STAT3)-dependent inhibition of REV-​ERBα expression in the IECs. Therefore, microbiota components may
further fine-​tune diurnal rhythms in the gut.

exhibited opposite effects between mice and humans in when administered at a more physiological dose that
generating intracellular reactive oxygen species, which is more likely to be encountered in nature23. Thus, this
subsequently regulated HIF1α expression67. This indi- heightened immune sensitivity would be an adapta-
cates that immune cells use reactive oxygen species as tion to potentially recurring environmental challenges,
their synchronization cue. Taken together, a complex with the additional effect of reducing detrimental
network of factors appears to be important for immune immune responses (such as a high activation status of
cells to be synchronized to external time. neutrophils) at times when they are not needed. Thus,
a timed immune response — with a refractory phase
Potential benefits of rhythmic immune responses in between, possibly for opposing immune processes
A key question in our field is why is the immune sys- to occur — may ultimately increase the survival of an
tem so tightly controlled by circadian oscillations? organism. Evidence for this lies in the fact that both
The most striking examples of timing in the immune immune cell-​i ntrinsic and cell-​e xtrinsic (such as
response are mice that succumb to high doses of pro-​ endothelial and stromal cell) rhythms are coordinated
inflammatory mediators or pathogen more promi- in a highly complex fashion to balance the recruitment
nently in the afternoon18,89. However, at the same time, and activation of immune cells. An alternative expla-
mice are able to fight off bacteria more effectively nation could be that rhythmic immune responses may

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have evolved as a secondary consequence of rhythmic
metabolic processing within the immune cell itself.
For the latter, the key benefit of circadian rhythmicity
would be the ability of cells to partition metabolic flux
and detrimental redox processes in time and space9.
Clearly, metabolic and immune processes are intricately
linked, and both affect each other also with respect to
circadian timing. For example, metabolic cues are crit-
ical for the entrainment of peripheral clocks, such as
those present in immune cells. In addition, disruption
of clock gene function results in a host of metabolic
disorders, which are often associated with immune
phenotypes90, such as mice with a myeloid cell-​specific
deletion of Bmal1 that present with a pro-​inflammatory
phenotype and over time become obese23. Thus, par-
titioning of immune cell function to specific times of
the day likely demonstrates an adaptation of the organ-
ism to pathogen exposure in addition to detrimental
endogenous processes.
Therapeutic implications
Numerous animal models show that genetic or envi-
ronmental disruption of the clock can expedite the
development of inflammatory disease or exacerbate
disease pathology91–94. This translates to humans, as
employment in rotating shift work has been associated
Box 3 | Circadian clock links to cancer
the circadian clock regulates daily rhythms in cell division, metabolism, inflammation
and DNa damage response, and disruption of the circadian clock is known to increase
the incidence of cancer122,123. specifically, the core clock machinery is tightly coupled to
metabolic cycles and is able to adapt to shifting metabolic rhythms, such as those
imposed by enforced feeding schedules, leading to reprogramming of circadian
transcriptional circuits and altered cyclic metabolism124–126. Both central and peripheral
circadian homeostasis are known to be disrupted in a number of pathological states,
including inflammation and cancer. Further insight into this process has been shown in
a recent study of lung adenocarcinoma in mice, which explored the impact of tissue
malignancy on the circadian organization of body organs127. Lung tumours driven by
conditional activation of KrasG12D mutation and loss of p53 tumour suppressor were
associated with widespread disruption of hepatic glucose production and altered
insulin signalling and lipid metabolism, mediated by alterations in aKt, aMPK and
sreBP pathways. surprisingly, these distal effects were not mediated by hepatic
malignancy, disruption of the core circadian clockwork or indeed behavioural
rhythmicity. rather, the authors proposed that circadian remodelling of the hepatic
transcriptome was mediated by the release of immune signals, such as tumour-​derived
cytokines (for example, iL-6) from malignant lung tissue, which then acted as distal
reorganizers of circadian hepatic metabolism.
the link between the circadian clock, inflammation and metabolism raises the
intriguing possibility as to whether pharmacological targeting of specific elements of
the core circadian clockwork may regulate metabolic and inflammatory pathways and
indirectly provides effective therapeutic options for the treatment of malignancy and
cancer. a recent study has shown that pharmacological activation of rev-​erB using
small-​molecule agonists is lethal in cancer and oncogene-​induced senescence128. these
circadian-​regulated rev-​erB proteins are constitutive repressors and nuclear hormone
receptors and act as important outputs coupling the core clock to physiology, playing a
central role in the regulation of rhythmic hepatic metabolism125 and inflammation31.
Sulli et al. showed that REV-​ERB agonists evoked an apoptotic response, confined to
malignant cells, and critically also revealed selective targeting of slowly proliferating
tumorigenic populations. importantly, this study now links for the first time key
hallmarks of cancer — de novo lipogenesis and apoptosis, important in meeting
metabolic demands of cancer cells — to a specific circadian clock circuit. this suggests
a hitherto unsuspected link between elements of the core clock, cellular metabolism
and malignancy.
Nature Reviews | Immunology
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Reviews
with an increased risk in developing inflammatory dis-
eases such as psoriasis95 and irritable bowel syndrome96.
In keeping with this, Cuesta et al. report desynchrony in
rhythmic immune parameters after simulated night-​shift
work97. Of course, rotating shift work affects not just
biological rhythms but also other lifestyle habits (sleep
quality and quantity, diet, mealtimes and exercise) that
may potentially affect the development of such disor-
ders. Nevertheless, this is a major concern for society.
Together, animal studies and clinical data corroborate
the notion that a healthy intact clock is necessary to
regulate immune homeostasis and to help prevent the
development of immune disorders. In addition to this
role under homeostatic conditions, the clock continues
to be critical in regulating inflammation once disease
is established. It is well recognized that human chronic
inflammatory diseases can show time-​of-day variation in
not only symptoms but also disease markers. Most often
cited is the nocturnal component to asthma, whereby
symptoms show exacerbations in the early morning98.
Similarly, patients suffering from rheumatoid arthritis
often report increased joint stiffness and pain in the early
morning99. These periods of increased symptoms corre-
late with peaks in disease markers, indicating that these
phenomena are not simply a consequence of sleep itself
(for example, caused by limited movement and altered
breathing patterns). There is an early morning peak in
the number of pulmonary eosinophils in patients with
asthma100 and in circulating cytokine levels in patients
with rheumatoid arthritis 101. The onset of cardio­
vascular events often shows time-​of-day variation, with
acute myocardial infarction and ventricular arrhythmias
more common in the morning102. This timing is likely a
consequence of circadian variation in blood pressure, the
coagulation cascade and vascular function. Furthermore,
there is evidence that in the days following the transition
to or from daylight savings time, there is an increase in
the incidence of acute coronary syndrome related to the
desynchrony between the internal clock and the external
environment103. Given the wide range of effects of the
circadian clock on immune function, it is not surprising
that in a chronic setting, there are rhythmic fluctuations
in inflammatory pathways, sufficient to cause physio­
logical effects. Our current understanding of these
interac­tions is very limited, but it is clearly important
that we gain deeper knowledge as this is likely to lead to
novel treatment options.
Seasonal diseases and underlying mechanisms.
There is emerging evidence that the immune system
also shows seasonal fluctuations in man and animals;
indeed, this may explain why some inflammatory dis-
eases have an additional seasonal component. Within
circulating immune cells, approximately 23% of the
genome shows seasonal variation in expression 104;
furthermore, the cellular makeup of the peripheral
immune system varies according to season104. It is not
yet established how annual changes in the external envi-
ronment modulate seasonal changes or to what extent
seasonal changes affect immune function over the year.
However, this new information may explain in part why
some immune disorders exhibit seasonal variation in
Reviews

the occurrence of exacerbations. Dopico and colleagues activity in patients with multiple sclerosis peaks in
noted a seasonal fluctuation in Bmal1 expression, with early spring and troughs in autumn106. This trend may
lower levels of expression during the winter months104. be attributed in part to seasonal variation in the noc-
Herpesviruses and influenza A viruses replicate more turnal pattern of secretion of pineal melatonin, with
efficiently in the absence of BMAL1, perhaps in part longer nocturnal durations in the autumn and winter61.
explaining why viral dissemination is more common These examples demonstrate that there may be impor-
in winter51. Individuals diagnosed with the debilitating tant seasonal changes in the phasing and exposure of
lung condition chronic obstructive pulmonary disease circadian-​regulated signals, tuned to the environment,
(COPD) are also more prone to exacerbation during the that can affect our physiology and health.
winter months105. This is thought to be the result of a
combination of increased prevalence of respiratory viral Chrono-​immunotherapy. Our knowledge regard-
infections, lifestyle changes during the colder months ing circadian variation in the expression and activ-
and reduced vitamin D levels. Conversely, disease ity of drug targets is steadily growing. A recent study
has shown that a high proportion of the best-​selling
drugs used in the treatment of inflammatory diseases
(as well as drugs for metabolic diseases, mental health
Targeting Timed disorders and cancer (Box 3)) directly target the prod-
the clock delivery ucts of genes that are themselves under clock control107.
Neurodegeneration Many of these drugs typically have short half-​lives
(multiple sclerosis) (less than 6 h); consequently, timed application in line
Melatonin decreases Vaccination
severity and/or incidence of Increased influenza antibody with target expression may markedly improve treatment
experimental autoimmune titres in morning versus efficacy (Fig. 5). Surprisingly, as yet there is very little use
encephalomyelitis132 evening in vaccinated elderly
population (≥ 65 years)77 of chronotherapy for the treatment of inflammatory
disorders in a clinical setting. One success involves the
Asthma use of glucocorticoids in the treatment of rheumatoid
Timed dosing increases arthritis. A slow-​release formula of prednisone has been
effectiveness of multiple manufactured, designed to be taken at bedtime, result-
treatments134
ing in release into the bloodstream 4 h later, and phased
Atherosclerosis to the peak in expression of inflammatory markers,
Anti-inflammatory effects
Chemotherapy of REV–ERB agonists112 which reduced the duration of morning stiffness of the
Timed treatment increases
therapeutic index133 joints108. We now know that the circadian clock (through
the actions of the CRY and REV-​ERB proteins) regu-
lates the response of the GR to its ligands109,110. Thus,
timed application of anti-​inflammatory glucocorticoids
may enable maximal therapeutic benefit while at the
same time reducing undesirable hepatic metabolic side
effects of these heavily used drugs109.
Bone marrow transplant
Arthritis
Slow-release prednisolone The clock as a therapeutic target for inflammation.
allows peak drug availability mouse model40 Our growing understanding of how components of the
at time of greatest need108
molecular clock interact with critical elements of inflam-
Cryptochrome-stabilizing matory pathways offers the exciting potential for the use
drugs are anti-inflammatory of pharmacological agents that target these clock pro-
(in vitro)116 teins as anti-​inflammatory agents. The challenge now
is to produce high-​affinity, high-​efficacy molecules that
enhance the activity of clock proteins. Recently, a num-
ber of tool compounds have been developed that show,
in principle, the benefits of pharmacological modula-
tion of clock proteins on the outcome of inflammatory
challenge. SR9009 is a synthetic REV-​ERB agonist111 that
Fig. 5 | Chronotherapy and the clock as a therapeutic target. The knowledge of has been shown to decrease atherosclerotic plaque bur-
circadian regulation of various aspects of immunity can inform treatment in two ways: den after chronic administration in mice112. Conversely,
modifying treatment regimens to the time of greatest efficacy (green boxes) and application of a synthetic REV-​E RB antagonist
targeting the clock itself (blue boxes).  Timed delivery of compounds has already been (SR82778)113 has detrimental effects in a mouse model of
shown to increase effectiveness of multiple drugs already in use, with the benefit of viral-​induced encephalitis114. In addition, the compound
increasing the therapeutic index77,108,132–134. Timing of surgical procedures such as bone KL001 acts to stabilize CRY proteins by preventing
marrow transplantation is also a key factor modulating success and survival and should
ubiquitin-​driven degradation115. Application of KL001
be carefully considered in clinical applications40. On the other hand, targeting
components of the molecular clock may yield novel treatment options in some diseases. in vitro has anti-​inflammatory effects on fibroblast-​like
Increasing the stability of the clock protein cryptochrome has shown anti-​inflammatory synoviocytes116, and this presents as another potential
effects in vitro116, and REV-​ERB agonists have also proved anti-​inflammatory in vivo112. anti-​inflammatory target. Taken together, the future will
However, given the broad influence of the clock upon physiology and pathology , care see the development of novel tools targeting the clock in
must be taken when developing compounds that manipulate this network. inflammatory disorders.

www.nature.com/nri
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
 Reviews

Conclusions in chronic responses manifested several days or even

The immune system is heavily influenced by time-​of-day
cues, both under steady-​state conditions and in response
to inflammatory challenges. The regulation of immune
responses according to the time of the day is likely opti-
mized to provide protection at specific times. In addi-
tion, time-​partitioning of metabolic and redox processes
in immune cells may help minimize detrimental effects
on the organism. Shifts of a few hours in the timing of
an inflammatory challenge targeting the innate or the
adaptive immune system have dramatic consequences
on subsequent immune responses and pathological
outcomes, not only in acute immune responses but also
weeks later. Despite the rapid recent progress made in
understanding the mechanisms involved, the role of the
circadian-​controlled expression signatures of specific
organs and crosstalk to other body systems remains
very poorly understood. An improved understand-
ing of these pathways is necessary in order to advance
chronotherapeutic options. From this, novel drugs and
treatments that could maximize therapeutic benefits at
specific phases while minimizing off-​target side effects
may then be developed.
Published online xx xx xxxx

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Acknowledgements Supplementary information
The authors thank V. Lavilla for creating the video and Supplementary information is available for this paper at
M. F. Loudon for providing the voice-​over to it. C.S. is funded https://doi.org/10.1038/s41577-018-0008-4.
by the German Research Foundation (DFG) (Emmy-​Noether
grant (SCHE 1645/2-1) and SFB914 projects B09 and Z03), Related links
the European Research Council (ERC; starting grant 635872, Manchester Biological Timing:
CIRCODE), the DZHK (German Centre for Cardiovascular https://www.bmh.manchester.ac.uk/research/biological-​timing/
Research) and the BMBF (German Ministry of Education and Loudon Laboratory:
Research). J.G. is an Arthritis Research UK Career http://www.manchester.ac.uk/research/Andrew.loudon/
Development Fellow (Ref. 20629). A.L. acknowledges the personaldetails
support of the Wellcome Trust (grant 107851/Z/15/Z). scheiermann Laboratory:
http://scheiermannlab.de/
Author contributions Gibbs Laboratory:
All authors contributed to the research, discussion of content, https://www.research.manchester.ac.uk/portal/Julie.Gibbs.html
writing and review of this manuscript.