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Organisms adapt to day-night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.
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Circadian clock regulates the host response
to Salmonella
Marina M. Bellet
a,1,2
, Elisa Deriu
b,1
, Janet Z. Liu
b
, Benedetto Grimaldi
a,3
, Christoph Blaschitz
b
, Michael Zeller
c
,
Robert A. Edwards
d
, Saurabh Sahar
a
, Satya Dandekar
e
, Pierre Baldi
c
, Michael D. George
e
, Manuela Raffatellu
b,4,5
,
and Paolo Sassone-Corsi
a,4,5
a
Center for Epigenetics and Metabolism, Department of Biological Chemistry, and
b
Department of Microbiology and Molecular Genetics, Institute for
Immunology, School of Medicine,
c
Department of Computer Science, Institute for Genomics and Bioinformatics, and
d
Department of Pathology, University of
California, Irvine, CA 92697; and
e
Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA 95616
Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 18, 2013
(received for review December 15, 2011)
Organisms adapt to daynight cycles through highly specialized
circadian machinery, whose molecular components anticipate and
drive changes in organism behavior and metabolism. Although
many effectors of the immune system are known to follow daily
oscillations, the role of the circadian clock in the immune response to
acute infections is not understood. Here we show that the circadian
clock modulates the inammatory response during acute infection
with the pathogen Salmonella enterica serovar Typhimurium (S.
Typhimurium). Mice infected with S. Typhimurium were colonized
to higher levels and developed a higher proinammatory response
during the early rest period for mice, compared with other times of
the day. We also demonstrate that a functional clock is required for
optimal S. Typhimurium colonization and maximal induction of
several proinammatory genes. These ndings point to a clock-reg-
ulated mechanism of activation of the immune response against an
enteric pathogen and may suggest potential therapeutic strategies
for chronopharmacologic interventions.
clock genes
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inammation
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gastroenteritis
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intestine
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microbes
The circadian (from the Latin circa diem)systemconsistsofan
internal clock, whose molecular components drive changes in
organism behavior, metabolism, and immune system, thus contrib-
uting to body homeostasis (1). Many immune functions follow
a circadian rhythm, including expression of several cytokines, the
number of lymphocytes, and activity of phagocytic cells (24). In
addition, a variety of chronic inammatory diseases, from rheu-
matoid arthritis to obesity, are linked to altered circadian regulation
(57), and clock disruption is associated with dysregulation of the
inammatory response (8). These studies underline the importance
of circadian rhythm in immune functions and inammation but do
not document its role in regulating the host response to infection.
The pathogen Salmonella enterica serovar Typhimurium (S.
Typhimurium) is one of the most common causes of foodborne
illness worldwide (9). Acute infection in humans is characterized
by a massive intestinal inammation with neutrophil migration to
the gut within 48 h after ingestion of contaminated food or water.
This infection can be modeled in mice pretreated with strepto-
mycin 24 h before infection (colitis model) (1013). Initiation of
the host response requires virulence factors, including two type III
secretion systems (T3SS-1 and -2), which mediate S. Typhi-
murium invasion of the intestinal mucosa and promote its
replication within antigen-presenting cells. Other bacterial com-
ponents, such as lipopolysaccharide (LPS) and agellin, activate
Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in
macrophages and/or epithelial cells, resulting in the secretion of
proinammatory cytokines that ultimately recruit neutrophils and
induce expression of antimicrobial proteins (14). Hence, the initial
interactions of host cells with secreted effector proteins, LPS and
agellin, are key events in the initiation of the host response to S.
Typhimurium. It is known that mice are susceptible to LPS-in-
duced endotoxic shock as well as to TNF-αtoxicity, depending
upon the administration time during the 24-h lightdark (LD)
cycle (15, 16). Furthermore, direct molecular links between
the circadian and innate immune systems have been recently
established (17, 18). Nonetheless, whether the circadian clock
also inuences the host response to a pathogen is not known.
Here we used the mouse colitis model to determine how the
circadian clock regulates the host response during acute
Salmonella infection.
Results and Discussion
Differential DayNight Response to Salmonella Infection. To de-
termine whether the circadian clock regulates the host response to
infection with S. Typhimurium, we infected wild-type (WT) mice
by oral gavage either at 10:00 AM (day, early rest phase; zeitgeber
time 4, ZT4) or at 10:00 PM (night, early active phase; ZT16). At
48, 60, 72, and 78 h postinfection (p.i.), mice werekilled, and tis sue
samples collected for bacteriology, histopathology, and gene
expression analyses (Fig. 1). We observed that S. Typhimurium
colonization signicantly changed with time of infection, espe-
cially at later time points. Notably, S. Typhimurium numbers were
signicantly increased in the colon content of mice infected at
10:00 AM in comparison with 10:00 PM at both 72 and 78 h p.i.
(Fig. 1A); colonization of Peyers patches and spleen was also
signicantly higher in mice infected during the day (Fig. S1A).
Next, we determined whether the degree of the host response to
infection changed with the time of inoculation. Histopathology
showed that ceca from mice infected during the day were on av-
erage more inamed than those from mice infected during the
night, at 48, 72, and 78 h p.i. (Fig. 1 Band C;Figs. S1Band S2). In
contrast, at 60 h p.i., a mild increase in inammation was observed
in mice infected at night, suggesting that the time of infection was
not the only variable inuencing the inammatory response. Re-
duced signs of cecal inammation were characterized by low-grade
submucosal edema and neutrophil inux (Fig. 1 Band Cand
Figs. S1Band S2). Major differences were also foundin the levels
Author contributions: M.M.B., E.D., B.G., M.R., and P.S.-C. designed research; M.M.B., E.D.,
J.Z.L., B.G., C.B., M.Z., R.A.E., S.S., and M.D.G. performed research; S.D. contributed new
reagents/analytic tools; M.M.B., E.D., J.Z.L., B.G., M.Z., R.A.E., P.B., M.D.G., M.R., and P.S.-C.
analyzed data; and M.M.B., E.D., M.R., and P.S.-C. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission.
Data deposition: The microarray data reported in this paper have been deposited in the
Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no.
GSE46356). The complete representation of our computational modeling approach to pre-
dict the transcriptional regulatory networks involved in the control of genes in each of the
four clusters identied in our genomic proling analysis is available at www.ics.uci.edu/
~baldig/CLOCK/salmonella.
1
M.M.B. and E.D. contributed equally to this work.
2
Present address: Dipartimento di Medicina Clinica e Sperimentale, Facoltà di Medicina,
Università degli Studi di Perugia, 06100 Perugia, Italy.
3
Present address: Drug Discovery and Development, Italian Institute of Technology, 16163
Genoa, Italy.
4
M.R. and P.S.-C. contributed equally to this work.
5
To whom correspondence may be addressed. E-mail: psc@uci.edu or manuelar@uci.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1120636110/-/DCSupplemental.
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of cryptitis and surface erosions (Fig. S1B), which constitute other
classical signs of inammation (1012). To ascertain whether
these differences were accompanied by changes in gene expres-
sion, we analyzed expression of proinammatory cytokine Tnfα,
neutrophil chemoattractant chemokine (C-X-C motif) ligand 1
(Cxcl-1), and antimicrobial peptide lipocalin-2 (Lcn-2) in the ce-
cum of infected and uninfected mice and found signicant differ-
ences during the course of infection (Fig. 1Dand Table S1).
Consistent with our previous ndings (1113), mice infected dur-
ing the day showed increased expression of these genes compared
with uninfected controls, and the expression was dependent both
on time of infection and time of death (Fig. 1D). Genes in mice
infected in the morning (ZT4) or at night (ZT16) showed maximal
expression when killed during the day (48 and 72 h for mice
infected at ZT4; 60 h for mice infected at ZT16). This nding was
particularly notable for Tnfαbecause its expression was increased
at both 48 and 72 h in mice infected at ZT4 (ZT4 death) relative to
those infected at ZT16 (ZT16 death), and the opposite trend was
observed at 60 h (ZT16 death for mice infected at ZT4; ZT4 for
mice infected at ZT16). In line with this observation is the absence
of signicant changes in expression in mice killed 78 h after in-
fection, corresponding to ZT10 for mice infected at ZT4 and ZT22
for mice infected at ZT16 (Fig. 1D). Additionally, the overall ex-
pression levels of the three proinammatory genes analyzed were
reduced in mice infected at ZT16 (yellow and gray graphs of Fig.
1D), consistently with the differences observed in colonization
level and pathology score.
To establish whether circadian transcription follows a normal
prole during infection we analyzed the expression of the clock gene
period 2 (Per2). Per2 oscillation in uninfected mice demonstrated
proper synchronization (Fig. 1D). Notably, Per2 expression was
progressively down-regulated after infection (Fig. 1D).
These results suggest that a circadian mechanism may regulate
components of the innate immune response to acute Salmonella
infection, also revealing a profound repression of circadian com-
ponents during infection.
Clock Mutation Affects Cytokine Production from LPS-Challenged and
Salmonella-Infected Bone Marrow-Derived Macrophages. Our nd-
ing that the inammatory response to S. Typhimurium infection is
time-of-daydependent in vivo prompted us to determine whether
we could reproduce similar results in vitro in a less complex system.
As detailed earlier, macrophages are involved in the rst response
to S. Typhimurium infection. Moreover, macrophages have an
efcient clock machinery, and LPS-activated pathways in these
cells are under tight circadian regulation at multiple levels (3, 4).
However, bone marrow-derived macrophages (BMDMs) do not
synchronously oscillate after 1 wk of differentiation in vitro (Fig.
S3A), and LPS stimulation at different circadian times leads to
similar levels of Il-6 expression in these asynchronous cultures (Fig.
S3B). We therefore tested whether BMDMs could be synchro-
nized by common methods such as dexamethasone or high-serum
treatments (Fig. S3 Cand D). Oscillation of circadian genes Per2
(Fig. S3 Cand D), cryptochrome 1 (Cry1) and brain and muscle
ARNT-like protein 1 (Bmal1)(Fig. S3C) conrmed entrainment
of these cells. To better evaluate the contribution of the circadian
system to the expression of proinammatory genes, we added LPS
(1 μg/mL) to synchronized macrophages at different times of their
circadian cycle and followed the expression prole of Il-6 (Fig. S3D).
As expected, we obtained different curves of expression depending
on the time of LPS administration, with minimal induction for ad-
ministration at T18 or T30 and major induction at T12 or T24,
where T0 is the time when synchronization began. Notably,
Fig. 1. The inammatory response is time-of-daydependent in the cecum of mice infected with S. Typhimurium. (A) Recovery of S. Typhimurium from colon
content of WT mice at 48, 60, 72, and 78 h p.i. at different circadian times (day, 10:00 AM or ZT4; night, 10:00 PM or ZT16). Each circle represents an individual
animal. Red bars indicate the geometric means (n7). (B) Cecal histopathology score of mice from A. Each bar represent the combined score of at least seven
mice. PMN, polymorphonuclear leukocytes. (C) Representative images (10×magnication) of cecal inammation in WT mice infected (Salmonella) or not at
day (ZT4) or night (ZT16). (D) mRNA expression of Tnfα,Cxcl-1,Lcn2,andPer2 in the cecum of mice from AC(n7). Data are represented as geometric means
of fold increases compared with uninfected WT (48 h) day ±SEM. Signicant daynight changes are shown. *P<0.05; **P<0.01; ***P<0.001. (Left)Graphs
with yellow and gray areas represent the progression of mRNA expression of Tnfα,Cxcl-1,andLcn2 depending on the time of infection.
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expression levels appear to be dependent both on the time of
treatment and on the time of collection, as suggested by our in
vivo data.
We next ascertained whether disruption of the circadian clock
could affect the function of some central component of the LPS
response. Circadian gene expression in mice carrying a mutation
of the master circadian regulator Clock [Clock mutant or clock/
clock mice (19)] is both phase-shifted by 8 h as well as reduced
compared with WT littermates (20). Clock mutant mice also fail
to rhythmically express a number of immunoregulatory genes in
the liver (21). Therefore, we followed the timing of expression of
different cytokines after LPS stimulation of BMDMs isolated
from either WT or Clock mutant mice (Fig. 2). Remarkably, we
observed an overall reduction in the fold induction of the
proinammatory genes Il-6,Il-1β,Tnfα,Cxcl-1,Ifn-β, and Che-
mokine (C-C motif) ligand 2 (Ccl2) in response to LPS stimu-
lation in BMDMs isolated from Clock mutant mice compared
with WT mice (Fig. 2A); BMDMs isolated from Clock mutant
mice also exhibited reduced secretion of IL-6 and TNF-αafter 24
h of LPS stimulation (Fig. 2B). A comparable reduction in the
levels of IL-6 in the supernatant of BMDMs isolated from Clock
mutant mice, compared with WT mice, was obtained after in-
fection with S. Typhimurium (Fig. 2C). Because the induction of
IL-6 is largely dependent on LPS stimulation of the TLR4
pathway, we also infected BMDMs with an isogenic S. Typhi-
murium strain carrying a mutation in the lipid A acylation
pathway (msbB mutant), which impairs signaling through TLR4
(11). As predicted, secretion of IL-6 was reduced in BMDMs
infected with the msbB mutant compared with infection with WT
S. Typhimurium. In contrast, both S. Typhimurium WT and the
msbB mutant elicited similar low levels of secretion of IL-6 (Fig.
2C) in BMDMs isolated from Clock mutant mice, indicating that
TLR4 signaling in response to S. Typhimurium was not induced
in the absence of a functional clock. Next we sought to in-
vestigate whether the secretion of the proinammatory cytokine
IL-1βwas also impaired in Clock mutant mice. IL-1βis directly
induced by TNF-α, causing its expression to peak later than Tnfα,
and mature IL-1βsecretion requires both TLR4 and NLR sig-
naling (22). Upon S. Typhimurium infection of BMDMs, LPS
activation of TLR4 induces proIL-1βexpression, which is fol-
lowed by NLR-mediated signaling through ice protease-activating
factor/NLR family CARD domain-containing protein 4 (Ipaf/
Nlrc4) in response to Salmonella T3SS-1 secretion of agellin into
the cytosol (22). As expected from these earlier studies, S. Typhi-
murium strains carrying either a mutation in msbB (unable to signal
through TLR4), a mutation in both agellin genes (iC jB mutant,
unable to signal through Ipaf/Nlrc4), or a mutation in T3SS-1 (invA
mutant, unable to signal through Ipaf/Nlrc4) induced secretion of
lower levels of IL-1βcompared with S. Typhimurium WT (Fig. 2D)
in BMDMs from WT mice. In contrast, infection of BMDMs from
Clock mutant mice with S. Typhimurium WT resulted in a marked
reduction of IL-1βsecretion. Moreover, all S. Typhimurium mutants
elicited similar low levels of IL-1βin Clock mutant BMDMs. Col-
lectively, we observed that secretion of proinammatory cytokines
in Clock mutant BMDMs was particularly low and comparable to
levels elicited by S. Typhimurium mutants designed to evade acti-
vation of particular components of the inammatory response. T hus,
an intact circadian machinery appears to be required for the
induction of proinammatory cytokines in vitro in response to
Salmonella infection.
Alteration of Time-Dependent Response to Salmonella in Clock Mutant
Mice. Because the circadian clock is necessary for a robust proin-
ammatory response in isolated macrophages, we explored its in-
volvement in the host response to Salmonella in vivo. We infected
Clock mutant mice at 10:00 AM (ZT4) or 10:00 PM (ZT16) (Fig.
3A). Differently from WT mice, similar numbers of S. Typhimu-
rium were recovered 72 h p.i. (Fig. 3A). Similar results were also
obtained when Clock-decient mice (Clock
/
) and their WT lit-
termates were infected, thus conrming that a functional CLOCK
protein is necessary for the observed effect. (Fig. S4A). Further-
more, a signicant difference was found in the colonization of
Peyers patches between WT mice infected at day and Clock mu-
tant mice, even if these lymphatic structures were signicantly
enlarged in Clock mutant mice during infection (Fig. S4B).
Moreover, the histopathology revealed higher inammation in
Clock mutant mice infected at night compared with mice infected
during the day (Fig. 3 Band C;Fig. S4 Cand D). Altogether, these
results suggest that a circadian mechanism regulates components
of the host response upon Salmonella infection and that clock
disruption largely affects this response.
Transcriptome of Cecum After Salmonella Infection. Because pre-
vious microarray studies revealed that 10% of genes follow cir-
cadian oscillation in almost all tissues (7), we set out to gain further
insight into the circadian clock regulation of the host response
to infection by analyzing global changes in gene expression by
microarray analysis. To determine whether circadian regulation
occurs at the transcriptional level in response to infection in vivo,
we analyzed the differential gene expression prole by microarray
analysis of the cecum in WT and Clock mutant mice 72 h p.i. with
S. Typhimurium or mock control at different circadian times
(day, ZT4; night, ZT16). Our analysis identied four main clusters
of transcripts with different patterns of expression, which we
Fig. 2. Reduced cytokine production from macrophages of
Clock mutant mice. (A) Time course of mRNA expression of
different cytokines after LPS stimulation of BMDMs. Time 0
(unstimulated cells) in both WT and Clock mutant cells was
set to 1. Bars represent mean ±SEM (n=3). (B) Supernatant
protein level of TNF-αand IL-6 from BMDMs of WT and
Clock mutant mice after 24 h of LPS stimulation. Bars rep-
resent means ±SEM (n=4). (C) IL-6 production in
BMDMs from WT and Clock mutant mice 24 h p.i. with S.
Typhimurium WT or with the msbB mutant.MOI,multi-
plicity of infection. (D) BMDMs from WT and Clock mu-
tant mice were infected with different strains of S.
Typhimurium as indicated. After 24 h, supernatants were
collected, and secretion of IL-1βwas measured. Data
represent means ±SEM (n=3).
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numbered clusters 14 (Fig. 4Aand Dataset S1). The gene ex-
pression prole in cluster 1 was characterized by the induction of
genes linked to inammation and immunity in response to in-
fection, including genes involved in immune cell activation and
antimicrobial responses. Genes in cluster 2, related to glycolipid
biosynthesis and protein transport, were up-regulated during in-
fection. In contrast, genes involved in lipid and steroid metabolism
(cluster 3) and dendritic cell maturation (cluster 4) were mostly
down-regulated in response to infection.
Notably, several genes in all four clusters were regulated in
atime-specic and clock-dependent manner during infection (Fig.
4A, Datasets S2,S3,andS4). Moreover, a detailed pathway analysis
identied several transcripts with a different daynight expression
in WT mice (Datasets S1 and S2). Specically, we observed the
most relevant changes of gene expression for transcripts involved
in the antimicrobial response (Fig. S5A), acute inammation (Fig.
S5B), leukocyte chemotaxis, and antigen presentation and pro-
cessing (Fig. 4Aand Dataset S1). Consistent with our observations,
the analysis of genes in cluster 1 revealed a general low level of
induction of the inammatory response in Clock mutant mice,
a response that was slightly higher at night (Fig. 4A,Fig. S5 Aand
B,and Dataset S3). These observations were further validated
through analysis of a subset of cluster 1 genes by quantitative real-
time PCR (Fig. 4 Band Cand Table S1). In particular, many of the
proinammatory (Tnfα,Il-17,Cxcl-1) and antimicrobial [Lcn2,
regenerating islet-derived 3 γand β(Reg3γand β)] genes analyzed
were up-regulated at night upon infection, but to lower levels than
during the day. In contrast, levels of expression in Clock mutant
mice were mostly similar between day and night, with onlya couple
genes [Tnfαand S100 calcium binding protein A8 (S100a8)] that
were expressed at higher levels at night than during the day.
Because expression of different cytokines follows a circadian
rhythm (24), we investigated whether intrinsic temporal changes
may inuence gene expression in the intestine of both WT and
Clock mutant mice (Fig. S6Aand Dataset S5). As expected,
a higher proportion of genes oscillated in the cecum from WT
uninfected mice compared with Clock mutant mice (Fig. S6 Aand
B), including circadian genes, metabolic genes, and genes involved
in the immune response (Fig. S6 Aand Cand Dataset S5). Because
we observed that some transcripts (Tnfα,Cxcl-1,andLcn2) did not
show differences between day and night, whereas other genes (Il-
17,Reg3β, and Reg3γ) were strongly up-regulated at night (Fig.
4C), we sought to determine the absolute expression levels of each
gene (Fig. S7). This analysis showed that the expression level of
the majority of genes analyzed was lower in Clock mutant mice in
both basal condition and after infection (Fig. S7). Together, these
results suggest that circadian regulation inuences cytokine expres-
sion at different levels, by modifying both basal and induced tran-
scription of pro- and anti-inammatory genes during acute infection.
Additional and Uncharacterized Circadian Regulations. Little is known
about the metabolic and physiological changes that occur during
the circadian cycle in infected mice. Our data suggest that the
rhythmicity of circadian and metabolic genes is suppressed or
attenuated during infection (Fig.4Aand Dataset S1). Furthermore,
we noticed that genes peaking at different circadian times also un-
dergo opposite transcriptional regulation after infection (Fig. 4A).
For instance, cluster 2 genes massively oscillate in basal conditions
and are strongly induced after infection in WT mice; however, their
cycle is inverted and their induction is completely abolished in Clock
mutant mice. Similarly, clusters 3 and 4 contain genes that are up-
regulated at basal levels at day or night, respectively, but are re-
pressed following infection (Fig. 4A). Within these categories are
the clock genes (Dataset S1 and Fig. S8). Consistent with previous
studies (23), circadian components [Bmal1,alsocalledArylhydro-
carbon receptor nuclear traslocator-like (Arntl), period proteins,
cryptochromes, neuronal PAS domain protein 2 (Npas2), nuclear
receptor subfamily 1, group D, member 1 (Nr1d1)] oscillated in
uninfected mice and showed either a marked reduction of the
oscillation or a phase-shifted rhythm in Clock mutant mice (Fig. S8
Aand B). Also, genes of the main metabolic pathways were found
in these clusters, as predicted by previous studies (24). Moreover,
the time-dependent changesobserved in the transcription of genes
involved in dendritic cell development and leukocyte function, as
well as other immune genes included in cluster 4 (Fig. 4Aand
Dataset S1), suggest a possible circadian control of the de-
velopment of innate and adaptive immunity following infection.
Computational Analysis Reveals Connections Between Circadian
Transcription and Inammatory Response. To extend our compre-
hension of the transcriptional pathways participating in the circa-
dian activation of the host defense against infection, we used
a computational modeling approach to predict the transcriptional
regulatory networks involved in the control of genes in each of
the four clusters identied in our genomic proling analysis (the
complete representation is available at www.ics.uci.edu/~baldig/
CLOCK/salmonella/). In cluster 1, we identied main synergistic
nodes connecting transcription driven by BMAL1:CLOCK
(ARNTL:CLOCK) to critical inammatory pathways (Fig. 4Dand
Fig. S9A). The gene node graphics show transcription factors with
signicant changes in expression between different conditions (blue,
WT infected vs. uninfected day; green, WT infected vs. uninfected
night; brown, Clock mutant infected vs. uninfected day; orange, Clock
mutant infected vs. uninfected night; P<0.05; Fig. 4Dand Fig. S9 A
and B). In agreement with previous reports, NF-κB and hypoxia in-
ducible factor 1, alpha subunit (HIF-1α) are the transcription factors
with the largest numbers of connections (25, 26). Both NF-κBand
HIF-1αshare many target genes with the transcription factor
BMAL1 (ARNTL), which cooperates with CLOCK in regulating
circadian transcription. Notably, HIF-1αappeared to mediate sig-
nicant changes in all four conditions that we analyzed. Nodes were
much larger in WT mice compared with Clock mutant mice, thus
indicating that many pathways regulated by HIF-1αalso require
a functional clock system. This observation is of particular interest
because cross-talk between hypoxic and circadian pathways has been
proposed, and Hif-1αis thought to be a clock-controlled gene (27).
Circadian Regulation of Antimicrobial Peptides Inuences Salmonella
Growth. We next sought to determine whether circadian changes
inuence Salmonella growth, which is known to be enhanced by
intestinal inammation, in part because Salmonella is resistant to
some antimicrobial proteins secreted in the inamed gut (11). As
shown in Fig. 1Dand Fig. 4Band Figs. S5Aand S7, the gene Lcn2,
which encodes for lipocalin-2, an antimicrobial peptide that
Fig. 3. Altered inammatory response in the intestine of Clock mutant mice
after in vivo infection with S. Typhimurium. (A) Tissue colonization in WT and
Clock mutant mice 72 h p.i. with S. Typhimurium. Day, 10:00 AM, ZT4; night,
10:00 PM, ZT16. Each circle represents an individual animal. Red bars indicate
the geometric means (n8). WT mice are as in Fig. 1Aand Fig. S1A.(B) His-
topathology of cecum of infected Clock mutant mice from A.Eachbarrep-
resent the combined score of at least eight mice. PMN, polymorphonuclear
leukocytes. (C) Representative images (10×magnication) of the ceca from
Clock mutant mice at 72 h p.i. (Salmonella)ormock,atdayornight.
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sequesters the essential nutrient iron and thereby reduces the
growth of susceptible bacteria (28), has a different daynight ex-
pression following infection. S. Typhimurium is resistant to LCN2
because it encodes for the synthesis and uptake of a high-afnity
iron chelator, termed salmochelin (29). High expression of
LCN2 in the inamed gut enhances Salmonella colonization over
competing microbes, whereas a LCN2sensitive mutant in the
salmochelin receptor IroN has a colonization defect and is
outgrown by Salmonella WT in mice expressing Lcn2, but not in
mice carrying a Lcn2 mutation (11). Thus, we hypothesized that
WT S. Typhimurium would have a greater colonization advan-
tage in competition with the iroN mutant during the day, when
Lcn2 is highly induced, whereas we would recover both strains at
similar levels at night, when Lcn2 is expressed at lower levels.
InfectionofWTmiceatZT4orZT16witha1:1mixtureofWT
S. Typhimurium and the iroN mutant conrmed this prediction.
Salmonella WT outcompeted the iroN mutant in the colon
content collected 72 h p.i. from mice infected during the day
(Fig. 4E). In contrast, S. Typhimurium WT had no signicant
advantage over the iroN mutant at night (Fig. 4E), when Lcn2
was induced at lower levels. These results further suggest that
circadian expression of Lcn2, and possibly other antimicrobial
peptides, inuences Salmonella colonization and its competitive
advantage over susceptible microbes.
Conclusions
Although in lower organisms a direct connection between the cir-
cadian system and susceptibility to infection has been established
(3031), this link has remained elusive in mammals. Here we show
that the host response to Salmonella infection changes between day
and night, corresponding to rest and active phase in mice, and that
this differential response is driven by the circadian clock. Immedi-
ately after infection, the inammatory response increases by fol-
lowing an oscillatory circadian course, with higher response during
the day and reduction at night. This effect is a consequence of the
clock-controlled expression of many genes whose products play
a fundamental role in host defense against infection. Among these,
genes encoding antimicrobial peptides display a robust circadian
oscillation during infection, resulting in the modulation of Salmonella
colonization and its competition with susceptible microorganisms
for a niche in the inamed gut. Although Salmonella is resistant to
several antimicrobial responses, including LCN2 and REG3γ,
commensal microbes are more susceptible. Therefore, circadian
regulation of antimicrobial proteins may be important to control
the overgrowth of the microbiota and prevent infection from
Fig. 4. Microarray analysis from cecum of mice infected with S. Typhimurium reveals a circadian mechanism modulating the response to acute bacterial
infection. (A) Heat diagram showing changes in gene expression detected in the ceca of mice 72 h p.i. with S. Typhimurium at day (D, ZT4) or night (N, ZT16) in
WT and Clock mutant mice, compared with uninfected controls (n3). Representative results from two animals are shown. Relative increase (red) or decrease
(green) of mRNA level is shown. A list of the most represented subcategories of genes from each cluster, the number of genes included in each subcategory,
and the relative Pvalue are shown. (Band C) Transcriptional proles of selected proinammatory/antimicrobial genes identied in cluster 1. Signicant
changes are shown. *P<0.05; **P<0.01; ***P<0.001. (D) Network of transcription factors involved in regulation of subsets of genes included in cluster 1.
Signicant changes (P<0.05) are shown as colored circles (blue, WT infected vs. uninfected day; green, WT infected vs. uninfected night; brown, Clock mutant
infected vs. uninfected day; orange, Clock mutant infected vs. uninfected night). (E) Competitive infection with a mixture of S. Typhimurium WT and iroN
mutant at two different times of the day (day, ZT4; night, ZT16). Bars indicate the average competitive index of bacteria recovered from colon contents. Data
represent geometric means ±SEM (n=10 each group).
Bellet et al. PNAS Early Edition
|
5of6
MEDICAL SCIENCES
susceptible microorganisms. Previous reports have shed light on
the effect that ablation of clock genes has on specicimmune
parameters (32). In i es, two circadian mutants are more sensi tive
to some bacterial pathogens than WT organisms (33). Further-
more, Per2-decient mice are more resistant to LPS-induced en-
dotoxic shock than WT mice (34), and disruption of the circadian
clockwork by targeting Bmal1 or Nr1d1 removed the circadian
gating of the endotoxin-induced cytokine response, both in vitro
and in vivo (17). Our results show that Clock mutant mice have an
altered timing of reaction following Salmonella infection, as well as
a signicant reduction in the expression of proinammatory genes.
Although a circadian clock-independent function of clock genes
cannot be formally excluded, recent ndings also suggest that al-
teration of circadian gene expression is likely to be responsible for
the phenotype of Clock mutant mice (17, 35).
In our experiments, rhythmicity of circadian and metabolic genes
is suppressed or attenuated at the site of infection, supporting the
concept of intimate bidirectional communication between the cir-
cadian and immune systems (36, 37). Our hypothesis is that immune
factors contribute to the daily coordination of the circadian system,
but powerful immunological challenges send signals to disrupt this
regulation, possibly by uncoupling some of the circadian outputs,
leading to reduction of the amplitude of the oscillations. Con-
versely, the circadian system dictates the right timing for the host
response to infection by regulating components of the immune
system both at the level of individual cells and at the systemic level,
through autonomic and endocrine outputs (18). The circadian
system modulates the activity of several transcription factors that
are important regulators of immune functions, including HIF1-α,
STAT1, STAT3, and NF-κB(3840). We were able to conrm that
these factors, together with many more, create the main tran-
scriptional regulatory networks during infection in our genomic
proling analysis. The results of our analysis point to connections
of the circadian clock to other functional systems, including met-
abolic and immune, during infection and will be instrumental for
future studies focused on elucidating these mechanisms.
Materials and Methods
Mouse Experiments. Mice kept in 12:12 light:dark conditions were pretreated
with streptomycin (0.1 mL of a 200 mg/mL solution in sterile water) intra-
gastrically, as described (1012) 24 h before inoculation with WT S. Typhi-
murium [100 μL containing 1×10
9
colony-forming units (cfu)] or with a 1:1
mixture of S. Typhimurium WT and iroN mutant strain, at two different
circadian times (10:00 AM, ZT4; 10:00 PM, ZT16). Mice were euthanized at
the time point indicated (4878 h p.i.). For enumerating bacterial cfu,
homogenates of colon content, Peyers patches, mesenteric lymph nodes,
and spleen were plated on agar plates containing the appropriate anti-
biotics [LB plus carbenicillin (Carb), LB plus naladixic acid (Nal)]. Competitive
index was calculated by dividing the output ratio (cfu of WT/cfu of iroN
mutant) by the input ratio (cfu of WT/cfu of iroN mutant). Mice used as
control were housed in the same conditions as infected mice, pretreated
with streptomycin, and left uninfected. Additional information can be
found in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank all members of the P.S.-C. and M.R.
laboratories for help and discussions; and G. Servillo, M. A. Della Fazia,
L. Romani, S.-P. Nuccio, and K. Eckel-Mahan for reagents, discussion, and
critical reading of the manuscript. Work in the P.S-C. laboratory was
supported by National Institutes of Health (NIH) Grant R01-GM081634 and
Sirtris Pharmaceuticals, Inc.; work in the M.R. laboratory was supported by
Public Health Service Grants AI083619 and AI083663; and work in the P.B.
laboratory was supported by NIH Grants LM010235-01A1 and 5T15LM007743
and National Science Foundation Grant MRI EIA-0321390. J.Z.L. was supported
by NIH Immunology Research Training Grant T32 AI60573 and by an American
Heart Association Predoctoral Fellowship.
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www.pnas.org/cgi/doi/10.1073/pnas.1120636110 Bellet et al.
Supporting Information
Bellet et al. 10.1073/pnas.1120636110
SI Materials and Methods
Animals. All experiments were approved by the Institutional An-
imal Care and Use Committee at the University of California,
Irvine. For the generation of Clock mutant mice, see ref. 1. Clock-
decient (Clock
/
) mice were a gift from S. Reppert (University
of Massachusetts Medical School, Worchester, MA) (2). For ex-
periments in Fig. S3, we used C57BL/6J wild-type (WT) mice of 8
12 wk of age. All experiments were conducted a minimum of two
times with similar results, even though some degree of variability
between WT mice was found, depending on mice housing con-
ditions (different vivaria, different degree of sterility of cages, and
food). For the microarray study, Clock mutant mice and their WT
littermates were used.
Bacterial Strains and Culture Conditions. We used Salmonella en-
terica serovar Typhimurium (S. Typhimurium) IR715, a fully vir-
ulent, nalidixic acid-resistant derivative of isolate ATCC14028 (3).
Mutants in invA,iC jB,msbB,andiroN were described (4, 5).
All strains were cultured aerobically in Luria Bertani (LB)broth at
37 °C, with the exception of the msbB mutant, which was grown in
low-salt LB medium (4). In the cfu counts, outliers were detected
with the Grubbs outlier test. All outliers with αcondence level
of 0.01 were removed from the counts.
RNA Extraction and Real-Time PCR. For gene expression analysis by
real-time PCR, total RNA was extracted with TRIzol Reagent
and processed according to the instructions of the manufacturer.
Next, 2 μg of RNA from each sample was retrotranscribed (Su-
perScript II Reverse Transcriptase; Invitrogen), and fourfold di-
luted cDNA was used for each real time reaction. For 20 μLof
PCR, 50 ng of cDNA was mixed with primers to a nal concen-
tration of 150 nM and 4 μLofRT
2
SYBR Green Fluor Fast master
mix (QIAGEN). The reaction was rstincubatedat9Cfor3min,
followed by 40 cycles at 95 °C for 30 s and 60 °C for 1 min. Each
quantitative real-time PCR was performed by using the Chromo4
real time detection system (Bio-Rad). All values are relative to
those of Gapdh or β-actin mRNA levels at each time point. A list of
the real-time primers used in this study is provided in Table S1.
Analysis was performed in Fig. 4Bby calculating the geometric
means of fold changes in gene expression of infected compared with
uninfected mice killed at the same time during the day. In Fig. 4C,
values in uninfected WT day were set to 1. Bars represent mean ±
SEM (n=714 for infected mice; n=35 for uninfected mice).
Bone Marrow-Derived Macrophage Preparations. Three Clock mu-
tant mice and three isogenic WT mice were killed between zeitgeber
time (ZT) 4 and ZT10, andbone marrow was extracted fromfemurs
of each animal. Differentiation of macrophages was obtained by
culturing the bone marrow in RPMI (GIBCO) supplemented with
10% (vol/vol) L-929 conditioned medium, 10% (vol/vol) FBS, 2 mM
glutamine, and antibiotics. After 7 d, cells were counted and plated
for the experiment. Bone marrow-derived macrophages (BMDMs)
were plated for the experiment and treated with lipopolysaccharide
(LPS) at a concentration of 1 μg/mL (Ultrapure LPS; Invivogen) or
infected with S. Typhimurium by using a gentamicin protection
assay as described (4). Cells were harvested, and samples were
processed for RNA extraction. For circadian experiments using the
dexamethasone (DEX) method, BMDMs were synchronized by
treatment with 100 nM DEX (Sigma) for 2 h. For circadian ex-
periments using the serum shock method, after 2 h of serum shock
with medium containing 50% (vol/vol) horse serum, cells were in-
cubated with serum-free medium for the indicated time. LPS
treatment (1 μg/mL) was performed at the indicated time points.
Enzyme-Linked ImmunoSorbent Assay. Supernatant from BMDMs
was collected 24 h poststimulation with LPS or after in vitro infection
with S. Typhimurium WT or mutants. Secretion of TNF-α,IL-6,and
-1βwas detected by using commercially available kits (eBioscience).
Histopathology. Tissue samples were xed in formalin, processed
accordingto standard procedures for parafn embedding, sectioned
at 5 μm, and stained with hematoxylin and eosin. The pathology
score of cecal samples was determined by blinded examinations of
cecal sections from a board-certied pathologist. Each section was
evaluated for the presence of neutrophils, mononuclear inltrate,
submucosal edema, surface erosions, inammatory exudates, and
cryptitis. Inammatory changes were scored from 0 to 4 according
to the following scale: 0, none; 1, low; 2, moderate; 3, high; 4, ex-
treme. The inammation score was calculated by adding up all of
the scores obtained for each parameter and interpreted as follows:
02, within normal limits; 35, mild; 68, moderate; 8+,severe.
Gene Expression Proling and Statistical Analysis. RNA extracted
from mice cecumwas isolated by using the RNeasy Mini Cleanup kit
(Qiagen). Amplication and labeling of mRNA, hybridization to
Mouse Gene 1.0 ST GeneChip arrays (Affymetrix), staining, and
scanning were performed according to protocols in the Affymetrix
Gene Expression Analysis Technical Manual. Analyses of micro-
array data were performed by using BRB Array tools (Version
4.1.0 Stable Release) developed by Richard Simon and the BRB
Developmental Team. For each condition, RNA from three or four
mice was analyzed. A minimum 2.0-fold difference (P0.05) be-
tween groups in the following comparisons was used as criteria for
identifying differentially expressed genes for hierarchical clustering:
(i) WT infected vs. WT uninfected (day); (ii)WTinfectedvs.WT
uninfected (night); (iii)Clock mutant infected vs. Clock mutant
uninfected (day); (iv)Clock mutant infected vs. Clock mutant un-
infected (night); (v)Clock mutant infected vs. WT infected (day);
and (vi)Clock mutant infected vs. WT infected (night). Genes
passing fold-change criteria were subjected to hierarchical cluster-
ing analysis, a nonbiased method of sorting the genes based on
similar patterns of expression. Pathway analysis was then performed
on each pattern of expression or subcluster. Ingenuity Pathway
Analysis software was used to identify pathways and processes
statistically overrepresented in subclusters of differentially ex-
pressed genes. The microarray dataset has been deposited in the
Gene Expression Omnibus database. (accession no. GSE46356).
Regulatory Network Analyses. Our network was initialized with the
proteins identied as belonging to cluster 1 from the analysis of
the microarray data using the BRB Array tools, together with the
clock protein aryl hydrocarbon receptor nuclear traslocator-like
(ARNTL) and the NF-κB proteins NF-kB1, -kB2, v-rel retic-
uloendotheliosis viral oncogene homolog A (RelA), and retic-
uloendotheliosis oncogene (cRel). The MotifMap (6) system was
used to search for putative transcription factor binding sites
within an 8-kb region centered on the translation start site of
each gene in our network, excluding exons. Using known posi-
tional-weight matrices for clock and NF-κB proteins, as well as
hypoxia inducible factor 1, alpha subunit (HIF1-α) (which was
already present in cluster 1), we assigned to every site the fol-
lowing two scores: (i) a motif matching score (Z-score); and (ii)
a conservation score [Bayesian Branch Length Score; BBLS (6)]
calculated by using a multiple alignment of 30 genomes from
Bellet et al. www.pnas.org/cgi/content/short/1120636110 1of12
mouse to zebrash. We ltered these sites by using a Z-score
threshold of 4.27 (P=0.00001), along with a modest amount of
conservation by using a BBLS cutoff of 1.0. Directed edges were
drawn between the transcription factors and the proteins whose
gene had at least one binding site satisfying the above criteria. The
resulting network was also further pruned (Fig. 4D) by pro-
gressively removing nodes that were not annotated as transcription
factors in either the JASPAR (7) or TRANSFAC 9.4 (8) data-
bases, which jointly comprise>800 binding matrices corresponding
to >400 distinct transcription factors in mouse. All networks were
visualized by using Cytoscape Web (Version 0.8) (ref. 9; www.ics.
uci.edu/~baldig/CLOCK/salmonella/).
Identication of Genotype- and Time-Dependent Genes. For the
expression analysis of genotype- and time-dependent genes
within cluster 1, we ran CyberT (10) across all pairs of treatments
to identify differentially expressed genes (BenjaminiHochberg
multiple test corrected, P<0.05). Time-dependent genes were
dened as genes that were signicantly differentially expressed
in both (i) WT uninfected day vs. WT uninfected night and (ii)
WT infected day vs. WT infected night. Genotype-dependent
genes were dened as genes that were signicantly differentially
expressed in all four of the following comparisons: (i)WTun-
infected day vs. CLOCK mutant uninfected day; (ii )WTun-
infected night vs. CLOCK mutant uninfected night; (iii)WT
infected day vs. CLOCK mutant infected day; and (iv)WTinfected
nightvs.CLOCKmutantinfectednight. A third gene list was created
by taking the intersection of the genotype- and time-dependent gene
lists (Datasets S1,S2,andS3).
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typhimurium: Nucleotide sequence, protein expression, and mutational analysis of
the cchA cchB eutE eutJ eutG eutH gene cluster. J Bacteriol 177(5):13571366.
4. Raffatellu M, et al. (2005) The Vi capsular antigen of Salmonella enterica serotype
Typhi reduces Toll-like receptor-dependent interleukin-8 expression in the intestinal
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enterica serotype Typhimurium for growth and survival in the inamed intestine. Cell
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regulatory motif sites. Bioinformatics 25(2):167174.
7. Bryne JC, et al. (2008) JASPAR, the open access database of transcription factor-
binding proles: New content and tools in the 2008 update. Nucleic Acids Res
36(Database issue):D1 02D106.
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regulation in eukaryotes. Nucleic Acids Res 34(Database issue):D108D110.
9. Lopes CT, et al. (2010) Cytoscape Web: An interactive web-based network browser.
Bioinformatics 26(18):23472348.
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Bellet et al. www.pnas.org/cgi/content/short/1120636110 2of12
Fig. S1. Time-dependent variations of the inammatory response in WT mice infected with S. Typhimurium. (A) Recovery of S. Typhimurium from Peyers
patches, mesenteric lymph nodes, and spleens of WT mice at 48 and 72 h following infection at different circadian times (10:00 AM, day or ZT4; 10:00 PM, night
or ZT16). Each circle represents bacterial numbers recovered from an individual animal, normalized for milligrams of tissue. Red bars indicate the geometric
means (n7). Statistically signicant (P<0.05) differences are indicated. Outliers were detected with the Grubbs outlier test. All outliers with a αcondence
level of 0.01 were removed from the counts. (B) Histopathology score of WT mice from experiment shown in Fig. 1B. Each bar represents the score of one
individual animal. Each group was divided in subgroups (WNL, within normal limits; mild; moderate; and severe) based on the severity of the inammation
(score from 0 to 8+).
Bellet et al. www.pnas.org/cgi/content/short/1120636110 3of12
Fig. S2. Cecal inammation after Salmonella infection at day or night. Representative images (40×magnication) of cecal inammation in WT mice 48, 60,
72, and 78 h after infection (Salmonella) or not (control), at day (ZT4) or night (ZT16).
Bellet et al. www.pnas.org/cgi/content/short/1120636110 4of12
Fig. S3. Circadian synchronization of BMDMs. (A) Bone marrow and ceca from WT mice were collected at four different times of the circadian cycle. Bone
marrow was cultured in vitro with differentiating medium for BMDM isolation. After 1 wk, BMDMs derived from each mouse were collected at four different
times of the day. Per2 mRNA expression prole was analyzed by quantitative PCR in both BMDMs and ceca. The values are relative to those of β-actin mRNA
levels at each circadian time. Bars represent mean ±SD (n=2). (B) Time course of Il-6 mRNA expression after 1, 2, or 4 h of LPS stimulation (1 μg/mL) of
macrophages derived from bone marrow of WT mice collected as in A, measured by quantitative real-time PCR. The values are relative to those of β-actin
mRNA levels at each circadian time. Bars represent mean ±SD (n=2). (C) Period 2 (Per2), cryptochrome 1 (Cry1), and brain and muscle ARNT-like protein 1
(Bmal1) mRNA expression proles in BMDMs synchronized by DEX treatment (10 μM) were analyzed by quantitative PCR. The values are relative to those of
Gapdh mRNA levels at each circadian time. All of the values are the mean ±SD (n=2). (D) BMDMs from WT mice were synchronized by high-serum treatment,
and RNA was collected at different circadian times. Per2 mRNA expression prole (Left) was analyzed by quantitative PCR. Colored arrows indicate different
times of treatment with LPS. Time course of Il-6 mRNA expression (Right) after LPS stimulation (1 μg/mL) started at different times of the circadian cycle in
BMDMs after serum shock synchronization, measured by quantitative real-time PCR. The values are relative to those of β-actin mRNA levels at each circadian
time. Bars represent mean ±SD (n=2).
Bellet et al. www.pnas.org/cgi/content/short/1120636110 5of12
Fig. S4. Altered inammatory response following Salmonella infection in Clock mutant mice. (A) Recovery of S. Typhimurium from colon content of Clock
+/+
and Clock
/
mice 72 h following infection at different circadian times (day or ZT4; night or ZT16). Each circle represents bacterial numbers recovered from an
individual animal, normalized for milligrams of tissue. Red bars indicate the geometric means (n=7). Statistically signicant (P<0.05) differences are in-
dicated. (B) Tissues from WT and Clock mutant mice were collected 72 h after S. Typhimurium infection and weighed. Data represent means ±SEM (n=28 for
WT; n=17 for Clock mutant). (C) Histopathology score of Clock mutant mice from experiment shown in Fig. 3B. Each bar represents the score of one individual
animal. Each group was divided in subgroups (WNL, within normal limits; mild; moderate; and severe) based on the severity of the inammation (score from
0to8+). (D) Representative images (40×magnication) of cecal inammation in Clock mutant mice 72 h after infection (Salmonella) or not (control) with S.
Typhimurium, at day (ZT4) or night (ZT16).
Bellet et al. www.pnas.org/cgi/content/short/1120636110 6of12
Fig. S5. Microarray analysis reveals major circadian differences in antimicrobial response and acute inammation. Changes detected by microarray analysis in
the expression of genes involved in antimicrobial response (A) and acute inammation (B) in WT and Clock mutant mice infected at two different circadian
times with S. Typhimurium, compared with uninfected mice.
Bellet et al. www.pnas.org/cgi/content/short/1120636110 7of12
Fig. S6. Basal gene expression in the cecum reveals a strong circadian regulation. (A) Heat diagram showing changes in gene expression detected by mi-
croarray analysis in the ceca of uninfected WT and Clock mutant mice at day (10:00 AM or ZT4) and night (10:00 PM or ZT16). Three samples in each group were
used for statistical analysis. Relative increase (red) or decrease (green) of mRNA level is shown. A list of the most represented subcategories of genes, the
number of genes included in each subcategory (istograms), and the relative Pvalue (solid lines) are shown. (B) Diagram showing the number of genes whose
basal levels are up- or down-regulated at night in WT and Clock mutant mice, identied from the microarray analysis. (C) Examples of genes of the defense
response category with differential basal levels of expression between day and night in WT mice and no changes in Clock mutant mice, identied from the
microarray analysis. Statistically signicant differences are indicated. *P<0.05; **P<0.01.
Bellet et al. www.pnas.org/cgi/content/short/1120636110 8of12
Fig. S7. Time- or genotype-dependent changes in cytokinesbasal levels inuence their expression during infection. Transcriptional proles as in Fig. 4 Band C
are shown. Analyses were performed by calculating the geometric means of gene expression values ±SEM (n=714). Value of uninfected WT mice killed at
ZT4 was set to 1 during the analysis. Signicant daynight changes are shown. *P<0.05; **P<0.01; ***P<0.001.
Fig. S8. Clock genestranscription is down-regulated during acute infection. (A) Changes detected by microarray analysis in the expression of circadian genes
in WT and Clock mutant mice left uninfected or infected with S. Typhimurium at two different circadian times (day, ZT4; night, ZT16). (B)Per2 and nuclear
receptor subfamily 1, group D, member 1 mRNA expression prole in cecum from WT and Clock mutant mice, infected or not with S. Typhimurium at day (ZT4)
or night (ZT16), 72 h after infection. Data are represented as geometric means of fold increases compared with uninfected WT day ±SEM (for infected mice n=
14 wt day; n=13 wt night; n=9 Clock mutant day; n=8 Clock mutant night; for uninfected mice n=35 in each condition). **P<0.01; ***P<0.001.
Bellet et al. www.pnas.org/cgi/content/short/1120636110 9of12
Fig. S9. Computational networks of transcription factors reveal a central role for NF-κB, HIF1α, and Arntl. (A) Network containing all genes, included in cluster 1,
with signicant correlations in WT infected vs. uninfected during the day. Signicant changes (P<0.05) are shown as colored circles (blue, WT infected vs.
uninfected day; green, WT infected vs. uninfected night; brown, Clock mutant infected vs. uninfected day; orange, Clock mutant infected vs. uninfected night).
(B) Network of transcription factors possibly involved in the regulation of subsets of genes included in clusters 2, 3, and 4. Signicant changes (P<0.05) are
shown as colored circles (blue, WT infected vs. uninfected day; green, WT infected vs. uninfected night; brown, Clock mutant infected vs. uninfected day;
orange, Clock mutant infected vs. uninfected night).
Bellet et al. www.pnas.org/cgi/content/short/1120636110 10 of 12
Table S1. List of real-time RT-PCR primers used in this study
Gene Primers
Lcn2 5-ACATTTGTTCCAAGCTCCAGGGC-3
5-CATGGCGAACTGGTTGTAGTCCG-3
Cxcl1 5-TGCACCCAAACCGAAGTCAT-3
5-TTGTCAGAAGCCAGCGTTCAC-3
S100A8 5-TGTCCTCAGTTTGTGCAGAATATAAA-3
5-TCACCATCGCAAGGAACTCC-3
Il-6 5-TTCCATCCAGTTGCCTTCTT-3
5-CAGAATTGCCATTGCACAAC-3
Tnf-α5-CCCATATACCTGGGAGGAGTCTTC-3
5-CATTCCCTTCACAGAGCAATGAC-3
Il-1β5-CTCTCCAGCCAAGCTTCCTTGTGC-3
5-GCTCTCATCAGGACAGCCCAGGT-3
Ifnβ5-CCCTATGGAGATGACGGAGA-3
5-CTGTCTGCTGGTGGAGTTCA-3
Ccl2 5-CTTCTGGGCCTGCTGTTCA-3
5-CCAGCCTACTCATTGGGATCA-3
Il-17 5-GCTCCAGAAGGCCCTCAGA-3
5-AGCTTTCCCTCCGCATTGA-3
Ifnγ5-TCAAGTGGCATAGATGTGGAAGAA-3
5-TGGCTCTGCAGGATTTTCATG-3
Reg3β5-ATGGCTCCTACTGCTATGCC-3
5-GTGTCCTCCAGGCCTCTTT-3
Reg3γ5-ATGGCTCCTATTGCTATGCC-3
5-GATGTCCTGAGGGCCTCTT-3
β-actin 5-GGCTGTATTCCCCTCCATCG-3
5-CCAGTTGGTAACAATGCCATGT-3
Gapdh 5-TGTAGACCATGTAGTTGAGGTCA-3
5-AGGTCGGTGTGAACGGATTTG-3
Per2 5-CGCCTAGAATCCCTCCTGAGA-3
5-CCACCGGCCTGTAGGATCT-3
Nr1d1 5-GGGCACAAGCAACATTACCA-3
5-CACGTCCCCACACACCTTAC-3
Cry1 5-CAGACTCACTCACTCAAGCAAGG-3
5-TCAGTTACTGCTCTGCCGCTGGAC-3
Bmal1 5-GCAGTGCCACTGACTACCAAGA-3
5-TCCTGGACATTGCATTGCAT-3
Lcn2, lipocalin 2; Cxcl1, chemokine (C-X-C motif) ligand 1; S100a8, S100
calcium binding protein; Ccl2, chemokine (C-C motif) ligand 2; Reg3β, regen-
erating islet-derived 3 β; Reg3γ, regenerating islet-derived 3 γ.
Dataset S1. Hierarchical clustering table with list of genes depicted in each cluster (Fig. 4A)
Dataset S1
Fold change differences between groups were calculated through comparisons of different conditions [WT infected day (wtid) vs. WT uninfected day (wtud);
WT infected night (wtin) vs. WT uninfected night (wtun); Clock infected day (ckid) vs. Clock uninfected day (ckud); Clock infected night (ckin) vs. Clock
uninfected night (ckun)]. Pvalues for the different comparisons are also shown. Both fold changes and Pvalues are color-coded. Fluorescence intensities
are shown but not color-coded.
Dataset S2. List of cluster 1 genes showing a timemain effect
Dataset S2
Dataset S3. List of cluster 1 genes showing a genotypemain effect
Dataset S3
Bellet et al. www.pnas.org/cgi/content/short/1120636110 11 of 12
Dataset S5. Hierarchical clustering table with list of genes depicted in each cluster (Fig. S6A)
Dataset S5
Fold change differences between groups were calculated through comparisons of different conditions [WT night vs. day; Clock mutant (CK) night vs. day; CK
day vs. WT day; CK night vs. WT night]. Pvalues for the different comparisons are shown. Both fold changes and Pvalues are color-coded.
Dataset S4. List of cluster 1 genes showing both a timemain and a genotypemain effect
Dataset S4
Bellet et al. www.pnas.org/cgi/content/short/1120636110 12 of 12
... ↓ expression of pro-inflammatory genes Il-6, Il-1β, Tnfα, Cxcl1, Ifnβ, and Ccl2 and ↓ TNFα and IL-6 response in BMDMs [63] Clock mutant mice Salmonella infection (in vivo) Impaired rhythmicity in bacterial colonization in the gut and reduced pro-inflammatory gene expression [63] ↓ TNFα and IL-12 production in challenged peritoneal macrophages and ↓ Tlr9 expression ...
... ↓ expression of pro-inflammatory genes Il-6, Il-1β, Tnfα, Cxcl1, Ifnβ, and Ccl2 and ↓ TNFα and IL-6 response in BMDMs [63] Clock mutant mice Salmonella infection (in vivo) Impaired rhythmicity in bacterial colonization in the gut and reduced pro-inflammatory gene expression [63] ↓ TNFα and IL-12 production in challenged peritoneal macrophages and ↓ Tlr9 expression ...
... These findings were supported by reduced activation of NF-κB in response to immune challenge in mouse embryonic fibroblasts (MEFs), as well as hepatocytes of Clock-deficient mice compared to wild-type controls [49]. Similarly, reduced induction of pro-inflammatory cytokines upon LPS challenge has been observed in MEFs and BMDMs from Clock-mutant mice [63,64]. Moreover, day/night differences in inflammatory response to Salmonella infection were eliminated in the gut of Clock mutants [63]. ...
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... Interestingly, it has been demonstrated that the expression of Per2 is progressively decreased in the caecum of mice infected with Salmonella enterica serovar Typhimurium [S. Typhimurium, a pathogenic Gram-negative bacteria found in the intestinal lumen with its toxicity determined by lipopolysaccharide (LPS) in its outer membrane] (Bellet et al., 2013). Synchronized treatment of LPS with high-serum (serum shock) alters the amplitude of Per2 gene expression in bone marrow-derived macrophages (BMDMs) (Bellet et al., 2013), indicating that LPS could be an effective regulator of the circadian clock (Mukherji et al., 2013). ...
... Typhimurium, a pathogenic Gram-negative bacteria found in the intestinal lumen with its toxicity determined by lipopolysaccharide (LPS) in its outer membrane] (Bellet et al., 2013). Synchronized treatment of LPS with high-serum (serum shock) alters the amplitude of Per2 gene expression in bone marrow-derived macrophages (BMDMs) (Bellet et al., 2013), indicating that LPS could be an effective regulator of the circadian clock (Mukherji et al., 2013). Pathogen infection can also reprogram the expression of circadian clock genes in extraintestinal tissues. ...
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... Besides, the microbiota rhythmicity also affected the susceptibility of a host to infection by pathogens. Bellet et al. (2013) found that the invasive capacity of Salmonella typhimurium has significant diurnal variation [90]. Meanwhile, antimicrobial peptide, which was supposed to be resistant to pathogen invasion and has fundamental implications for innate immunity, showed a rhythmic pattern [91]. ...
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... The circadian immune response against Salmonella Typhimurium has recently been demonstrated to be driven by oscillations in feeding behavior and the activity of the gut microbiota. Mice pretreated with streptomycin are more susceptible to oral Salmonella Typhimurium when infected early during the day (ZT4; higher bacterial count in the colon and mesenteric LNs) compared with the night (ZT16) (94). In contrast, nonantibiotic-treated mice infected with Salmonella Typhimurium orally at ZT12 exhibit an increased bacterial burden in the SI 24 hours later and suffer from significantly increased mortality compared with those infected at ZT0 (morning) (89). ...
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