INNATE IMMUNE REGULATION OF METABOLIC PHYSIOLOGY ...
Transcript of INNATE IMMUNE REGULATION OF METABOLIC PHYSIOLOGY ...
INNATE IMMUNE REGULATION OF
METABOLIC PHYSIOLOGY & INFLAMMATORY RHYTHM
A DISSERTATION
SUBMITTED TO THE PROGRAM IN IMMUNOLOGY
AND THE COMMITTEE ON GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
KHOA DINH NGUYEN
September 2013
http://creativecommons.org/licenses/by-nc/3.0/us/
This dissertation is online at: http://purl.stanford.edu/km773gy2297
© 2013 by Khoa Dinh Nguyen. All Rights Reserved.
Re-distributed by Stanford University under license with the author.
This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 United States License.
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I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Ajay Chawla, Co-Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Lawrence Steinman, Co-Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Edgar Engleman
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Elizabeth Mellins
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost for Graduate Education
This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.
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ABSTRACT
Monophagocytes are a frontline defense against anything that should not be
present in the body. Being highly mobile, they infiltrate almost every tissue to consume
and dispose of material that might be damaging. To fight pathogens, monocytes and
macrophages are transformed into pro-inflammatory machines that secrete
catecholamines. However, monocytes and macrophages also exist in alternatively
activated, anti-inflammatory forms that have a wide range of physiological roles. Unlike
classically activated cells, which exhibit high pro-inflammatory potential, alternatively
activated monocytes and macrophages (which are promoted by the TH2-type cytokines
IL-4 and IL-13) are less pro-inflammatory and have distinct secretory and functional
capacities.
The inherent functional plasticity as well as the omnipresence of monocytes and
macrophages in all tissues enables them to sense environmental changes. My dissertation
will highlight two physiological settings in which, monocytes and macrophages, act as
the sensors of perturbations in the environment to activate distinct physiological
programs. The first part of my dissertation will discuss the role of adipose tissue
alternatively activated macrophages in sensing changes in environmental temperature and
its subsequent involvement in the maintenance of body temperature. The second part of
my dissertation will provide evidences to show that inflammatory monocytes can sense
change in the daily light dark cycle via their interaction with the circadian clock system
to generate diurnal oscillation in monocyte-mediated inflammation.
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ACKNOWLEDGEMENTS
I would like to thank my thesis advisor, Dr. Ajay Chawla, for his scientific
guidance and my PhD reading and oral committee members, Drs. Edgar Engleman,
Elizabeth Mellins, Kari Nadeau, and Lawrence Steinman for their critical evaluations of
my research findings and their continued support during my graduate study. In addition, I
would also like to express my gratitude towards the members of the Chawla laboratory
for their help with various aspects of my research. Lastly, I am grateful to Stanford
University and the American Heart Association for their financial support via the
Stanford Graduate Fellowship and the American Heart Association Pre-doctoral
Fellowship.
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TABLE OF CONTENTS
LIST OF TABLES……………………………………………………………………...vii
LIST OF FIGURES………………………………………………..…………………..viii
CHAPTER 1……………………………………………………………………………….
INTRODUCTION.…………...……………………………………………….....1
MATERIALS AND METHODS………………………………………..............2
RESULTS……………………………..……………………………………….....8
CONCLUSIONS………….………………………………………..…………...16
FIGURE LEGENDS……………...…………………………………………….17
FIGURES………………………………………………………………………..27
CHAPTER 2……………………………………………………………………………….
INTRODUCTION.…………...………………………………………………...49
MATERIALS AND METHODS………………………………………............50
RESULTS……………………………..………………………………………...55
CONCLUSIONS………….………………………………………..…………...66
FIGURE LEGENDS……………...…………………………………………….67
FIGURES………………………………………………………………………..84
FUTURE DIRECTIONS………………………………………………………….......111
REFERENCES………………………………………………………………………...114
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LIST OF TABLES
Table S1.1..……………………..……………………..………………………………..31
Table S1.2..……………………..……………………..………………………………..32
Table S1.3..……………………..……………………..………………………………..33
Table S2.1..……………………..……………………..………………………………..85
Table S2.2..……………………..……………………..………………………………..86
Table S2.3..……………………..……………………..………………………………..87
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LIST OF FIGURES
Figure 1.1……………………………………………………………………………..…27
Figure 1.2……………………………………………………………………………..…28
Figure 1.3……………………………………………………………………………..…29
Figure 1.4……………………………………………………………………………..…30
Figure S1.1…..………………………………………………………………………..…34
Figure S1.2…..………………………………………………………………………..…35
Figure S1.3…..………………………………………………………………………..…36
Figure S1.4…..………………………………………………………………………..…37
Figure S1.5…..………………………………………………………………………..…38
Figure S1.6…..………………………………………………………………………..…39
Figure S1.7…..………………………………………………………………………..…40
Figure S1.8…..………………………………………………………………………..…41
Figure S1.9…..………………………………………………………………………..…42
Figure S1.10..…………………………………………..………………………………..43
Figure S1.11..…………………………………………..………………………………..44
Figure S1.12..…………………………………………..………………………………..45
Figure S1.13..…………………………………………..………………………………..46
Figure S1.14..…………………………………………..………………………………..47
Figure S1.15..…………………………………………..………………………………..48
Figure 2.1……………………………………………………………………………..…80
Figure 2.2……………………………………………………………………………..…81
Figure 2.3……………………………………………………………………………..…82
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Figure 2.4……………………………………………………………………………..…83
Figure 2.5..…..………………………………………………………………………..…84
Figure S2.1…..………………………………………………………………………..…88
Figure S2.2…..………………………………………………………………………..…89
Figure S2.3…..…………………………………………………………………………..90
Figure S2.4…..…………………………………………………………………………..91
Figure S2.5…..…………………………………………………………………………..92
Figure S2.6…..…………………………………………………………………………..93
Figure S2.7…..…………………………………………………………………………..94
Figure S2.8…..…………………………………………………………………………..95
Figure S2.9…………………………………………………………………………..…..96
Figure S2.10..…………………………………………..………………………………..97
Figure S2.11..…………………………………………..………………………………..98
Figure S2.12..…………………………………………..………………………………..99
Figure S2.13..…………………………………………..………………………………100
Figure S2.14..…………………………………………..………………………………101
Figure S2.15..…………………………………………..………………………………102
Figure S2.16..…………………………………………..………………………………103
Figure S2.17..…………………………………………..………………………………104
Figure S2.18..…………………………………………..………………………………105
Figure S2.19..…………………………………………..………………………………106
Figure S2.20..…………………………………………..……………………………....107
Figure S2.21..…………………………………………..………………………………108
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Figure S2.22..…………………………………………..………………………………109
Figure S2.23..…………………………………………..………………………………110
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CHAPTER 1 - INTRODUCTION
All homeotherms use thermogenesis to maintain their core body temperature,
ensuring that cellular functions and physiological processes can continue in cold
environments (1-3). In the prevailing model of thermogenesis, when the hypothalamus
senses cold temperatures it triggers sympathetic discharge, resulting in the release of
noradrenaline in brown adipose tissue and white adipose tissue (4, 5). Acting via the β3-
adrenergic receptors, noradrenaline induces lipolysis in white adipocytes (6), whereas it
stimulates the expression of thermogenic genes, such as PPAR-γ coactivator 1a
(Ppargc1a), uncoupling protein 1 (Ucp1) and acyl-CoA synthetase long-chain family
member 1 (Acsl1), in brown adipocytes (7-9). However, the precise nature of all the cell
types involved in this efferent loop is not well established.
Here we report in mice an unexpected requirement for the interleukin-4 (IL-4)-
stimulated program of alternative macrophage activation in adaptive thermogenesis.
Exposure to cold temperature rapidly promoted alternative activation of adipose tissue
macrophages, which secrete catecholamines to induce thermogenic gene expression in
brown adipose tissue and lipolysis in white adipose tissue. Absence of alternatively
activated macrophages impaired metabolic adaptations to cold, whereas administration of
IL-4 increased thermogenic gene expression, fatty acid mobilization and energy
expenditure, all in a macrophage-dependent manner. Thus, we have discovered a role for
alternatively activated macrophages in the orchestration of an important mammalian
stress response, the response to cold.
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CHAPTER 1 - MATERIALS AND METHODS
1.1 Animals and in vivo studies
Male mice, 8–12 weeks old, were used in all experiments. Breeding pairs of wild-
type and Stat6−/− mice on a BALB/cJ background were purchased from the Jackson
Laboratory, and Il4−/−/Il13−/−, Il4raLoxP/LoxP and LysmCre mice on the BALB/cJ
background were obtained from the Locksley or Brombacher laboratories. For cold
challenge experiments, mice were fed ad libitum and individually housed in cages that
had been pre-chilled at 4 °C (8). Core body temperature was monitored hourly by a rectal
temperature probe (Physitemp). For the thermoneutrality experiments, mice were adapted
to 30 °C in a laboratory incubator (Darwin Chambers) for 2–4 weeks before
experimentation. For rescue experiments, the β3-adrenergic agonist CL-316243 (Sigma)
was injected intraperitoneally at 0.1 mg kg−1 30 min before the cold challenge. Tissues
were collected at the end of a 6-h cold challenge, and processed for RNA and protein
analyses. To deplete macrophages, mice were injected intraperitoneally with two doses of
clodronate-containing or empty liposomes (400 µl and 100 µl at 24 h and 30 min,
respectively, before initiation of experiment) (22). Depletion was confirmed by flow
cytometric analysis of monocytes and macrophages in blood, adipose tissues and spleen.
Cohorts of ≥4 mice per genotype or treatment were assembled for all in vivo studies,
which were repeated 2–3 independent times.
1.2 Flow cytometry and immunoblot analysis
Adipose tissues were minced and digested with collagenase I (3 mg ml−1,
Worthington) for 45 min at 37 °C in a shaker (400 r.p.m.). The digested cell suspension
was centrifuged at 1,600 r.p.m. for 5 min to separate stromal-vascular fraction from
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adipocytes. Pelleted cells were re-suspended in FACS buffer (PBS containing 5% FBS
and 1% L-glutamine) and passed through a 40 µm strainer (BD Biosciences) to remove
large cellular debris. Antibodies directed against mouse CD3, B220, Ly6G, CD45,
CD49b, CD11c and F4/80 (Biolegend), Siglec F (BD Biosciences), FcεR1 (eBioscience),
tyrosine hydroxylase (Origene), Ly6C, dopamine β-hydroxylase and dopa decarboxylase
(Abcam), arginase-1 (Santa Cruz Biotech), and anti-rabbit and anti-mouse IgG
(Invitrogen) were used for flow cytometric analysis. Samples were stored in FACS buffer
with 1% paraformaldehyde at 4 °C before analysis. Data was acquired on LSRII (BD
Biosciences) and data analysis was performed using FlowJo (Treestar). For analysis of
mitochondrial proteins in muscle, soleus muscle from mice housed at 22 °C and 4 °C was
lysed using TissueLyser II (Qiagen), and antibodies directed against Cox1 (Invitrogen) or
Cpt1b (Alpha Diagnostic International) were used to detect mitochondrial proteins.
1.3 Macrophage culture and stimulation
Bone-marrow-derived macrophages (BMDMs) were cultured as previously
described (23). Classical or alternative activation was induced in BMDMs by stimulation
with LPS (10 ng ml−1) or IL-4 (10 ng ml−1) for 24 h, respectively. Macrophages were
elicited into the peritoneal cavity by injection of thioglycollate (3 ml, BD Biosciences).
To promote alternative activation of elicited macrophages, mice were given a single
injection of IL-4 (2 µg) complexed to anti-IL-4 antibody (10 µg) 3 days after injection of
thioglycollate, and elicited macrophages were recovered 24 h later. After washing two
times, elicited macrophages were cultured in low glucose (LG)-DMEM with 3% BSA
and macrophage-conditioned medium was collected 24 h later. Human monocytes, which
were isolated from whole blood by magnetic purification with CD14 microbeads
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(Miltenyi Biotech), and the human macrophage cell line U937 were cultured in RPMI
with 5% FBS and 1% L-glutamine and stimulated with LPS (10 ng ml−1) or IL-4 (10
ng ml−1) for 24 h to induce classical or alternative activation, respectively. Conditioned
media was collected and supplemented with 3% BSA for lipolysis assays. In some
experiments, α-methyl-p-tyrosine (AMPT, 2 mM, Sigma) was added to cultured
macrophages to inhibit tyrosine hydroxylase.
1.4 Quantitative RT–PCR
Tissues were homogenized in Trizol (Invitrogen) and total RNA was isolated
using the RNeasy kit (Qiagen) and used as template for cDNA synthesis (Origene).
Quantitative PCR reactions were carried out in triplicate using the CFX384 real-time
PCR detection system (Bio-Rad). Relative expression level of mRNAs was calculated by
the comparative threshold cycle method using 36B4 as an internal control24. The
following primers were used in these studies: Il1 forward 5′-
GAAGAAGAGCCCATCCTCTG-3′, reverse 5′- TCATCTCGGAGCCTGTAGTG-3′; Il6
forward 5′-AGTCCGGAGAGGAGACTTCA-3′, reverse 5′-
TTGCCATTGCACAACTCTTT-3′; Acsl1 forward 5′-TGGGGTGGAAATCATCAGCC-
3′, reverse 5′-CACAGCATTACACACTGTACAACGG-3′; Acox forward 5′-
GGTGGACCTCTGTCTTGTTCA-3′, reverse 5′-AAACCTTCAGGCCCAAGTGAG-3′;
Ucp1 forward 5′-GTGAAGGTCAGAATGCAAGC-3′, reverse 5′-
AGGGCCCCCTTCATGAGGTC-3′; Ppargc1a forward 5′-
CAACATGCTCAAGCCAAACCAACA-3′, reverse 5′-
CGCTCAATAGTCTTGTTCTCAAATGGG-3′; Tnf forward 5′-
CCAAGGCGCCACATCTCCCT-3′, reverse 5′-GCTTTCTGTGCTCATGGTGT-3′;
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Nos2 forward 5′-ACCTTGGTGAAGGGACTGAG-3′, reverse 5′-
TCCGTTCTCTTGCAGTTGAC-3′; Arg1 forward 5′-AGACCACAGTCTGGCAGTTG-
3′, reverse 5′-CCACCCAAATGACACATAGG-3′; Mrc1 forward 5′-
TGATTACGAGCAGTGGAAGC-3′, reverse 5′-GTTCACCGTAAGCCCAATTT-3′;
Clec10a forward 5′-CTCTGGAGAGCACAGTGGAG-3′, reverse 5′-
ACTTCCGAGCCGTTGTTCT-3′; 36B4 forward 5′-
GAGACTGAGTACACCTTCCCAC-3′, reverse 5′-ATGCAGATGGATCAGCCAGG-3′.
1.5 Catecholamines and lipids
Catecholamines (Rocky Mountain Diagnotics), free fatty acids (Biovision) and
glycerol (Abcam) were quantified in duplicate as per the manufacturers’ protocols. For
catecholamine ELISAs, tissues were homogenized by sonification in homogenization
buffer (1 N HCl, 0.25 M EDTA, 1 mM Na2S2O5), and cellular debris was pelleted by
centrifugation at 13,000 r.p.m. for 15 min at 4 °C. The cleared homogenates were
collected and stored at −80 °C before quantification. All samples were normalized to total
tissue protein concentration.
1.6 Adipocyte differentiation and lipolysis
The 3T3-L1 pre-adipocytes were grown in high glucose Dulbecco’s modified
Eagle’s medium (HG-DMEM) supplemented with BCS (10%). Two days after
confluence, differentiation was induced with insulin (10 µg ml−1), dexamethasone (1
µM), and 3-isobutyl-1-methylxanthine (0.5 mM) in HG-DMEM containing FBS (10%).
All subsequent media changes (every 2 days) were performed using HG-DMEM
supplemented with FBS (10%) and insulin (10 µg ml−1). For the lipolysis studies,
differentiated adipocytes were cultured in low glucose (LG)-DMEM supplemented with
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BSA (3%) for 16 h before stimulation with vehicle, CL-316243 (1 µM), IL-4
(10 ng ml−1) or macrophage conditioned medium for 15 min or 6 h to quantify phospho-
HSL, phospho-perilipin or glycerol release, respectively. Basal lipolysis was quantified in
the presence of N6-phenylisopropyl adenosine (PIA, 1 µM). Glycerol release into the
culture medium was quantified using the Free Glycerol Assay Kit (Abcam). For
immunoblot analysis, treated adipocytes were lysed in lysis buffer (20 mM Tris-HCl,
pH 7.5, 100 mM KCl, 0.1% Nonidet P-40, 1 mM EDTA, and 10% glycerol containing
1 mM phenylmethylsulphonyl fluoride, 1% protease inhibitor cocktail and 1%
phosphatase inhibitor cocktails I/II) for 30 min at 4 °C. Total cellular protein extracts
were separated on SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose
membrane (Bio-Rad), and incubated with antibodies directed against HSL, serine-660
phosphorylated HSL (Cell Signaling), perilipin, or serine-552-phosphorylated perilipin
(Vala Sciences). After incubation with the appropriate secondary antibodies, proteins
were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific).
1.7 Energy expenditure
Oxygen consumption, RER and activity were quantified in 12-week-old male
mice of various genotypes fed ad libitum using CLAMS (Columbus Instruments).
Following acclimatization to CLAMS cages for 48 h, mice were given an intraperitoneal
injection of recombinant IL-4 (45.5 µg kg−1 body weight) at 11:00. Consumption rates of
O2 (VO2) and release of CO2 (VCO2) were monitored for ~8 h every 14 min. Locomotor
activity—the number of x-axis beam breaks—was monitored every minute. Data was
collected during light cycle (9:00 to approximately 19:00).
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1.8 Statistical analysis
All data are presented as mean ± s.e.m. and analysed using Prism (Graphpad).
Statistical significance was determined using the Student’s t-test and two-way analysis of
variance test. A P value of <0.05 was considered to be statistically significant, and is
presented as * (P < 0.05), ** (P < 0.01), or *** (P < 0.001).
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CHAPTER 1-RESULTS
1.1 Cold induces IL-4/IL-13 dependent alternative activation of macrophages
Mice housed at the thermoneutral temperature of 30 °C do not require adaptive
thermogenesis, whereas those housed in colder environments depend on brown adipose
tissue (BAT) thermogenesis to maintain their body temperature (10). Thus, to understand
the relationship between temperature and macrophage activation, we profiled the status of
BAT and white adipose tissue (WAT) macrophages in mice chronically housed at 30 °C
(thermoneutrality), 22 °C (normal housing temperature), or after an acute challenge to
4 °C. Gene expression profiling revealed a progressive increase in the expression of
alternative activation messenger RNAs (11, 12), including Arg1, Mrc1 andClec10a, in
BAT and WAT of mice exposed to colder temperatures (Fig. 1.1a, b). In contrast,
expression of classical activation markers was unchanged by cold exposure (Fig. 1.1a, b).
This correlation between alternative macrophage activation and exposure to colder
environments was further verified using flow cytometry. In wild-type mice, exposure to
progressively lower temperatures increased expression of CD206 (encoded by Mrc1),
CD301 (Clec10a) and arginase 1 (Arg1) proteins in BAT and WAT macrophages (Fig.
1c–e and Supplementary Fig. 1.1a–f). Notably, disruption of IL-4/IL-13 signalling, as
in Il4/Il13−/− and Stat6−/− mice (13), completely abrogated the cold-induced increase in
alternative activation of BAT and WAT macrophages, as assessed by expression of
CD206, CD301 and Arg1 (Fig. 1.1c–e and Supplementary Fig. 1.1a–f). This was a
specific defect in cold-induced alternative activation because loss of IL-4/IL-13
signalling did not introduce a classical activation bias in BAT and WAT macrophages
(Supplementary Fig. 1.2a–d). Finally, acute exposure of mice to 4 °C failed to induce
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alternative macrophage activation in other tissues, including skeletal muscle and liver
(Fig. 1.1f), indicating that BAT and WAT alternative activation is an adaptive response
for acclimation to cold.
1.2 Alternatively activated macrophages are required for adaptive thermogenesis
To investigate the importance of alternative macrophage activation in cold-
induced thermogenesis, we challenged mice lacking alternatively activated macrophages
to cold temperatures. Unlike wild-type mice, Il4−/−/Il13−/− and Stat6−/− mice showed a
drop in core body temperature when exposed to temperatures of 4 °C (Fig. 1.2a). In wild-
type mice, to counteract the change in environmental temperature, thermogenic genes
(Ppargc1a and Ucp1) and the β-oxidation genes (Acox1 andAcsl1) were induced in BAT.
This induction of thermogenic genes was blunted in BAT
of Il4−/−/Il13−/− and Stat6−/− mice (Fig. 1.2b, c). To determine whether the observed
defects in cold-induced thermogenesis were a direct consequence of the loss of
alternatively activated macrophages, we disrupted IL-4/IL-13 signalling in myeloid cells
by breeding conditional Il4raLoxP/LoxP with LysmCre mice (14). BAT macrophages
in Il4raLoxP/LoxPLysmCre mice displayed impairment in alternative activation at 22 °C and
4 °C (Fig. 1.2d), which was sufficient to render mutant mice susceptible to cold-induced
hypothermia (Fig. 1.2e). Il4raLoxP/LoxPLysmCre mice also showed defects in expression of
cold-inducible thermogenic genes, including Ucp1, Acox1, Acsl1 and Ppargc1a (Fig.
1.2f). Comparable results were obtained in a second model when macrophages were
pharmacologically depleted in BAT using clodronate-containing liposomes
(Supplementary Fig. 1.3a–e), which selectively deplete tissue macrophages and
circulating monocytes but not neutrophils (Supplementary Fig. 1.4a, b). Moreover,
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expression of skeletal muscle mitochondrial genes implicated in thermogenesis was
unaltered (Supplementary Fig. 1.5a), indicating a primary defect in non-shivering
thermogenesis. Serum triglyceride levels and expression of lipogenic genes in liver were
similarly unchanged across the genotypes and temperatures (Supplementary Table
1.1 and Supplementary Fig. 1.5b). Finally, defects in cold-induced thermogenesis were
also observed inStat6−/− mice on the C57BL/6J background (Supplementary Fig. 1.5c,
d).
1.3 Alternatively macrophages mobilize free fatty acids to fuel adaptive
thermogenesis
During cold exposure, β-adrenergic signalling in white adipocytes stimulates the
release of free fatty acids to fuel uncoupled respiration in BAT (1, 6). Because WAT
macrophages also undergo alternative activation upon cold challenge (Fig. 1.1b, e), we
examined whether a defect in alternative macrophage activation was associated with
impaired release of free fatty acids. Indeed, compared to wild-type mice, circulating
levels of free fatty acids were reduced by ~75% in Il4/Il13−/− and Stat6−/− mice (Fig.
1.2g). Serum free fatty acid levels were similarly reduced by ~65% in
Il4raLoxP/LoxPLysmCre mice at 4 °C (Fig. 1.2h). Consistent with reduced release of
fatty acids, gross and microscopic histology revealed that all mutant mice impaired in
alternative macrophage activation had exhausted their lipid stores in BAT (Fig. 1.2i, j).
Correspondingly, mice deficient in IL-4/IL-13 signalling or alternatively activated
macrophages lost less weight during the cold challenge (Supplementary Table 1.1).
To explore whether factors released by alternatively activated macrophages work
in trans to stimulate lipolysis of stored triglycerides, we used differentiated 3T3-L1 cells
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to study triglyceride lipolysis in vitro. Treatment of adipocytes with conditioned medium
from alternatively activated macrophages induced phosphorylation of perilipin and
hormone sensitive lipase (HSL), lipases that are phosphorylated by protein kinase A in
response to adrenergic signalling (Fig. 1.2k) (15). The phosphorylation of perilipin A
releases CGI-58, allowing it to interact with Pnpla2 to enhance the lipolysis of stored
triglycerides (16, 17). Indeed, paralleling the increase in perilipin phosphorylation,
triglyceride lipolysis, as quantified by glycerol release, was increased by ~4.5-fold in
adipocytes treated with conditioned medium from alternatively activated macrophages
(Fig. 1.2l). No significant increase in phosphorylation of perilipin, HSL, or triglyceride
lipolysis was observed when adipocytes were exposed to conditioned medium from
Stat6−/− macrophages (Fig. 1.2k, l). Together, these data indicate that alternatively
activated macrophages coordinate the thermogenic response during cold exposure by
increasing the thermogenic capacity of BAT and mobilizing fatty acids to fuel uncoupled
respiration.
1.4 Alternatively activated macrophages produce catecholamines
The requirement for alternatively activated macrophages in fatty acid
mobilization and thermogenic gene induction prompted us to investigate whether WAT
and BAT macrophages might be an important source of catecholamines. In this regard,
catecholamine production by classically activated macrophages has previously been
shown to promote inflammation-induced injury
(18, 19). Intracellular staining for tyrosine hydroxylase (Th), dopa decarboxylase (Ddc)
and dopamine β-hydroxylase (Dbh) revealed that all three catecholamine-synthesizing
enzymes were induced in macrophages upon stimulation with IL-4 (Supplementary Fig.
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1.6a–f). This induction was a bona fide part of alternative activation because IL-4 failed
to induce Th, the rate-limiting step in the synthesis of catecholamines (20), in
macrophages lacking STAT6 (Fig. 1.3a and Supplementary Fig. 1.6g). Congruent with
this, stimulation of macrophages with IL-4, but not lipopolysaccharide (LPS), increased
secretion of noradrenaline and adrenaline into the culture medium in a STAT6-dependent
manner (Fig. 1.3b and Supplementary Fig. 1.7a). Furthermore, treatment of wild-type
macrophages with α-methyltyrosine, a specific inhibitor of tyrosine hydroxylase (18),
inhibited secretion of noradrenaline into the culture medium and abrogated its lipolytic
activity on cultured adipocytes (Fig. 1.2k and Supplementary Fig. 1.7b, c).
1.5 Cold induces catecholamine synthesis in adipose tissue macrophages
Next, we examined catecholamine synthesis by adipose tissue macrophages. At
thermoneutrality (30 °C), expression of Th in BAT and WAT macrophages was the
lowest (Fig. 1.3c, e). Th expression progressively increased as mice were exposed to
colder temperatures (Fig. 1.3c, e), and was restricted to Ly6Clo-midCD301+ alternatively
activated BAT and WAT macrophages (Supplementary Fig. 1.8a, b). Consistent with
this, loss of IL-4/IL-13 signaling abrogated cold-induced expression of Th in BAT and
WAT macrophages (Fig. 1.3c, e) and reduced noradrenaline content of these adipose
tissues by ~50–60% in Il4−/−/Il13−/− and Stat6−/− mice (Fig. 1.3d, f and Supplementary
Table 1.2). This decrease in BAT and WAT catecholamine content was a direct
consequence of loss of alternative activation because similar changes were observed
in Il4raLoxP/LoxPLysmCre mice. Specifically, cold exposure failed to induce Th protein in
BAT and WAT macrophages (Supplementary Fig. 1.9a, c), resulting in a 70–80%
reduction of noradrenaline content in BAT and WAT ofIl4raLoxP/LoxPLysmCre mice
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(Supplementary Fig. 1.9b, d). These data prompted us to investigate whether the β3-
adrenergic agonist CL-316243 can rescue the thermogenic defect in Il4−/−/Il13−/− mice
(21). Indeed, a single injection of CL-316243 increased core body temperature and
thermogenic gene expression in Il4−/−/Il13−/− mice (Supplementary Fig. 1.11a, b). The
restoration of core body temperature by CL-316243 also normalized weight loss and
BAT histology in Il4−/−/Il13−/− mice housed at 4 °C (Supplementary Fig. 1.11c–e),
including the reappearance of lipid droplets in brown adipocytes. The increased
accumulation of lipid droplets probably resulted from enhanced mobilization of free fatty
acids and induction of lipogenic genes, such as Lpl, Hmgcs1 and Dgat1, in BAT
of Il4−/−/Il13−/− mice treated with CL-316243 (Supplementary Figs 1.11f, g and 1.12a,
b). Hence, alternatively activated macrophages are an unexpected source of noradrenaline
that sustains the metabolic adaptation to cold.
1.6 IL-4 induces catecholamine synthesis in adipose tissue macrophages
A hallmark of cold-induced thermogenesis is an increase in uncoupled respiration
and energy expenditure by noradrenaline (10). Because we observed that IL-4 driven
alternatively activated macrophages release noradrenaline in BAT and WAT in response
to cold, we next examined the metabolic effects of IL-4 in wild-type mice. Injection of
IL-4 induced alternative activation and Th expression in BAT and WAT macrophages
(Supplementary Fig. 1.13a, b). As expected, the strongest effects of IL-4 were observed
at thermoneutrality, when basal alternative activation and Th expression was lowest.
However, administration of IL-4 was sufficient to augment alternative activation and Th
expression in mice housed at 22 °C and 4 °C (Supplementary Fig. 1.13a, b).
Concomitant with the induction of alternative activation, noradrenaline content and
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thermogenic gene expression in BAT, as well as fatty acid levels in serum, increased after
administration of IL-4 (Supplementary Fig. 1.13c–e). Finally, administration of IL-4
enhanced expression of Th in alternatively activated macrophages taking residence in
other tissues, including liver, spleen, lung and bone marrow (Supplementary Figs
1.14a–f and 1.15a, b), albeit to a much lower degree than Th expression in BAT and
WAT macrophages.
1.7 IL-4 induces energy expenditure in a macrophage dependent manner
We next investigated whether acute administration of IL-4 to adapted animals
could enhance oxygen consumption in a macrophage-dependent manner. As shown
in Fig. 1.4a, administration of IL-4 promoted alternative activation of BAT and WAT
macrophages in Il4raLoxP/LoxP mice but notIl4raLoxP/LoxPLysmCre mice. This was
accompanied by an increase in expression of Th in BAT and WAT macrophages,
resulting in induction of thermogenic genes and release of free fatty acids (Fig. 1.4b–d).
Furthermore, quantification of energy expenditure revealed that injection of IL-4 rapidly
increased oxygen consumption in Il4raLoxP/LoxP but not Il4raLoxP/LoxPLysmCre mice (Fig.
1.4e). Importantly, consistent with a shift from carbohydrate to fatty acid metabolism,
administration of IL-4 decreased the respiratory exchange ratio (RER)
in Il4raLoxP/LoxP mice (Fig. 1.4f). These changes in energy expenditure were independent
of alterations in locomotor activity (Supplementary Fig. 1.15c). Furthermore, in wild-
type mice, the stimulatory effect of IL-4 on energy expenditure showed a marked
dependence on macrophages, as IL-4 failed to raise oxygen consumption or decrease
RER in mice treated with clodronate-containing liposomes (Fig. 1.4g,
h and Supplementary Fig. 1.15d). These findings provide direct evidence that actions of
15
alternatively activated macrophages in BAT and WAT orchestrate the metabolic
programs that constitute adaptive thermogenesis.
16
CHAPTER 1-CONCLUSIONS
The data presented here show that alternatively activated macrophages
participate in vivo in the regulation of adaptive and facultative aspects of non-shivering
thermogenesis. In a macrophage-dependent manner, the administration of IL-4 raises
energy expenditure in a facultative manner, whereas adaptation to lower temperatures is
associated with polarization of BAT and WAT macrophages to the alternative state.
Moreover, the secretion of noradrenaline by alternatively activated macrophages allows
these cells to coordinate the thermogenic response in animals experiencing cold stress.
Thus, we propose that, in addition to the sympathetic nerves, cells of the haematopoietic
system, such as alternatively activated macrophages, constitute a second, parallel circuit
for controlling non-shivering thermogenesis.
17
CHAPTER 1-FIGURE LEGENDS
Supplementary Table 1.1 Metabolic characteristics of mice exposed to a cold
challenge. *P < 0.05, **P < 0.01.
Supplementary Table 1.2 Catecholamine content of BAT and WAT in various
strains of mice at 22 ºC and 4 ºC. *P < 0.05, **P < 0.01.
Supplementary Table 1.3 Catecholamine content of tissues in various strains of mice
at 22 ºC and 4 ºC.
Figure 1.1 Exposure to cold environment induces alternative activation of adipose
tissue macrophages. a, b, Real-time PCR analysis of markers of alternative and classical
activation in BAT (a) and WAT (b) of wild-type (WT) mice chronically housed at 30 °C,
22 °C, or acutely subjected to a 4 °C challenge from 22 °C (n = 4 per temperature).
Expression of all genes is normalized to their relative expression at 30 °C in wild-type
mice. c–e, Expression of alternative activation markers Arg1, CD206 and CD301 was
monitored by flow cytometry in BAT (c, d) and WAT (e) macrophages of wild-
type, Il4−/−/Il13−/− and Stat6−/− mice housed at 30 °C, 22 °C and 4 °C (n = 4–5 per
genotype and temperature). MFI, median fluorescence intensity. f, Alternative activation
of tissue macrophages was monitored at 22 °C and 4 °C by quantifying expression of
CD301. BM, bone marrow. *P < 0.05, **P < 0.01, ***P < 0.001 comparison between
wild-type mice at 30 °C and 22 °C, or between 22 °C and 4 °C. †P < 0.05, †††P < 0.001
comparison between wild-type and various knockout mice at the same temperature. All
data are presented as mean ± s.e.m.
Figure 1.2 Cold-induced metabolic adaptations require alternatively activated
macrophages. a, Core body temperature of wild-type, Il4−/−/Il13−/− and Stat6−/− mice
18
during a cold challenge at 4 °C (n = 8 per genotype and temperature). b, c, Real-time
PCR analysis of thermogenic genes in BAT of wild-type,Il4−/−/Il13−/− and Stat6−/− mice
housed at 30 °C, 22 °C or subjected to 4 °C cold challenge (n = 4–5 per genotype and
temperature). Expression of all genes is normalized to their relative expression at 30 °C in
wild-type mice. d, Expression of alternatively activated mRNAs in BAT
of Il4raLoxP/LoxP andIl4raLoxP/LoxPLysmCre mice housed at various temperatures (n = 5 per
genotype and temperature). e, Core body temperature
of Il4raLoxP/LoxP and Il4raLoxP/LoxPLysmCre mice during exposure to 4 °C (n = 5–6 per
genotype and temperature). f, BAT of Il4raLoxP/LoxP and Il4raLoxP/LoxPLysmCre mice was
analysed by real-time PCR for expression of thermogenic and β-oxidation genes (n = 5
per genotype and temperature). Expression of all genes is normalized to their relative
expression at 30 °C in Il4raLoxP/LoxP mice. g, Serum free fatty acid (FFA) levels in wild-
type, Il4−/−/Il13−/− and Stat6−/− mice housed at 30 °C, 22 °C and 4 °C (n= 5–8 per
genotype). h, Serum FFAs in Il4raLoxP/LoxP and Il4raLoxP/LoxPLysmCre mice housed at the
three temperatures (n = 5–11 per genotype). i, Representative gross and microscopic
(haematoxylin and eosin staining) histology of BAT from wild-
type, Il4−/−/Il13−/− and Stat6−/− mice at 22 °C and after exposure to 4 °C for 6 h. j,
Representative gross and microscopic (haematoxylin and eosin staining) histology of
BAT from Il4raLoxP/LoxP and Il4raLoxP/LoxPLysmCre mice at 22 °C and after 6 h exposure to
4 °C. k, Immunoblot analysis for serine-phosphorylated perilipin, total perilipin, serine-
phosphorylated HSL and total HSL in 3T3-L1 adipocytes treated with PIA (N6-
phenylisopropyl adenosine), CL-316243 (CL), IL-4 or macrophage conditioned medium
(with/without IL-4 and AMPT (α-methyl-p-tyrosine)) for 15 min. l, Glycerol release by
19
3T3-L1 adipocytes after 6-h treatment with PIA, CL-316243, IL-4 or macrophage
conditioned medium (n = 5–7). *P < 0.05, **P < 0.01, ***P < 0.001 compared to
comparison between wild-type or Il4raLoxP/LoxP mice at 30 °C and those at 22 °C, or at
22 °C and 4 °C. †P < 0.05, ††P < 0.01, †††P < 0.001 comparison between knockouts and
wild-type or Il4raLoxP/LoxP mice at the same temperature. All data are presented as
mean ± s.e.m.
Figure 1.3 Alternatively activated macrophages produce catecholamines. a,
Expression of tyrosine hydroxylase in wild-type and Stat6−/− peritoneal macrophages
treated with vehicle (Veh.) or IL-4 (n = 5 per genotype and condition). b, Noradrenaline
secretion by wild-type andStat6−/− bone-marrow-derived macrophages stimulated with
IL-4 or LPS (n = 5 per genotype and condition). c, e, Tyrosine hydroxylase expression in
BAT (c) and WAT (e) macrophages of wild-type andStat6−/− mice at 30 °C, 22 °C and
4 °C (n = 5 per genotype and temperature). d, f, Noradrenaline content of BAT (d) and
WAT (f) at 22 °C and 4 °C of wild-type and Stat6−/− mice (n = 4–5 per genotype and
temperature). *P < 0.05, **P < 0.01, ***P < 0.001 compared to wild
type. †P < 0.05, ††P < 0.01, †††P < 0.001 compared to wild type with IL-4 at 4 °C
samples. All data are presented as mean ± s.e.m.
Figure 1.4 Alternative activation of macrophages increases energy expenditure. a, b,
Expression of alternative activation marker CD301 (a) and Th (b) in adipose tissue
macrophages fromIl4raLoxP/LoxP and Il4raLoxP/LoxPLysmCre mice treated with vehicle (Veh.)
or IL-4 for 6 h at 22 °C (n = 4–5 per genotype and condition). c, Real-time PCR for
thermogenic genes in BAT of Il4raLoxP/LoxP andIl4raLoxP/LoxPLysmCre mice treated with
Veh. or IL-4 for 6 h at 22 °C (n = 4–5 per genotype and condition).d, Serum free fatty
20
acid (FFA) levels in Il4raLoxP/LoxP and Il4raLoxP/LoxPLysmCre mice treated with Veh. or IL-4
for 6 h at 22 °C (n = 4–5 per genotype and condition). e, f, Quantification of energy
expenditure inIl4raLoxP/LoxP and Il4raLoxP/LoxPLysmCre mice treated with vehicle (Veh.) or
IL-4 (n = 7–9 per genotype and condition). e, f, Oxygen consumption (e; VO2) and
respiratory exchange ratio (f; RER). g, h, Quantification of energy expenditure in wild-
type mice after macrophage depletion (n = 8 per condition). Mice were injected with
empty liposomes (Lipo.) or clodronate-containing liposomes (Clod.) 24 h before energy
expenditure studies. All data were collected during the light cycle. *P < 0.05, **P < 0.01,
***P < 0.001 compared to Il4raLoxP/LoxP with Veh. †P < 0.05, ††P < 0.01, †††P < 0.001
compared to Il4raLoxP/LoxP with IL-4. All data are presented as mean ± s.e.m.
Supplementary Figure 1.1 Expression of alternative activation markers in adipose
tissue macrophages. a, b, Representative FACS plots of CD206 in BAT (a) and WAT
(b) macrophages of WT, IL4/IL13-/-, and STAT6-/- mice housed at 30 ºC, 22 ºC or 4ºC.
c, d, Representative FACS plots of CD301 in BAT (c) and WAT (d) macrophages of
WT, IL4/IL13-/-, and STAT6-/- mice housed at 30 ºC, 22 ºC or 4 ºC. e, f, Representative
FACS plots of Arg1 in BAT (e) and WAT (f) macrophages of WT, IL4/IL13-/-, and
STAT6-/- mice housed at 30 ºC, 22 ºC or 4 ºC.
Supplementary Figure 1.2 Expression of inflammatory genes representative of
classical macrophage activation in adipose tissue. a, b BAT (a) and WAT (b) from
WT, IL4/IL13-/-, and STAT6-/- mice housed at 30 ºC, 22 ºC or 4 ºC were analyzed by
real-time PCR for expression of Il1, Il6, and Tnfa (n=4 per temperature). c, d BAT (c)
and WAT (d) from IL4RαL/L and IL4RαL/LLysMCre mice housed at 30 ºC, 22 ºC, and
21
4 ºC were analyzed by real-time PCR for expression of Il1, Il6, and Tnfa (n=4 per
temperature).
Supplementary Figure 1.3 Macrophages are required for adaptation to cold
temperatures. a, BAT macrophage content in mice treated with empty (Veh) or
clodronate-containing (Clod) liposomes (n=13-15 per treatment). b, Real-time PCR
analysis of alternative activation markers in BAT of mice treated with Veh or Clod and
then housed at 22 ºC and 4 ºC (n=5 per treatment and temperature). c, Core body
temperature of mice treated with Veh or Clod after exposure to 4 ºC (n=7-8 per
treatment). d, e, Real-time PCR analysis of Ppargc1a (d), Ucp1, and Acsl1(e) in BAT of
mice treated with Veh or Clod and housed at 22 ºC or 4ºC (n=4-5 per treatment and
temperature). **P < 0.01, ***P < 0.001 compared to Veh. ΦP < 0.05, ΦΦP < 0.01
compared to Veh at 4 °C.
Supplementary Figure 1.4 Depletion of macrophages and monocytes by clodronate-
containing liposomes. a, Representative FACS plots (left) and frequencies (right) of
Ly6G+ neutrophils and F4/80+ macrophages in total splenocytes from mice treated with
empty (Veh) or clodronate-containing (Clod) liposomes (n=2-3 per treatment). b,
Representative FACS plots (left) and frequencies (right) of Ly6G+ neutrophils and
CD115+ monocytes in total white blood cells from mice treated with Veh or Clod (n=2-3
per treatment).
Supplementary Figure 1.5 a, Immunoblot analysis of muscle cpt1 (Cpt1b) and
cytochrome c oxidase (Cox1) in solelus muscles of WT, IL4/IL13-/-, and STAT6-/- mice
housed at 22 ºC or 4 ºC. b, Expression of lipogenic genes in liver of WT, IL4/IL13-/-, and
22
STAT6-/- mice housed at 30 ºC, 22 ºC or 4 ºC (n=3 per genotype and temperature). c, d,
Core body temperature of C57BL/6J WT and STAT6-/- mice housed at 4 ºC for 6 hours
(n=5 per genotype and temperature). d, Real-time PCR of Acox1, Ppargc1a, and Ucp1
mRNA levels in BAT of C57BL/6J WT and STAT6-/- mice housed at 22 ºC and 4 ºC
(n=5 per genotype and temperature). **P < 0.01, ***P < 0.001 comparison between WT
and STAT6-/- at the same temperature.
Supplementary Figure 1.6 Alternative activation induces expression of
catecholamine synthesizing enzymes in macrophages. a-c, Representative FACS plots
demonstrating intracellular staining for tyrosine hydroxylase (Th), dopa decarboxylase
(Ddc), and dopamine β-hydroxylase (Dbh) in wild type peritoneal macrophages treated
with vehicle (Veh) or IL4 (10 ngml-1). Background staining was quantified using an
appropriate isotype (Iso) control antibody. d-f, Th (d), Ddc (e), and Dbh (f) expression in
wild type peritoneal macrophages stimulated with vehicle (Veh) or IL4 (10 ngml-1) for
24 hours (n=5 per condition). g, Representative FACS plots of tyrosine hydroxylase
expression in WT and STAT6-/- peritoneal macrophages treated with IL4 (10 ngml-1).
*P<0.05, **P < 0.01 compared to Veh.
Supplementary Figure 1.7 Macrophage conditioned medium regulates lipolysis. a,
Adrenaline secretion by WT and STAT6-/- bone marrow-derived macrophages
(BMDMs) stimulated with IL4 or LPS (n=5 per genotype and condition). b,
Noradrenaline production by IL4-treated BMDMs in the presence or absence of α-
methyl-p-tyrosine (AMPT 2 mM, Sigma). c, Glycerol release by 3T3-L1 adipocytes after
treatment with PIA, CL, IL4 or macrophage conditioned medium (± IL4 and AMPT) for
23
6 hours, n=4-6 per genotype and condition. ***P < 0.001 compared to WT with Veh.
ΦΦΦP < 0.001 compared to WT with IL4.
Supplementary Figure 1.8 Flow cytometric gating strategy for tyrosine hydroxylase
expression in adipose tissue macrophages. a, b, Stromal vascular fractions were
isolated from BAT (a) and WAT (b), and gated for side- and forward-scatter (SSC/FSC),
doublets, and live cells prior to the analysis of the CD45+F4/80+ macrophages. Tyrosine
hydroxylase (Th) colocalizes with CD301 in BAT and WAT macrophages.
Supplementary Figure 1.9 Tyrosine hydroxylase expression and catecholamine
production by alternatively activated adipose tissue macrophages. a, c, Tyrosine
hydroxylase expression in BAT (a) and WAT (c) macrophages of IL4RαL/L and
IL4RαL/LLysMCre mice housed at 22 ºC or 4 ºC (n=4-5 per genotype and temperature).
b, d, Noradrenaline content of BAT (b) and WAT (d) of IL4RαL/L and
IL4RαL/LLysMCre mice at various temperatures (n=4-5 per genotype and temperature).
**P < 0.01 comparison of values at 22ºC and 4 ºC in IL4RαL/L mice. ΦP < 0.05, ΦΦP <
0.01, ΦΦΦP < 0.001 comparison between IL4RαL/L and IL4RαL/LLysMCre mice at the
same temperature.
Supplementary Figure 1.10 Human monocytes and macrophages produce
catecholamines. a, e, Expression of tyrosine hydroxylase and CD206 in primary
monocytes (a) and human macrophage cell line U937 (e) treated with vehicle (Veh), IL4
(10 ngml-1), or lipopolysaccharide (LPS, 10 ngml-1), n=4-5 per condition. b, f,
Catecholamine secretion by primary monocytes (b) and human macrophage cell line
U937 (f) stimulated with IL4 or LPS, (± AMPT), n=4 per condition. c, g, Immunoblot
analysis for phosphorylated-HSL and total HSL, phosphorylated-perilipin and total
24
perilipin in 3T3-L1 adipocytes treated with PIA (1mM), CL-316243 (1 mM), IL4 (10
ngml-1), primary monocyte (c) or U937 cell (g) conditioned medium (± IL4 and AMPT)
for 15 min. PIA (N6-phenylisopropyl adenosine), AMPT (a-methyl-p-tyrosine). d, h,
Glycerol release by 3T3-L1 adipocytes after 6h treatment with PIA, CL, IL4, primary
monocyte (d) or U937 cell (h) conditioned medium (n=5 per condition). *P < 0.05, **P <
0.01 compared to Veh.
Supplementary Figure 1.11 Characteristics of IL4/IL13-/- mice treated with β3-
adrenergic agonist CL-316243. a, Core body temperature of WT, IL4/IL13-/- and
IL4/IL13-/- mice treated with CL-316243 (n=5 per genotype and treatment). b, Real-time
PCR analysis of thermogenic genes in BAT of WT, IL4/IL13-/- and IL4/IL13-/- mice
treated with CL-316243 (n=4-5 per genotype and treatment). c, Cold (4 °C) induced
weight loss in WT, IL4/IL13-/- and IL4/IL13-/- mice treated with CL-316243 (n=4-5 per
genotype and treatment). d-e, Representative histology of BAT from WT, IL4/IL13-/-
and IL4/IL13-/- mice treated with CL- 316243 after exposure to 4 °C; gross (d) and
haematoxylin and eosin stained sections (e). f, Serum free fatty acid (FFA) levels in
WT, IL4/IL13-/-, and IL4/IL13-/- mice treated with CL-316243 housed at 22 ºC or 4 ºC
(n=4-5 per condition and genotype). g, Real-time PCR of lipogenic genes (Lpl, Dgat1,
Hmgcs1) in BAT of WT, IL4/IL13-/-, and IL4/IL13-/- mice treated with CL-316243
housed at 22 ºC or 4 ºC (n=4-5 per genotype and temperature). *P < 0.05, **P < 0.01,
*** P < 0.001 compared to WT.
Supplementary Figure 1.12 Cold challenge induces lipogenic gene expression in
brown adipose tissue. a, Real-time PCR of lipogenic genes (Lpl, Dgat1, Hmgcs1) in
BAT of WT, IL4/IL13-/-, and STAT6-/- mice housed at 22 ºC or 4 ºC (n=4-5 per
25
genotype and temperature). b, Real-time PCR of lipogenic genes (Dgat1, Hmgcs1) in
BAT of IL4RαL/L and IL4RαL/LLysMCre mice housed at 22 ºC or 4 ºC (n=4-5 per
genotype and temperature). ΦP < 0.05, ΦΦP < 0.01, ΦΦΦP < 0.001 compared to WT or
IL4RαL/L at 4ºC.
Supplementary Figure 1.13 Effects of IL4 in wild type mice housed at various
temperatures. a, b, Expression of alternative activation marker CD301 (a) and tyrosine
hydroxylase (b) in adipose tissue macrophages of WT mice injected with vehicle (Veh)
or IL4 at 30 °C, 22 °C or 4°C (n=4-5 per condition). c, Real-time RT-PCR analysis of
thermogenic genes (Acox1, Acsl1,Ppargc1a, and Ucp1) in BAT of WT mice treated with
Veh or IL4 for 6h at various temperatures (n=4-5 per condition). d, Serum free fatty acid
(FFA) levels of WT mice treated with Veh or IL4 for 6h at 30°C, 22°C, and 4°C (n=4-5
per condition). e, Noradrenaline content of serum and various tissues 30 minutes after
injection of IL4. *P < 0.05, **P < 0.01, *** P < 0.001 compared to Veh.
Supplementary Figure 1.14 Expression of CD301 and tyrosine hydroxylase in wild
type mouse tissues at various temperatures. a, c, e, Expression of CD301 was
quantified by flow cytometry in wild type mice 6 hours after administration of vehicle
(Veh) or IL4 at 30°C (a), 22°C (c), and 4°C (e) (n=4-5 per condition). b, d, f, Tyrosine
hydroxylase expression was quantified in mouse tissue macrophages by flow cytometry 6
hours after injection of vehicle (Veh) or IL4 at 30°C (d), 22°C (e), and 4°C (f) (n=4-5 per
condition). Bone marrow (BM). *P < 0.05, **P < 0.01, *** P < 0.001 compared to Veh.
Supplementary Figure 1.15 Effects of IL4 in IL4RαL/L and IL4RαL/LLysMCre
mice. a, b, Expression of CD301 (a) and tyrosine hydroxylase (b) in tissue macrophages
of IL4RαL/L and IL4RαL/LLysMCre mice treated with vehicle (Veh) or IL4 at 22°C
26
(n=4-5 per genotype and condition). c, d, Locomotor activity of IL4RαL/L and
IL4RαL/LLysMCre mice (c), and liposome (Lipo) or clodronate-containing liposome
(Clod) treated mice (d) after IL4 injection (n=7-8 per genotype and condition). *P < 0.05,
**P <0.01, ***P < 0.001 compared to IL4RαL/L with Veh. ΦP < 0.05, ΦΦP < 0.01,
ΦΦΦP < 0.001 compared to IL4RαL/L with IL4.
27
Figure 1.1
28
Figure 1.2
29
Figure 1.3
30
Figure 1.4
31
Supplementary Table 1.1
32
Supplementary Table 1.2
33
Supplementary Table 1.3
34
Supplementary Figure 1.1
35
Supplementary Figure 1.2
36
Supplementary Figure 1.3
37
Supplementary Figure 1.4
38
Supplementary Figure 1.5
39
Supplementary Figure 1.6
40
Supplementary Figure 1.7
41
Supplementary Figure 1.8
42
Supplementary Figure 1.9
43
Supplementary Figure 1.10
44
Supplementary Figure 1.11
45
Supplementary Figure 1.12
46
Supplementary Figure 1.13
47
Supplementary Figure 1.14
48
Supplementary Figure 1.15
49
CHAPTER 2- INTRODUCTION
The circadian clock is a timekeeping system that allows organisms to adapt their
physiological and behavioral rhythms to anticipatory changes in their environment (1, 2).
In mammals, the circadian timing system has a hierarchical architecture, consisting of the
light-responsive central clock in the suprachiasmatic nuclei and the peripheral clocks that
are present in virtually all cells of the body (3). Whereas the central clock entrains and
synchronizes the peripheral clocks with the day-night cycle, the peripheral clocks
regulate tissue-specific programs in an anticipatory manner. Although the peripheral
clock has been identified in macrophages (4–9), its role in anticipatory immune responses
remains poorly understood.
In simple terms, the inflammatory response can be expressed as a product of inducible
gene expression in an innate cell multiplied by the number of infiltrating innate cells.
When examined from this viewpoint, circadian oscillations could potentially regulate
inflammatory responses by modulating rhythmic expression of inflammatory genes in
tissue macrophages or by controlling rhythmic trafficking of Ly6Chi inflammatory
monocytes (10, 11). Because the cumulative cost incurred by rhythmic expression of
inflammatory genes is likely to be high (in terms of tissue inflammation and damage), we
postulated that rhythmic mobilization of Ly6Chi monocytes provides a better means of
mounting anticipatory inflammatory responses. In this scenario, the rhythmic
mobilization of Ly6Chimonocytes would fortify the host’s innate immune defenses in
anticipation of environmental challenges, a process we term anticipatory inflammation.
50
CHAPTER 2-MATERIALS AND METHODS
2.1 Animals
8-to-12-week-old mice, fed ad libitum and housed at 22ºC under 12 hour light:dark cycle
were used in these experiments. ArntlLoxP/LoxP mice on C57BL6/J background
(B6.129S4(Cg)-Arntltm1Weit/J) were backcrossed for 10 generations onto C57BL/6J
(Nnt-/-) background, and then subsequently crossed with Lyz2Cre mice (B6.129P2-
Lyz2tm1(cre)Ifo/J) to generate ArntlLoxP/LoxPLyz2Cre mice. Ccr2-/- mice on the
C57BL/6J background were obtained from the Charo laboratory.
2.2 Monocyte isolation and macrophage culture
Blood monocytes and peritoneal macrophages were isolated using magnetic microbeads
(Miltenyi) coupled to anti-CD115 antibody (clone AFS98, Biolegend). Prior to magnetic
isolation, each blood sample (pooled from 5 mice) was centrifuged over a Ficoll gradient
to remove granulocytes and RBCs. The purity of isolated cells was confirmed by flow
cytometry and was routinely >95%. To activate circadian cycling in cultured cells, bone
marrow-derived macrophages (BMDMs) were stimulated with 50% horse serum for 2
hours, as described previously (38). Subsequently, synchronized BMDMs were harvested
at the indicated intervals for gene expression and immunoprecipitation studies.
Flow cytometry and ELISAs
Blood samples were subjected to RBC lysis, and spleens were homogenized and filtered
through a 40 µm strainer (BD) to remove large cellular debris. eWAT and BAT were
homogenized and digested with Collagenase I (2 mgml−1, Worthington) for 20 minutes
at 37°C in a shaker (250 rpm). The digested cell suspensions were passed through a 40
µm strainer, and subjected to centrifugation to separate the stromal vascular cells from
51
adipocytes. Pelleted cells were re-suspended in FACS buffer (PBS, 5% FBS, 5 mM
EDTA) for antibody staining and analyses. The following antibodies directed against
mouse antigens were used: CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-
6.7), B220 (clone RA3-6B2), Ly6C (clone HK1.4), Ly6G (clone 1A8), CD11b (clone
M1/70), CD11c (clone N418), CD45 (clone 30-F11), CD49b (clone DX5), CD115 (clone
AFS98), F4/80 (clone BM8), TNFα (clone MP6-XT22), and IFNγ (clone XMG1.2, all
from Biolegend); Siglec-F (clone E50-2440) and CCL2 (clone 2H5, BD Biosciences);
FcεR1 (clone MAR-1, eBioscience); CCL8 (bs1985R) and S100A8 (bs2696R, Bioss);
CD301 (clone ER-MP23, AbdSerotec); iNOS (sc-7271, Santa Cruz); anti-rabbit
(A21246) and anti-mouse IgG (A21235, Invitrogen). Samples were fixed (FACS buffer
plus 1% paraformaldehyde) and stored at 4ºC prior to analysis. Data was acquired on
FACSVerse (BD) and analyzed using FlowJo (Treestar). Inflammatory cytokines (IL1β,
IL6, IL12, TNFα, and IFNγ) and chemokines (CCL2 and CCL8) in serum samples,
peritoneal fluid, and culture media were detected by cytometric bead arrays (BD) and
ELISAs (R&D), as per manufacturers’ protocol. Cytokine and chemokine concentration
in peritoneal fluid was normalized to total protein.
2.3 Thioglycollate elicitation
Mice were intraperitoneally injected with 2 ml of thioglycollate broth (BD), and the
peritoneal cavity was flushed with 5 ml of PBS 30 mins or 2 hours later. Cells were
pelleted for flow cytometric analysis, and the supernatants were analyzed for cytokine
and chemokines.
52
2.4 Listeria infection
L. monocytogenes 10403S expressing green fluorescent protein (DHL1252) was grown
to mid-log (OD600 0.25-0.5) in brain-heart infusion (BHI) medium (Difco) (39). Adult
male and female mice were infected with L. monocytogenes via intraperitoneal injection
with the stated number of bacteria. Serial dilutions of all inocula were plated onto BHI
agar plates for enumeration. At intervals following infection, peritoneal fluid, liver, and
spleen were homogenized for colony forming unit (CFU) determination.
2.5 Immunoprecipitation
Chromatin immunoprecipitations were performed using serum shocked BMDMs, and
antibodies against BMAL1 (ab3350) and CLOCK (ab3517, Abcam); EZH2 (clone
D2C9), tri-methylated H3K4 (clone C42D8), tri-methylated H3K27 (clone C36B11), and
Rbp1 (clone 4H8, all from Cell Signaling) with chromatin immunoprecipitation assay kits
(Cell Signaling). Primers used to amplify the precipitated chromatin are listed in tables
S1 and S2. E-boxes were identified at positions -2428 (CATCTG for Ccl2); -223 and -
293 (CAGATG for Ccl8); and -4381 (CACCTG for S100a8). For the co-
immunoprecipitation experiments, serum shocked BMDMs (ZT8) were lyzed in 20 mM
Tris-HCl, 10 mM KCl, 1 mM EDTA, 0.1% NP40, 10% Glycerol, 1:200 Protease
Inhibitor Cocktail (pH 7.5), and pre-cleared with agarose beads (Cell Signaling) before
immunoprecipitation with 5 µg anti-BMAL1 antibody (ab3350, Abcam) per 1 ml lysate.
Immunoprecipitated proteins were analyzed using BMAL1(ab3350), CLOCK (ab3517,
Abcam); EZH2 (clone D2C9) and SUZ12 (clone D39F6, Cell Signaling); and EED
antibodies (17-663, EMD Millipore).
53
2.6 Quantitative RT-PCR
RNA was isolated from cells or tissues using the TRIzol reagent (Invitrogen). Reverse
transcription was carried out using First-strand cDNA Synthesis kit (Origene), and
quantitative PCR reactions were performed on CFX384 real-time PCR detection system
(Bio-Rad). Relative expression level of mRNAs was calculated using the comparative CT
method using 36B4 as an internal control. Primers used for qRTPCR analysis are listed in
table S3.
2.7 Diet-induced obesity
6-week-old male mice were fed with 60% kcal fat diet (Research Diets, D12492) to
promote obesity. Body composition was assessed by DEXA, whereas Oxygen
consumption, RER, and total activity were quantified using the CLAMS system
(Columbus Instruments). Intraperitoneal glucose (1 gkg-1) and insulin (1 Ukg-1)
tolerance tests were performed in overnight-fasted and 6-hour-fasted mice, respectively.
For analysis of insulin signaling, mice were injected with insulin (1 Ukg-1) through the
inferior vena cava, and liver, eWAT, and quadriceps were isolated after 2, 5, and 7
minutes, respectively. Tissues lysates were immunoblotted for total (9272S) and
phosphorylated AKT (clone 193H12, Cell Signaling). Total JNK (clone 56G8) and pJNK
(9251S) were detected in total lysates using antibodies from Cell Signaling. For short
term HFD feeding, mice were housed at 30ºC and fed HFD for one week. Immune cell
numbers were subsequently quantified in blood and adipose tissues, as described
previously (40). For time restricted feeding, mice were given free access to food only
during the 12-hour light:dark cycle for 14 days.
54
2.8 Statistical analysis
All data are presented as means ± SEMs and analyzed using Prism (GraphPad). Statistical
significance is determined using the two-tailed Student’s t-test, one-way and two-way
analysis of variance tests, and log-rank test for pair-wise comparisons, multiple-group
comparisons, and survival analyses, respectively. A p-value of <0.05 was considered to
be statistically significant.
55
CHAPTER 2-RESULTS
2.1. Diurnal Oscillations and Trafficking of Ly6Chi Monocytes
To investigate this hypothesis, we examined whether blood monocytes exhibited
diurnal variation in expression of clock genes. An analysis of monocytes obtained from
mice kept under a 12-hour light-dark cycle revealed rhythmic expression of messenger
RNA (mRNA) encoded by the clock gene Bmal1 (Arntl), whose oscillation was
antiphasic to its two target genes, Nr1d1 and Dbp (Fig. 2.1a). A similar rhythm was
observed for luciferase protein in monocytes derived from Per2Luc knock-in mice (12),
which express PERIOD2 as a luciferase fusion protein (Supplementary Fig. 2.1a).
Furthermore, serum synchronization of THP-1 cells, a human monocytic cell line,
revealed a 24-hour oscillation of clock genes under constant culture conditions
(Supplementary Fig. 2.1b). Together, these data demonstrate that blood monocytes
exhibit diurnal variation in clock genes, which might impart rhythmicity to their
functions.
Recent studies have demonstrated rhythmic trafficking of immune cells into
tissues (6, 8), prompting us to ask whether monocyte frequency in the three major
reservoirs (blood, spleen, and bone marrow) also varies in a diurnal manner. Indeed,
between the peak and nadir Zeitgeber time (ZT, where ZT0 refers to lights on and ZT12
refers to lights off), there was ~twofold difference in the total number of monocytes
present in blood and spleen (Supplementary Fig. 2.2 and 2.3a, b), with bone marrow
displaying a reciprocal diurnal rhythm (Supplementary Fig. 2.3c).
We next examined whether the observed oscillations in total monocytes resulted
from rhythmic changes in Ly6Chi (inflammatory) or Ly6Clow (patrolling) monocytes
56
(13, 14). The peak and nadir of Ly6Chi monocytes in blood and spleen mirrored the cyclic
pattern of total monocytes in these reservoirs (Fig. 2.1b, c and Supplementary Fig.
2.3a, b), whereas Ly6Clow monocytes did not display strong oscillatory behavior in blood
or spleen (Supplementary Fig. 2.3d, e). Moreover, the expression of Ccr2 mRNA in
monocytes did not correlate with Ly6Chi monocyte numbers in blood (Supplementary
Fig. 2.3f). Because Ly6Chi monocytes are recruited to sites of inflammation (15, 16), we
investigated the relation between diurnal variations in Ly6Chi monocyte numbers and
monocyte-driven inflammation by using the thioglycollate model of sterile peritonitis
(17). The numbers of Ly6Chi monocytes recruited to the inflamed peritoneum at ZT8
were ~threefold higher than at ZT0 (Fig. 2.1d), which resulted in ~3 to 3.5-fold higher
inflammation, as quantified by the release of interleukin (IL)–1β and IL-6 (Fig. 2.1e and
Supplementary Fig. 2.3g). On a per cell basis, expression of IL-1 and IL-6 did not
exhibit diurnal oscillations (Supplementary Fig. 2.3h, i), suggesting that diurnal
variation in Ly6Chi monocyte numbers dictates the magnitude of the innate inflammatory
response. Moreover, monocyte-attracting chemokines CCL2 and CCL8, whose
concentrations were higher (~two- to threefold) in the inflamed peritoneum at ZT8
(Supplementary Fig. 2.3j, k), were primarily secreted by recruited monocytes and
resident macrophages but not neutrophils (Supplementary Fig. 2.3l).
2.2 Diurnal Rhythms of Ly6Chi Monocytes During Listeria monocytogenes Infection
Ly6Chi monocytes provide the first-line defense against L. monocytogenes (18),
leading us to posit that anticipatory oscillations in Ly6Chi monocyte numbers might
modulate innate responses to infection. To investigate this hypothesis, we
intraperitoneally infected C57BL/6J mice with L. monocytogenes at ZT0 or ZT8. Two
57
days postinfection (dpi), the peritoneum, spleen, and liver of mice infected at ZT8 had
significantly fewer bacteria than those infected at ZT0 (Fig. 2.2a-c). Improved bacterial
clearance at ZT8 was associated with ~2.2- to 3.8-fold higher numbers of TNF (tumor
necrosis factor)/iNOS (inducible nitric oxide synthase)–producing (Tip)–dendritic cells
(DCs), which differentiate from Ly6Chi monocytes to control the growth of this pathogen
(Fig. 2.2d-f and Supplementary Fig. 2.4) (19). Moreover, serum and, to an even greater
extent, peritoneum concentrations of chemokines and cytokines necessary for the
mobilization and activation of Ly6Chi monocytes were much higher at ZT8 than at ZT0
(Supplementary Fig. 2.5a and Fig. 2.2g). This was associated with higher recruitment
of Ly6Chi monocytes but not neutrophils to the peritoneum (Supplementary Fig. 2.5b,
c), findings that are consistent with previous observations that neutrophils are dispensable
for defense against L. monocytogenes (20).
Although Ly6Chi monocytes and Tip-DCs limit bacterial growth during the early
phase of infection, efficient clearance of L. monocytogenes requires adaptive immunity
(18), prompting us to investigate the activation of adaptive immunity 6 dpi. Although the
peritoneal cavity was below the limit of detection, mice inoculated at ZT8 continued to
demonstrate enhanced clearance of L. monocytogenes in secondary sites, such as the liver
(Supplementary Fig. 2.5d). In contrast, bacterial burden in the spleen was not
significantly different between mice inoculated at ZT0 and ZT8, especially after
normalization for spleen size (Supplementary Fig. 2.5e-g). Infection at ZT8 resulted in
stronger adaptive immune response in the spleen and liver, as evidenced by ~3- to 4.5-
fold higher numbers of interferon-γ (IFN-γ)–producing CD4+ and CD8+ T cells at ZT8
58
(Supplementary Fig. 2.4 and 2.5h, i). Tip-DCs were also more numerous in spleen and
liver, but not the peritoneum, at 6 dpi (Supplementary Fig. 2.5j, l).
Infection with a high inoculum of pathogens often results in overactivation of the
immune system, causing systemic inflammation and death (11). Because previous studies
have demonstrated circadian influence on sepsis-induced mortality (8, 21, 22), we
investigated whether the host response to infection with a higher dose ofL.
monocytogenes might exhibit similar diurnal variation. Intraperitoneal infection of mice
with 1 × 107 L. monocytogenes resulted in significantly higher mortality rate at ZT8 than
at ZT0 (Fig. 2.2h). This increase in mortality was not associated with a higher bacterial
burden (Supplementary Fig. 2.6a-c) but rather with an enhanced inflammatory response
(Fig. 2.2i). These results demonstrate that the host response to L. monocytogenesexhibits
diurnal rhythms that parallel the rhythms of Ly6Chi monocytes.
2.3 BMAL1 Regulates Rhythmic Oscillations of Ly6Chi Monocytes
To determine whether clock genes in monocytes regulate their diurnal rhythms,
we generated myeloid-specificBmal1 knockout mice
using ArntlLoxP/LoxP and Lyz2Cre mice (designated ArntlLoxP/LoxPLyz2Cre) (23).
Immunoblot analysis confirmed loss of BMAL1 protein in blood monocytes
of ArntlLoxP/LoxPLyz2Cre mice (Supplementary Fig. 2.7a). Quantitative reverse
transcription polymerase chain reaction (RT-PCR) analysis of mRNAs provided further
verification that diurnal variations of core clock genes, including Arntl and Nr1d1, were
abolished in blood monocytes of ArntlLoxP/LoxPLyz2Cre mice (Supplementary Fig. 2.7b
and Fig. 2.3a). Remarkably, the disruption of BMAL1 expression in myeloid cells was
sufficient to impair the diurnal variations in Ly6Chi monocyte numbers in blood, spleen,
59
and bone marrow (Fig. 2.3b-d). The normal diurnal rhythm of total monocytes was
similarly disrupted in the blood and spleens of ArntlLoxP/LoxPLyz2Cre mice
(Supplementary Fig. 2.7c,d ). In contrast, we failed to detect rhythmic changes in the
numbers of Ly6Clow monocytes (Supplementary Fig. 2.7e, f) and neutrophils
(Supplementary Fig. 2.7g, h) in control (ArntlLoxP/LoxP) and ArntlLoxP/LoxPLyz2Cre mice.
These results demonstrate that BMAL1 regulates the rhythmic oscillations of
Ly6Chi monocyte numbers in all three monocyte reservoirs.
We next tested whether disruption of the diurnal rhythms of Ly6Chi monocytes
alters their trafficking patterns. Unlike the diurnal recruitment of Ly6Chi monocytes
in ArntlLoxP/LoxP mice, the inflamed peritoneum ofArntlLoxP/LoxPLyz2Cre mice had higher
numbers of Ly6Chi monocytes, which lacked rhythmicity (Supplementary Fig. 2.8a).
These changes were specific for Ly6Chi monocytes because recruitment of total
monocytes did not exhibit a diurnal pattern (Supplementary Fig. 2.8b). Moreover, there
were no significant differences between the genotypes in the numbers of total monocytes,
Ly6Chi monocytes, neutrophils, or macrophages in the uninflamed peritoneum
(Supplementary Fig. 2.8c-f). However, the increased recruitment of Ly6Chi monocytes
did amplify the local inflammatory response inArntlLoxP/LoxPLyz2Cre mice, as quantified
by the release of CCL2, CCL8, IL-1β, and IL-6 (Supplementary Fig. 2.9a-d). This
increase in peritoneal inflammation was again independent of diurnal changes in the
expression of Il1b and Il6 (Supplementary Fig. 2.9e,f).
The amplification of inflammation in ArntlLoxP/LoxPLyz2Cre mice suggested that
these animals might be predisposed to developing infection-induced systemic
inflammation. To test this hypothesis, we
60
infectedArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice at ZT0 and ZT8 with a nonlethal
dose of L. monocytogenes and monitored their survival. Compared
with ArntlLoxP/LoxP mice, all ArntlLoxP/LoxPLyz2Cre mice exhibited greatly reduced survival
with median survival times of 77 to 91 hours (Fig. 2.3e), which could not be accounted
for by differences in expression of Tlr2 or Tlr5 (Supplementary Fig. 2.9g, h), two
pattern recognition receptors that have been implicated in the recognition of L.
monocytogenes (24, 25). However, ArntlLoxP/LoxPLyz2Cre mice infected at ZT8 were
slightly more susceptible to infection-induced lethality than those infected at ZT0
(Fig. 2.3e), perhaps reflecting incomplete depletion of BMAL1 protein in the newly
recruited bone marrow monocytes (18).
An analysis of sera 2 dpi confirmed that ArntlLoxP/LoxPLyz2Cre mice had higher
circulating concentration of inflammatory cytokines and chemokines, including IL-1β,
IL-6, IFN-γ, and CCL2 (Fig. 2.3f-i). This increase in systemic inflammation occurred in
the absence of worsening infection because bacterial colony-forming units (CFUs) in the
spleens and livers of ArntlLoxP/LoxPLyz2Cre mice were lower or unchanged, respectively
(Supplementary Fig. 2.10a, b), whereas those in peritoneum were marginally higher
(Supplementary Fig. 2.10c). Congruent with the CFU data, spleens rather than livers
of ArntlLoxP/LoxPLyz2Cre mice exhibited a more robust increase in numbers of Tip-DCs and
IFN-γ–producing CD4+ and CD8+ T cells (Supplementary Fig. 2.10d-k). These data
show that BMAL1-dependent diurnal rhythms of Ly6Chi monocytes confers a survival
advantage during an infectious challenge with L. monocytogenes.
61
2.4 BMAL1 Recruits PRC2 Complex to Repress Chemokine Genes
The recruitment of Ly6Chi monocytes to inflammatory sites is mediated by the
chemokine receptor CCR2 and its ligands, such as CCL2 and CCL8 (26). We observed
that expression of CCL2, CCL8, and S100A8, a small calcium-binding protein implicated
in monocyte chemotaxis (27), is regulated in a diurnal manner in monocytes recruited to
sites of inflammation (Supplementary Fig. 2.11a-c). These observations led to us to ask
whether BMAL1/CLOCK heterodimers might directly regulate chemokine gene
expression in monocytes and macrophages. Indeed, deletion of Arntl resulted in higher
expression of all three chemokine genes (Ccl2, Ccl8, and S100a8) in monocytes and
peritoneal macrophages (Fig. 2.4a and Supplementary Fig. 2.12a-e), which contributed
to increased concentrations of CCL2 and CCL8 in the serum (Supplementary Fig. 2.12f,
g). These data suggest that repression by BMAL1 is necessary to generate diurnal
rhythms in chemokine expression. Furthermore, bioinformatic analyses confirmed that
promoter regions of Ccl2, Ccl8, and S100a8 contained E-box motifs to which both
BMAL1 and CLOCK were recruited in a rhythmic manner (Fig. 2.4b and
Supplementary Fig. 2.13a-e). This rhythmic recruitment of BMAL1 or CLOCK to the
chemokine promoters was absent in bone marrow–derived macrophages (BMDMs)
lacking BMAL1 (Fig. 2.4b and Supplementary Fig. 2.13a-e), suggesting that
BMAL1/CLOCK heterodimers might recruit a repressor complex to silence chemokine
gene expression.
Previous studies have demonstrated that histone acetylation and methylation is
important in circadian gene expression (28, 29). Among the epigenetic marks that
regulate clock-controlled genes (CCGs), trimethylation of histone H3 at
62
Lys27 (H3K27Me3) by polycomb repressive complex 2 (PRC2) has been implicated in
the silencing of CCGs (30), prompting us to ask whether BMAL1 can interact with
members of PRC2 in BMDMs. Immunoprecipitation of endogenous BMAL1 not only
pulled down CLOCK but also members of PRC2, including the histone methyltransferase
EZH2 (enhancer of zeste), EED (extra-sex comb), and SUZ12 (suppressor of zeste) (Fig.
2.4c). This interaction was specific because we failed to pull down CLOCK or members
of PRC2 in BMAL1-deficient BMDMs (Fig. 2.4c). Chromatin immunoprecipitation
(ChIP) experiments revealed that EZH2 was rhythmically recruited to the proximal
promoter of Ccl2 gene in a BMAL1-dependent manner (Fig. 2.4d), which temporally
coincided with its silencing by H3K27Me3 (Fig. 2.4e). Moreover, in the absence of
BMAL1, the chromatin state of the Ccl2 gene was more active, as evidenced by the
presence of H3K4Me3 activation marks and the constitutive recruitment of RNA
polymerase II (Pol II) to the promoter (Fig. 2.4f, g). Similar patterns of EZH2 and RNA
Pol II recruitment and the associated chromatin modifications were observed
on Ccl8 (Supplementary Fig. 2.14a-d) and S100a8 promoters (Supplementary Fig.
2.15a-d).
We next tested the importance of the CCL2-CCR2 chemokine axis in the
maintenance of Ly6Chi diurnal rhythms. Consistent with published reports, the number of
total and Ly6Chi monocytes were lower in blood and spleens of Ccr2−/− mice (Fig.
4h and Supplementary Fig. 2.16a-c) (31). However, loss of CCR2 also abolished the
diurnal variation of total and Ly6Chi monocytes in the blood and spleen (Fig. 2.4h and
Supplementary Fig. 2.16a-c). Because bone marrow monocyte content is antiphasic to
that of the periphery, Ccr2−/− mice had a higher number of Ly6Chimonocytes throughout
63
the time course (Supplementary Fig. 2.16d). In contrast, administration of CCL2 to
C57BL/6J mice was sufficient to disrupt the diurnal oscillations of Ly6Chi and total
monocytes in all three reservoirs (Fig. 2.4h and Supplementary Fig. 2.16a-d). These
data indicate that the rhythmic recruitment of the PRC2 complex by BMAL1/CLOCK
heterodimers imparts diurnal variation to chemokine expression that is necessary to
sustain Ly6Chi monocyte rhythms.
2.5 Myeloid Cell BMAL1 Deficiency Worsens Metabolic Disease
Having established a physiological role for the diurnal rhythms of
Ly6Chi monocytes during acute infection, we investigated whether their disruption
contributes to pathogenesis of chronic inflammatory diseases. Our initial studies focused
on diet-induced obesity and insulin resistance, because low-grade chronic inflammation
has been shown to modulate the expression of these disease phenotypes (32, 33). We thus
fed ArntlLoxP/LoxP andArntlLoxP/LoxPLyz2Cre mice a high-fat diet (HFD) for 1 week and
monitored the recruitment of Ly6Chi monocytes to metabolic tissues. Compared
with ArntlLoxP/LoxP mice, short-term HFD feeding induced Ly6Chi monocytosis and
increased the Ly6Chi macrophage content of epididymal white adipose tissue (eWAT)
and brown adipose tissue (BAT) of ArntlLoxP/LoxPLyz2Cre mice (Supplementary Fig.
2.17a-c). Moreover, in ArntlLoxP/LoxPLyz2Cre mice, the newly recruited Ly6Chi eWAT and
BAT macrophages expressed higher levels of monocyte-attracting chemokines
(Supplementary Fig. 2.17d-f), suggesting that disruption of the diurnal rhythms of
monocytes might potentiate metabolic inflammation and disease.
To explore this hypothesis, we fed ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice a
HFD and monitored the development of metabolic disease. Compared
64
with ArntlLoxP/LoxP mice, ArntlLoxP/LoxPLyz2Cre mice gained ~30% more weight on HFD,
which contributed to their higher total body adiposity and increased tissue weight (Fig.
2.5a-c). This increase in weight gain likely resulted from a decrease in energy
expenditure, as reflected in the lower oxygen consumption rate (~12%)
of ArntlLoxP/LoxPLyz2Cre mice during the day cycle (Fig. 2.5d). Food intake, total activity,
and substrate utilization were not different
between ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cremice (Supplementary Fig. 2.18a-c).
We next investigated whether chronic exposure to HFD exacerbated inflammation
in metabolic tissues ofArntlLoxP/LoxPLyz2Cre mice. Flow cytometric analysis revealed
that ArntlLoxP/LoxPLyz2Cre mice had higher numbers of total and Ly6Chi (~2.5- and ~1.8-
fold higher than ArntlLoxP/LoxP mice, respectively) macrophages in their eWAT (Fig. 2.5a
and Supplementary Fig. 2.19). Macrophage subset analysis further showed that absolute
numbers of CD11c+ and CD301+ macrophages were both higher in eWAT
of ArntlLoxP/LoxPLyz2Cre mice (Supplementary Fig. 2.19a, b). These inflammatory
changes were not restricted to eWAT because we observed similar increases in
macrophage content of BAT in ArntlLoxP/LoxPLyz2Cre mice (Supplementary Fig. 2.19c,
d). Congruent with these observations, there was evidence for increased local and
systemic inflammation in ArntlLoxP/LoxPLyz2Cre mice (Supplementary Fig. 2.20a-c),
including increased infiltration of eWAT and BAT by adaptive immune cells
(Supplementary Fig. 2.20d, e) and higher expression of monocyte-attracting
chemokines (Supplementary Fig. 2.20f-h). In contrast, body weight and macrophage
content of eWAT and BAT were not significantly different in mice fed normal chow
(Supplementary Fig. 2.21a-c). These data demonstrate that deletion of Arntl in myeloid
65
cells prevents diurnal oscillations between Ly6Chi and Ly6Clow monocytes, which
potentiates the inflammatory response to obesity.
On the basis of these findings, we assessed whether ArntlLoxP/LoxPLyz2Cre mice
were more prone to developing obesity-associated insulin resistance and metabolic
disease. Glucose tolerance testing showed impaired clearance of glucose
in ArntlLoxP/LoxPLyz2Cre mice (Fig. 2.5f), which likely resulted from a decrease in
systemic insulin action (Fig. 2.5g). Congruent with this postulate, insulin-induced serine
phosphorylation of Akt was markedly impaired in eWAT, liver, and skeletal muscle
of ArntlLoxP/LoxPLyz2Cre mice (Fig. 2.5h and Supplementary Fig. 2.22a, b). In addition,
histological analysis demonstrated ectopic deposition of triglycerides in liver and BAT
(Supplementary Fig. 2.22c-f), and leukocytic infiltration in eWAT (Supplementary
Fig. 2.22g), thus providing further evidence for worsening insulin resistance and
metabolic dysfunction in ArntlLoxP/LoxPLyz2Cre mice.
Because previous studies have demonstrated that changes in feeding period can
entrain the peripheral cellular clocks (34), we lastly investigated whether monocyte
diurnal rhythms are responsive to changes in nutrient intake. As expected, restricting
feeding to the daytime induced a 12-hour phase shift in liver diurnal gene expression
(Supplementary Fig. 2.23a, b), whereas the expression of clock genes in peritoneal
macrophages remained unchanged (Supplementary Fig. 2.23c, d). Accordingly, the
diurnal oscillations of Ly6Chi and total monocytes in all three reservoirs displayed similar
rhythms irrespective of the feeding regimen (Supplementary Fig. 2.23e-i), suggesting
that Ly6Chimonocyte rhythms primarily fortify host defenses against anticipatory changes
in the environment.
66
CHAPTER 2-CONCLUSIONS
Previous studies have demonstrated bidirectional cross-talk between circadian
clocks and metabolism (1, 2, 28). For instance, the peripheral clocks in metabolic tissues
anticipate feeding-fasting cycles, and, conversely, feeding rhythms are strong Zeitgebers
that can entrain peripheral clocks of metabolic tissues. In contrast, we found that the
diurnal rhythms of myeloid cells do not anticipate metabolic rhythms and cannot be
entrained by time-restricted feeding cycles. This suggests that the primary function of
diurnal oscillations in myeloid cell numbers is not in anticipatory regulation of
metabolism but perhaps in host defense. In support of this idea, both the time of infection
and the myeloid-specific BMAL1 are critical determinants of the pathology associated
with infectious challenge with L. monocytogenes. Moreover, the ability of CCL2, whose
expression is induced upon sensing of pathogens, to override the diurnal oscillations in
myeloid cell numbers provides a mechanism by which the host defenses can shift from
anticipatory to pathogen-directed responses.
Although diurnal variation in monocyte numbers is not entrained by feeding cues,
its disruption does render mice susceptible to diet-induced obesity and insulin resistance.
The increased susceptibility of ArntlLoxP/LoxPLyz2Cremice to metabolic disease likely
results from Ly6Chi monocytosis and the subsequent recruitment of these cells into
metabolically stressed adipose tissue. This potentiates the chronic inflammatory response
both locally and systemically, resulting in insulin resistance and hyperglycemia. Last,
because chronic inflammatory diseases, such as myocardial infarction, asthma, and
rheumatoid arthritis, exhibit diurnal clustering in humans (35–37), rhythmic oscillations
of inflammatory monocytes might also contribute to their pathogenesis.
67
CHAPTER2-FIGURE LEGENDS
Supplementary Table 2.1 Primers for chemokine E-boxes
Supplementary Table 2.2 Primers for proximal promoter region of chemokines.
Supplementary Table 2.3 Primers for core clock genes and chemokines.
Figure 2.1. Diurnal variation of Ly6Chi monocytes.
(a) Quantitative RT-PCR analysis of clock-controlled genes (Arntl, Nr1d1, and Dbp) in
blood monocytes during a 12-hour light-dark cycle (n = 3 or 4 samples per time point).
AU, arbitrary units. (b, c) Ly6Chi monocyte numbers in blood (b) and spleen (B) during a
12-hour light-dark cycle (n = 5 mice per time point). (d, e) Recruitment of
Ly6Chi monocytes to inflamed peritoneum. Ly6Chi monocyte number (d) and
concentration of IL-1β (e) were quantified in the peritoneal fluid 2 hours after elicitation
with thioglycollate (n = 5 mice per time point). Pooled data (a) or representatives [(b) to
(e)] of two independent experiments are shown as mean ± SEM. Two-tailed
Student’s t tests (a) and one-way analysis of variance (ANOVA) (b-e) were used for
statistical analyses (comparisons were made between the acrophase and other time
points). *P < 0.05; **P< 0.01; ***P < 0.001.
Figure 2.2 Diurnal variation in the pathogenicity of L. monocytogenes.
(a-c) Mice kept under a 12-hour light-dark cycle were intraperitoneally inoculated with 1
× 106 L. monocytogenes at ZT0 and ZT8, and CFUs recovered from the peritoneal cavity
(a), spleen (b), and liver (c) were quantified 2 dpi (n = 14 or 15 mice per time point). (d-
f) Numbers of iNOS+CD11c+ and TNFα+CD11c+ cells in peritoneal cavity (d), spleen (e),
and liver (f) of mice 2 dpi with 1 × 106 L. monocytogenes at ZT0 and ZT8 (n = 15 mice
per time point). (g) Concentration of chemokines and cytokines in peritoneal fluid 2 dpi
with 1 × 106 L. monocytogenes at ZT0 and ZT8 (n = 15 mice per time point). (h)
68
Survival curves of mice after infection with 1 × 107 L. monocytogenes at ZT0 and ZT8
(n = 25 mice per time point). (i) Serum concentration of chemokines and cytokines 2 dpi
with 1 × 107 L. monocytogenes at ZT0 and ZT8 (n = 10 mice per time point). Pooled data
from two or three independent experiments are presented as mean ± SEM. Statistical
significance (*P < 0.05; **P < 0.01; ***P < 0.001) was assessed by using two-tailed
Student’s t-test [(A) to (G) and (I)] and log-rank test (H).
Figure 2.3 BMAL1 regulates rhythmic oscillations of Ly6Chimonocytes in a cell-
autonomous manner.
(a) Quantitative RT-PCR kinetic analysis of Nr1d1 mRNA in blood monocytes isolated
from ArntlLoxP/LoxP andArntlLoxP/LoxPLyz2Cre mice kept on a 12-hour light-dark cycle (n= 3
or 4 samples per genotype and time point). (b-d) Ly6Chimonocyte numbers in blood (b),
spleen (c), and bone marrow (d) of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice kept
under a 12-hour light-dark cycle at various ZTs (n = 5 mice per genotype and time point).
(e) Survival curves of ArntlLoxP/LoxPand ArntlLoxP/LoxPLyz2Cre mice after infection with 1 ×
106 L. monocytogenes at ZT0 and ZT8 (n = 10 or 11 mice per genotype and time point).
(f-i) Serum concentrations of IL-1β (f), IL-6 (g), IFN-γ (h), and CCL2 (i)
in ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice 2 dpi with 1 × 106 L. monocytogenes at
ZT0 and ZT8 (n= 4 to 6 mice per genotype and time point). Pooled data [(a) and (e)] and
representative (b-d) of two to three independent experiments are shown as mean ± SEM
and analyzed by using two-tailed Student’s ttests [(a-d) and (f-i)] and log-rank test (E).
*P < 0.05; **P < 0.01; ***P < 0.001.
Figure 2.4 BMAL1 recruits PRC2 to repress expression of Ccl2.
69
(a) Quantitative RT-PCR analysis of Ccl2 expression in blood monocytes
of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice kept on a 12-hour day-light cycle (n = 3 or
4 samples per genotype and time point). (b) ChIP analysis of BMAL1 binding to
the Ccl2 promoter (n = 4 samples per genotype and time point). (c)
Coimmunoprecipitation of BMAL1 and with members of PRC2. Nuclear lysates from
serum-shocked BMDMs were immunoprecipitated (IP) with BMAL1 antibody and
immunoblotted (IB) for BMAL1, CLOCK, EZH2, EED, and SUZ12. Ig, immunoglobulin
G. (d and g) ChIP analysis for the recruitment of EZH2 (d) and Pol II (g) to the proximal
promoter of the Ccl2 gene (n = 4 samples per genotype and time point). (e-f) ChIP
analysis for H3K27Me3 (e) and H3K4Me3 (f) at the proximal promoter of Ccl2 gene (n =
4 samples per genotype and time point). (H) Ly6Chi monocyte numbers in the blood of
wild-type or Ccr2−/− mice during a 12-hour light-dark cycle. Wild-type mice were
intraperitoneally injected with phosphate-buffered saline (Veh) or CCL2 (20 µg kg−1) 24
hours before quantification of Ly6Chimonocytes (n = 4 or 5 mice per genotype/treatment
and time point). Pooled data (a-g) from two independent experiments are shown as mean
± SEM and analyzed by using two-tailed Student’s t tests [(a), (b), and (d-g)] and two-
way ANOVA (H). *P < 0.05, **P < 0.01, and ***P < 0.001 represent comparison
between ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre or between wild-type–treated Veh versus
CCL2 or Ccr2−/− at each time point.
Figure 2.5 Myeloid cell-specific deletion of BMAL1 exacerbates metabolic disease.
(a -d) Body weight (a), adiposity (b), tissue weight (c), and oxygen consumption (d)
in ArntlLoxP/LoxP andArntlLoxP/LoxPLyz2Cre mice kept on a 12-hour light-dark cycle fed a
HFD for 19 weeks (n = 4 or 5 mice per genotype). (e) Total and Ly6Chi macrophage
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content in eWAT of ArntlLoxP/LoxP andArntlLoxP/LoxPLyz2Cre mice fed a HFD (n = 5 mice
per genotype). (f-g) Glucose (f) and insulin tolerance (g) tests
of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice fed a HFD (n = 5 to 8 mice per genotype).
(h) Immunoblots of total and phosphorylated Akt (pAKT) in eWAT of
obeseArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre administered intraportal insulin. HSP90, heat
shock protein 90. Representative data of two to four independent experiments [(a-c) and
(e-h)] are shown as mean ± SEM and analyzed by using two-tailed Student’s t tests. *P <
0.05; **P < 0.01; ***P < 0.001.
Supplementary Figure 2.1 Diurnal variation of clock genes in monocytes
(a) Expression of luciferase protein in blood monocytes isolated from Per2Luc mice (n = 3
samples per time point). (b) Quantitative PCR analysis of Arntl, Nr1d1, and Dbp mRNAs in
serum-shocked THP1 over 24 hours (n = 4-5 samples per time point). Pooled data from two
independent experiments are presented as mean ± SEM and statistically analyzed using two-
tailed Student’s t-tests (a) and one-way ANOVA (b) (comparisons were made between the
acrophase and other time points). *P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.2 Gating strategy of monocytes in different tissues.
Mononuclear cells from blood or tissues were gated for forward and side-scatter (FSC/SSC),
doublets, and live/dead prior to identification of blood (CD45+CD115+Ly6G-), splenic
(CD45+CD11b+CD11c-F4/80-Ly6G-), bone marrow (CD45+CD11b+CD11c-F4/80-Ly6G-
Ly6C+), and peritoneal (CD45+CD115+Ly6G-F4/80-) monocytes.
Supplementary Figure 2.3 Diurnal variation in monocyte number and inflammation.
(a-c) Total monocyte numbers in blood (a), spleen (b), and bone marrow (c) as quantified
during a 12 hour light-dark cycle (n = 5 mice per time point). (d-e) Numbers of Ly6Clow
monocytes in blood (d) and spleen (e) during a 12 hour light-dark cycle (n = 5 mice per time
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point). (f) Quantitative RT-PCR analysis of Ccr2 mRNA expression in blood monocytes
during a 12 hour light-dark cycle (n = 3-4 samples per time point). (g-k) Concentration of
IL6 (g), CCL2 (B), and CCL8 (k) in the peritoneal fluid 2 hours after elicitation with
thioglycollate. Intracellular staining for IL1 (h) and IL6 (i) in Ly6Chi monocytes 2 hours after
elicitation with thioglycollate expression (n = 5 mice per ZT). (l) Intracellular staining for
CCL2 in Ly6Chi monocytes, macrophages, and neutrophils 30 mins after injection of
thioglycollate (n = 5 mice). Representative (a-e) or pooled data (f and g, j and k) of two
independent experiments are shown as mean ± S.E.M and statistically analyzed using one-
way ANOVA. Comparisons were made between the acrophase and other time points
*P<0.05; **P<0.01.
Supplementary Figure 2.4 Gating strategy for identifying cytokine producing cells in
mice infected with L. monocytogenes.
Mononuclear cells were gated for forward and side-scatter (FSC/SSC), doublets, and
live/dead prior to analysis of CD4+IFNγ+ T cells, CD8+IFNγ+ T cells, CD11c+TNFα+, and
CD11c+iNOS+ dendritic cells.
Supplementary Figure 2.5 Diurnal variation in host response to L. monocytogenes.
Wild type mice kept on a 12 hour light-dark cycle were intraperitoneally inoculated with
1x106 L. monocytogenes at ZT0 and ZT8. (a) Concentration of chemokines and cytokines in
serum 2 dpi (n = 15 mice per time point). (b, c) Numbers of Ly6Chi monocytes (b) and
neutrophils (c) in different tissues of mice 2dpi (n=15 mice per time point). (d, e) L.
monocytogenes colony forming units (CFUs) recovered from the liver (d), and spleen (e)
were quantified 6 dpi (n = 8-10 mice per time point). (F) Spleen weight at 6 dpi (n = 8-10
mice per time point). (g) CFUs normalized to spleen weight at 6 dpi (n = 8-10 mice per time
point). (h-i) Numbers of IFNγ+ CD4+ and CD8+ T cells in spleen (h) and liver (i) 6 dpi (n =
10-15 mice per time point). (j-l) Numbers of iNOS+CD11c+ and TNFα+CD11c+ cells in
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peritoneal cavity (j), spleen (k), and liver (l) of mice 6 dpi (n = 8-10 mice per time point).
Pooled data from two to three independent experiments are shown as mean ± S.E.M. Two-
tailed Student’s t-tests are used for statistical analyses. *P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.6 High dose intraperitoneal infection with L. monocytogenes.
(a-c) Wild type mice kept on a 12 hour light-dark cycle were intraperitoneally inoculated
with 1x107 L. monocytogenes at ZT0 and ZT8, and CFUs recovered from peritoneal cavity
(a), liver (b), and spleen (c) were quantified 2 dpi (n = 10 mice per time point). Pooled data
from two independent experiments are shown as mean ± S.E.M and statistically analyzed
using two-tailed Student’s t-test. *P<0.05.
Supplementary Figure 2.7 Deletion of Arntl in myeloid cells disrupts monocyte rhythms.
(a) Immunoblot of BMAL1 protein in blood monocytes isolated from ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice at ZT0. (b) Quantitative RT-PCR analysis of Arntl mRNA in blood
monocytes of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice kept on a 12 hour light-dark cycle
at the various ZTs (n = 3-4 samples per genotype and time point). (c-d) Total monocyte
numbers in blood (c) and spleen (d) of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice during the
12 hour light-dark cycle (n = 5 mice per genotype and time point). (e-f) Ly6Clow monocyte
numbers in blood (e) and spleen (f) of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice at various
ZTs (n = 5 mice per genotype and time point). (g-h) Neutrophil numbers in blood (g) and
spleen (h) of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice during the 12 hour light-dark cycle
(n = 5 mice per genotype and time point). Pooled data (a-b) or representative (c-f) from two
to three independent experiments are shown as mean ± S.E.M. Two-tailed Student’s t-tests
were used for statistical analyses. *P<0.05; **P<0.01 comparison between ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre at each time point.
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Supplementary Figure 2.8 Loss of diurnal variation in thioglycollate-induced sterile
peritonitis in ArntlLoxP/LoxPLyz2Cre mice
(a-b) Numbers of Ly6Chi (a) and total (b) monocytes in the peritoneum of ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice kept under a 12 hour light-dark cycle 2 hours after injection with
thioglycollate at different ZTs (n = 5 mice per genotype and time point). (c-f) Resident
numbers of Ly6Chi (c) and total (d) monocytes, neutrophils (e), and macrophages (f) in
peritoneal cavity of mice kept under a 12 hour light-dark cycle at various ZTs (n = 4 mice per
genotype and time point). Representative data (a-b) from two independent experiments are
shown as mean ± S.E.M. Two-tailed Student’s t-tests were used for statistical analyses.
*P<0.05; **P<0.01 comparison between ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre at each time
point.
Supplementary Figure 2.9 Characteristics of acute inflammation in
ArntlLoxP/LoxPLyz2Cre mice. (a-d) Concentration of CCL2 (a), CCL8 (b), IL1β (c), and
IL6 (d) in the peritoneal fluid of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice kept on a 12
hour light-dark cycle 2 hours after elicitation with thioglycollate at ZT0, ZT8, and ZT12
(n = 5 mice per genotype and time point). (e-h) Quantitative PCR analysis of Il1b (e), Il6
(f), Tlr2 (g), and Tlr5 (h) mRNAs in blood monocytes of ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice kept on a 12 hour light-dark cycle at the various ZTs (n = 3-4
samples per genotype and time point). Pooled data from two independent experiments are
shown as mean ± S.E.M and statistically analyzed by two-tailed Student’s t-tests.
*P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.10 Quantification of bacterial burden and cytokine
producing cells in ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice infected with L.
monocytogenes. (a-c) ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice kept on a 12 hour light-
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dark cycle were intraperitoneally inoculated with 1x106 L. monocytogenes at ZT0 and
ZT8, and colony 18 forming units (CFUs) recovered from the spleen (a), liver (b), and
peritoneal cavity (c) were quantified 2 dpi (n = 4-6 mice per genotype and time point). (d to
k) Numbers of TNFα+CD11c+ (d and h), iNOS+CD11c+ (e and i), IFNγ+ CD4+ (f and j), and
CD8+ T (g and k) cells in spleens and livers, respectively, of ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice kept on a 12 hour light-dark cycle 2 dpi with 1x106 L.
monocytogenes at ZT0 and ZT8 (n = 4-6 mice per genotype and time point). All data are
presented as mean ± S.E.M and statistically analyzed by two-tailed Student’s t-tests.
*P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.11 Chemokine expression in thioglycollate-elicited peritoneal
monocytes.
(a-c) Intracellular staining for CCL2 (a), CCL8 (b), and S100A8 (c) in peritoneal
monocytes of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice kept on a 12 hour light-dark
cycle 30 mins after injection of thioglycollate at various ZTs (n = 4-5 mice per genotype
and time point). Representative data of two independent experiments are shown as mean
± S.E.M and analyzed using two-tailed Student’s t-test. *P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.12 Chemokine expression in monocytes and macrophages
of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice.
(a-b) Quantitative RT-PCR analysis for Ccl8 (a) and S100a8 (b) mRNA expression in
blood monocytes of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice during a 12 hour light-
dark cycle (n = 3-4 samples per genotype and time point). (c-e) Quantitative RT-PCR
analysis for Ccl2 (c), Ccl8 (d), and S100a8 (e) mRNA expression in peritoneal
macrophages of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice during a 12 hour light-dark
cycle (n = 4-5 mice per genotype and time point). (f-g) Serum CCL2 (f) and CCL8 (g)
75
concentrations in ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice during a 12 hour light-dark
cycle (n = 4-19 mice per genotype and timepoint). Pooled data from two to four
independent experiments are presented as mean ± S.E.M and statistically analyzed with
two-tailed Student’s t-test. *P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.13 Circadian recruitment of BMAL1 and CLOCK to the
chemokine promoters.
(a-b) ChIP analysis of BMAL1 binding to the E-boxes in Ccl8 (a) and S100a8 (b) promoters
(n = 4 samples per genotype and time point). (c-e) ChIP analysis of CLOCK binding to the E-
boxes in promoters of Ccl2 (c), Ccl8 (d), and S100a8 (e) genes (n = 4 samples per genotype
and time point). Pooled data from two independent experiments are presented as mean ±
S.E.M and statistically analyzed using two-tailed Student’s t-test. *P<0.05; **P<0.01;
***P<0.001.
Supplementary Figure 2.14 Circadian recruitment of PRC2 to the promoter of Ccl8
gene.
(a-d) ChIP analysis for the recruitment of EZH2 (a) and Pol II (d) to the proximal promoter
of the Ccl8 gene (n = 4 samples per genotype and time point), and for H3K27Me3 (b) and
H3K4Me3 (c) at the proximal promoter of Ccl8 gene (n = 4 samples per genotype and time
point). Pooled data from two independent experiments are presented as mean ± S.E.M and
statistically analyzed using two-tailed Student’s t-test. *P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.15 Circadian recruitment of PRC2 to the promoter of S100a8
gene.
(a-d) ChIP analysis for the recruitment of EZH2 (a) and Pol II (d) to the proximal promoter
of the S100a8 gene (n = 4 samples per genotype and time point), and for H3K27Me3 (b) and
H3K4Me3 (c) at the proximal promoter of S100a8 gene (n = 4 samples per genotype and
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time point). Pooled data from two independent experiments are presented as mean ± S.E.M
and statistically analyzed using two-tailed Student’s t-test. *P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.16 CCL2-CCR2 axis regulates diurnal variation in trafficking
of monocytes. (a-b) Monocyte numbers in the blood (a) or spleens (b) of wild type or Ccr2-/-
mice during a 12 hour light-dark cycle. Wild type mice kept on a 12 hour light-dark cycle
were administered PBS (Veh) or CCL2 (20 µgkg-1) 24 hours prior to quantification of
monocytes (n = 4-5 mice per genotype/treatment and time point). (c) Ly6Chi monocyte
numbers in spleens of various groups during a 12 hour light-dark cycle (n = 4-5 mice per
genotype/treatment and time point). (d) Monocyte numbers in bone marrow of various
groups during a 12 hour light-dark cycle (n = 4-5 mice per genotype/treatment and time
point). All data are presented as mean ± S.E.M and statistically analyzed using two-way
ANOVA. *P<0.05; **P<0.01; ***P<0.001 represent comparison between wild type Veh
treated vs. CCL2 or Ccr2-/- at each time point.
Supplementary Figure 2.17 BMAL1 regulates monocyte trafficking during HFD
feeding.
ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice kept on a 12 hour light-dark cycle were housed at
30°C and fed HFD for 1 week. (a-c) Numbers of monocytes in blood (a), macrophages in
eWAT (b), and macrophages in BAT (c) of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice (n =
5 mice per genotype). (d-f) Expression of CCL2 (d), CCL8 (e), and S100A8 (f) in Ly6Chi
blood monocytes and adipose tissue macrophages of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre
mice (n = 5 mice per genotype). Representative data (a-c) from two independent experiments
are shown as mean ± S.E.M and statistically analyzed using two-tailed Student’s t-test.
*P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.18 Behavioral characteristics of ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice on HFD.
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(a-c) Food intake (a), total activity (b), and RER (c) of obese ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice kept on a 12 hour light-dark cycle fed HFD (n = 4 mice per
genotype). Data are presented as mean ± S.E.M and statistically analyzed using two-tailed
Student’s t-test.
Supplementary Figure 2.19 Gating strategy for analysis of immune subsets in adipose
tissue.
(a) Mononuclear cells were gated for forward and side-scatter (FSC/SSC), doublets, and
live/dead prior to analysis of CD3+ T cells, B220+ B cells, Siglec F-F4/80+ macrophages,
Siglec F+ eosinophils, Ly6G+ neutrophils, FcεRI+ basophils/mast cells, and CD49b+ NK cells.
Macrophages subsets were identified as being Ly6Chi, CD301+, or CD11c+. (b-d)
Macrophage content and subtypes in eWAT (b) and BAT (c-d) of ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice fed high fat diet (n = 5 mice per genotype). Representative data (b-
d) from two independent experiments are shown as mean ± S.E.M and statistically analyzed
using two-tailed Student’s t-tests. *P<0.05; **P<0.01.
Supplementary Figure 2.20 Evaluation of tissue inflammation in obese ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice.
(a-b) Immunoblots of total and phosphorylated JNK (pJNK) in eWAT (a) and livers (b) of
obese ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice fed HFD for 19 weeks and kept on a 12
hour light-dark cycle. (c) Serum concentration of chemokines and cytokines of obese
ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice (n = 4-5 mice per genotype). (d-e) Immune cell
repertoire of eWAT (d) and BAT (e) of obese ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice (n
= 4-5 mice per genotype). (f-h) Expression of CCL2 (f), CCL8 (g), and S100A8 (h) in blood
monocytes and adipose tissue macrophages of obese ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre
mice (n = 4-5 mice per genotype). Pooled data (a-c) or representative (d-e) of two
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independent experiments are shown as mean ± S.E.M and statistically analyzed using two-
tailed Student’s t-test. *P<0.05; **P<0.01; ***P<0.001.
Supplementary Figure 2.21 Characteristics of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre
mice on normal chow diet.
(a) Body weight of ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice kept on a 12 hour light-dark
cycle fed normal chow diet (NC) for 26 weeks (n = 4-5 mice per genotype). (b-c) Total and
Ly6Chi macrophage content in eWAT (b) and BAT (c) of ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice on various diets (n=3-5 mice per genotype). Representative data of
two independent experiments are shown as mean ± S.E.M and statistically analyzed using
two-tailed Student’s t-test. *P<0.05; **P<0.01.
Supplementary Figure 2.22 Metabolic characteristics of ArntlLoxP/LoxP and
ArntlLoxP/LoxPLyz2Cre mice on HFD.
(a-b) Immunoblots of total and phosphorylated AKT (pAKT) in liver (a) and quadriceps (b)
of obese ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre administered intraportal insulin. (c-d) Liver
(c) and BAT (d) triglyceride content of obese ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice (n
= 5 mice per genotype). (e-g) Representative sections of liver (e), BAT (f), and eWAT (g) of
obese ArntlLoxP/LoxP and ArntlLoxP/LoxPLyz2Cre mice stained with hematoxylin and eosin
(H&E). Pooled data (a-d) and representative of two independent experiments (e-g) are shown
as mean ± S.E.M and statistically analyzed using two-tailed Student’s t-tests. *P<0.05.
Supplementary Figure 2.23 Time restricted feeding does not entrain the diurnal
rhythms of monocytes.
(a-d) Quantitative RT-PCR analysis of Arntl and Nr1d1 mRNAs in liver (a-b) and peritoneal
macrophages (c-d) of C57BL/6J mice fed ad libitium or exclusively during the light phase of
a 12 hour light-dark cycle (n = 4-5 mice per treatment and time point). (e-g) Numbers of total
monocytes in blood (e), spleen (f), and bone marrow (g) of C57BL/6J mice fed ad libitium or
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exclusively during the light phase of a 12 hour light-dark cycle (n = 4-5 mice per treatment
and time point). (h-i) Numbers of Ly6Chi monocytes in blood (h) and spleen (i) of C57BL/6J
mice fed ad libitium or exclusively during the light phase of a 12 hour light-dark cycle (n = 4-
5 mice per treatment and time point). In e to i, rhythms of time-restricted animals are
overlayed on data presented in Fig. 1 and Supplementary Fig. 3. All data are presented as
mean ± S.E.M and statistically analyzed using two-tailed Student’s t-test. *P<0.05;
**P<0.01.
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Figure 2.1
81
Figure 2.2
82
Figure 2.3
83
Figure 2.4
84
Figure 2.5
85
Supplementary Table 2.1
86
Supplementary Table 2.2
87
Supplementary Table 2.3
88
Supplementary Figure 2.1
89
Supplementary Figure 2.2
90
Supplementary Figure 2.3
91
Supplementary Figure 2.4
92
Supplementary Figure 2.5
93
Supplementary Figure 2.6
94
Supplementary Figure 2.7
95
Supplementary Figure 2.8
96
Supplementary Figure 2.9
97
Supplementary Figure 2.10
98
Supplementary Figure 2.11
99
Supplementary Figure 2.12
100
Supplementary Figure 2.13
101
Supplementary Figure 2.14
102
Supplementary Figure 2.15
103
Supplementary Figure 2.16
104
Supplementary Figure 2.17
105
Supplementary Figure 2.18
106
Supplementary Figure 2.19
107
Supplementary Figure 2.20
108
Supplementary Figure 2.21
109
Supplementary Figure 2.22
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Supplementary Figure 2.23
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FUTURE DIRECTIONS
The findings above highlighted the functional dynamics of monocytes and
macrophages at the intersection between the host and the environment. In response to
cold stress, mono-phagocytes can adopt an anti-inflammatory phenotype and secrete
catecholamines to activate the thermogenic machinery essential for the generation of heat
to protect against cold induced hypothermia. On the other hand, these myeloid cells could
also adopt a rhythmic inflammatory phenotype under the control of the circadian clock to
anticipate changes in environmental light-dark cycle. More importantly, these findings
have implications for the involvement of the catecholaminergic pathways in myeloid
cells and their circadian clock system in general stress responses and the development of
aging-associated inflammation, respectively.
1. Role of macrophage-derived catecholamines in stress responses:
In 1664, Thomas Willis reported the the biological importance of the adrenergic
system has long been appreciated in cardiovascular biology. For instance, work over the
last 6 decades has demonstrated that the catecholamines secreted by the SNS exert first
anatomic description of sympathetic nerves, thereby laying the groundwork for our
current understanding of how the sympathetic nerves mediate their pressor and
chronotropic effects on the vasculature and viscera. Based on these and other studies, the
medical textbook model of the mammalian stress response is: environmental stress →
activation of SNS → norepinephrine release by SNS in tissues → physiologic
adaptations → survival. In contrast to this well appreciated model, our studies which
identified a hematopoietic circuit comprising of alternatively activated macrophages
mediates an organisms stress response to environmental cold by releasing norepinephrine
112
in brown and white adipose tissue. These findings lead us to ask whether the newly
identified hematopoietic circuit might also play a more general role in mammalian stress
response. We thus propose to test this hypothesis using a model of chemical
sympathectomy, which selectively ablates sympathetic nerves in adult animals. Our
central hypothesis is: environmental stress → mobilization and activation of innate
immune cells → norepinephrine release → physiologic adaptations → survival. We
will test this hypothesis in two main studies. First, we will investigate how environmental
stress leads to mobilization and activation of the innate immune system. Secondly, we
will investigate the contribution of myeloid cell-derived catecholamines to mammalian
stress responses. The successful completion of these studies should provide fundamental
insights into the mechanisms by which stress modulates various aspects of cardiovascular
biology.
2. Role of monocyte specific clock in the development of aging related pathologies:
Aging is a major risk factor for many of the degenerative diseases that afflict our
society, including atherosclerosis, obesity, diabetes, cancer, Alzheimer’s and
neurodegenerative diseases. Thus, it is of paramount importance that we gain better
understanding of how aging increases the susceptibility of individuals to age-related
disorders. Over the last decade, low-grade chronic inflammation has emerged as a
hallmark of the age-related diseases in mice and humans. For instance, aging results in a
gradual increase (2-4-fold) in serum levels of various inflammatory cytokines and
chemokines, including Ccl2, Ccl11, Ccl7, Ccl12, IL-6, Tnf, and IL-1. This increase in
tissue and systemic inflammation likely results from aberrant activation of the innate
immune system because adaptive immunity, both cellular and humoral, declines with age.
113
In support of this idea, casual relationship between low-grade innate inflammation and
age-associated disorders has now been firmly established using rodent models of disease.
While a number of factors have been implicated in the initiation of age-associated
inflammation, such as environmental stress, antigenic stress, hormonal imbalance and
tissue damage, cell intrinsic regulatory pathways that contribute to age-induced innate
activation remain unexplored. Based on the findings above on the role of monocyte
specific clock in the regulation of inflammatory rhythm, we will test the hypothesis
decline in circadian function contributes to inflammatory activation of monocytes,
resulting in tissue inflammation and metabolic pathology. These studies will not only
provide novel insights into the pathogenesis of age-related inflammation and metabolic
disorders but also suggest new strategies for combating the decline in tissue function
associated with aging.
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