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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http://purl.stanford.edu/km773gy2297

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


Lawrence Steinman, Co-Adviser


Edgar Engleman


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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iv

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.

vi

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

vii

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

viii

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

x

Figure S2.22..…………………………………………..………………………………109

Figure S2.23..…………………………………………..………………………………110

1

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

3

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

4

(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′;

5

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

6

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).

7

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).

8

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

11

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.

12

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

13

(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

14

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


33


34

Supplementary Figure 1.1

35


36


37


38


39


40


41


42


43


44


45


46


47


48


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)

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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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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

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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.

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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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