cousins center for psychoneuroimmunology ucla semel institute for neuroscience
TRANSCRIPT
Cousins Center for Psychoneuroimmunology
UCLA Semel Institute for Neuroscience
www.cousinsPNI.org
Sleep, Immunity, and the Central Nervous System: Concepts in
Psychoneuroimmunology
Michael R. Irwin, MDNorman Cousins Professor
Cousins Center for PsychoneuroimmunologySemel Institute for Neuroscience
David Geffen School of Medicine at UCLA
?Biological
mechanisms
MortalitySleepfactors
1. Kripke DF, Garfinkel L, Wingard DL, Klauber MR, Marler MR. Arch Gen Psychiatry. 2002;59:131-136. 2. Dew M A, Hoch C C, Buysse D.J, et al. Psychosomat Med. 2203;65:63-73.
Outline of the Problem:
• Up to 25% of adults report sleep problems with over 10% having insomnia– Disordered sleep is a risk factor for mortality
• Correlational epidemiology1
• Prospective studies2
• Experimental animal studies
• Biological mediators of these effects are poorly understood
Model of Sleep-Immune Interactions
Primary InsomniaSleep ApneaMajor DepressionAlcohol Dependence
Proinflammatory Cytokine Levels & Production
Sleep Disturbances
Natural & Cellular Immunity
Inflammatory & Cardiovascular Disorders
Infectious Disease Risk
Objectives
• Address relevant questions:– Does sleep influence immune responses with
consequences on health?– Do immune changes occur in clinical
populations with sleep disturbance? – Does the immune system cause changes
in sleep?
Bryant PA, Trinder J, Curtis N. Nat Rev Immunol. 2004;4(6):457-467.
EEG Sleep Stages
Does Sleep Influence Immunity?
• Experimental approaches
• Naturalistic studies
Born J, Lange T, Hansen K, Molle M, Fehm H L. J Immunol. 1997;158:4454-4464. Open circles WS-WS; Closed WW:WS
Dynamic Changes of Immune Cell Numbers During Sleep
Dynamic Expression of Cellular Adhesion Molecules during
Sleep
Redwine L, Dang J, Irwin MR. Brain Behavior and Immunity. 2004:;65(1):75-85.
60
40
20
0Baseline PSD-E
Lym
ph
oki
ne
Act
ivat
edK
iller
-Cel
l Act
ivit
y(L
ytic
Un
its)
Sleep Loss Decreases Killer Cell Activity
Irwin M, McClintick J, Costlow C, Fortner M, White J, Gillin JC. FASEB J. 1996;10:643-653.
Redwine L, Dang J, Hall M, Irwin MR. Psychosom Med. 2003;65(1):75-85.
23:00 03:00 06:30
Time
NK
-20
Rat
io
10
9
8
7
6
5
0
Controls
Alcoholics
4
3
2
1
Alcoholics With Sleep Disturbance Fail to Show Nocturnal Increases of NK
Activity
20
15
10
5
0BaselineSleep
PSD-E Recovery
IL2
Pro
du
ctio
n(n
g/m
L)
Sleep Loss Suppresses Cellular Immunity
Irwin M, McClintick J, Costlow C, Fortner M, White J, Gillin JC. FASEB J. 1996;10:643-653.
Redwine L, Dang J, Hall M, Irwin M. Psychosom Med. 2003;65(1):75-85.
60
50
40
30
20
10
023:00 03:00 06:30
Controls
Alcoholics
Time
TH
1/T
H2
Shift in TH1 vs TH2 Cytokine Balance During Sleep
*P=0.018
Lange T, Perras B, Fehm HL, Born J. Psychosom Med. 2003;65(5):831-835.
800
600
400
200
0
0 5 6 7 8 9 10 11 12 13 14 28 d
HA
V A
nti
bo
dy Sleep
Wake
mlU/mL
*
Sleep Loss Impairs Viral Specific Immune Responses
Redwine L, Hauger RL, Gillin JC, Irwin M. J Clin Endocrinol Metab. 2000;85:3597-3603.
Sleep Regulates Nocturnal Increases of IL-6
Primary Insomniacs Show Elevated Awake and Early-night Levels of IL-6
00
.51
1.5
22
.5
22:00 0:00 2:00 4:00 6:00
Time (hrs)
IL-6
(p
g/m
L)
Controls
Insomniacs
Irwin MR. (Unpublished data).
Sleep Deprivation Induces Differential Increases of Nocturnal IL-6 in Alcoholics as Compared to
Controls
Irwin MR, Rinetti G, Redwine L, Motivala S, Dang J, Ehlers C. Brain Behav Immun. 2004;18(4):349-360.
0
1
2
3
4
Baseline PSD-E Recovery
Time
IL-6
Alcoholics
Controls
* *
Immune Activation and Daytime Fatigue
LN
(IL
-1ra
)(pg
/ml)
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Non-fatigued (n=18)
Fatigued (n=32)
P=0.05
sIL
-6R
in p
lasm
a (n
g/m
l)
p = .00019
20
30
40
50
Non-fatigued (n=18)
Fatigued (n=32)
1 a 1 b
Immune Activation and Daytime Fatigue
Shift toward TH1 cytokine balance
Sleep
Increase in proinflammatory cytokines
Decrease in lymphocyte numbers
Increase in cellular adhesion molecule expression
Summary of the Effects of Sleep on Immunity
Model of Sleep-Immune Interactions
Primary InsomniaSleep ApneaMajor DepressionAlcohol Dependence
Proinflammatory Cytokine Levels & Production
Sleep Disturbances
Natural & Cellular Immunity
Inflammatory & Cardiovascular Disorders
Infectious Disease Risk
Sleep Loss Alters Proinflammatory Cytokine
Activity
Shearer WT, Reuben JM, Mullington JM, et al. J Allergy Clin Immunol. 2001;107(1):165-170.
2.2
2.0
1.8
1.6
1.4
1.2
1.00 1 2 4 53
Day
TSD
PSD
Mea
n in
pg
/mL
± S
EM
Sleep Loss Alters Proinflammatory Cytokine Activity
Meier-Ewert HK, Ridker PM, Rifai N, et al. J Am Coll Cardiol. 2004;43(4):678-683.
0.10
0.08
0.06
0.04
0.02
0.00
BL2 Dep 1 Dep 2 Dep 3 Recovery
Mild Risk
Low Risk
CR
P (
mg
/dL
)
*
*
**
Sleep Loss Increases C-Reactive Protein:A Marker of Systemic Inflammation
Sleep restriction increases IL-6 levels
Vgontzas A, et al. J Clin Endocrin Metab. 2004;80(1):2119-2126.
Sleep Deprivation and Activation of Morning Levels of Cellular and
Genomic Markers of Inflammation
Irwin MR, Wang M, Campomayor C, Collado-Hidalgo A, Cole SW: Arhc Intern Med. 2006;1661756-1762.
Questions
To clarify the cellular source of
increases in inflammatory responses
after sleep loss
- production of IL-6 and TNF by
monocytes
- ligation of the Toll-like receptor
Questions
To identify upstream sources by testing
impact of sleep loss on gene expression
- transcription of IL-6 and TNF mRNA
Questions
Does increased inflammatory gene
expression constitute one element of a
more general genomic response to sleep
loss?
- DNA microarray analyses
- bioinformatic analyses for
characterization of
transcription control pathways
Methods
30 healthy volunteers
4 days/nights of testing
3 baseline periods
1 partial night sleep deprivation
Repeated sampling for monocyte production of IL-6/TNF
Real-time RT-PCR for IL-6/TNF mRNA
High density oligonucleotide microarrays
40.00
45.00
50.00
55.00
60.00
65.00
70.00
8:00 12:00 16:00 20:00 23:00
Time
% m
on
ocy
te e
xpre
ssin
g T
NF
or
IL-6
Baseline 1,2,3
PSD
*
Sleep Loss Increases Morning Levels of Monocyte Production of IL-6
IL-6 IL-6
TN
F
TN
F
CDSC080603.024CDSC080603.024
100
101
102
103
104
FL2-H
0 1 2 3 4
FL2-H
CDSC080803.024CDSC080803.024
100
101
102
103
104
FL2-H
100
101
102
103
104
FL
4-H
100
101
102
103
104
FL
4-H
Baseline PSD
Monocyte Intracellular Production of TNF and IL-6
0
1
2
3
4
5
1 2
IL-6 TNF
Fo
ld In
cre
as
e in
mR
NA
Co
pie
s/G
AP
DH
Baseline PSD Baseline PSD
Sleep Loss Induces Increases in Transcription of IL-6 and TNF
Genes
(Taken from http://www.affymetrix.com)
Microarray
http://www.umanitoba.ca/faculties/afs/plant_science/COURSES/bioinformatics/lec12/lec12.1.html
PSDBaseline
Microarray
Sleep Loss Activates Multiple Genomic Markers of Inflammation and Energy Metabolism
Elements of the “up-regulated” gene ensemble
master circadian regulator PER1
“immediate early genes” FOSB, IER3, PMAIP
Growth factor genes EGFL5, HBEGF
Inflammatory response genes IL-1beta, IL-8, CD83,
CCL3, and
TNFAIP3
http://gostat.wehi.edu.au
Find statistically over-represented functional group of genes
Functional gene ontology (GO) analysis
Beissbarth et al (2004) Bioinformatics, 20, 1464
List of significant GO terms List of genes and GO associations
Functional gene ontology (GO) analysis
Best GOs Genes P-value Description
GO: 0051243 RHOB; IL1B;PP1R15A; KLF10;FOSB; IL8;IER3
0.000124 Negative regulation of cellular processes
GO: 0006950 CD83; IL1B; CCL3; PPP1R15A; IL8; DUSP1
0.00428 Stress response
GO: 0001525; 0048514; 0001568
RHOB; IL8 0.0114 Angiogenesis
GO: 0012501; 0006915; 0008219; 0016265
RHOB; IL1B; PPP1R15A; IER3
0.0114 Apoptosis
GO: 0048513 KLF10; IL8; RHOB; HBEGF 0.0114 Growth Factor
GO: 0000074; 0051726
RHOB; IL1B; PPP1R15A; FOSB; IL8
0.00118 Cell cycle regulation
GO: 0006954; 0006959
IL1B; CCL3; CD83; IL8 0.0108 Immune response and inflammation
GO: 0009613 CD83; IL1B; CCL3; IL8 0.0114 Wound healing
GOStat Results:
Functional gene ontology (GO) analysis
www.telis.ucla.edu
Cole et al (2005) Bioinformatics, 21, 803
Reverse inference of transcription factor activity
Move from gene expression (microarrays) to transcription factors
by inverting the normal flow of biological information.
Transcription Element Listening System
Environment NF-BPromotersequence
Expression
Transcription Element Listening System
Environment NF-BPromotersequence
Expression
Environment NF-BPromotersequence
Expression
?
Transcription Element Listening System
Sp1
CREB
NF-B
Transcription Element Listening System
Sp1
CREB
NF-B
Transcription Element Listening System
Sp1
CREB
NF-B
Transcription Element Listening System
Sp1
CREB
NF-B
Transcription Element Listening System
Transcription Factor
P value
CREB/ATF Family P< 0.0001
AP-1 Family P<0.0001
NF-B/Rel Family P= 0.021
ELK-1 Family P=0.0011
Transcription Element Listening System
TELiS Results:
Environment:Hormones, pro-inflammatory cytokines, growth factors
Conclusions
•After sleep loss, ligation of TLR4 triggered greater monocyte IL-6 and TNF production
•Sleep loss induced increases in IL-6 and TNF gene expression
Conclusions
•Microarray analyses showed that sleep loss induced increased expression of early response genes, signal transduction mediators, and growth factor-related genes
•Bioinformatic analyses suggested transcriptional responses to sleep loss involve the NFKB inflammatory signaling system and classical hormone pathways (CREB/ATF)
Conclusions
•Loss of sleep during part of the night activates cellular and genomic markers of inflammation
•Testing of interventions that target sleep might identify new strategies to constrain inflammation and to promote health in aging
Bryant PA, Trinder J, Curtis N. Nat Rev Immunol. 2004;4(6):457-467.
Model of Sleep Immune Interactions
Model of Brain-immune Interactions
• Bi-directional interactions between the brain, autonomic, neuroendocrine, and immune systems
Tracey KJ. Nature. 2002;420:853-859.
Mullington J, Korth C, Hermann DM, et al. Am J Physiol Regul Integr Comp Physiol. 2000;278(4):R947-955.
0.2 mg/kg 0.4 mg/kg 0.8 mg/kg
Wa
ke
+ S
tag
e 1
(m
in)
No
n-R
EM
(m
in)
Infection and Cytokines Alter Sleep Behaviors
Does the Immune System Cause Changes in Sleep?
Irwin M, Rinetti G, Redwine L, Motivala S, Dang J, Ehlers C. Brain Behav Immun. 2004;18(4):349-360.
-0.25
5
3.25
1.5
-1.5 -0.5 0.5 1.5 2.5
Lo
g S
leep
Lat
ency
PS
D N
igh
t
Log IL-6 at 23:00
Elevated Awake, Pre-sleep Levels of IL-6 Correlate With Sleep Initiation
Difficulties
In conversation with M.R. Irwin, MD (May 2006).
The Immune System Causes Changes in Sleep
• Infectious challenge induces increased arousals and “light” sleep in humans
• Cytokines have a role in the regulation of sleep with effects dependent on the cytokine, cytokine dose, and time of administration
• Elevated levels of proinflammatory cytokines prior to sleep onset are associated with delays in sleep initiation
Question
Does sleep influence immune responses
with consequences for health?
• Bi-directional interactions between the brain, autonomic, neuroendocrine, and immune systems
Acknowledgements and Research Collaborators
Steve Cole, Ph.D.
Capella Campomayor
Alicici Collado-Hidalgo, Ph.D.
Minge Wang
NIAAA 13239NIDA 16541
T32-MH19925M01-RR00865
General Clinical Research Centers Program
Cousins Center for PNIUCLA NPI
Primary InsomniaSleep ApneaMajor DepressionAlcohol Dependence
Proinflammatory Cytokine Levels & Production
Sleep Disturbances
Natural & Cellular Immunity
Inflammatory & Cardiovascular Disorders
Infectious Disease Risk
Sympathovagal balance
Proinflammatory cytokine activity
Chronic insomnia
Irwin MR. (Unpublished data).
Model of Sleep Immune Interactions (cont’d)
Patients With Primary Insomnia Show Elevated Nocturnal Levels of
Norepinephrine
0
50
100
150
200
250
300
22:00 24:00 02:00 04:00 06:00
Time
Nor
ephi
neph
rine
(pg
/mL
)
Controls
Depressed
Insomniacs
Irwin MR, Clark C, Kennedy B, Christian Gillin J, Ziegler M. Brain Behav Immun. 2003;17(5):365-372.
Sleep Deprivation Induces Greater Increases in Heart Rate and Plasma Catecholamines in
Alcoholics
• Findings persist after recovery sleep
140
160
180
200
220
23:00 6:30 23:00 6:30 23:00 6:30
Time
Nor
epin
ephr
ine
(pg/
ml)
Alcoholics
Controls
Sleep Deprivation
**
Irwin MR, Ziegler M. Hypertension. 2005;45(2):252-257.
Sympathetic activity Proinflammatory cytokine levels
Sleep
Vagal tone Proinflammatory cytokine production? ?
Model of Sleep Immune Interactions (cont’d)
Irwin MR. (Unpublished data).
Control Alcoholic Patient
Valladares EM, Irwin MR. Sleep Med. 2006;7(1):79-80.
Low- and High-frequency Spectral Power in Alcoholic vs Control Subject: Representative
Findings
Low Vagal Tone Correlates With Poor Sleep Quality
Irwin MR, Valladares EM, Motivala S, Thayer JF, Ehlers CL. Psychosom Med. 2006;68:159-166.
14
10
8
6
4
2
0
12
16
500 1000 1500 2000 2500 30000
Controls
Alcoholics
High Frequency Power(Awake)
Sle
ep Q
ual
ity
(PS
QI –
To
tal S
core
)
Low Vagal Tone During Awake Before Sleep Correlates With Loss of Delta Power
0
5
10
15
20
25
0 5 10 15 20 25
High Frequency Power (Awake)
Del
ta P
ow
er (
All
Nig
ht)
Control
Alcohol Dependent
Irwin MR, Valladares EM, Motivala S, Thayer JF, Ehlers CL. Psychosom Med. 2006;68:159-166.