Sleep and Healthy Aging Research on Depression for Younger Women
Phase: PHASE1
Conditions: Anhedonia, Depression
Sponsor: University of California, Los Angeles

Official Title: Sleep and Healthy Aging Research on Depression for Younger Women 
 
 
NCT03848715; IRB#16-000583-AM-00016 
 
 
Date: 5/23/2019 
 
 
 
 
Study Officials:  
 
 
Chloe C Boyle, PhD 
Study Principal Investigator 
University of California, Los Angeles 
 
Michael R Irwin, MD 
Study Principal Investigator 
University of California, Los Angeles   
 
 
 

SPECIFIC AIMS 
Depression is a major public health burden that is associated with increased morbidity and mortality,1 
and current antidepressant therapies have limited effectiveness2. Thus, biologically plausible models that 
would translate into new interventions are being pursued. Inflammation is hypothesized to play a significant 
role in the pathophysiology of depression in at least a subset of individuals. Approximately 30% of patients 
treated with high doses of interferon (IFN)-α develop major depressive episodes,3 and when endotoxin is 
experimentally administered inducing robust systemic inflammation, there are substantial increases in 
depressive symptoms.4 However, not all individuals exposed to increased inflammation develop depression.5 
Thus, investigating who becomes depressed at exposure to heightened inflammatory states—e.g., aging, 
obesity, infections, IFN-α, or endotoxin—and why one becomes depressed in such states informs future 
studies of depression prevention regarding identification of at-risk individuals and actual interventions. In 
particular, risk for depression is nearly twice as high in females as in males from puberty through adulthood,6,7 
while this female preponderance and the potentially related psychobiological mechanisms change in old age. 
The overarching objective of this study is to evaluate affective mechanisms underlying the sex- and 
age-related differences in inflammation-induced depression. 
We have demonstrated in younger adults that low-dose endotoxin induces transient increases in 
depressive symptoms,8-12 which correlate with activation of brain regions implicated in depression10,11,13 and 
predict subsequent depressive symptoms over the following 2 years (Preliminary Data). Notably, depressive 
responses to endotoxin are far more accentuated in females than males, which suggests one potential 
mechanism underlying enhanced risk for depression in females.14,15 
A key unanswered question is whether female sex increases vulnerability to endotoxin-induced 
depression across the adult lifespan. The transition to late adulthood, particularly in women, is marked by a 
number of substantial psychobiological changes, including alterations in physiology (e.g., dopaminergic 
function19, major hormonal shifts) and psychological processes (e.g., coping strategies15). Most notably, late 
adulthood is characterized by both increased systemic inflammation20 and decreased depression prevalence16. 
Rates of depression in women drop precipitously after age 6517; one hypothesis is that this “aging paradox” 
may reflect age-related decreases in affective sensitivity to inflammation among women. However, no studies 
have directly compared effects of inflammation on affective processes in younger and older females.  
Indeed, the psychobiological mechanisms underlying any differential risk conferred by sex and age in 
the context of an acute inflammatory challenge have not been systematically interrogated. Depression is 
increasingly recognized as a heterogeneous disorder comprised of dysfunction in a variety of domains, 
including increased negative affective processes (e.g., dysphoria) and decreased positive affective processes 
(e.g., anhedonia). While there is some evidence for sex- and age-related shifts in the predominance of such 
alterations, the extent to which inflammation differentially alters these domains is unclear. Characterizing these 
processes may clarify the mechanisms that contribute to the disproportionate prevalence of depression in 
women likely across the adult lifespan, which will inform prevention and intervention efforts.   
The current study proposes to interrogate these mechanisms and how they differ as a function of sex 
and age. We will first leverage our ongoing experimental study examining endotoxin-induced depression in 
older adults (R01AG051944, PI Irwin) to test for sex differences in endotoxin-induced alterations in negative 
and positive affective processes among older adults. This study is expected to recruit 160 subjects aged 60-80 
years (80 females vs. 80 males). We next propose to recruit an additional 40 healthy premenopausal women 
(age 30-40) to participate in this supplemental study, which will allow us a complementary test of age as a 
vulnerability factor for endotoxin-induced depression and associated affective responding mechanisms in 
females. Specifically, in this randomized placebo-controlled experimental study, we aim to: 
1. Examine the effect of female sex on affective responses to endotoxin in older adults. We hypothesize 
that, compared to males, females will present with higher negative affective responses and lower positive 
affective responses to endotoxin. 
2. Examine the effect of age on affective responses to endotoxin in females. We hypothesize that, 
compared to older females, younger females will present with higher negative affective responses and lower 
positive affective responses to endotoxin. 
3. Examine the correlation between inflammatory response and affective response to endotoxin 
according to sex and age. We hypothesize that the correlations between circulating proinflammatory 
cytokines and affective responses following endotoxin administration will be stronger in older females than in 
older males and in younger females than in older females. 
Exploratory Aim: Examine the prospective associations between acute affective responses to endotoxin and 
long-term development of depressive disorders over 2 years, extending our preliminary findings. 

RESEARCH STRATEGY 
A. SIGNIFICANCE 
A.1. Female Sex: A Vulnerability Factor for Inflammation-Induced Depression? 
Compelling evidence suggests that inflammation plays a significant role in the pathophysiology of 
depression.16-18 Endotoxin is a model of systemic inflammation that can be used to experimentally interrogate 
the role of inflammation in inducing depressive symptoms. Endotoxin resembles a pathogen-induced, naturally 
occurring inflammatory immune response, which is driven by a complex interplay of various cytokines, all with 
distinct kinetics and locally differing concentrations.19,20 Injection of a single cytokine does not model this 
response, nor does acute laboratory-based stress, which does not specifically target the inflammatory cascade 
and only induces modest inflammatory and depressive responses that are less robust than endotoxin. 
Increasingly, depression is viewed along a continuum of affective responses that lead to symptom expression, 
as opposed to being a categorical, diagnostic construct,21,22 and endotoxin affects the two cardinal affective 
symptoms that constitute depression: depressed mood and anhedonia. Endotoxin induces depressed mood 
(Profile of Mood Scores23, POMS>3) and mild depression (Montgomery-Asberg Depression Rating Scale24, 
MADRS>7)25 along with increased activity in neural areas related to depressed mood and threat detection.11  
Similarly, evidence suggests endotoxin also increases anhedonia, the lack of interest or pleasure,25 and alters 
activity in reward-related brain regions like the ventral striatum (VS).10 Thus, endotoxin can be used to probe 
for changes in both negative and positive affective processes and their associated neural substrates.  
Despite the important contribution of inflammation to depression, not all individuals exposed to 
increased inflammation develop depression.5 Less than 30% of patients treated with high doses of IFN-α 
develop major depressive episodes3; and even when endotoxin is experimentally administered inducing robust 
systemic inflammation, largely variable increases in depressive symptoms are found.4 Indeed, we have shown 
that depressive responses to endotoxin are far more accentuated in females.14 In line with our experimental 
data, the female preponderance in the prevalence of depression is a well-established finding, indicating a 
higher depression risk in females than males.6,7 Moreover, women show higher levels of inflammatory 
markers,26 and there are well-known sex differences in the prevalence of inflammatory disorders, with females 
being between 2 and 9 times as likely as males to develop autoimmune disorders.27 
However, the female preponderance of depression prevalence decreases in the post-menopausal 
period,28 and females show a nearly 3-fold decrease in depression incidence from ages 20 to 60. Rates of 
depression in males, by contrast, remain stable.29 Likewise, levels of systemic inflammation are higher in 
females in males, but this sex gap decreases with age.26 Thus, it is unknown whether the vulnerability to 
inflammation-induced depression evident among younger women remains in later life. If this study shows older 
females to be more vulnerable to endotoxin-induced depression than older males, the finding will be significant 
as it would direct prioritization of female older adults for depression prevention. If this study shows no sex 
differences, the finding will also be significant as it would suggest age-related changes in sex differences and 
inform future studies on underlying inflammatory and affective mechanisms. 
An institute of medicine (IOM) report in 2001 stated: “Sex matters. Sex, that is, being male or female, is 
an important basic human variable that should be considered when designing and analyzing studies in all 
areas and at all levels of biomedical and health-related research … .The study of sex differences is evolving 
into a mature science. There is now sufficient knowledge… to allow the generation of hypotheses. The next 
step is to move from the descriptive to the experimental…”30 This supplemental study is significant by 
examining sex differences in inflammation-induced depression leveraging our highly controlled experimental 
study (R01AG051944, PI Irwin), and particularly because, while sex differences in depression and 
inflammation decrease with aging, it is unknown whether female sex remains a vulnerability factor for 
inflammation-induced depression in late life. Thus, this application addresses the following strategic goals of 
the new Trans-NIH Strategic Plan for the Health of Women "Advancing Science for the Health of Women": 1) 
To advance rigorous research that is relevant to the health of women; 2) To develop methods and leverage 
data sources to consider sex and gender influences that enhance research for the health of women. 
A.2. Using Age to Interrogate Sex Differences in Vulnerability to Inflammation-Induced Depression 
No studies have specifically tested for age differences in vulnerability to inflammation-induced 
depression. Our prior work has shown that young females (i.e., below the age of 30) are more affectively 
sensitive to the effects of inflammation than young males, but it is not known if this vulnerability persists into 
late adulthood. Because late adulthood is characterized by both increased systemic inflammation20 and 

decreased depression prevalence,16, 17 we hypothesize there may be potential age-related decreases in 
affective sensitivity to inflammation in older compared to younger females. Notably, major hormonal shifts (e.g., 
menopause) render older females more biologically similar to older males, which could help explain changes in 
affective sensitivity to inflammation across the lifespan. If results are consistent with this hypothesis, we will 
further characterize underlying affective mechanisms by testing sensitivity to both negative and positive 
affective processes in younger and older women. 
If, conversely, results are not consistent with our hypothesis, and sex differences in vulnerability to 
inflammation induced depression remain evident in late adulthood, we will have identified a potentially 
vulnerable subgroup (i.e., female older adults) and we will be poised to interrogate affective mechanisms that 
enable this vulnerability to persist. Furthermore, it is also possible that both older female and older male adults 
will evidence greater vulnerability to inflammation-induced depression than younger women. Regardless of 
sex, there are alterations in cognition and dopaminergic function (e.g., the dopaminergic vulnerability 
hypothesis)1931 that could confer increased sensitivity to inflammation and increased risk for inflammation-
induced depression among older versus younger adults. The current study includes assessment of cognition 
and dopaminergic activity. Thus, results from this study will facilitate identification of at-risk subgroups and 
potential mechanisms underlying these vulnerabilities.  
A.3. Negative and Positive Affective Mechanisms: A Dimensional Approach to Depression 
Clinical presentation of depression is enormously heterogeneous, composed of both negative and 
positive psychological processes that have distinct neurobiological substrates that can further dynamically 
interact with the environment over time. Consistent with the articulated goals of the National Institute of Mental 
Health (NIMH), the current study adopts a dimensional approach to the study of depression, focusing on 
dysregulation in fundamental processes, such as excess negative affect and a deficit in positive affect. To 
date, the role of negative processes in depression has received a great deal of attention, but there is some 
evidence that among older adults, presence of anhedonia, but not dysphoria, is associated with elevated risk 
for disability and death.32 Further, anhedonia predicts poor treatment response and is less successfully treated 
than other symptoms of depression, likely because therapies and pharmacology have traditionally addressed 
dysfunction in negative valence systems.33 Indeed,  dysregulation in the negative and positive valence systems 
likely benefit from different psychological and pharmacological treatment approaches.34 Thus, addressing 
disruption in both negative and positive valence  systems is critical and may have particular relevance for older 
adults. As detailed in the approach section below, we employ a vertically integrated assessment of 
dysregulation in the negative and positive valence systems, incorporating self-report questionnaires, observer 
ratings, and behavioral tasks.  
A.4. Prospective and Clinical Validity of Endotoxin Model of Depression 
Acute inflammatory reactivity predicts acute increases in depressive symptoms35,36 as well as increases 
in depressive symptoms over the following year.35 Our data also show that acute depressive reactivity (i.e., 
transient increases in depressed mood during 6 hours following endotoxin administration) predicts subsequent 
depressive symptoms over 2 years (Preliminary Data). However, the predictive ability of this experimental 
model—either inflammatory or depressive reactivity—for clinical depressive disorders has never been 
examined. Inflammation predicts the onset of depressive disorders.37 Affective mechanisms such as 
hyposensitivity to reward38,39 and negative bias in facial emotion recognition40 also predict future depressive 
disorders. However, such affective mechanisms potentiated by an inflammatory challenge have never been 
examined for depression risk prediction. In our preliminary study, only depressed mood enhanced by 
endotoxin, but not depressed mood observed after placebo infusion, was predictive of subsequent depressive 
symptoms (Preliminary Data). Similar to a cardiac stress test for coronary heart disease, inflammatory and 
affective reactivity to endotoxin stress may be able to uncover latent vulnerability to depression that cannot be 
identified by conventional assessments. If the predictive ability of the endotoxin depression model for clinical 
depression is confirmed in this 2-year longitudinal design, we will have further justification to test interventions 
that target such inflammatory and affective mechanisms in prevention trials. This experimental study followed 
by a 2-year prospective observation will be significant because of its potential to provide prior and future 
endotoxin studies of depression with empirical validation and clinical significance. Importantly, it also has the 
potential to provide further justification to test interventions that target inflammatory and affective mechanisms 
in large prevention trials of depression. 
A.5. Summary of Significance 
 
The proposed study is significant by providing experimental insight into the impact of age and sex on 

negative and positive affective mechanisms underlying vulnerability to inflammation-induced depression, which 
can inform the development of adjunctive psychological treatment components that target specific processes 
related to emotion processes (i.e., social rejection sensitivity, negative bias in facial emotion recognition, 
reward processing). Depression is a major public health burden across the lifespan, and effective screening 
methods and interventions for prevention are needed. Female sex may identify older adults at high depression 
risk to be prioritized for prevention efforts. Alternatively, aging may confer resilience among females; if so the 
current study assesses potential mechanisms that may underlie this resilience and could be leveraged at 
efforts for depression prevention and treatment. This study is also significant in using a 2-year longitudinal 
observation period to probe whether acute inflammatory and depressive responses as a function of sex and 
age predict depression risk over time. The findings will inform future trials to test pharmacologic and 
psychobehavioral interventions for depression prevention based on inflammatory and affective mechanisms. 
B. INNOVATION 
This research is highly innovative in using an experimental model of inflammation-induced depression 
to develop a framework for prevention of depression across the lifespan, integrating behavioral (i.e., reward, 
socio-emotional responses) and biologic (i.e., inflammation, inflammatory signaling, gene expression) 
processes to probe mechanisms that may underlie sex-differences in depression prevalence, focusing on how 
age may modulate affective sensitivity to inflammation. Furthermore, this study substantially advances our prior 
work for several reasons: 1) Older adults show higher rates of inflammation and depression, yet prior research 
using this experimental model of inflammation has been performed only in young- or middle-aged adults; 2) No 
studies have directly compared response to an inflammatory challenge in younger and older females, which 
will inform on sex differences in sensitivity to inflammation-induced depression; 3) Objective assessment of 
negative and positive affective responding informs the development of treatments that target these affective 
pathways, yet our prior research has relied exclusively on single item self-report measures and related neural 
substrates. 
B.1. Use of a highly controlled experimental model to understand depression risk mechanisms 
Low-dose endotoxin administration (0.8 ng/kg) is a highly innovative experimental model to understand 
the causal role of inflammation in the induction of depressive symptoms. Endotoxin induces increases in 
proinflammatory cytokines to levels similar to those found in an inflammatory disorder (e.g., rheumatoid 
arthritis) and chronic infections,41 and induces replicable increases in depressive symptoms.8-12,19,20,42 Other 
models such as interferon- treatment and typhoid vaccination have also been proposed. However, the effects 
of interferon- do not usually occur until 8-12 weeks of treatment,43 and this model cannot be double-blind nor 
applied to healthy volunteers. Administration of the typhoid vaccine induces only slight increases in IL-6, and is 
not robustly associated with induction of depressed mood.44,45 Laboratory stress (i.e., Trier Social Stress Task) 
can also activate inflammation, and such reactivity is associated with depressive symptoms 1 year later35; 
however, laboratory stress does not target the inflammatory cascade, and inflammatory responses are 
transient and less robust than endotoxin. Despite its advantages, endotoxin is a procedure that has only been 
implemented by a few laboratories in the world with only a small number of studies conducted to date 
(n=12).19,20 Furthermore, no research has been conducted in older adults.12 Moreover, no study has attempted 
to evaluate key clinical characteristics (i.e., sex and age) that might explain differential increases in depressed 
mood and anhedonic symptoms following inflammatory challenge.19,20,46 Here, we address these substantial 
gaps by evaluating differences in affective responding as a function of sex as well as age among females. 
B.2. Use of task based multi-dimensional methods to evaluate affective processes 
Negative and positive affect mechanisms have been examined in young adults, but there is a paucity of 
such research in older adults; and experimental or longitudinal studies evaluating such mechanisms using task 
based multi-dimensional methods in older adults are even more limited. Given that social disconnection plays a 
critical role in the onset and perpetuation of depression,36,47 we will examine distress following an experimental 
episode of social rejection, as a function of prior depression history and inflammatory challenge.  We will 
examine the impact of sex and age and inflammatory challenge on accurate judgment of facial emotions with 
implications for perceived threat (i.e., angry face) or reward value (i.e., happy face).48 The facial emotion 
recognition task is used to identify negatively biased processing of emotional faces and is associated with 
depression and risk of relapse in remitted patients.40,49 Finally, this study is novel by evaluating positive 
affective responses, using a series of behavioral reward tasks that assess reward reactivity, learning, and 
motivation.50-52 Understanding how sex and age influence affective responding, at baseline and in response to 
inflammatory challenge, has the potential to refine psychobehavioral interventions to prevent depression, and 

to inform the design of neuroimaging studies aimed at identifying the neural sensitivities related to 
inflammation. 
B.3. Use of systemic, cellular and genomic markers of inflammation: predictors of depression  
The vast majority of research that has examined the associations between inflammation and 
depression has assessed C-reactive protein (CRP) and circulating levels of interleukin (IL)-6 and tumor 
necrosis factor (TNF).53 We have pioneered the use of cellular and genomic methods to evaluate the upstream 
molecular pathways of inflammation: inflammatory gene expression,54 inflammatory transcription factor, 
nuclear factor (NF)-B,55 and signal transducer and activator of transcription (STAT) family proteins,56 which 
regulate IL-6 and the subsequent induction of CRP. We will implement these novel methodologies to 
understand the impact of endotoxin administration, in the context of female sex and age, on genes involved in 
inflammation, and the extent that these genes and related transcriptional pathways map onto increases in 
inflammation and depressive symptoms. Integrated analyses of inflammatory and affective responding will 
inform targets of future translational studies. 
C. APPROACH 
C.1. Preliminary Data  
C.1.1. Inflammation induces increases in depressive 
symptoms. 
Using an experimental inflammatory challenge (i.e., 
endotoxin vs. placebo), increases in proinflammatory 
cytokines are associated with increases in depressed mood 
and anhedonia.8-12 
C.1.1.1 Inflammatory challenge induces increases 
in depressed mood, especially in females.14  Aim: To 
evaluate the effects of endotoxin on depressed mood and 
sex differences in depressive responses to endotoxin. 
Methods: In a randomized, double-blind, placebo-controlled 
design, 115 participants (69 females, mean age 24.1 ± 6.3 
years) received either low-dose endotoxin (0.8 ng/kg of body weight) or placebo (same volume of 0.9% saline). 
Blood sampling for IL-6 and TNF was obtained approximately every hour post-injection for the next six hours 
(T1-T6), along with assessment of social disconnection and depressed mood using the POMS.  Results: 
Endotoxin (vs. placebo) led to significant increases in IL-6 (P<0.001) and TNF levels (P<0.001). Endotoxin (vs. 
placebo) also led to significantly greater increases in depressed mood (P<0.001), and these effects did not 
change when controlling for sickness symptoms (P<0.05) (Fig. 1). Additional analyses found that endotoxin 
induced increases in IL-6 and TNF which were similar between the two sexes, but suggested that women 
showed significantly greater increases in depressed mood than men in response to endotoxin (P<0.05). Similar 
results were found for feelings of social disconnection.  Conclusion: Endotoxin induces increases in depressed 
mood, which are not due to sickness symptoms (i.e., headaches, muscle pain, shivering, nausea, breathing 
difficulties, and fatigue). Additional analyses suggested that women are more sensitive to an inflammatory 
challenge and show greater increases in depressed mood and social disconnection as compared to men. 
However, these analyses were limited to younger adults. Given the different hormonal milieu of younger vs. 
older women, and the regulation of inflammation by endocrine hormones, this study will evaluate inflammatory 
and affective responses in older adults stratified by sex and examine whether there are sex differences in 
inflammatory and affective responses. 
C.1.1.2. Increases in inflammation correlate with depressive symptoms following endotoxin.  
Aim:  To evaluate whether increases in inflammation account for increases in depressed mood following an 
inflammatory challenge.  Methods: See C.1.3.1.  Results:  There were significant correlations between 
changes in IL-6 or TNF and social disconnection (r = 0.27, P<0.05; r = 0.37, P<0 0.01), and between changes 
in IL-6 or TNF and depressed mood (r = 0.25, P<0.08; r = 0.30, P<0.05). Additional analyses suggested that 
women showed more robust correlations than men.  Conclusion: Levels of inflammation following endotoxin 
are associated with social affective responses, and these relationships appear to be stronger in younger 
women than men despite similar increases in inflammation in the two groups. This study will examine there are 
sex differences in affective sensitivity to inflammatory challenge in older adults. 
Figure 1. Changes in depressed mood over 6 hours 
following endotoxin administration (vs. placebo) 

C.1.1.3. Endotoxin safely induces increases in depressed mood in older females.  Aim: To 
evaluate the safety and feasibility of endotoxin administration in older females.  Methods: In our ongoing study 
(K23AG049085, PI Cho), healthy older females aged 60-70 years (N=35) were entered into the previously 
described endotoxin protocol, which included hourly assessments of depressed mood using the POMS.  
Results: None of the subjects had significant adverse events. Similar to prior results in younger adults, 
endotoxin was found to induce a significant increase in depressed mood (P=0.017; effect size 0.51), with peak 
increases in depressed mood at 2 hours; this timepoint of maximal increase is identical to our prior results 
(C.1.1.1).  Conclusion: These data show that endotoxin can be safely administered to older females and 
induces increases in depressed mood with a similar time course of response. 
C.1.2. Endotoxin-induced depressed mood predicts 
subsequent depressive symptoms over 2 years.  Aim: To 
examine whether transient increases in depressed mood during 6 
hours following endotoxin administration predict subsequent 
depressive symptoms over a 2-year follow-up.  Methods: In our 
ongoing study (K23AG049085, PI Cho), healthy older females 
aged 60-70 years who completed the above-described endotoxin 
protocol (N=22) were followed up for 21 months with an 
assessment of depressive symptoms every 3 months using the 
telephone-administered 9-item Patient Health Questionnaire 
(PHQ-9).  Results: Endotoxin and placebo groups did not differ in 
subsequent depressive symptoms over 21 months (P=0.37), 
indicating that the intervention itself did not affect subsequent 
depressive symptoms (Fig. 2). In the endotoxin group, depressed 
mood assessed during 6 hours of experimental session at 
baseline strongly correlated with subsequent depressive 
symptoms over 21 months (r = 0.71, P<0.0001) (Fig. 3). However, 
in the placebo group, depressed mood assessed during 6 hours of 
experimental session did not correlate with subsequent 
depressive symptoms (r = 0.07, P=0.74).  Conclusion: Transient 
increases in depressed mood following endotoxin administration 
strongly predict subsequent depressive symptoms over 2 years. 
C.1.3. Increases in inflammation following influenza vaccine 
are associated with changes in reward processing.  Aim: The 
aim of this observational study was to assess whether increases 
in the proinflammatory cytokine IL-6 would increase pre- to post-
influenza vaccine, and whether these changes would correlate 
with changes in performance on reward tasks assessing 
motivation (monetary reward), learning (monetary reward) and sensitivity (monetary and general social 
reward).  Methods: 41 healthy UCLA undergraduate students participated in this pre-post within-subjects study. 
Participants completed baseline assessment of reward tasks, a week of daily diary assessment, and were then 
scheduled to receive the annual influenza vaccine. Participants provided blood samples prior and 24-29 hours 
after the vaccine. Participants completed behavioral reward tasks 24-29 hours after the vaccine; this 24-29 
hour period was based on prior research showing an IL-6 peak one day post-influenza vaccine.  Results: 
Levels of IL-6 increased significantly from pre-to post-vaccine, but performance on the reward tasks did not 
significantly differ from pre- to post-vaccine. However, as hypothesized, increases in IL-6 were correlated with 
decreases in reward motivation for monetary reward, and decreases in attentional bias to positive faces (an 
index of sensitivity to general social reward). Changes in sensitivity to monetary reward, as operationalized 
through parameters in the reward motivation task, were not associated with changes in IL-6.  Increases in IL-6 
correlated with increases in reward responsiveness on a standardized learning task, although sample size for 
computational analyses was not sufficient to identify whether this was driven by increases in learning or 
sensitivity.  Conclusion: Consistent with hypotheses, mild increases in IL-6 following influenza vaccination 
correlated with decreases in motivation for monetary reward, and decreases in sensitivity to general social 
stimuli. Contrary to hypotheses, increases in IL-6 were not correlated with change in sensitivity for monetary 
reward and were associated with increases in reward responsiveness on a reward learning task. These data 
suggest an association between the proinflammatory cytokine IL-6 and performance on reward tasks in 
multiple reward dimensions for both non-social and general social reward. 
Figure 2. Changes in depressive symptoms over 
2 years after endotoxin protocol (vs. placebo) 
Figure 3. Correlations between endotoxin-induced 
depressed mood at baseline and subsequent 
depressive symptoms over 2 years (vs. placebo) 

C.1.3. Summary of Preliminary Data 
 
Inflammatory challenge induces depressed mood, especially in females; and increases in depressed 
mood correlate with increases in circulating markers of inflammation. Importantly, preliminary data suggest that 
older adults show similar increases in depressed mood in response to endotoxin as found in younger adult 
samples. Transient increases in depressed mood following endotoxin administration strongly predict 
subsequent depressive symptoms over 2 years. Prediction of clinical depression should be examined in a 
larger sample with appropriate diagnostic assessments in order to validate the endotoxin study procedures as 
a clinically relevant experimental model of depression. 
C.2. RESEARCH DESIGN AND METHODS 
C.2.1. Summary of the parent project 
 
“Depression, one of the most common diseases in older adults, carries significant risk for morbidity and 
mortality. Because of the burgeoning population of older adults, the enormous burden of late-life depression, 
and the limited efficacy of current antidepressants in older adults, biologically plausible models that translate 
into depression prevention efforts are needed. Insomnia predicts depression recurrence, and is a modifiable 
target for depression prevention. Yet, it is not known how insomnia gets converted into biological- and affective 
risk for depression, which is critical for identification of molecular targets for pharmacologic interventions, and 
for refinement of insomnia treatments that target affective responding to improve efficacy. This study will use 
an inflammatory challenge (i.e., endotoxin) to probe acute inflammatory- and depression responses (primary 
outcome) in older adults as a function of insomnia. Older adults with insomnia show chronic inflammation; 
sleep disturbance also activates inflammatory signaling; chronic inflammation primes acute inflammatory 
responses; chronic inflammation, as well as acute inflammatory reactivity, predict depression over the following 
year; and finally, endotoxin induces acute inflammation along with depressive symptoms, with preliminary 
evidence that “two-hits” (i.e., sleep disturbance and inflammatory challenge) are associated with exaggerated 
increases in depression, especially in women. In this placebo-controlled, randomized, double-blind study of low 
dose endotoxin in older adults (60-80 y; stratified by sex) with insomnia (n=80) vs. comparisons without 
insomnia (n=80), we hypothesize that older adults with insomnia will show heightened inflammatory- and 
affective responding to inflammatory challenge as compared to those without insomnia. We aim to: 1) examine 
differences in depressive symptoms and measures of negative affect responding as a function of insomnia and 
inflammatory challenge; 2) examine differences in measures of positive affect responding as a function of 
insomnia and inflammatory challenge; and 3) examine differences in experimentally-induced inflammation in 
relation to depressive symptoms and measures of negative- and positive affect responding as a function of 
insomnia. If the hypotheses are confirmed, older adults with two “hits”, insomnia and inflammation, would 
represent a high risk group to be prioritized for monitoring and for depression prevention efforts using 
treatments that target insomnia or inflammation. Moreover, this study will inform the development of 
mechanism-based treatments that target affect responses in addition to sleep behaviors, and which might also 
be coupled with efforts to reduce inflammation to optimize efficacy of depression prevention.” 
C.2.2. Overview of study design 
To address Aim 1, examining sex differences in affective sensitivity to inflammation among older adults, 
we will select a total of 80 healthy older adults aged 60-80 years (40 males and 40 females), who are absent of 
current psychiatric illnesses, insomnia, and medical conditions (e.g., inflammatory disorders), into a placebo-
controlled, randomized, double-blind, experimental inflammatory challenge protocol. Participants will be 
randomly assigned to receive either endotoxin (n=40) or placebo (n=40) in a between-subjects manner, 
stratified by sex. To address Aim 2, examining age differences in affective sensitivity to inflammation among 
females, we will recruit an additional 40 healthy premenopausal adult women (age 30-40) to be randomly 
assigned to receive endotoxin (n=20) or placebo (n=20). 
For all participants, administration of endotoxin (0.8 ng/kg body weight vs. placebo), will be followed by 
self-report measures of depressed mood, negative and positive affect, and sickness symptoms (e.g., fatigue) 
assessed hourly over a 12-hour period; objective ratings of depressive symptoms by an observer blind to 
condition will be made every 2 hours. Behavioral tasks will be administered to coincide with peak onset of 
depressed mood, which we have previously shown to occur at 2 hours in both older and younger adults. Our 
prior protocol duration was 6 hours, but the current proposal will test for the presence of delayed termination of 
response57,58 and extend protocol duration to 12 hours. At baseline and starting two hours post-injection, at 
peak cytokine- and depressed mood responses, participants will complete the Probabilistic Reward Learning 
task50-52, the Effort Expenditure for Reward Task,59 the Social Choice task (i.e., intersection of reward 

motivation and sensitivity for social reward),60,61 the Emotional Face Recognition task,62 and assessment of 
cognitive function.63 At two hours post-injection only, participants will complete the Cyberball Social Exclusion 
task (i.e., self-reported sensitivity to social rejection).11,64 Proinflammatory cytokines levels will be repeatedly 
assessed through hourly blood draws, and measures of activation of the inflammatory transcription factor (NF-
B) and inflammatory gene expression, as well as assessment of dopaminergic activity, will be obtained during 
the first half of the protocol prior to and immediately after peak cytokine responses. A between-subject parallel 
design will be used for the following reasons: 1) the proposed study involves endotoxin administration, and in a 
hypothetical crossover design, subjects who experience aversive sickness symptoms from endotoxin (possibly 
also from placebo due to expectation) might drop out, which would introduce bias and invalidate the results; 2) 
the effect of endotoxin on inflammation might not resolve between sessions; 3) repeated exposure to 
behavioral tests of social exclusion might alter responses; 4) two sessions have high subject burden; 5) distinct 
endotoxin and placebo groups are required for the 2-year follow-up. 
Because the administration of endotoxin in this experimental protocol is designed to mimic the 
increases in plasma cytokines reported in chronic low-grade inflammatory conditions (i.e., 2-10 fold increases 
in IL-6 and TNF), we will use low-dose endotoxin (0.8 ng/kg) similar to what we have used in our prior 
studies.9,11,12,42,65 An endotoxin dose of 0.8 ng/kg yields significant increases in proinflammatory cytokines, 
increases in depressed mood and anhedonia.9,11,12,42,65 Our preliminary data indicate that older adults show 
similar response to endotoxin without adverse events. We have extensive experience with all aspects of the 
endotoxin vs. placebo inflammatory challenge, and have administered endotoxin dose 0.8 ng/kg in over 170 
participants with no adverse events, including older adults (20-70 years). We have also not observed any 
clinically significant fever or hyper- or hypotension at the 0.8 ng/kg dose. Of note, we have elected to use this 
low-dose rather than higher endotoxin dose (2.0 ng/kg; results in a 2.5-fold increase in proinflammatory 
cytokines) because the higher dose has been shown to induce greater fever responses, and hypotension in 
elderly subjects (60-69 years) as compared to young adults (20-27 years).106,107  
C.2.3. Subjects 
As of January 2019, a total of 74 participants have completed all the study procedures in the parent 
project, which is expected to complete the recruitment of 160 participants by July 2020. Thus, the parent 
project will include 40 insomniac older females, 40 non-insomniac older females, 40 insomniac older males, 
and 40 non-insomniac older males. For this supplemental study, we will select 40 non-insomniac older females 
and 40 non-insomniac older males to examine sex differences (Aim 1). We will use the basic demographic 
information in the sample of 40 healthy older females to guide recruitment of the younger female sample (Aim 
2). Specifically, we will ensure that the samples are comparable in terms of BMI, years of education, and 
ethnicity. As with the parent project, compensation for completion of all study components will be $1,000.  
Inclusion Criteria: Participants will be healthy older adults aged 60-80 years. For the younger sample, 
inclusion criteria will be healthy, biologically female adults between the ages of 30 and 40. By using this age 
range we will focus on women prior to the perimenopause or menopause transition who are also at high risk 
(median age of onset for mood disorders is 30; of note most studies using endotoxin have a mean age of 
participants well below age 30).66,67 
Exclusion Criteria: Participants will be excluded with a current medical or psychiatric disorder or who 
report current use of medications such as antidepressants and anti-inflammatory drugs, consistent with prior 
endotoxin protocols involving elderly subjects.41,58,68 For the younger sample, males will be excluded for the 
following reasons: feasibility, reduction of variability and subsequent enhanced power, and to build upon our 
prior observation of accentuated affective sensitivity to inflammation among women. Other exclusion criteria 
include pregnancy or planning to become pregnant; presence of chronic mental or physical illness, history of 
allergies, current and regular use of prescription medications, and nightshift work or time zone shifts (>3 hours) 
within the previous 6 weeks, or previous history of fainting during blood draws. See Protection of Human 
Subjects section for full details. 
C.2.4. Experimental Procedures 
Recruitment and Enrollment:  Recruitment for the younger female sample will use existing infrastructure 
in place for the parent project, including identification of potential participants through the GENESYS Sampling 
System (Fort Washington, PA), which records telephone numbers and mailing addresses of households across 
the United States. This will allow us to select for households with at least one female person aged 30-40 years 
and living in the greater Los Angeles area; age information is based on known age-related data or a statistical 
estimate of age, predicted using individual household characteristics and Census demographic information. 

Research staff are in place to send out recruitment letters and respond to inquiries by phone. Interested 
individuals will go through a two-step screening and assessment procedure to ensure that they are eligible for 
participation in the study. The initial screening process will be completed by phone; eligible and interested 
participants will then be scheduled for an in-person morning screening and assessment session (Visit 1).  
Baseline Visit 1:  Participants will be scheduled for this baseline session near the last day of their 
menstrual cycle to minimize variability due to hormonal status. They will have their height and weight 
measured, a urine sample to check for substance use, and an EKG to check for signs of heart disease. They 
will complete the Structured Clinical Interview for DSM-5 Disorders (SCID-5) with trained research staff. After 
this, participants will complete baseline assessment of several behavioral tasks, including tasks assessing 
cognition, reward, and emotion processing. These tasks will be administered between the hours of 10am and 
2pm to match the time of administration during the experimental endotoxin vs. placebo session.  
Experimental study session - Endotoxin vs. placebo administration:  Similar to our previous research,8-
12 the experimental session will be conducted at the UCLA Clinical Translational Research Center (CTRC) 
using a randomized, double-blind, placebo controlled design. Before each session beginning at 8am, a CTRC 
nurse blind to the randomization schedule will assess height and weight as well as vital signs (blood pressure, 
pulse, temperature). Participants will be excluded if: (a) blood pressure less than 90/60 or greater than 
160/120, (b) pulse less than 50 beats/minute, or (c) temperature greater than 99.5°F. An indwelling venous 
catheter with a heparin lock will be inserted into the participant’s dominant forearm (right) for hourly blood 
draws and one into the non-dominant forearm (left) for a continuous saline flush (150 cc/h) for endotoxin vs. 
placebo administration. Baseline blood samples, self-report questionnaires, and experimental affective 
response tasks will then be completed. CTRC pharmacy will maintain the randomization assignment and 
prepare the endotoxin vs. placebo. After 90 minutes, participants will randomly receive either low-dose 
endotoxin (0.8 ng/kg of body weight) or placebo as an intravenous bolus over 30-60 seconds. NIH will provide 
reference endotoxin humans (E. coli group O:113).68 Previous research has demonstrated the safe use of this 
reference endotoxin across many different samples41,68 57,58. Throughout the study protocol lasting 12 hours, 
vital signs and blood sampling will be obtained every half hour for the first two hours, and then hourly. 
Placement of an intravenous catheter for this duration does not induce nonspecific increases in circulating 
levels of IL-6.9 Human Subjects section details safety monitoring. Although we are extending the protocol for 
12 hours to accommodate a possible delayed termination of response in the older adult participants,57,58 
subject burden precludes in person follow-up the next day; however, participants will receive a safety follow-up 
phone call 1 and 7 days after the CTRC session. 
C.2.5. Baseline Measures: Prior to the Experimental Protocol 
Baseline assessment procedures (prior to experimental protocol):  In a face-to-face interview, the 
following domains will be assessed: psychiatric history, insomnia, negative and positive valence, biobehavioral 
and medical factors (i.e., demographic; medical/medication histories; health status) and psychosocial stress 
and social support. The SCID-5 interview will determine the presence of past history of depression, and the 
absence of current depressive disorder and other psychiatric diagnoses. We have over two decades of 
experience in the administration of the SCID, train to criterion validity, obtain diagnoses in a weekly consensus 
meeting to maintain reliability and criterion validity, and use videotaped interviews for quarterly monitoring of 
validity/reliability of interviewers. Depressive/anxiety symptoms will be assessed using Beck Depression 
Inventory (BDI-II), Beck Anxiety Inventory (BAI),69,70 and Inventory of Depressive Symptomatology.71 
Negative and positive affect valence:  Affect valence characteristics might be related to variable affect 
response following inflammatory challenge, and will be assessed by self-report questionnaires including the 
Behavioral Inhibition and Activation Systems (BIS/BAS) Scale72 (i.e., punishment sensitivity; drive, fun seeking, 
reward responsiveness); Temporal Experience of Pleasure Scale 73 (i.e., anticipatory, consummatory 
pleasure); and Response Style Questionnaire Ruminative Responses Scale74 (i.e., tendency to ruminate in 
response to symptoms of negative emotion). 
Biobehavioral and medical factors will be assessed that might be associated with inflammation, 
depression risk, or both, including demographic information; biobehavioral confounds of inflammation 75 such 
as alcohol use, smoking, BMI, and physical activity (i.e., Godin Leisure-Time Exercise Questionnaire76); and 
medical factors (i.e., Charlson Co-Morbidity Index, Chronic Disease Score77) and health functioning (i.e., 
Medical Outcomes Study Short-form [SF-36]78-80). We also assess menstrual history and status.  
Psychosocial factors will be assessed including number and perceived threat of recent life stresses 
(i.e., Perceived Stress Scale81) and subjective social isolation that is associated with depression, sleep 

disturbance and inflammation (i.e., 10-item Revised UCLA Loneliness Scale,82 Interpersonal Support 
Evaluation List,83 short form Social Support Questionnaire [SSQ]84). 
C.2.6. Experimental Measures 
Negative Affect Responding 
 
Self-report and observer based measures of depressed mood: Depressed mood, a core symptom of 
depression, will be assessed hourly using the Profile of Mood States (POMS).23 The Depression Adjective 
Check List (DACL) will also be administered hourly as the DACL, used in another endotoxin study,65 correlating 
well with the Beck Depression Inventory (BDI). We will also use the Montgomery-Asberg Depression Rating 
Scale (MADRS)24 as it has been found to be sensitive to acute changes in depressed mood and anhedonia 
following endotoxin.19 Because feelings of social disconnection and loneliness predict depression and co-occur 
with depressed mood following inflammatory challenge, feelings of social disconnection (Feelings of Social 
Disconnection Scale9) and loneliness (adapted UCLA Loneliness Scale82) will also be assessed. To 
complement prior research that perceived social support is a risk factor for cytokine-induced depression, 
participants will complete the short form of the SSQ84 at baseline and 2-hours post injection. Finally, an 
interviewer, blind to the experimental condition, will make ratings of items of depressed mood, feelings of guilt, 
loss of interest, retardation/agitation, anxiety, and somatic symptoms using items from the Hamilton 
Depression Rating Scale,85 one of the most widely used scales in depression research, which will be adapted 
to evaluate acute changes in depressive symptoms. 
 
Experimental task: subjective sensitivity to social rejection (Cyberball Social Exclusion Task)  Socio-
emotional mechanisms of depression will be examined in this study as they are particularly significant to older 
adults, who often experience objective and subjective social isolation, which is associated with depression 
risk.86 Feelings of ‘social disconnection’ play a critical role in the onset and perpetuation of (non-inflammatory 
forms of) depression.87 Inflammatory processes can trigger social withdrawal88 and increase feelings of 
distress and related neural sensitivity (i.e., activation of the dorsal anterior cingulate cortex).9 Using the 
Cyberball Social Exclusion Task,64 we will examine feelings of social distress. Briefly, participants will be told 
that they will play a virtual ball-tossing game with two other players over the Internet. In reality, they will play 
with a preset computer program. In the first round (inclusion), participants will play with the two other players 
for the entire period. In the second round (exclusion), participants will receive the ball for seven throws and 
then will be excluded for the rest of the round when the two players will stop throwing the ball to the participant. 
Following this task, they will complete a self-report measure of social distress in response to social exclusion.89 
 
Experimental task: negative bias to emotional face recognition (Emotion Recognition Task)  Depressed 
subjects have a negative bias in the perception of emotions expressed in faces as they tend to interpret neutral 
faces as sad and happy faces as neutral.49 Furthermore, such a bias appears to play a role in the etiology and 
maintenance of depression as it predicts relapse in currently depressed and remitted patients.40 Emotional 
Face Recognition Task62 consists in showing participants a series of black and white photographs (Ekman 
Pictures of facial affect), in which facial expression is morphed from Neutral to either Sad, Angry, or Fearful.90 
For each image, they will be asked to make a forced choice about the emotion expressed, and rate their 
certainty on a 0-9 scale. Earlier recognition of a Sad face with certainty would be indicative of negative bias. 
Positive Affect Responding 
 
Self-report measures of positive mood. Participants will self-report positive mood each hour using items 
from the Profile of Mood States (POMS), and items that assess anticipatory (i.e., future oriented) and 
consummatory (i.e., present oriented) pleasure and interest in activities and social interaction (e.g., the Snaith 
Hamilton Pleasure Scale; the Affective Forecasting Questionnaire).91 
Experimental task: intersection of reward motivation and sensitivity for social and non-social reward 
(Social Choice Task and cartoon task). A social choice61 and semi-structured social interaction60 task will be 
used to assess motivation and sensitivity for social reward. The social choice task will be administered at 
baseline (visit 1) and during the experimental session (2 hour post-injection). Participants will be asked to rate 
their desire to engage in three 10-minute activities on a 1-10 Likert scale. These include a social activity 
(talking with another person) and two solitary activities (solving word problems, sitting quietly). Participants are 
told that this preference along with an element of chance will determine which they actually do. At the baseline 
visit, a neutral option will always be chosen. During the experimental session, the social interaction will always 
be selected. For this semi-structured social interaction task, participants will be asked to spend 5 minutes 
talking about an important person in their life to a research assistant trained in reflective listening. The 

discussion will be recorded and later transcribed and scored for percentage of positive and negative emotional 
words using Linguistic Inquiry and Word Count Software. Perceptions of the RA will also be assessed (e.g., 
“S/he was truly interested in me”). A “Cartoon Effort Task”41 that has been used in samples with depressed 
adults as well as healthy controls, will be used to assess the degree to which reward motivation and sensitivity 
coincide. Past work suggests dissociation is a feature of depression. The task uses humorous and 
nonhumorous single-panel cartoons as reward and non-reward stimuli. Motivation is the amount of effort 
participants are willing to exert to view funny vs non-funny cartoon. Participants also rate their enjoyment when 
viewing funny cartoons. The task is 20-30 minutes long, and has been piloted in our lab previously.    
Experimental tasks: reward learning (Probabilistic Reward Task, PRT; Probabilistic Selection Task), motivation 
(Effort Expenditure for Rewards Task (EEfRT), and sensitivity (Emotional Dot Probe, Face Morphing Task) risk 
(BART).  Anhedonia is a core feature of major depressive disorder and includes a reduction in experienced 
pleasure (liking reward), motivation (wanting reward) and reward learning.50-52 We have found that 
inflammatory challenge reduces neural sensitivity to reward anticipation10, and endotoxin has also been shown 
to alter reward motivation.92,93 We will use a series of laboratory based tasks to probe these dimensions of 
reward. To assess reward learning, we will use the PRT, which objectively measures participants’ ability to 
modulate behavior as a function of reward, because this task identifies reduced reward learning in depressed 
patients which is state-dependent and also predicts the persistent diagnosis of depression in the midst of 
treatment.50-52 This computerized reward-learning task is extensively described in Pizzagalli et al.94 The 
probabilistic selection task is a 15-minute trial and error (procedural) implicit learning task that assesses 
reinforcement learning for both positive and negative monetary cues; this task has also been used by the 
Pizzagalli group and was developed by Michael Frank (Science, 2004). Prior work suggests that dopaminergic 
function is associated with learning from both types of cues via different neurobiological mechanisms. Lower 
dopamine levels are associated with decreased sensitivity to positive reinforcement, but increased sensitivity to 
negative reinforcement. A similar trend is expected following the inflammatory stimulus.  
To assess reward motivation, we will employ the EEfRT,59 an established task in the depression 
literature that assesses motivation to work for monetary reward at varying degrees of probability of winning and 
varying degrees of potential reward magnitude. Reduction in reward sensitivity/liking will be assessed with two 
common emotion processing tasks, the emotional dot probe and face morphing task,95,96 which assess 
sensitivity to positive social reward cues; specifically, reduced sensitivity to rewarding social stimuli is indicated 
by slower identification of happy emotional faces or less attentional vigilance for happy emotional faces.  The 
Balloon Analogue Risk task (BART) is used to assess reward sensitivity and risk. In this brief task, participants 
view a computer screen with three items: a balloon with a button labeled Click here to pump, button labeled 
Collect $$$, and a box where total earnings were tallied in every trial. The goal during the BART is to earn as 
many dollar points as possible. Participants are shown a mock list of high scores to provide a frame of 
reference for their performance, but no monetary reward was offered. Every time the subject clicked on the 
“pump” button, the balloon increased slightly in size. When the Collect $$$ button was pressed, the total 
earnings display added 5 cents for the current balloon. Each balloon in the 30 trials was set to explode at a 
random pump. If a balloon was pumped past its individual explosion point, a “pop” sound effect played and the 
participant did not earn any money for that balloon. At any point during a trial, participants could cash out by 
clicking the Collect $$$ button and their earnings would be updated while a slot machine “payoff” sound 
emphasized the payment. The number of pumps before an explosion occurred ranged from 1–128. For every 
balloon, the first pump had 1/128 probability of exploding and a potential gain of 100% (i.e., from 5 cents to 10 
cents), the second pump had a 1/127 probability of exploding and a potential gain of 50% (i.e., from 10 cents to 
15 cents), and so on until the 128th pump which carried a 1/1 probability of exploding and a potential gain of 
0%. Thus, with each additional pump on a particular balloon the risk of losing increased and the relative gain 
decreased. In this way, some risk taking was necessary to make gains but excessive risk was associated with 
diminishing returns.  
 
Dopaminergic Function. Given that inflammation can influence dopaminergic function, which underlies 
reward motivation and learning, this proposal includes an assessment of resting state eye blink rate (EBR). 
EBR is used as an indicator of striatal dopaminergic (D2 receptor) activity and correlates with performance on 
reward motivation and learning tasks. Alterations in dopaminergic function activity have been proposed to play 
a key role in mediating effects of inflammation on reward motivation, and EBR has been shown to decrease 
with greater age. However, EBR has not been assessed following endotoxin administration. With the addition 
of this brief and simple measure, we will test for changes in central dopaminergic activity following endotoxin, 
and examine whether these changes are correlated with reward task performance among women in early and 
late adulthood. EBR will be assessed prior to infusion, and 1.5, 3, 4.5, and 6 hours post-infusion. Eye blinks will 

be video recorded for a 5-minute period. The participant will be asked to sit quietly, look ahead, and refrain 
from visually fixating on any objects. The video will be coded by two research assistants (blinded to condition).   
Daily Diary Assessment 
 
Daily diaries will be completed for 7 days prior to the experimental session and 7 days after the 
session. Participants will receive prompts by text or email that will direct them to the survey site at 6 random 
points during the day (range of time between prompts = 30 to 180 minutes). Participants will have 1 hour to 
respond to a single prompt; this is to ensure that participants are given sufficient time to complete an 
assessment during a working environment (e.g., business meetings) or family obligations (e.g., driving children 
to school). Data from a second or third submission in the same 30-minute window will be discarded. 
Completing each prompt will take 2 minutes. Participants first indicate the extent to which they enjoyed 10 
types of activities since the last prompt (or since waking) on a 0-100 visual analogue scale. From the same list 
of activities, participants then rate how much they are currently looking forward to each activity. For each 
activity, they rate their motivation (hedonic, eudaimonic, or not applicable if their “wanting” is sufficiently low). 
Each evening, participants will complete one additional prompt assessing positive and negative mood, daily 
rumination and sleep quality (estimated time: 2 minutes). 
 
Cognition and Executive Function. Behavioral tasks will be administered by computer to assess key 
aspects of executive function at baseline (Visit 1) and 3-hours post-injection during the experimental session.63 
The Spatial 2-Back task is used to assess Updating, the ability to monitor and replace information in working 
memory. The Color-Shape task is used to assess Shifting, or the ability to switch between mental sets. The 
Antisaccade task is used to assess Inhibition. Participants are tasked with inhibiting a reflexive response 
towards a visual cue in order to correctly identify a target stimulus presented elsewhere.  
Emotion Regulation. Participants will complete questionnaires assessing dispositional and situational 
emotion regulation strategies and attitudes towards emotions. Participants will also complete a 20-30 minute 
standardized emotion regulation task44. The task includes two phases: a reactivity phase and a regulation 
phase, and assesses the ability to down-regulate negative emotional response to negative images and/or film 
clips using reappraisal strategies. Participants receive instructions as to how to reappraise (e.g., thinking about 
the “silver lining” or imagining oneself at a distance from the negative emotion). The dependent variable is the 
degree to which self-reported emotion changes when reacting to versus reappraising negative stimuli.  The 
task also includes assessment of the ability to up-regulate positive emotion using cognitive strategies (e.g., 
thinking about a positive image/film clip in such a way that more positive emotion is felt). The stimuli used in 
the emotion regulation task are drawn from standardized databases and have been used in many previous 
studies, including studies conducted in our group with younger breast cancer survivors. 
Physical Sickness Symptoms (i.e., muscle pain, shivering, nausea, breathing, difficulties, and fatigue) 
will be assessed hourly by self-report, and controlled in analyses to ensure that the social or affective 
consequences of inflammatory challenge are not due simply to individuals feeling more ‘sick.’ 
 
Inflammatory Cytokine, Signaling Pathway, Gene Expression Analysis: Blood samples will be 
collected at baseline, every half hour for the first two hours, and hourly for the remainder of the 12 h. Samples 
will be immediately processed and stored at -800C. Plasma samples (all timepoints) will be assayed for pro- 
and anti-inflammatory cytokines (IL-1, IL-6, IL-8, TNF, and IL-10) by means of a high sensitivity bead-based 
multiplex immunoassay (Performance High Sensitivity Human Cytokine, R& D Systems, Minneapolis, MN) and 
a Bio-Plex 200 (Luminex) Instrument, with excellent intra- and inter-assay reproducibility97 and very strong 
correlations (r ≥ 0.94) with high sensitivity ELISA.98 We are examining IL-1, TNF, and IL-6 as these cytokines 
are increased in response to endotoxin and correlate with increases in depressed mood. IL-8 is also assayed 
because endotoxin activates the TLR-4 receptor and IL-8 is a proinflammatory cytokine that is released with 
TLR-4 activation. We also explore changes in IL-10, an anti-inflammatory cytokine that is thought to counter-
regulate the inflammatory responses; no study has examined IL-10 responses to endotoxin and determined 
whether its expression might alter magnitude/duration of inflammatory response. Baseline levels of CRP will be 
assessed, as CRP is a robust marker of systemic inflammation. Baseline levels of CRP may moderate 
increases in inflammation and affective responding. However, repeated measures of CRP will not be obtained. 
Increases in IL-6 induce CRP over several hours or longer; hence, the experimental protocol is too short in 
duration to evaluate changes in CRP. Peripheral blood mononuclear cell (PBMC) nuclear extracts (baseline, 
30 min, 1 h, 2 h or peak cytokine response) will be prepared and assayed to quantify the amount of activated 
NF-κB p65 present in the nucleus utilizing recombinant p65 (Active Motif, Carlsbad, CA) as the reference 

standard (range 0.08-5.00ng),99 as we have previously described.100 At baseline, 2- and 4 hours post-injection, 
blood samples will be drawn in PaxGene RNA tubes, which preserve RNA integrity. Expression of genes 
involved in proinflammatory pathways (IL1B, IL6, IL8, CD83, CCL3, TNFAIP3, and NF-κB/Rel family) will be 
assayed by quantitative real-time RT-PCR using established TaqMan Gene Expression Assays. Genome-wide 
transcriptional profiling will be performed using Illumina HT-12 BeadArrays. 
C.2.7. Two-Year Longitudinal Follow-up 
Participants who complete the experimental session will be followed up by telephone over 2 years for 
the assessment of depressive symptoms and disorders. A research assistant will call participants every 3 
months for administration of the PHQ-9. If their responses suggest depression (i.e., PHQ-9 ≥ 5), the project 
coordinator will conduct the SCID-5-RV over telephone. We have successfully conducted such a telephone 
follow-up in an ongoing depression prevention Study (R01AG026364, PI Irwin). Telephone administration of 
the SCID has been found acceptable and comparable to in-person administration.101,102 
C.2.8. Statistical Analyses 
All measured variables will be assessed for distributional qualities and transformed if necessary for use 
in the selected statistical models. Adequacy of random assignment and matching will be tested using t-tests 
and chi-square tests as appropriate. All pre-classification variables (age, BMI, insomnia, medical comorbidity, 
prior depression history) and baseline values of outcomes will be compared; those that are significantly 
different between females and males (at P<0.15) will be considered for inclusion as covariates in the main 
analyses. Missing data patterns will be analyzed to assess the potential for biased parameter estimates; 
missing data will not be imputed as the methods selected allow for missing data under common missingness 
assumptions. The basic design for Aim 1 is a 2 group (females vs. males) by 2 condition (endotoxin vs. 
placebo) independent balanced analysis of variance (ANOVA) with one or repeated outcome measures (times: 
Thr where hr = 0 for baseline, 0.5 to 12 post-infusion; peak cytokine concentration at T212), conducted with 
linear mixed models (LMM). The number of repeated measures varies by assessment. The key results are the 
main effects of sex, condition, and their interaction; for those analyses with repeated measures, the two main 
effects and interaction will further interact with the time variable. Other planned analyses include lagged (time 
series) relationships between cytokine outcomes and affect measures via general linear modeling (GLM). 
Standard pharmacokinetic parameters (e.g., AUC, CMAX, TMAX) may also be used to create summary cytokine 
and affect profile outcomes. Finally, covariates (i.e., pre-classification variables such as prior depression 
history, insomnia, or baseline CRP) will be tested if appropriate (i.e., p<0.15) to assess the robustness of the 
results and further explicate the findings. Aim 1 is tested with LMM for the following outcomes (timepoints): 
POMS Depression, DACL, Social Disconnection (T0, T1 – T12), Observer rated depression (T0, T2,4,6,8,12), 
Cyberball Social Exclusion Task (T2), Emotion Tasks (T0, T2), Social Support Questionnaire (T0, T2), Social 
Choice Task (T0, T2), Reward Response (T0, T2), Reward Learning (T0, T2), and Reward Motivation (T0, T2). 
The basic design for Aim 2 is identical to Aim 1 but using age as the group variable (young females vs. older 
females) instead of sex. Aim 3 is restricted to the endotoxin condition to examine the correlations between 
inflammatory responses and affective responses to endotoxin according to sex and age. These correlations will 
be examined using mixed-effects model linear regression, and cytokines and depressed mood, including 
excess negative affect and deficits in positive affect, will be entered in the regression models in their 
longitudinal format, thus reflecting all the measurements performed during the study (T0, T1 – T12). Exploratory 
Aim is tested using mixed-effects logistic regression, separately in the endotoxin and placebo groups, because 
the outcome (depressive episodes) is a binary variable repeatedly measured over 2 years. The predictor 
variables include the sum of POMS depression during 12 hours of experimental session, Cyberball Social 
Exclusion Task (T2), Emotion Tasks (T2), Social Choice Task (T2), and Reward Tasks (Response, Learning, 
Motivation, Sensitivity) (T2). 
Finally, we will also evaluate the changes of gene expression in response to endotoxin as a function of 
sex and age and evaluate a priori whether changes in the inflammatory transcriptome are associated with 
affective response; these analyses will focus upon specialized genomic analyses (T0, T2, T4), and utilize 
methods previously reported by us.54,103,104 Quantile-normalized gene expression values will be log2-
tranformed and subject to GLM analysis to provide maximum likelihood point estimates of differential transcript 
abundance across group (females vs. males; younger females vs. older females) and condition (endotoxin vs. 
placebo), which provide maximally replicable inputs into the higher-order set-based bioinformatics analyses. 
TELiS (Transcription Element Listening System www.telis.ucla.edu) promoter-based bioinformatics analyses 
will test the hypothesis that PBMC will show alterations by group and condition in global gene expression 
profiles consistent with increased activity of the proinflammatory transcription factors NF-κB (assessed by 

prevalence of the TRANSFAC V$NFKAPPAB65_01 nucleotide motif in differentially expressing promoters) and 
AP-1 (V$AP1FJ_Q2) in the female groups and the endotoxin condition. We will explore whether female sex, 
younger age, and endotoxin decrease activity of Type I interferon signaling pathways (V$ISRE_01, 
V$IRF2_01); decrease activity of the anti-inflammatory glucocorticoid receptor (GR) (V$GR_Q6); and increase 
activity of CREB transcription factors involved in β-adrenergic signaling by the sympathetic nervous system 
(V$CREB_01). Both depression and inflammation are implicated in the regulation of these pathways.105,106 
Moreover, the human immune system appears to have developed a conserved transcriptional response to 
adversity (CTRA) that involves up-regulating pro-inflammatory gene expression (e.g., cytokine genes) while 
conversely down-regulating anti-viral gene expression (e.g., interferon response genes) whenever 
environmental conditions are experienced as threatening, stressful, or uncertain for an extended period of 
time.107 The ratio of response element frequencies in the promoters of up- vs. down-regulated genes are 
assumed to be a measure of differential activity of transcription control pathways, and (log) ratios will be 
averaged over 9 different parametric combinations of promoter length (-300, -600, and -1000 to +200 bp 
upstream of RefSeq-designated transcription start site) and motif detection stringency (TRANSFAC mat_sim 
values of .80, .90, and .95) to ensure robust results.108 To identify the primary cellular sources of differentially 
expressed genes, we will carry out Transcript Origin Analysis.109 In both TELiS and Transcript Origin Analyses, 
standard errors will be estimated by 2000 cycles of bootstrap resampling of residual vectors from the linear 
models used to estimate differential gene expression across groups (sex and age) and condition (controlling 
for correlated expression across genes). 
C.2.9. Justification of Sample Size 
In our endotoxin study of younger adults, the effect of endotoxin on depressed mood was large in 
females (d=0.70) while minimal in males (d=0.003).14 Among older adults, we expect to see a smaller but still 
substantial difference of effect between sexes. In our endotoxin study of older females (K23AG049085, PI 
Cho), as shown in C.1.1.3, the effect size of endotoxin on depressed mood was still large but expectedly 
smaller (d=0.51) than in younger females. For Aim 1, based on these estimates, the assumed minimal effect 
size in older males (d=0.003), and the use of matching and multivariate regression analyses, with 40 older 
healthy females and 40 older healthy males, we expect a power >80% (α=.05, two-tailed). For Aim 2, 
assessing differences between younger and older females, our previous work with several of the behavioral 
reward tasks has yielded moderate effect sizes when assessing the relationship between increases in IL-6 and 
task performance (e.g., f2 = 0.22, 0.32). Given this range of effect sizes, a multiple regression analysis with 
three predictors of interest (age, group, age x group interaction) and two covariates (BMI, education level) 
would require a sample size of 38 to 75 to detect effects with 0.80 power. This work has relied on very mild 
increases in IL-6, and we expect endotoxin to yield stronger effects. Indeed, prior work with the endotoxin 
model has shown significant differences on a reward motivation task with a sample of 29 participants (15 saline 
placebo, 14 endotoxin),92 and greater feelings of social disconnection following endotoxin vs. placebo were 
observed in a small sample of women (n=20; age 18-36) in our previous study. Thus, the current study aims to 
recruit 40 premenopausal women with equal randomization between the endotoxin and saline group, and an 
additional 40 females will be drawn from the parent study for a total sample of 80. 
C.3. Interpretation of Results and Potential Concerns 
Endotoxin elicits sickness symptoms (e.g., nausea, achiness) that will be assessed as potential 
covariates in statistical analyses. The battery of administered tasks could lead to excessive fatigue and non-
compliance. This is not currently a concern in the parent study, but we will monitor participants’ subjective 
response to the tasks, as well as current fatigue, and assess these variables either as potential covariates or 
as indicators that the experimental session requires modification. While we do not anticipate strong emotional 
response to any of the tasks, there is the potential for carryover effects without task counterbalancing. 
However, since the order is the same for the experimental and the control group, such effects should not 
impact the primary research questions assessing group differences. 
 

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