An Experimental Treatment Approach for Inflammation-Induced Depression
Phase: EARLY_PHASE1
Conditions: Depression
Sponsor: University of California, Los Angeles

An Experimental Treatment Approach for Inflammation-Induced Depression 
 
Short Title: Leucine for Depression (L-DEP) Study 
 
ClinicalTrials.gov ID: NCT03557684 
 
Document Date: 4/30/2019 
 
Joshua Hyong-Jin Cho, MD, PhD, Michael R. Irwin, MD, Richard Olmstead, PhD, Robert Dantzer, DVM, PhD 
 
 
ABSTRACT 
 
Depression is a major public health burden, and is even more prevalent and disabling in individuals with 
heightened inflammatory states due to certain diseases or treatments. Compelling evidence suggests that 
inflammation plays a causal role in the onset or perpetuation of some forms of depression including those in 
the context of interferon treatment, chronic diseases, aging, and obesity. Antidepressant medications—the 
mainstay of the current depression treatments, with limited efficacy, substantial adverse effects, and poor 
adherence—have consistently shown an even more limited efficacy in this inflammatory subtype of depression. 
Consequently, anti-inflammatory drugs (nonsteroidal anti-inflammatory drugs, cytokine antagonists) have been 
tested for the treatment of inflammation-associated depression. However, these drugs have shown limited 
efficacy, substantial risk profile, and prohibitive cost. Thus, novel and safe pharmacologic strategies that target 
mood-specific mechanisms of inflammation-associated depression are needed. One of the potential 
mechanisms by which inflammation causes depression is via kynurenine metabolism. Inflammation activates 
the tryptophan-kynurenine metabolic pathway, converting tryptophan to kynurenine. Kynurenine produced in 
excess at the periphery can be transported into the brain, where it is metabolized into neurotoxic kynurenine 
metabolites responsible for the development of depressive symptoms. As this transport system across the 
brain-blood barrier makes use of the large amino acid transporter LAT1, it is theoretically possible to block 
kynurenine entry into the brain by administering competing amino acids such as leucine. Preclinical 
experiments confirmed the inhibitory effects of leucine on inflammation-induced depression as a result of the 
blockade of excess kynurenine from entering the brain. The overarching objective of this innovative proof-of-
concept trial is to test whether leucine pretreatment abrogates acute depressive responses to an inflammatory 
challenge (i.e., endotoxin) in humans, relative to placebo pretreatment. This study hypothesizes that, 
compared to placebo pretreatment, leucine pretreatment will attenuate depressive responses to endotoxin. In 
this double-blind randomized placebo-controlled study of healthy volunteers (N=90; 18-65 y), we aim to 
examine the effects of leucine vs. placebo pretreatment on: 1) endotoxin-induced depressive symptoms 
(primary outcome); 2) negative and positive affect responses to endotoxin (respectively, feelings of social 
rejection, negative bias in facial emotion recognition; and feelings of social acceptance, reward learning); and 
3) cognitive responses to endotoxin (verbal and non-verbal memory, executive function, attention). If the 
hypotheses are confirmed, the involvement of the kynurenine pathway in the induction of depressive symptoms 
will be demonstrated for the first time in a human experimental study. This study will also provide instrumental 
information in designing future clinical trials of leucine to treat or prevent inflammation-associated depression. 
 
 
RELEVANCE TO PUBLIC HEALTH 
 
Depression is highly prevalent (~20%) and poses a huge disease burden. Among individuals with heightened 
inflammatory states due to certain diseases or treatments, depression is even more prevalent and disabling, 
and antidepressant and anti-inflammatory medications show limited efficacy and substantial adverse effects. 
This study—the premise of which is supported by the rigor of our preliminary data in animals—is significant 
because it will examine whether a novel treatment, leucine, will mitigate depression in response to 
inflammatory exposure, with a potential for a safe and efficacious application in clinical practice. 

 
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LAY SUMMARY 
 
Depression is very common and poses a huge disease burden. About 20% of the US population suffers from 
depression at least once in their lifetime. Inflammations that are hidden inside our body as a result of aging, 
obesity, chronic diseases, or certain treatments (e.g., interferon for hepatitis C) appear to cause depressive 
symptoms and even clinical depression. Individuals with such inflammations are more likely to suffer from 
depression and are less likely to respond to currently available antidepressant medications. This study will test 
leucine, an amino acid, as a new way to mitigate depressive symptoms in response to such inflammations. 
This study begins with a 90-minute screening session to determine whether participants are eligible to join the 
main study. Those who meet the eligibility criteria will then join the main study, which will consist of taking 
leucine or maltodextrin (i.e., oral placebo) for 2 weeks at home and an 8-hour session at the UCLA Medical 
Center. A brief telephone follow-up every 3 months for 2 years with questions on mood is also planned. 
Approximately 90 healthy adults will be recruited for participation in the study. During the course of the study, 
participants will take leucine or maltodextrin for 2 weeks at home and then will be injected either 
lipopolysaccharide (LPS) or saline (i.e., intravenous placebo) at the UCLA Medical Center. LPS is a bacterial 
substance that can initiate chemical reactions that are similar to those seen in individuals with mild sickness 
symptoms, such as a slight increase in body temperature, muscle aches, or tiredness. It is a safe way of 
investigating the body’s response to inflammation and how these changes may alter cognitive, emotional, or 
neural function. It has been given thousands of times to healthy volunteers – both younger and older adults – 
without any serious side effects. 
 
 

 
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SPECIFIC AIMS 
Depression is highly prevalent (~20%1) and is a leading cause of worldwide disease burden.2 However, 
antidepressants—the mainstay of the current depression treatments—have a limited efficacy typically 
achieving remission rates of 30% or less3; burden of adverse effects is substantial and adherence is poor.4-6 
Among individuals with heightened inflammatory states due to certain diseases or treatments, depression is 
even more prevalent and disabling.7-9 Likewise, the efficacy of antidepressants is even more limited.10-12 
Compelling evidence suggests that inflammation plays a causal role in the onset or perpetuation of some 
forms of depression including those in the context of chronic diseases (cardiovascular, infectious, rheumatic, 
autoimmune), aging, and obesity.13-15 Elevated biomarkers of inflammation predict depression risk,16,17 and 
inflammatory challenge (e.g., interferon, endotoxin) leads to the development of depressive symptoms.18-23 
Given the poor treatment response to conventional antidepressants in the inflammatory subtype of depression, 
recent interest has focused on the use of drugs that are anti-inflammatory (i.e., nonsteroidal anti-inflammatory 
drugs [NSAIDs], cytokine antagonists).24-26 However, these drugs have shown limited efficacy, substantial risk 
profile, and prohibitive cost.  Thus, novel and safe pharmacologic strategies that target mood-specific 
mechanisms of inflammation-associated depression are needed.  
One of the potential mechanisms by which inflammation 
causes depression is via kynurenine metabolism.27,28 
Inflammation activates indoleamine 2,3 dioxygenase (IDO), 
which converts tryptophan to kynurenine (Fig. 1). Kynurenine 
produced in excess at the periphery can be transported into the 
brain, where it is metabolized into neurotoxic kynurenine 
metabolites responsible for the development of depressive 
symptoms. As this transport system across the brain-blood 
barrier (BBB) makes use of the large amino acid transporter 
LAT1, it is theoretically possible to block kynurenine entry into brain by administering competing amino acids 
such as leucine. Preclinical experiments confirmed the inhibitory effects of leucine on inflammation-induced 
depression as a result of the blockade of excess kynurenine from entering the brain (Preliminary Data). This 
effect appears clinically meaningful as the effect of leucine was 60% larger than that of ketamine, which is 
known to have potent antidepressant effects in humans.29 Leucine, with few adverse effects, has been studied 
for muscle mass preservation, weight loss, and glucose homeostasis.30-32 
The overarching objective of this innovative proof-of-concept trial is to test whether leucine 
pretreatment abrogates acute depressive responses to an inflammatory challenge (i.e., endotoxin) in 
humans, relative to placebo pretreatment. Endotoxin administration has been used as a novel experimental 
model of depression.19 We have demonstrated that low-dose endotoxin induces depressive symptoms in 
humans,21-23 which correlate with activation of brain regions implicated in depression.33-35 Low-dose endotoxin 
also induces transient cognitive impairment,20 a common symptom of depression that is often overlooked and 
associated with poor response to treatment.36 Furthermore, we have found that endotoxin increases 
tryptophan-kynurenine conversion rate, which correlates with depressed mood (Preliminary Data). 
This study hypothesizes that, compared to placebo pretreatment, leucine pretreatment will 
attenuate depressive responses to endotoxin. In this double-blind randomized placebo-controlled four-arm 
study of 90 healthy volunteers aged 18-65 years (n1=35: oral [PO] leucine for 2 weeks followed by single 
intravenous [IV] endotoxin; n2=35: PO placebo [i.e., maltodextrin] + IV endotoxin; n3=10: PO leucine + IV 
placebo [i.e., saline]; n4=10: PO placebo + IV placebo), we aim to: 
1. Examine the effects of leucine vs. placebo pretreatment on endotoxin-induced depressive symptoms 
(primary outcome). 
2. Examine the effects of leucine vs. placebo pretreatment on negative and positive affect responses to 
endotoxin (respectively, feelings of social rejection, negative bias in facial emotion recognition; and feelings 
of social acceptance, reward learning). 
3. Examine the effects of leucine vs. placebo pretreatment on cognitive responses to endotoxin (verbal and 
non-verbal memory, executive function, attention). 
Hypothesis: Compared to placebo pretreatment, leucine pretreatment for 2 weeks will attenuate endotoxin-
induced depression outcomes including depressive symptoms, negative affect, and positive affect. 
Impact Statement: If the hypotheses are confirmed, this proof-of-concept study will provide instrumental 
information in designing future clinical trials of leucine to treat or prevent inflammation-associated depression. 

 
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RESEARCH STRATEGY 
A. SIGNIFICANCE 
 
A.1. Burden of Depression and Limitation of Current Treatments: Depression is a major public 
health burden: highly prevalent (~20% in US1) and projected to become the leading cause of disease burden.2 
However, antidepressants, the mainstay of the current depression treatments, have a limited efficacy typically 
achieving remission rates of 30% or less when used as a monotherapy.3 They are also associated with poor 
adherence and substantial adverse effects.4-6 Depression is even more prevalent and disabling in individuals 
with inflammatory conditions such as chronic diseases (cardiovascular, infectious, rheumatic, autoimmune), 
aging, obesity, and interferon treatment.7-9 Moreover, poor treatment response to antidepressants has been 
consistently shown in those with inflammatory disorders, inflammation-related gene variants, and elevated 
levels of inflammatory markers.10-12 This study is significant as it will examine novel inflammation-related 
mechanisms with translational potential for treatment or prevention of this major public health burden. 
 
 
A.2. Inflammatory Mechanisms of 
Depression: Compelling evidence suggests that 
inflammation plays a causal role in the onset or 
perpetuation of some forms of depression.13-15 
Elevated biomarkers of inflammation, including C-
reactive protein (CRP) and proinflammatory 
cytokines, predict depression risk,16,17 and 
inflammatory challenge (interferon, endotoxin) 
leads to the development of depressive 
symptoms.18-23 Given the poor treatment response 
to conventional antidepressants in the inflammatory 
subtype of depression, recent research has 
focused on the use of drugs that are anti-
inflammatory (e.g., NSAIDs, cytokine 
antagonists).24-26 However, these drugs show 
limited efficacy and also have a broad range of 
actions that can adversely affect other than mood 
domain with serious side effects (e.g., 
gastrointestinal bleeding, severe infection). Also, 
the cost of cytokine antagonists is prohibitive (~$3K per dose). 
Thus, novel and safe pharmacologic strategies that target mood-
specific mechanisms of inflammation-associated depression are 
needed, as proposed here. 
 
 
A.3. Kynurenine Metabolism and Leucine: Kynurenine 
metabolism is increasingly recognized as a key neurochemical 
pathway in the link between inflammation and depression.27 
Inflammatory mediators activate IDO, an enzyme that metabolizes 
tryptophan (TRP) to kynurenine (KYN); hence KYN/TRP ratio 
represents a measure of IDO activity (Fig. 2). Circulating KYN is 
transported into brain by the large amino acid transporter LAT1, 
which is expressed in endothelial cells of the BBB (Fig. 3).28 KYN 
is further metabolized into 3-hydroxykynurenine (3HK) by the 
enzyme kynurenine monooxygenase (KMO), leading to an 
increase in quinolinic acid (QA) on the putatively neurotoxic 
branch of the kynurenine pathway. Of note, besides IDO, KMO is also upregulated under inflammatory 
conditions (Fig. 2),29,37 and endotoxin has been shown to almost exclusively increase neurotoxic metabolites 
including 3HK and QA but not kynurenic acid (KynA) on the putatively neuroprotective branch.29 3HK is a free 
radical generator, and QA, an N-methyl-D-aspartate (NMDA) receptor agonist, has been associated with a 
plethora of neurotoxic effects.38,39 KynA is an NMDA receptor antagonist and is protective against the 
excitotoxic action of QA.40,41 Hence, a shift in the balance of these neurotoxic and neuroprotective kynurenine 
metabolites is believed to be responsible for the development of depressive symptoms.42-48 
One way to block the shift in the balance of kynurenine metabolites is to block the entry of KYN into 
brain. KYN is transported into brain via LAT1. It is therefore theoretically possible to decrease the amount of 

 
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KYN transported into brain by using competing amino acids at the level of LAT1. Leucine is the amino acid that 
has the highest affinity for LAT1. Preclinical experiments in a murine model of inflammation-induced 
depression show that leucine competitively inhibits LAT1 transport of excess KYN into brain in inflamed mice 
(Preliminary Data). Through this competitive inhibition of KYN transport, leucine is capable of abrogating the 
development of depressive symptoms in response to inflammatory challenge, but without affecting 
neuroinflammation or inflammation-induced sickness. Endotoxin is known to induce or increase the secretion 
of cytokines from the cells which comprise the BBB and to activate IDO at this level.49 However, as leucine 
does not affect peripheral inflammatory responses or neuroinflammation (Preliminary Data), its specific effects 
on mood may be unaltered by the endotoxin-induced immune activation of the BBB itself. Furthermore, given 
the observation that endotoxin upregulates the neurotoxic branch of the kynurenine pathway without altering 
the neuroprotective branch,29 the blockade of KYN transport by leucine appears to affect primarily the 
neurotoxic branch. Notably, the effect of leucine was about 60% larger than that of ketamine.29 Ketamine, an 
NMDA receptor antagonist, has a rapid and robust antidepressant effect in humans.50 This study is significant 
by evaluating the potential antidepressant effects of leucine for the first time in humans. 
 
A.4. Complexity of Kynurenine System Balance and Inflammatory Depression: As in 
neurodegenerative diseases, an increased influx of KYN from the periphery to CNS can increase the 
neurotoxic metabolites (3HK and QA) and decrease the neuroprotective KynA, augmenting neuronal 
vulnerability.38 Thus, this neurotoxic shift is believed to contribute to the development of depressive symptoms. 
In line with this hypothesis, a recent meta-analysis has reported decreased serum and CSF levels of KynA in 
depressed patients compared to controls.51 KynA is an NMDA receptor antagonist like ketamine, which has a 
rapid and robust antidepressant effect in humans. On the other hand, the observation that ketamine produces 
schizophrenia-like symptoms in normal individuals and exacerbates psychotic features in schizophrenic 
patients has led to the idea that the excessive KynA might have a role in schizophrenia pathology. Increased 
CNS levels of KynA have been found in schizophrenic patients, and further studies in animals and humans 
indicate that increased KynA levels may be especially relevant for the cognitive deficits present in 
schizophrenia.38 As cognitive deficit is also seen in depression, a more complex kynurenine system balance in 
depression is indeed possible; e.g., the key for mental health might be an optimal balance between 3HK+QA 
and KynA rather than a simple predominance of KynA. However, the existing data largely support that a 
neurotoxic shift of the kynurenine pathway is involved at least in depression pathophysiology51; and most 
importantly, the key premise of our project focuses on the blockade of the BBB transport of KYN, a step 
upstream to the aforementioned balance mechanism between 3HK+QA and KynA. Thus, the premise of our 
project overcomes the controversy regarding whether a deficient or an excessive presence of KynA leads to 
depression. Furthermore, the increase of KYN relative to TRP in the periphery, indicating an increased IDO 
activity, has been consistently associated with inflammatory depression.52 Importantly, our previous human 
endotoxin study also showed a robust increase in the ratio KYN/TRP after endotoxin administration and a 
significant correlation between this ratio and depressive symptoms.53 Thus, although there is indeed a 
complexity of kynurenine system balance in relation to psychopathology, our proposed intervention targets an 
upstream level that has been consistently associated with inflammatory depression. 
 
A.5. Human Experimental Studies of Endotoxin-Induced Depression: Our and other groups have 
demonstrated that low-dose endotoxin induces depressive symptoms in humans as well19-23, which correlate 
with activation of brain regions implicated in depression.33-35 We have also found that endotoxin increases 
TRP-KYN conversion rate, which correlates with depressed mood (Preliminary Data). Increases of 
inflammation in response to low-dose endotoxin are comparable with the magnitude of increases in acute 
inflammation (e.g., infection) and chronic inflammation (e.g. cardiovascular disease, cancer, aging processes, 
sleep disturbance, or obesity). Chronic inflammation predicts depression risk.54,55 Chronic inflammation primes 
inflammatory and depressive responses to challenges that acutely activate inflammation (e.g., infection, 
interpersonal stress).56-64 Moreover, acute inflammatory reactivity predicts acute increases in depressive 
symptoms,58,65 as well as increases in depression over the following year.65 Although this model is specific to 
inflammation-associated depression, it should be noted the burden of inflammation-associated depression 
itself is huge for two reasons. First, a high proportion of depressed individuals identified in community present 
with elevated inflammation. In the National Health and Nutrition Examination Survey (NHANES), 47% of 
individuals with the Patient Health Questionnaire (PHQ-9) scores indicative of significant depression (≥ 10) had 
CRP levels ≥ 3 mg/L and 29% had CRP levels ≥ 5 mg/L.66 Second, poor treatment response to 
antidepressants has been consistently shown in those with inflammatory disorders, inflammation-related gene 
variants, and elevated levels of inflammatory markers 10-12. Furthermore, the findings of this study may provide 

 
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some justification to test leucine in a broader context of clinical depression. This study is significant as a unique 
opportunity to advance translational depression research using a novel human experimental model and 
examining a potential biological mechanism of depression identified by rigorous pre-clinical research. 
 
A.6. Depressive vs. Sickness Symptoms in Endotoxin Model: Low-dose endotoxin can induce not 
only depressive symptoms but also some sickness symptoms (i.e., lethargy, fatigue, muscle pain, headache, 
shivering, and nausea). Although in clinical practice depressed individuals experience both emotional and 
somatic symptoms of depression in varying degrees, a possible critique on the endotoxin model would be that 
depressed mood might be majorly influenced by sickness symptoms. To minimize this possibility, we have 
previously used a depression scale that only assesses emotional symptoms such as “unhappy”, “sad,” and 
“hopeless” (Profile of Mood Scale [POMS]67).21-23 Furthermore, we have used analytical procedures to ensure 
the effects of endotoxin on depressed mood are independent of sickness symptoms (Preliminary Data). In this 
study, beyond these two measures, we will use experimental tasks of negative and positive affect to assess 
mood-specific processes (see C.2.6. Experimental Tasks). This innovative approach is particularly important 
for this study as we hypothesize that leucine pretreatment—which interferes with the transport of excess KYN 
into brain without modifying inflammatory responses to endotoxin—will specifically abrogate depressed mood 
but not sickness symptoms induced by endotoxin. This study is significant because it will examine depressive 
vs. sickness symptoms in a human endotoxin model of depression by employing mood-specific assessments 
and especially by testing a treatment that mat act on a mood-specific mechanism without modifying 
inflammatory responses to endotoxin. 
 
A.7. Transient Cognitive Impairment in Endotoxin Model of Depression: Cognitive impairment is a 
common symptom of depression, experienced by two-thirds of depressed patients and associated with poor 
treatment response, but often overlooked by clinicians.36 Low-dose endotoxin transiently impairs verbal and 
nonverbal memory functions in humans.20 Independent preclinical data demonstrate that peripherally 
administered endotoxin induces memory deficit through IDO-dependent neurotoxic kynurenine metabolism; 
furthermore, a single peripheral injection of KYN is sufficient to induce memory deficit.68 Notably, human 
studies have shown that the supplementation with branched-chain amino acids (leucine, isoleucine, and valine) 
has protective effects against cognitive deficits during strenuous exercises.69-72 Based on the evidence from 
animal and human research, it is expected that the blockade of the BBB transport of KYN by leucine 
pretreatment will attenuate endotoxin-induced cognitive impairment. 
A.8. Potential Impact of the Current Study and Future Directions: If the hypotheses are confirmed, 
the involvement of the kynurenine pathway in the induction of depressive symptoms, including cognitive 
impairment, will be demonstrated for the first time in a human experimental study. Furthermore, this proof-of-
concept study will provide instrumental information in designing future trials of leucine to treat or prevent 
inflammation-associated depression. Given the minimal adverse effects and the additional benefits such as 
muscle mass preservation, leucine will be a prime candidate for a pharmacologic strategy to treat or prevent 
inflammation-associated depression. 
 
B. INNOVATION 
 
B.1. A highly controlled experimental model of depression to examine a novel mood mechanism 
Low-dose endotoxin administration is a highly innovative experimental model to understand the causal role of 
inflammation in the induction of depressive symptoms; yet, it 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).19,73 Using this 
experimental model, in addition to a randomized controlled trial design, we will evaluate the kynurenine 
pathway, beyond the inflammatory cascade, as a potential mechanism of inflammation-associated depression. 
To our knowledge, this will be the first human experimental study to evaluate the involvement of the kynurenine 
pathway in the induction of depressive symptoms. 
 
B.2. A novel pharmacologic agent shown to act on a novel mood mechanism in animal model 
Leucine has been shown to abrogate endotoxin-induced depression-like behavior in mice by specifically acting 
on blood-to-brain transport of excessively produced KYN without affecting inflammatory responses (Preliminary 
Data). Although leucine has been studied in humans for other purposes, it has never been tested for 
depression. This study will employ this novel paradigm for the first time in humans. 
 
B.3. Task based multi-dimensional methods to evaluate affective processes 
No prior experimental research on inflammation and depression has used innovative objective measures to 
assess negative and positive affective processes. Given that social disconnection plays a critical role in the 

 
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onset and perpetuation of depression,58,74 we will examine distress following an experimental episode of social 
rejection as a function of pretreatment conditions. Further, we will examine the impact of pretreatments on 
accurate judgment of facial emotions with implications for perceived threat (i.e., angry face) or reward value 
(i.e., happy face).75 Facial emotion recognition task is used to identify negatively biased processing of 
emotional faces and is associated with depression and risk of relapse in depressed patients.76,77 This study is 
novel also by evaluating positive affective responses, using reward-based tasks.78-80 Use of these experimental 
affect tasks is strategic in distinguishing the effects of endotoxin on depressive symptoms from its effects on 
sickness symptoms. Furthermore, understanding how leucine pretreatment influences negative and positive 
affect in response to endotoxin has the potential to refine pharmacologic strategies of depression treatment 
and to inform the design of neuroimaging studies aimed at identifying the neural sensitivities related to 
inflammation. For example, findings related to reward deficits could direct attention toward pharmacologically 
targeting neurotransmitters (i.e., dopamine) and toward scanning reward-related brain regions. 
 
C. APPROACH 
 
C.1. Preliminary Studies 
 
C.1.1. Leucine abrogates inflammation-induced depression-like behavior 
Aim: We examined 
whether L-leucine 
(hereafter referred to as 
leucine), which has a 
high affinity for LAT1, 
competitively inhibits 
KYN binding to LAT1, 
reduces blood-to-brain KYN transport, and blocks 
endotoxin-induced depression-like behavior in mice.  
Methods: CD-1 mice (N=20; n=5 per group) were 
injected lipopolysaccharide (LPS = endotoxin, 0.83 
mg/kg, intraperitoneal [IP]) or saline and treated with 
leucine (50 mg/kg, IP) or vehicle administered before and 
6 h after LPS (Fig. 4). Depression-like behavior was 
measured at 24 h by increased duration of immobility in 
the forced swim test (FST) and decreased sucrose 
preference.  Results: Leucine blocked LPS-induced 
depression-like behaviors (p’s<0.05) (Fig. 5). Leucine 
had no effect on sickness behavior and 
neuroinflammation. Consistent with our hypothesis that 
leucine would competitively inhibit blood-to-brain KYN 
transport, leucine had no effect on LPS-induced 
increases in plasma KYN/TRP ratio (i.e., IDO activity) or brain TRP levels but significantly reduced brain KYN 
levels. Using an in vitro model of BBB transport based on primary cultures of brain microvasculature 
endothelial cells, we confirmed the ability of leucine to block KYN influx via LAT1 and demonstrated it did not 
interfere with KYN efflux.  Conclusion: Leucine abrogated LPS-induced depression-like behavior without 
affecting LPS-induced sickness, inflammation, and IDO activation. Leucine reduced blood-to-brain transport of 
KYN and decreased brain KYN levels. These data suggest that targeting KYN transport mechanisms at the 
BBB level using leucine is a viable approach to attenuating inflammation-induced depression in humans. 
 
C.1.2. Inflammatory challenge (i.e., endotoxin) induces increases in depressed mood in humans 
In a randomized double-blind placebo-controlled design, 115 participants (69 female, mean age: 24.1± 6.3 
years) received either low-dose endotoxin (0.8 ng/kg of body weight, n=61) or placebo (saline, n=54). 
Circulating interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured hourly post-injection for the 
next six hours (T1-T6), along with assessment of depressed mood using the POMS. Endotoxin (vs. placebo) 
led to significant increases in IL-6 (p<0.001) and TNF-α 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).22 Endotoxin induced increases in depressed mood, which were not due to 
sickness symptoms. These data show the feasibility and safety of the human endotoxin model. 
 
C.1.3. Endotoxin induces changes in KYN metabolism, which correlates with depressed mood 

 
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Aim: In the aforementioned study in C.1.2. (N=115), we examined whether endotoxin induces changes in 
kynurenine metabolism and whether these changes correlate with inflammation-induced depressed mood.  
Methods: Circulating kynurenine metabolites (TRP, KYN, QA, KynA) were measured at T0, T2, and T6. The 
primary outcome was IDO activity assessed using KYN/TRP.  Results: Participants exposed to endotoxin (vs. 
placebo) showed greater increases in IDO activity (p<0.0001). Among endotoxin-exposed participants (N=61; 
38 females), the correlation between IDO activity and depressed mood was highly significant (p=0.004).  
Conclusion: Inflammatory challenge increased IDO activity, which in turn was significantly correlated with 
inflammation-induced depressed mood. These data confirm endotoxin-induced increases in peripheral 
conversion of TRP to KYN as well as increases in depressed mood, and thus show the feasibility of inhibiting 
blood-to-brain transport of KYN as well as inflammation-induced depressed mood. 
 
C.2. Research Design and Methods 
 
C.2.1. Overview of Study Design (Fig. 6): In 
this double-blind randomized placebo-controlled four-
arm study, a total of 90 healthy volunteers (ages 18-65) 
will be randomly assigned to a two-week pretreatment 
of L-leucine (hereafter referred to as leucine) or PO 
placebo (i.e., maltodextrin). After the pre-treatment 
period, they will receive low-dose endotoxin or IV 
placebo (i.e. saline): n1=35 leucine + endotoxin; n2=35 
PO placebo + endotoxin; n3=10 leucine + IV placebo; 
n4=10 PO placebo + IV placebo. (see Justification of Sample Size for the rationale for smaller IV placebo 
arms) 
 
C.2.2. Subjects: Participants will be recruited from: 1) flyers that will be posted at the UCLA campus 
including the UCLA Medical Center; 2) advertisements posted in local university and community newspapers 
and websites (Daily Bruin, Brentwood News, Santa Monica Daily Press, www.bruinwalk.com and 
www.dailybruin.com); and 3) lists of telephone numbers and mailing addresses purchased from GENESYS 
Sampling Systems (Fort Washington, PA), a company that maintains a bimonthly updated database of all 
available listed telephone households in the US, following our previously successful recruitment strategies. A 
list of households with at least one adult aged 18 to 65 years within a 10-mile radius of UCLA Westwood 
Campus will be purchased every other month. Age information for each household is based on either known 
age-related data or a statistical estimate of age, predicted on individual household characteristics and Census 
demographic information. We have used these lists for our previous telephone survey of community-dwelling 
older adults (R01AG034588). A letter will be sent to the individuals listed by GENESYS Sampling Systems, 
briefly describing the study and inviting them to participate in the study. Participants interested in the study will 
contact the project coordinator by email or phone. All interested participants will then go through a two-step 
screening procedure, consisting of a structured telephone interview and an in-person screening session. The 
initial screening process will be completed over the phone by the project coordinator. In addition to the 
telephone screening, we will use a web survey (https://tinyurl.com/UCLA-LDEP). When potential participants 
type this link on their Internet browser or click the advertisement banner on one of the websites, they will be 
directed to the web survey. Lastly, we will include a QR code on the flyers to facilitate the enrollment. By 
scanning the QR code, the prospective participants will be directed to the web survey as a screening 
procedure. The second screening will be done in person by one of the study physicians (Dr. Joshua Hyong-Jin 
Cho, Dr. Michael Irwin, or Dr. Ariya Mahbod). Most of the assessment will be conducted by the study 
physician, especially for medical and psychiatric comorbidities. The other interview components including C-
SSRS and SCID will be conducted by the project coordinator and reviewed by the study physician. 
Participants’ eligibility will ultimately be determined by the study physician, based on the results from the blood 
and urine tests as well as the medical and psychiatric evaluation obtained during the in person screening 
processes. To maximize the participant retention, they will be regularly contacted by phone throughout the 
study, especially during the 2-week pretreatment period. 
Inclusion Criteria: Participants will be required to be in good general health (as evaluated during the phone and 
in-person screening sessions) and aged 18-65 years. 
Exclusion Criteria: Individuals with the following conditions will be excluded: history of maple syrup urine 
disease (a contraindication to leucine treatment), chronic diseases; current use of prescription medications 
such as steroids, NSAIDs, immune modifying drugs, opioid analgesics, and psychotropics; Axis I psychiatric 
disorders including current major depressive disorder; current depressive symptoms assessed by the Patient 

 
9
Health Questionnaire (PHQ-9 ≥ 5); heavy smoking (1 pack or more per day) or excessive caffeine use (>600 
mg/day); BMI > 35 due to the effects of obesity on cytokine activity; evidence of recreational drug use or 
pregnancy from urine test; evidence of clinically significant rhythm abnormality on a resting electrocardiogram 
(ECG); clinically significant abnormalities on screening laboratory tests. (see Protections of Human Subjects 
section for the full criteria) 
 
C.2.3. Procedures 
Pretreatment: Leucine is commercially available in US as dietary supplements. For this study, we will use oral 
preparation of the pharmaceutical grade leucine (12 g/day in 2 divided doses for 2 weeks; Ajinomoto, Raleigh, 
NC) mixed with effervescent powder. PO placebo will resemble the compounded mixture of leucine and 
effervescent powder. Clinical studies of leucine have used doses ranging from 2 to 18 g/day.81-87 According to 
a meta-analysis, the long-term supplementation dose (i.e., 10 days to 6 months) ranged from 2.8 to 16.1 g/day 
with a weighted mean of 8.5 g/day.30 Casperson et al. found that leucine 12 g/day for 2 weeks improved 
muscle protein synthesis with a concomitant increase in phosphorylation of mechanistic target of rapamycin 
(mTOR) following the stimulus of a meal.87 As this above-average dose given for 2 weeks caused increases in 
post-prandial protein synthesis and mTOR activation, both mechanisms also observed in preclinical studies of 
leucine after the stimulus of endotoxin,88 we will use 12 g/day for 2 weeks. Participants will be given leucine or 
PO placebo on the day of in-person screening and will take it for 2 weeks including the morning of endotoxin 
administration, hence a total of 28 doses. At the end of pretreatment, subjects will indicate the presence or 
absence of a number of possible adverse effects in a questionnaire using a 4-point scale from 'not at all' to 
'very often': symptoms include not feeling well, headache, weakness, fatigue, nausea, vomiting, burping, 
stomachache, bloating, flatulence, constipation, diarrhea, dry mouth, change of taste, cough, skin complaints, 
muscle pain, weight gain, weight loss, dizziness, sweating, and shaking. The adherence will be assessed using 
the Patient Adherence Questionnaire (PAQ)89 and the count-back method (i.e., counting doses remaining in 
returned packets)90 at the end of the pretreatment (i.e., immediately before endotoxin). 
Endotoxin Administration (Main Study Session): The main study session including endotoxin administration 
and experimental tasks will be conducted at the UCLA Clinical & Translational Research Center (CTRC). 
Participants will arrive at the CTRC at 07:00. Upon arrival, a urine sample will be collected and tested for drug 
use and pregnancy in order to ensure continued eligibility for study participation. A CTRC nurse will assess 
vital signs (blood pressure, pulse, temperature). An indwelling venous catheter with a heparin lock will be 
inserted into the participant’s dominant forearm for hourly blood draws and one into the non-dominant forearm 
for endotoxin administration. Oral hydration with water or juice will be offered hourly in order to minimize the 
negative side effects of endotoxin (hydration reduces risk of diastolic blood pressure decreases). Following 
these procedures, participants will eat a light breakfast before undergoing experimental tasks and taking the 
last dose of study drug at 08:30. At 09:00 (T0), participants will receive endotoxin (0.8 ng/kg of body weight) or 
IV placebo as an intravenous bolus over 30-60 seconds. Endotoxin to be used in this study is derived from 
Escherichia coli (E. coli group O:113) and will be provided by the NIH Clinical Center.91 
Safety Monitoring and Discharge from Study: Participants will be monitored as per the protocol (see 
Protections of Human Subjects section) and discharged following the last blood draw upon approval from the 
study physician. Briefly, in addition to the continuous observation, scheduled nursing assessments including 
vital sign check will be performed every 30 minutes for the first 2 hours after LPS administration and then 
hourly. If any potential adverse reactions are noted as detailed in the protocol, the study physician will be 
contacted and the pre-determined actions will be taken as appropriate such as ECG, verbal or manual 
stimulation, relieving medications, or discontinuation of the study procedures. 
 
C.2.4. Baseline Measures (Prior to Pretreatment): During the in-person screening session, the following 
measures will be assessed: sociodemographic information, sleep (DSM-5 insomnia using the SCID-592 and 
sleep disturbance using the Pittsburgh Sleep Quality Index93), depressive/anxiety symptoms (Inventory of 
Depressive Symptomatology94, Beck Depression Inventory95, Beck Anxiety Inventory96, POMS67, and the 
Montgomery-Asberg Depression Rating Scale [MADRS]97), cognitive functions (estimated IQ using Test of 
Premorbid Functioning from Advanced Clinical Solutions™; verbal memory, visual memory, executive function, 
and attention using computerized tests from CNS Vital Signs™ including Verbal Memory Test, Visual Memory 
Test, Stroop Test, Shifting Attention Test, and Continuous Performance Test), systemic inflammation (CRP), 
and kynurenine metabolism (TRP, KYN, 3HK, QA, and KynA). Baseline measures such as sex, sleep 
disturbance, systemic inflammation, and KYN metabolism will be tested as potential moderators of the 
pretreatment effects on endotoxin-induced depression outcomes. In particular, we expect stronger effects of 
leucine pretreatment in females compared to males because our previous studies indicate that: 1) whereas 

 
10 
endotoxin increases circulating proinflammatory cytokines and IDO activity (i.e., KYN/TRP ratio) equally in 
males and females,22,53 it increases depressed mood much more robustly in females22; 2) the correlations 
between cytokines and depressed mood are stronger in females22; and 3) the correlations between IDO activity 
and depressed mood are stronger in females53. Furthermore, before and after pretreatment, blood samples will 
be drawn for the assays of intracytoplasmic cytokine expression (IL-1, IL-6 and TNF-α) and nuclear signaling 
of inflammation (nuclear factor [NF]-κB) using ex-vivo stimulation with endotoxin (also known as 
lipopolysaccharide [LPS] stimulation). These assays will measure the cellular production of cytokines in 
response to LPS stimulation and thus the sensitivity of peripheral blood mononuclear cells (PBMC) to an 
inflammatory challenge before and after pretreatment. 
 
C.2.5. Repeated Measures (Main Study Session) 
Behavioral Measures: Depressed mood, a core symptom of depression, will be assessed immediately before 
endotoxin administration (T0) and then hourly for the next 6 hours (T1-T6) using the POMS67 (primary 
outcome). We will also use the MADRS97 hourly (T0-T6) as it is sensitive to acute changes in depressed mood 
and anhedonia following endotoxin19 and is less oriented towards somatic symptoms than the Hamilton 
Depression Rating Scale. 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 Scale21) and loneliness (adapted UCLA Loneliness Scale98) will also be assessed. 
Participants will also complete self-report measures of sickness symptoms (e.g., muscle pain, fatigue). 
Cognitive Measures: Three items from the POMS-Confusion subscale (unable to concentrate, forgetful, 
confused) will be assessed at T0 and then hourly (T1-T6).67 Verbal memory, visual memory, executive 
function, and attention be assessed at T0 and then between T2 and T3 using computerized tests from CNS 
Vital Signs™: Verbal Memory Test, Visual Memory Test, Stroop Test, Shifting Attention Test, and Continuous 
Performance Test. Reichenberg et al. assessed cognitive impairment at 1, 3, and 9 hours after endotoxin and 
found it to be worst at 3 hours.20 The order of tests will be counterbalanced across subjects to avoid any non-
random version-dependent bias.99 
Cytokines, Kynurenine Metabolites & Gene Expression Analysis: Blood samples will be collected immediately 
before endotoxin administration (T0) and then hourly (T1-T6). Samples will then be appropriately processed 
and stored for later assessment of proinflammatory cytokines (IL-1 receptor antagonist, IL-6, TNF-α, soluble 
TNF receptor) and KYN metabolites (TRP, KYN, 3HK, QA, KynA). Depressed mood and biomarkers will be 
hourly assessed even after the time of peak responses (T2) because depressed mood can linger and has been 
shown to correlate with cytokines and kynurenine metabolites.16,42 At baseline prior to pretreatment, 
immediately before endotoxin injection (T0), and 30 minutes post-injection (T0.5), blood samples will be drawn 
in PaxGene RNA tubes, which preserve RNA integrity. Genome-wide transcriptional profiling will be conducted 
using Illumina HT-12 BeadArrays. Gene expression analysis will provide information on the underlying 
biological mechanisms of inflammatory and affective responses, and in particular, will be used to independently 
replicate the findings of our prior endotoxin studies. 
 
C.2.6. Experimental Tasks (Main Study Session) 
For Negative Affect: Subjective Sensitivity to Social Rejection (Cyberball Social Exclusion Task): Feelings of 
‘social disconnection’ play a critical role in the onset and perpetuation of (non-inflammatory forms of) 
depression.100 Inflammatory processes can trigger social withdrawal101 and increase feelings of distress and 
related neural sensitivity (i.e., activation of the dorsal anterior cingulate cortex).21 Using the Cyberball Social 
Exclusion Task,102 we will examine feelings of social distress. 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 7 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 measure of social distress in response to social exclusion.103 
Negative Bias in Facial Emotion Recognition (Emotional Face 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.76 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.77 Emotional Face Recognition 
Task104 consists in showing participants a series of black and white photographs (Ekman Pictures of facial 
affect), in which the facial expression is morphed from neutral to either Sad, Angry, or Happy.105 For each 
image, participants 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. 
 

 
11 
For Positive Affect: Subjective Sensitivity to Social Acceptance (Positive Social Feedback Task): Decreased 
sensitivity to positive social experiences, like social acceptance, is another mechanism that might account for 
cytokine-induced feelings of social disconnection and depressed mood. The Positive Social Feedback Task 
was developed based on Izuma et al.’s study involving monetary and social rewards, which demonstrated that 
positive social feedback robustly activates reward-related brain areas, notably the striatum, and these overlap 
with the areas activated by monetary rewards.106 Prior to the experimental session, participants will be asked to 
complete a survey that contains several personality questionnaires (NEO Five Factor Inventory),107 as well as 
several open-ended questions (“What do you do in your free time?” “Briefly describe your personality.” “What is 
your main goal in the future?”). Then, participants will be video-recorded for 2-5 minutes as they discuss what 
they like about themselves. Participants will be told that 8 people (4 male, 4 female) will form impressions of 
them—based on their answers to the personality questionnaires and the video-taped self-introduction—by 
selecting personality traits to describe them. Participants will then see a photograph of themselves along with a 
descriptive word underneath (supposedly provided by the evaluators), which will be pre-rated based on 
desirability, and will be asked to rate subjective happiness when each of the feedback items is presented, 
using a 7-point scale. 
Reward (Reward Learning Task): Anhedonia is a core feature of major depressive disorder and includes a 
reduction in experienced pleasure (liking reward) and dysfunction in reward anticipation reward learning.78-80 
We have found that inflammatory challenge reduces neural sensitivity to reward anticipation.108 We will use a 
laboratory based probabilistic reward task that 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.78-80 This 
computerized reward-learning task is extensively described in Pizzagalli et al.109 
Order and Timing of Experimental Tasks: The order of the tasks will be first a randomized sequence of Reward 
Learning and Emotional Face Recognition, then followed by a randomized sequence of Positive Social 
Feedback and Cyberball Social Exclusion. Because the latter two tasks involve deception, they will be 
completed only once after the former two tasks, at T2, time of peak cytokine responses. The former two tasks 
will be completed at T0 and T2. 
 
C.2.7. Interpretation of Results and Potential Pitfalls 
As leucine may not inhibit endotoxin-induced inflammation, unopposed sickness symptoms could mask the 
antidepressant effects of leucine assessed using the POMS. Thus, the results will be statistically adjusted for 
sickness symptoms. Furthermore, we will use experimental tasks of negative and positive affect to assess 
mood-specific processes. Although the proposed leucine dose is above average,30,87 the adequate 
antidepressant dose is unknown. Thus, if an interim data analysis for the first 30 participants using 12 g/day 
shows no signal of an antidepressant effect, we will consider increasing the dose to 16 g/day for the same 
period of 14 days. Of note, 16 g/day was the maximum dose of leucine used in long-term supplementation 
studies according to a meta-analysis.30 
 
C.2.8. Statistical Analysis 
The basic design is a 2 group (leucine vs. PO placebo) by 2 condition (endotoxin vs. IV placebo) independent 
analysis of variance (ANOVA) with one or more repeated outcome measures analyzed with linear mixed 
models (LMM) with primary focus upon the two endotoxin-exposed groups. The number of repeated measures 
varies by assessment. The key results are the main effects of pretreatment (i.e., group) while the main effects 
of group, condition, and their interaction will be tested. For analyses with repeated measures, the two main 
effects and interaction will further interact with the time variable. POMS will be the primary outcome measure. 
Simes correction for multiple testing will be applied for the secondary outcome measures as they are expected 
to be correlated.110 
 
C.2.9. Justification of Sample Size 
The effect sizes for the reduction of endotoxin-induced depressive symptoms by leucine within the animal data 
are very large (d=.90), but we have assumed some attenuation in humans (d=.70). Assuming an effect of this 
size, with n=35 per group, there is >80% power to detect differences between the leucine and PO placebo 
pretreatments with endotoxin exposure for the single time point outcomes (e.g., Cyberball Social Exclusion); 
comparisons for repeated measures assessments will have more power (>90%) and able to detect more subtle 
effects at >80% power. Both ‘leucine + IV placebo’ and ‘PO placebo + IV placebo’ arms are expected to show 
minimal depression with means over time to be very near zero but exist as reference points to the endotoxin 
condition, which our previous work suggests to have large effects. With repeated measures, n=10 can detect 

 
12 
medium sized effects (d=.50) with power >80%. Thus, there is more than adequate power with 35 vs. 10 to 
show the effect of endotoxin exposure within pretreatment groups. 
 
 

 
13 
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