REGISTERED REPORT PROTOCOL
Identifying causative mechanisms linking
early-life stress to psycho-cardio-metabolic
multi-morbidity: The EarlyCause project
Nicole Mariani
ID
1‡
*, Alessandra Borsini
1‡
, Charlotte A. M. Cecil
2,3
, Janine F. Felix
4,5
,
Sylvain Sebert
6,7,8
, Annamaria Cattaneo
9,10
, Esther Walton
11
, Yuri Milaneschi
12
,
Guy Cochrane
13
, Clara Amid
ID
13,14
, Jeena Rajan
13
, Juliette Giacobbe
1
, Yolanda Sanz
15
,
Ana Agustı
´
15
, Tania Sorg
16
, Yann Herault
16
, Jouko Miettunen
6,17
, Priyanka Parmar
6
,
Nadia Cattane
9
, Vincent Jaddoe
4,5
, Jyrki Lo
¨
tjo
¨
nen
18
, Carme Buisan
18
, Miguel A. Gonza
´
lez
Ballester
ID
18,19
, Gemma Piella
18
, Josep L. Gelpi
20
, Femke Lamers
11
, Brenda W. J.
H. Penninx
11
, Henning Tiemeier
21
, Malte von Tottleben
ID
22
, Rainer Thiel
22
, Katharina
F. Heil
23
, Marjo-Riitta Ja
¨
rvelin
6,24,25,26
, Carmine Pariante
1
, Isabelle M. Mansuy
27
,
Karim Lekadir
23
1 Department of Psychological Medicine, Stress, Psychiatry and Immunology Laboratory, Institute of
Psychiatry, Psychology & Neuroscience, King’s College London, London, United Kingdom, 2 Department of
Epidemiology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands,
3 Department of Child and Adolescent Psychiatry, Erasmus MC, University Medical Center Rotterdam,
Rotterdam, The Netherlands, 4 Generation R Study Group, Erasmus MC, University Medical Center
Rotterdam, Rotterdam, The Netherlands, 5 Department of Pediatrics, Erasmus MC, University Medical
Center Rotterdam, Rotterdam, The Netherlands, 6 Faculty of Medicine, Center for Life Course Health
Research, University of Oulu, Oulu, Finland, 7 Medical Research Council Integrative Epidemiology Unit,
Bristol Medical School, University of Bristol, Bristol, United Kingdom, 8 Faculty of Medicine, Department of
Metabolism, Digestion and Reproduction, Genomic Medicine, Imperial College London, London, United
Kingdom, 9 IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Biological Psychiatry Laboratory,
Brescia, Italy, 10 Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan,
Italy, 11 Department of Psychology, University of Bath, Bath, United Kingdom, 12 Department of Psychiatry,
Amsterdam UMC/Vrije Universiteit & GGZinGeest, Amsterdam Public Health and Amsterdam Neuroscience
Research Institutes, Amsterdam, The Netherlands, 13 European Molecular Biology Laboratory, European
Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom, 14 Department
of Viroscience, Erasmus Medical Center, Rotterdam, Netherlands, 15 Microbial Ecology, Nutrition and Health
Research Group, Institute of Agrochemistry and Food Technology, National Research Council (IATA-CSIC),
Valencia, Spain, 16 Centre Europe
´
en de Recherche en Biologie et Me
´
dicine, Institut de Ge
´
ne
´
tique et de
Biologie Mole
´
culaire et Cellulaire, PHENOMIN-ICS, Universite
´
de Strasbourg, CNRS, INSERM, Strasbourg,
France, 17 Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland,
18 Department of Information and Communication Technologies, Universitat Pompeu Fabra, Barcelona,
Spain, 19 Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain,
20 Department of Biochemistry and Molecular Biomedicine, Universitat de Barcelona, Barcelona, Spain,
21 Department of Social and Behavioral Science, Harvard T.H. Chan School of Public Health, Boston,
Massachusetts, United States of America, 22 Empirica Communication and Technology Research, Bonn,
Germany, 23 Departament de Matemàtiques i Informàtica, Universitat de Barcelona, Barcelona, Spain,
24 Department of Epidemiology and Biostatistics, MRC-PHE Centre for Environment and Health, School of
Public Health, Imperial College London, London, United Kingdom, 25 Unit of Primary Health Care, Oulu
University Hospital, OYS, Oulu, Finland, 26 Department of Life Sciences, College of Health and Life
Sciences, Brunel University London, London, United Kingdom, 27 Medical Faculty of the University of Zu¨rich
and Department of Health Science and Technology of the ETH Zu¨rich, Laboratory of Neuroepigenetics, Brain
Research Institute, Zu¨rich Neuroscience Center, Zu¨rich, Switzerland
‡ These authors share first authorship on this work
* nicole.mariani@kcl.ac.uk
PLOS ONE
PLOS ONE | https://doi.org/10.1371/journal.pone.0245475 January 21, 2021 1 / 18
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
This is a Registered Report and may have
an associated publication; please check the
article page on the journal site for any
related articles.
OPEN ACCESS
Citation: Mariani N, Borsini A, Cecil CAM, Felix JF,
Sebert S, Cattaneo A, et al. (2021) Identifying
causative mechanisms linking early-life stress to
psycho-cardio-metabolic multi-morbidity: The
EarlyCause project. PLoS ONE 16(1): e0245475.
https://doi.org/10.1371/journal.pone.0245475
Editor: Alessandro Bartolomucci, University of
Minnesota, UNITED STATES
Received: June 25, 2020
Accepted: November 27, 2020
Published: January 21, 2021
Copyright: © 2021 Mariani et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data from
this study will be made available upon study
completion.
Funding: This work is supported by the European
Union’s Horizon 2020 research and innovation
programme (grant n˚ 848158). The funders had
and will not have a role in study design, data
Abstract
Introduction
Depression, cardiovascular diseases and diabetes are among the major non-communicable
diseases, leading to significant disability and mortality worldwide. These diseases may
share environmental and genetic determinants associated with multimorbid patterns.
Stressful early-life events are among the primary factors associated with the development
of mental and physical diseases. However, possible causative mechanisms linking early life
stress (ELS) with psycho-cardio-metabolic (PCM) multi-morbidity are not well understood.
This prevents a full understanding of causal pathways towards the shared risk of these dis-
eases and the development of coordinated preventive and therapeutic interventions.
Methods and analysis
This paper describes the study protocol for EarlyCause, a large-scale and inter-disciplinary
research project funded by the European Union’s Horizon 2020 research and innovation
programme. The project takes advantage of human longitudinal birth cohort data, animal
studies and cellular models to test the hypothesis of shared mechanisms and molecular
pathways by which ELS shapes an individual’s physical and mental health in adulthood. The
study will research in detail how ELS converts into biological signals embedded simulta-
neously or sequentially in the brain, the cardiovascular and metabolic systems. The
research will mainly focus on four biological processes including possible alterations of the
epigenome, neuroendocrine system, inflammatome, and the gut microbiome. Life-course
models will integrate the role of modifying factors as sex, socioeconomics, and lifestyle with
the goal to better identify groups at risk as well as inform promising strategies to reverse the
possible mechanisms and/or reduce the impact of ELS on multi-morbidity development in
high-risk individuals. These strategies will help better manage the impact of multi-morbidity
on human health and the associated risk.
1. Introduction
1.1 Early life stress and psycho-cardio-metabolic multi-morbidity
The World Health Organisation has identified mental disorders, including depression, cardio-
vascular diseases and diabetes among the six major non-communicable diseases [1]. Individu-
ally, each of these groups of diseases represents a burden at the individual and population
level. Depression alone is the single largest contributor to global disability, accounting for 12%
of total years lived with disability [2] with more than 300 million individuals affected per year.
Cardiovascular diseases (CVDs) remain the prime cause of mortality worldwide, accounting
for about a third of annual deaths [3]. Finally, type 2 diabetes and related metabolic dysfunc-
tions, including obesity, are a major public health challenge, with an average prevalence of
over 8% in the general population [4]. In addition to their separate complexity, existing
research has shown important multi-morbidity between these diseases, where multi-morbidity
is defined as the co-occurrence of two or more chronic conditions [5]. Epidemiological studies
have indeed shown that for example patients experiencing depression are more likely to have
comorbid CVD [6], type 2 diabetes [7], or both [8]. However, the specific causative mecha-
nisms leading to psycho-cardio-metabolic (PCM) multi-morbidity are not well understood,
which limits the development of effective preventive and therapeutic measures.
PLOS ONE
Causative mechanisms linking early-life stress to psycho-cardio-metabolic multi-morbidity
PLOS ONE | https://doi.org/10.1371/journal.pone.0245475 January 21, 2021 2 / 18
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Recent evidence suggests that many mental and physical conditions find their origins in
exposure to stress early in life, otherwise defined as early-life-stress (ELS) [9]. ELS can be both
prenatal, such as exposure to clinically-significant depression in utero, and postnatal, such as
emotional, physical and sexual abuse or neglect in childhood, parental psychopathology and
separation, prepubertal bullying, as well as victimisation or violence by peers [10]. Growing
evidence has supported an association between ELS (both prenatal and postnatal) and the
development of the PCM conditions. Specifically, patients with a history of ELS have higher
vulnerability for depression [11], and higher risk of developing cardiovascular disease [12],
obesity [13] and type 2 diabetes [14] later in life. Prenatally, the overarching hypothesis is that
the maternal stress response is passed to the fetus, via stress hormones crossing the placenta,
which affects subsequent brain and physical development of the fetus and newborn [15]. Dur-
ing childhood, exposure to excessive levels of stress early in life can cause several biological
alterations which can ultimately favour the development of PCM multi-morbidity [16]. As
suggested by Barker’s work on the developmental origins of chronic diseases, including PCM
conditions [17], the exact predictors of the development of these diseases are to be linked with
variations of key systems during the developmental stage. Examples of key biological system
alterations due to responses to stress include hypothalamic-pituitary-adrenal (HPA) axis
changes as a response to stress [18], changes in the inflammatory response [19], microbiome
dysbiosis [20,21] and overall bio-psycho-social axis dysfunction [22]. While ELS has also been
linked to resilience in adulthood [23], our research will focus on understanding the mecha-
nisms leading to the negative consequences, in particular PCM multi-morbidity. Considering
that the prevalence of ELS, both in utero and postnatally, whether mild or severe, has reached
alarming heights [15], this area of research is essential for future disease prevention and health
promotion.
The objective of this paper is to present the EarlyCause project, a large-scale interdisciplin-
ary research that aims to infer evidence for causative mechanisms linking pre- and postnatal
ELS to PCM multi-morbidity, even decades after the exposure itself has ceased. To explain this
enduring effect, EarlyCause will seek to identify both biological mediators and environmental
moderators. With regards to biological mediators, the hypothesis is that the enduring effects of
ELS may reflect in part a “biologically embedding”, whereby ELS alters biological development
and function in a way that engenders latent vulnerability for poor health outcomes and
increased susceptibility. This is supported by the identification of numerous biological corre-
lates of ELS, and previously mentioned, including neuroendocrine dysregulation, heightened
inflammatory response, changes in gut microbiota composition, and, more recently, alter-
ations to the epigenome. The ambition of EarlyCause is to go well beyond reductionist
approaches which have traditionally investigated new biomedical knowledge while treating
disease classes, biological scales and temporal domains (e.g. childhood vs. adulthood) sepa-
rately. We will thus investigate more holistic models of the causative pathways by building on
the experience of our consortium in advanced methods such as Mendelian randomisation,
structural equation modelling, multi-omics, and machine/deep learning. We will detail below
EarlyCause objectives and hypotheses, as well as the methods that will be implemented to test
these hypotheses (see Method section).
Furthermore, the EarlyCause consortium fully understands that given the prevalence of
ELS affecting millions of mothers and children, important efforts need to be dedicated from
day one to maximising the clinical and socioeconomic impacts. As one of its core objectives,
EarlyCause will ensure that each impact is adequately assessed from clinical and non-clinical
perspectives, as to allow future exploitation of the innovation outputs. This work will be done
while taking into account the current gaps and limitations due to the single-disease frame-
works that have dominated for a very long-time research, biotech innovation and healthcare.
PLOS ONE
Causative mechanisms linking early-life stress to psycho-cardio-metabolic multi-morbidity
PLOS ONE | https://doi.org/10.1371/journal.pone.0245475 January 21, 2021 3 / 18
1.2 Rationale and overview of the EarlyCause project
EarlyCause is the product of a collaboration between 14 participating institutions across
Europe (Table 1) and is supported by the European Union’s Horizon 2020 research and inno-
vation programme (SC1-BHC-01-2019).
EarlyCause will investigate the hypothesis that ELS, as a risk factor for depressive, cardio-
vascular and metabolic disorders individually, is a cause of multi-morbidity between these
conditions. From a biological point of view, the main hypothesis is that ELS activates a chain
of events leading to cellular, molecular, epigenetic and microbial changes from the norm. This
causative chain would ultimately trigger specific cellular and tissue phenotypes and comorbid
pathological traits in the mental, cardiovascular and metabolic domains.
To this end, EarlyCause’s overarching concept is to build upon a unique repertoire of longi-
tudinal data in humans across the lifespan and conduct mechanistic studies in established ani-
mal and cellular models to:
i. Identify the causal mechanisms linking exposure to ELS to the risk of multi-morbid symp-
toms across life;
ii. Delineate the potential molecular mechanisms underlying these causal associations;
iii. Discover new biomarkers tapping into multiple biological domains;
iv. Build integrative computational models and proof-of-concept tools for multi-morbidity
assessment.
The project will focus on four candidate families of biological pathways that have been
linked to ELS, specifically:
1. Epigenetic alterations are a presumed link between stress exposure and phenotypes. Clear
associations between early-life adverse exposure and epigenetic processes (e.g. DNA meth-
ylation) and between these epigenetic modifications and later health outcomes have been
shown both in humans [24–26] and mouse models [27].
2. HPA changes have been associated with ELS exposure [18]. Molecular components of the
HPA axis provide a relay chain across the body from the brain to the periphery, and some
Table 1. Participating institutions in the EarlyCause project.
Participant no. Participant organisation name Acronym Country
1 (Coordinator) Universitat de Barcelona UB ES
2 European Molecular Biology Laboratory EMBL DE
3 Erasmus Medical Centre Rotterdam EMC NL
4 University of Zurich UZH CH
5 King’s College London KCL UK
6 Consejo Superior de Investigaciones Cientificas CSIC ES
7 Centre Europe
´
en de Recherche en Biologie et Me
´
dicine CERBM FR
8 University of Oulu OULU FI
9 Fatebenefratelli Institute IRCCS IT
10 University of Bath UOB UK
11 VU Medical Centre, Amsterdam VUMC NL
12 Empirica Communication and Technology Research EMP DE
13 Combinostics Oy COMBI FI
14 Universitat Pompeu Fabra UPF ES
https://doi.org/10.1371/journal.pone.0245475.t001
PLOS ONE
Causative mechanisms linking early-life stress to psycho-cardio-metabolic multi-morbidity
PLOS ONE | https://doi.org/10.1371/journal.pone.0245475 January 21, 2021 4 / 18
of the final products (glucocorticoid hormones) are potent regulators of glucose and lipid
metabolism. Thus, this represents a central candidate mechanistic player in the aetiology of
multi-morbid symptoms.
3. Inflammatory pathways are a form of cellular response to ELS [19] reflecting activation of
white blood cells in the circulation and peripheral tissues such as the spleen, lymph nodes
and adipose tissue. Inflammatory components may have profound effects on the cardiovas-
cular system, endothelial accumulation and activation of plaques, and adipose tissue metab-
olism, whose dysfunction has been associated with stress-related diseases including
depression, cardiovascular disease and diabetes.
4. Gut microbiome is a major contributor to health and disease [20,21], which plays a key
role in modulating immune, neuroendocrine and behavioural responses to ELS, as proven
mainly in mouse models [28].
In addition to these biological factors, EarlyCause will test the potential moderating role of
key factors such as sex, socioeconomics, and lifestyle in the association between ELS and
multi-morbidity development. Evidence for causality, mediation and moderation will be used
to identify potential targets for intervention acting on the causative mechanisms to reduce the
impact of ELS on multi-morbidity development in high-risk individuals.
Specifically, using longitudinal human data, as well as animal and cellular models we aim to
address the following hypotheses (Table 2).
2. Methods
EarlyCause’s methodology is divided into multiple steps. As shown in Fig 1, the study protocol
aims to triangulate evidence based on (1) longitudinal human data, (2) animal and cellular
Table 2. Table of hypotheses for longitudinal human data, animal and cellular models.
Model Hypotheses
Longitudinal human data
(section 2.1)
Hypothesis 1: Early-life stress, including sexual, physical, psychological abuse and/
or neglect in early life, is a causal factor in the development of PCM multi-
morbidity. Specifically, ELS is a shared risk factor for pre-clinical psychological and
cardiometabolic symptoms in childhood and PCM multi-morbidity in adulthood;
Hypothesis 2: Specific alterations in DNA methylation inflammation,
neuroendocrine function, and/or the gut microbiota mediate the ELS effects on
PCM multi-morbidity;
Hypothesis 3: The association between ELS and PCM is modifiable by lifestyle
factors. We hypothesise positive moderation (prevention) by physical activity,
dietary factors and sleep. In contrast, we hypothesise that the association is
exacerbated by smoking and alcohol consumption;
Hypothesis 4: PCM multi-morbidity is partly heritable and the individual genetic
determinants of depression, and cardiometabolic diseases form a joint genetic
factor of PCM symptoms in child- and adulthood.
Animal model (section 2.2) Hypothesis 1: Exposure to pre- and postnatal stress induces behavioural,
cardiovascular and metabolic changes across adulthood in mice;
Hypothesis 2: The effect of pre- and postnatal stress on the above outcomes
(Hypothesis 1) is mediated by similar epigenetic and molecular changes associated
with PCM multi-morbidity symptoms previously identified in the human model.
Cellular model (section 2.3) Hypothesis 1: Exposure of cells from the brain and peripheral organs to stress-
related insults (cortisol and cytokines) induces cellular changes relevant to PCM
multi-morbidity;
Hypothesis 2: Mechanisms underlying the effect of the stress-related insults on the
aforementioned cell types (Hypothesis 1) are mediated by molecular and gene
expression changes previously identified both in the human and animal models.
https://doi.org/10.1371/journal.pone.0245475.t002
PLOS ONE
Causative mechanisms linking early-life stress to psycho-cardio-metabolic multi-morbidity
PLOS ONE | https://doi.org/10.1371/journal.pone.0245475 January 21, 2021 5 / 18
