PERSPECTIVE

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Perspective: The Role of Human Breast-Milk
Extracellular Vesicles in Child Health and Disease
Daniel O’Reilly,1,2 Denis Dorodnykh,3 Nina V Avdeenko,3 Nikita A Nekliudov,3 Johan Garssen,4 Ahmed A Elolimy,5,6
Loukia Petrou,7 Melanie Rae Simpson,8,9 Laxmi Yeruva,5,6,10 and Daniel Munblit3,11,12,13
1 Children’s Health Ireland at Temple Street, Dublin, Ireland; 2 SPHERE Research Group, Conway Institute, University College Dublin, Dublin, Ireland;
3 Department of Pediatrics and Pediatric Infectious Diseases, Institute of Child’s Health, Sechenov First Moscow State Medical University (Sechenov University),

Moscow, Russia; 4 Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, Netherlands;
5 Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, USA; 6 Arkansas Children’s Nutrition Center, Little Rock, AR, USA;
7 Department of Bioengineering, Imperial College London, London, United Kingdom; 8 Department of Public Health and Nursing, Norwegian University of

Science and Technology, Trondheim, Norway; 9 Clinic of Laboratory Medicine, St. Olavs Hospital , Trondheim, Norway; 10 Arkansas Children’s Research Institute,
Little Rock, AR, USA; 11 Inflammation, Repair, and Development Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London,
London, United Kingdom; 12 inVIVO Planetary Health, Group of the Worldwide Universities Network, West New York, NJ, USA; and 13 Research and Clinical
Center for Neuropsychiatry, Moscow, Russia

ABSTRACT
Human breast milk (HM) contains multiple bioactive substances determining its impact on children’s health. Extracellular vesicles (EVs) are a
heterogeneous group of secreted nanoparticles that are present in HM and may be partially responsible for its beneficial effects. The precise roles
and content of EVs in HM remain largely unknown. To examine this, we performed a short narrative review on the literature focusing on HM EVs to
contextualize the available data, followed by a scoping review of MEDLINE and Embase databases. We identified 424 nonduplicate citations with
19 original studies included. In this perspective, we summarize the evidence around HM EVs, highlight some theoretical considerations based on
existing evidence, and provide an overview of some challenges associated with the complexity and heterogeneity of EV research. We consider how
the existing data from HM studies conform to the minimal information for studies of EVs (MISEV) guidelines. Across the studies a variety of research
methods were utilized involving both bench-based and translational methods, and a range of different EV contents were examined including RNA,
proteins, and glycopeptides. We observed a variety of health outcomes in these studies, including allergy and atopy, necrotizing enterocolitis, and
HIV. While some promising results have been demonstrated, the heterogeneity in outcomes of interest, methodological limitations, and relatively
small number of studies in the field make comparison between studies or further translational work problematic. To date, no studies have examined
normative values of HM EVs in a large, diverse population or with respect to potentially important influencing factors such as timing (hind- vs.
foremilk), stage (colostrum vs. mature milk), and infant age (preterm vs. term), which makes extrapolation from bench or “basic”research impossible.
Future research should focus on addressing the current inadequacies in the literature and utilize MISEV guidelines to inform study design. Adv Nutr
2021;12:59–70.

Keywords: breast milk, extracellular vesicles, exosomes, human milk, microvesicles, nanovesicles

Introduction this to the variation in the constituents of HM within and

Human breast milk (HM) is a unique body fluid containing between individuals (1). Although PUFAs, HMOs, and the
a plethora of constituents with multiple functions (1). HM microbiome remain the main focus of HM research, recent
can be described as a combination of immunologically active studies suggest that HM extracellular vesicles (EVs) may
factors, PUFAs, human milk oligosaccharides (HMOs), and a also contribute to the short- and long-term benefits of
complex microbiome, which may potentially influence health breastfeeding (8–10).
outcomes in infancy and early childhood (1–4). Epidemio- EVs are a heterogeneous group of secreted nanoparticles,
logical studies of breastfeeding have shown apparent benefits often referred to as distinct subsets, such as “exosomes,”
for both the infant and mother (5–7). However, the exact “apoptosomes,” “oncosomes,” “microvesicles,” and “platelet
HM constituents responsible for the protective effect remain dust,” with a wide range of roles in human health and
unknown. Breastfeeding has not been consistently observed disease (11, 12). Although originally believed to be derived
to be protective across the studies and some experts attribute from megakaryocytes and platelets, recent studies have

Copyright 
C The Author(s) on behalf of the American Society for Nutrition 2020. Adv Nutr 2021;12:59–70; doi: https://doi.org/10.1093/advances/nmaa094. 59
demonstrated that they are produced by most cell types evidence is very sparse, we reviewed available knowledge to
(12). EVs can be identified in various biological fluids, such highlight unmet needs for future research.
as urine, blood, cerebrospinal fluid, and breast milk (13–
15), and several studies have demonstrated that they play Methodological Approach
a role in both infectious and noncommunicable diseases This Perspective is the result of a narrative review and
(12, 16, 17). subsequent systematically conducted scoping review. We
Both human and animal milk have been examined for followed the methods outlined in the PRISMA-ScR (Pre-
the presence and function of EVs (18–21). A number of ferred Reporting Items for Systematic reviews and Meta-

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these studies have had a strong focus on the possible impact Analyses extension for Scoping Reviews) (33). We conducted
of these particles on immune system and gastrointestinal a comprehensive search in the bibliographic databases
tract development in infancy (8, 22–26), highlighting the role MEDLINE and Embase using both free-text and MeSH
of mammalian breast-milk EVs in immunity, metabolism, (medical subject heading) terms to identify bibliographic
and infant development (19, 27–32). Yet, methodological records involving HM and EVs. To reduce the potential
differences in approaches to EV analysis make it difficult to for selection bias, titles, abstracts, and full-text articles were
compare and combine the available data. Recently published screened by 2 reviewers (DO and DD) independently for
minimal information for studies of EVs (MISEV) guidelines eligibility, and any disagreements were resolved through
outlined a methodological framework for EV research to discussion involving additional reviewers (MRS and DM)
allow for reproducibility of results given the current technical until consensus was reached. A full description of the search
limitations in examining EVs and their contents (11). The strategy, study selection, and exclusion process may be found
guidelines encourage the use of a standardized collection in Supplemental Figure 1. The search resulted in 598
and processing method and utilizing multiple methods to titles, of which 19 studies were eligible for inclusion in the
describe the EVs of interest in both their structure and qualitative analysis. The heterogeneity of these studies, both
function. in terms of their aims and methods, does not lend itself to
A systematic evaluation of available data on EVs in HM is any meta-analysis of their results. In the following sections
lacking. This Perspective aims to first evaluate the available we summarize and discuss the findings of these 19 studies
evidence on the role of HM EVs on infant and child health into HM EVs, placing them within the broader context of EV
defined by the authors as conditions or diseases that occur research. Details of methodology used may be found in the
or first develop in the neonatal, infant, or childhood part of Supplemental Methods.
the human life cycle [e.g., necrotizing enterocolitis (NEC),
bronchopulmonary dysplasia (BPD), and allergic diseases]. Characteristics of the Studies Examining EVs in
Second, we aimed to critically appraise the concordance of HM
published data with the MISEV guidelines (11). Although A comparable number of studies used a translational ap-
approaches to EV assessment in HM are variable and existing proach (9/19) (8, 26, 34–40) and a descriptive or basic science
AE and LY are supported by USDA-ARS project 6026–51000–010–06S. In addition, LY is
approach (10/19) (22–25, 41–46). Most research was carried
supported by NIH P20GM121293 and NIH R21AI146521. DM acknowledges the receipt of out in the United States (8 studies) or used US subjects
funding from the “5–100” Russian Academic Excellence Project. (42%) (22, 23, 25, 26, 35, 36, 41, 45), 4 studied Swedish
Author disclosures: DM received funding from the European Cooperation in Science and
Technology (COST) and the “5-100” Russian Academic Excellence Project and is involved in
populations (21%) (8, 34, 42, 44), and the remaining studies
expert activities within the ILSI Europe expert group. He also has given paid lectures for Merck, took place in the Netherlands (43), China (24, 37), Israel (38),
Sharp & Dohme (MSD) and Bayer. MRS receives funding from the Liaison Committee between Japan (40), Poland (46), and Norway (39). While the number
the Central Norway Regional Health Authority (RHA) and the Norwegian University of Science
and Technology (NTNU). All the other authors report no conflicts of interest.
of participants and/or samples greatly varied between the
Perspective articles allow authors to take a position on a topic of current major importance or studies, the sample size in most of the studies was relatively
controversy in the field of nutrition. As such, these articles could include statements based on small, with between 1 and 61 subjects included (41, 44) and
author opinions or point of view. Opinions expressed in Perspective articles are those of the
author and are not attributable to the funder(s) or the sponsor(s) or the publisher, Editor, or
between 1 and 54 samples analyzed (41, 44). Eight studies did
Editorial Board of Advances in Nutrition. Individuals with different positions on the topic of a not provide details on the number of subjects or samples (25,
Perspective are invited to submit their comments in the form of a Perspectives article or in a 26, 34–36, 38, 42, 45).
Letter to the Editor.
Supplemental Methods, Supplemental Figure 1, and Supplemental Table 1 are available from
Milk-collection time differed both within and between
the “Supplementary data” link in the online posting of the article and from the same link in the identified studies with timing of the “mature milk” varying
online table of contents on https://academic.oup.com/advances. from day 4 of life (8) to 11 months (40), and 4 studies had no
DO, MRS, LY, and DM contributed equally to this work.
Address correspondence to DM (daniel.munblit08@imperial.ac.uk) or DO (e-mail:
description of the infant’s age at the time of milk collection
danieloreilly@alumnircsi.com). (27%) (26, 35, 36, 42). Colostrum was inconsistently defined,
Abbreviations used: ALADDIN, Allergic Disease During INfancy; BPD, bronchopulmonary with 1 study collecting samples in the first 3 d of life (37), a
dysplasia; CD, cluster of differentiation; DANCR, differentiation antagonizing non-protein
coding RNA; EV, extracellular vesicle; GAS5, growth arrest-specific 5; HLA, human leukocyte
second study describing milk collected during the first 4 d of
antigen; HM, human breast milk; HMO, human milk oligosaccharide; HSC, heat shock cognate; life as colostrum (8), and a third describing HM collected at
IEC-6, intestinal cell line 6; MDDC, monocyte-derived dendritic cell; miR, microRNA; MISEV, 1 to 3 mo as “early milk” (23) (Table 1).
Minimal Information for Studies of Extracellular Vesicles; MSC, mesenchymal stem cell; MUC-1,
mucin 1 cell surface–associated; NEC, necrotizing enterocolitis; PBMC, peripheral blood
Mechanical expression or expression into sterile tubes
mononuclear cell; ProPACT, Probiotics in the Prevention of Allergy among Children in using a breast pump were the most commonly described
Trondheim; SRA1, steroid receptor RNA activator 1; Treg, regulatory T cell. methods of sample collection (8, 22–25, 34, 38, 41, 44, 46).

60 O’Reilly et al.
 TABLE 1 Summary of characteristics of the studies and EVs1

Study (reference) Subjects, Number of Collection Milk-collection Size of EVs,

(year) Location n samples procedure times nm Protein/RNA/glycosome content
Admyre et al. (8) Sweden 17 25 (8 samples Manual breast pump 4 d–6 mo 50 Protein: HLA-DR, CD63, CD81, HLA-ABC,
(2007) colostrum, 17 in sterile tubes CD40, CD54, CD80, lactadherin
samples mature (MFG-E8). Unique: CD36, Butyrophilin
milk) and polymeric Ig receptor, MUC-1
Batista et al. (41) USA 1 1 Mechanical 27 d N.R. Protein: CD63/CD81 used to determine
(2011) expression "microvesicles"; glycome: no clear
description of breast-milk glycome,
focused on cell line glycomes
Golan-Gerstl et al. (38) Israel N.R. N.R. Manual breast pump 1 mo N.R. RNA: miR-148–3p, miR-6073, let-7a-5p,
(2017) in sterile tubes miR-30a-5p, miR-99a-5p, miR-146–5p,
miR-200a, miR-21–5p, miR-30d
Kahn et al. (22) (2018) USA 20 10 term samples, Manual breast pump 5 d–4 wk (preterm N.R. Protein: CD54/lactoferrin; RNA: 21 preterm
10 preterm in sterile tubes samples collected specific miRNAs miR-22–3p most
samples 25–32 wk abundant, miR-148a-3p, miR-146a
(digested and corrected
undigested) gestational age)
Karlsson et al. (25) USA/Sweden 30 30 Manual breast pump 0–2 mo 100 Protein: CD63/CD9 used to determine EVs;
(2016) RNA: lncRNAs: CRNDE, DANCR, GAS5,
SRA1, ZFAS1 (90–100%) HOTAIRM1,
NCBP2-AS2, OIP5-AS1, PRKCQ-AS1, SHNG8,
TUG1 (50%)
Kosaka et al. (40) Japan 8 24 samples, N.R. 4 d–11 mo N.R. Protein: CD63; RNA: miR-155, miR-181a and
(2010) different time miR-181b, miR-17, miR-92 cluster,
points for each miR-125b, miR-146b, miR-223, let-7I
subject, 1
subject with no
timings
provided
Lässer et al. (42) Sweden N.R. N.R. N.R. N.R. 50–80 Protein: CD9/CD63/CD81 used to
(2011) determine EVs; RNA: mRNA qualitatively
assessed but N.R., nil specific constructs
Leiferman et al. (45) USA 5 N.R. N.R. 2–10 mo 128.3–169.9 Protein: CD63/CD9/Alix; RNA: miR-30d-5p,
(2019) let-7b-5p, let-7a-5p, miR-125a-5p,
miR-423–5p, miR-21–5p, miR-423–5p,
let-7g-5p, let-7f-5p, miR-30a-5p,
miR146b-5p
Liao et al. (23) (2017) USA 12 12 Breast pump into 1.5–8 mo 30–120 Protein: CD9/lactoferrin; RNA: 288 miRNA
sterile tubes with identified; noted immune active miRNA
RNAse inhibitor miR-22–3p, miR-181c-5p, miR-181a-5p,
miR-16–5p, miR-148a-3p
Lukasik et al. (46) Poland 6 6 split into Mechanical 1.5–8 mo N.R. RNA: specifically examined 5 plant-based
(2018) whole-milk and expression RNA constructs, 2 identified miR168a,
“exosome” miR156a
fraction

Breast-milk EVs in child health 61

(Continued)

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62
TABLE 1 (Continued)

O’Reilly et al.
Study (reference) Subjects, Number of Collection Milk-collection Size of EVs,
(year) Location n samples procedure times nm Protein/RNA/glycosome content
Martin et al. (26) USA N.R. N.R. Leftover samples of N.R. 74.6(26.6–122.6) Protein: CD81/clathrin used to determine
(2018) breast milk from "exosomes"
NICU
Näslund et al. (34) Sweden N.R. N.R. Manual breast pump N.R. N.R. Protein: MUC-1, LFA-1
(2014) in sterile tubes
Sims et al. (35) (2017) USA N.R. N.R. Leftover samples of N.R. 116.2 Protein: CD9/CD63
breast milk from
NICU
Sims et al. (36) (2018) USA N.R. N.R. Scavenged samples N.R. 94 ± 3.4 Protein: CD81/clathrin
of breast milk from
the NICU
Simpson et al. (39) Norway 54 54 samples: 32 Collected into sterile 3 mo N.R. RNA: miR-148a-3p, miR-22–3p, miR-30d-5p,
(2015) from probiotic tubes, method not let-7b-5p, and miR-200a-3p
arm; 22 from reported
placebo arm
Torregrosa Paredes et Sweden 61 22 paired samples; Manual breast pump Early: days 3–8; late: N.R. Protein: early: high HLA-DR, low HLA-ABC,
al. (44) (2014) 47 comparing in sterile tubes 2 mo MUC-1 higher in sensitized vs.
impact of nonsensitized, MUC-1 lower in
anthroposophic anthroposophic vs.
lifestyle (8 of nonanthroposophic, high CD63
which paired anthroposophic
samples)
van Herwijnen et al. Netherlands 7 7 (10–35 mL) Breast pump mixed 3–9 mo N.R. (sucrose Protein: unique to this study: OLAH (oleal
(43) (2016) and 10–50 density) acp hydrolase), parathyroid
mL aliquoted hormone–related protein, MPZL1, ehd3,
3331 unique proteins total
Wang et al. (37) (2019) China N.R. N.R. N.R. N.R. 50–100 Protein: CD9/CD63, different expression of
70 peptides between preterm and term
EVs
Zhou et al. (24) (2012) China 4 4 (20–50 mL) Manual breast pumps 60 d N.R. RNA: miR-148–3p (most common and with
in sterile tubes TGIF2, PXR, DNMT3B as known targets),
miR-30b-5p (immunosuppression),
miR-182–5p, miR-200a-3p, let-7 family
(let-7f-1–5p, let-7f-2-5p, let-7a-2–5p, and
let-7a-3–5p)
1
CD, cluster of differentiation; CRNDE, colorectal neoplasia differentially expressed; DANCR, differentiation antagonizing non-protein coding RNA; EV, extracellular vesicle; GAS5, growth arrest-specific 5; HLA, human leukocyte antigen; miR,
microRNA; MUC-1, mucin 1 cell surface–associated; NICU, neonatal intensive care unit; N.R., not reported; SRA1, steroid receptor RNA activator 1; ZFAS1, ZNFX1 antisense RNA 1.

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Details on type of breast pump used were not provided in and extended storage (40, 49, 50). EVs confer protection by
any of the above studies. Other methods included utilizing enclosing miRNAs and preventing ribonuclease degradation,
remaining expressed milk following feeding from neonatal thus enabling cell-to-cell communication (51, 52). Through
intensive care (26, 35–37) (Table 1). Substantial variation in our search, we found 5 studies that investigated the general
the size of EVs in HM samples collected using the same RNA content of EVs (25, 38, 40, 42, 46). Another 5 papers
methodology in the same clinical settings was reported in considered the RNA content of HM EVs in association with
some studies (26, 35, 36) (Table 1). A single study described digestion (22–24), allergic outcomes (39, 44), and preterm
the timing of milk collection as “mid-stream” (46). No infants (22). These papers are described in more detail in later

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differentiation was made on any of the studies between sections.
foremilk or hindmilk. Lässer et al. (42) examined EVs in saliva, plasma, and
Overall, we also observed a wide range of outcomes breast milk. This study concluded that breast-milk EVs con-
considered across these studies. Two studies examined tain RNA, while not defining which specific RNA constructs
digestion survival of EVs (22, 23). A small number of were involved. As per MISEV 2018 guidelines, authors as-
studies examined atopy specifically (39, 44), with 3 studies sessed EVs using multiple methodologies, including electron
examining HIV transmission with conflicting results (34– microscopy, flow cytometry, and Western blot (11). They also
36). While many researchers examined predictive immune- investigated the ability of HM EVs to be taken up by recipient
regulatory RNAs in breast-milk–derived EVs (8, 22–25, 38, cells (cultured human macrophages) using fluorescent labels
42), only a single study on this topic demonstrated an effect (42). RNA content of HM-derived EVs was examined
of these EVs in a cell culture model (8). further by Karlsson et al. (25) who assessed long noncoding
RNAs and identified a range of long noncoding RNAs
Challenges in Characterizing and Studying EVs implicated in the immune system [e.g., growth arrest-specific
Similar to other areas, the field of EV biology is subject to 5 (GAS5), ZNFX1 antisense RNA 1 (ZFAS1), steroid receptor
rapid change where evolving technologies and terminologies RNA activator 1 (SRA1), and differentiation antagonizing
make comparison between studies difficult (11). Analysis non-protein coding RNA (DANCR)] and metabolism [e.g.,
of EVs and their contents is inherently difficult as their GAS5, SRA1, colorectal neoplasia differentially expressed
small size means their contents can become dilute relative (CRNDE)] (Table 1). This study demonstrated a robust
to the surrounding fluid. While several studies included characterization of EV content utilizing electron microscopy,
in this review were cognizant of this, many used only a with labeling using anti-CD63/CD9 and nanoparticle track-
single method of confirming that the contents they described ing analysis used in addition to a commercially available
were truly EV-derived [e.g., cluster of differentiation (CD) EV isolation kit (25). Kosaka et al. (40) used anti-CD63
63 fractions of HM] (40). As described, the MISEV guidelines antibody–coated magnetic beads to extract EVs. Although
provide us with useful advice about how to approach EV the study did not demonstrate similar rigor when defining
research. While many of the articles we identified were EV population, high expression levels of immune-related
published before the most recent version of these guidelines, miRNAs were observed in the CD63-positive fraction during
we considered the recommendation of the MISEV guidelines first 6 mo of lactation. The authors reported HM miRNAs
as we reviewed the studies characterizing HM EV contents, being stable even upon acidic (pH 1) solution treatment. The
their capacity to survive digestion, as well as their potential presence of microRNA (miR)-181a and miR-17 in a CD63-
role in allergic and other immunological outcomes, preterm positive fraction of milk supported the presence of EVs as
infants, and NEC, and in the context of future therapeutics. delivery vehicles (40). While the exact functions of these HM
EVs are largely unknown, it appears that miRNA in HM EVs
Studies Characterizing HM EV Content are reasonably conserved with overlapping miRNA profiles
We identified 10 papers that characterized the nucleomic, found across different human populations and mammalian
proteomic, or glycomic contents of HM EVs. However, there species.
was heterogeneity in the techniques used to isolate EVs, as Looking to the animal world, existing data suggest
well as how they were characterized (Supplemental Table 1). that bovine-milk EVs are enriched in miRNAs that are
The details of the studies’ methodologies are outlined in brief involved in both the adaptive and innate immune responses
as we consider this information useful for any potential future (53). Porcine-milk EV–associated miRNAs are also thought
EV research. to target genes within immune system, metabolism, and
development, including through members of the let-7 family,
Ribonucleic Acids in EVs miR-181a and miR-320 (19). HM EVs and HM-associated
EVs are enriched with microRNAs (miRNAs), which are miRNAs may also play a role in the regulation of metabolism.
short nucleic acids that are involved in post-transcriptional Although this possibility is poorly understood in humans, ev-
gene regulation (47). miRNAs have been shown to be very idence for the role of mammalian-milk EVs and metabolism
stable in HM despite the prevalence of ribonucleases (40, in other mammals is better established (18, 23). For instance,
48). The stability or concentration of miRNAs has been porcine EVs contain miRNAs associated with regulation
shown to persist even after HM was subjected to extreme of key metabolic pathways including the citrate cycle and
temperature and pH changes, multiple freeze-thaw cycles, glycerophospholipid metabolism (19). Both rat and mouse

Breast-milk EVs in child health 63

models showed differences in muscle physiology in those Zhou et al. (24) provided evidence of miRNA cargo preserva-
that received an oral gavage of EVs that were RNA sufficient tion after treatment with RNAse, multiple freeze-thaw cycles,
versus those treated with RNA-depleted EVs (30, 54). Some and incubation at 100◦ C. The miRNA cargos identified
mechanistic evidence was also found to support a role of EV- in all 3 studies were predominantly immune-associated,
associated miRNAs in the regulation of the mammalian tar- with a good agreement between the authors (22–24). Liao
get of rapamycin complex (mTORC) pathway, a key regulator et al. (23) also characterized cargo at different stages of
of both growth and development (55). Additionally, bovine- lactation, providing exosomes and miRNA profile for “early,”
milk EVs have been shown to survive in vitro digestion “mid,” and “late” lactation, and showed significant variation

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(56), suggesting they can survive the transit through the in exosome concentrations and miRNA counts with the
intestinal tract and deliver their various protein and nucleic relative abundance of miRNAs changing according to timing,
acid contents. and further validating the importance of timing in breast-
It has also been speculated that exogenous dietary milk composition. These findings suggest the physiological
miRNAs could be transferred via breast milk in addition relevance of miRNA and protein readings in undigested
to the mother’s endogenous miRNAs. Lukasik et al. (46) HM (22, 23). However, the physiological concentration
isolated EVs using an EV precipitation solution with no of HM EVs remains largely unknown, with only 1 study
other specific methodology used to characterize EVs. Two identifying the concentration of EVs per milliliter in a range
of the plant food–derived miRNAs were present in both of HM (23). Further studies pertaining to EV concentra-
whole milk and the EV fraction of HM (miR-156a, miR- tions would benefit future research and allow for design
168a), although no significant difference in expression was of better dose–response studies to examine whether an
found between vegetarian and nonvegetarian mothers in the average infant ingests sufficient quantities of these particles
EV fraction. Leiferman et al. (54) examined not only the to generate the responses demonstrated in experiments to
stability of HM EVs in storage but also set out to identify date.
miRNA profiles in both HM and infant formula. While
successfully demonstrating the presence of miRNA in HM HM EVs’ Role in Allergic and Immunological
EVs, they could not replicate this with infant formula. They Outcomes
suggested that EV-like particles in formula milk represent HM has been shown to play a role in protection from upper
casein micelles. It is also possible that miRNAs are depleted respiratory tract, ear, and gastrointestinal infection, and is
during the processing of cow milk to infant formula. thought to have longer lasting effects on the development
of the immune system. The role of EVs in these outcomes
has been recently appreciated. For example, Admyre et al. (8)
Proteins in HM EVs
studied the role of HM EVs in immunomodulation. Differ-
Using proteomics analysis of HM EVs, van Herwijnen et al.
ential ultracentrifugation was utilized to enrich a sample of
(43) revealed >600 proteins not previously detected in HM.
HM EVs, described in the paper as “exosome-like particles.”
While non-EV milk proteins are involved in metabolism and
The EVs were analyzed using electron microscopy and flow
the “immune response,” EV milk proteins were associated
cytometry. Trypsinization of EVs followed by Western blots
with signal transduction, controlling inflammatory signaling
demonstrated EV markers human leukocyte antigen–DR
pathways, suggesting that EVs could support the newborn’s
isotype (HLA-DR), heat shock cognate (HSC) 70, and CD81.
gut and immune system (43). In particular, many HM EV
This was further confirmed by flow cytometry; interestingly,
proteins are associated with the “cytoskeleton” and “plasma
HLA-DR was higher in “colostrum derived” (0–4 d of life)
membrane.” This is the only study to date that has used
EVs than in “mature milk.” Additionally, proteomics analysis
a high-throughput method to assess the protein content
identified additional proteins [e.g., lactadherin, mucin 1 cell
of HM EVs, with other studies analyzing specific proteins
surface–associated (MUC-1)] and confirmed EV markers
or surface markers in association with digestion or infant
such as CD81, HSC, and major histocompatibility complex
outcomes. This is also the case for animal studies where,
II. Finally, the function of EVs was assessed by an in
for example, bovine-milk EVs have been found to contain
vitro assay using both autologous and allogeneic maternally
transforming growth factor β (TGF-β), a cytokine known for
derived peripheral blood mononuclear cells (PBMCs) by co-
its importance in both mucosal barrier formation and T-cell
culturing with HM EVs for 4 d. This showed a reduction in
regulation promoting a T-helper 17 (Th17) phenotype (57).
IL-2, IFN-γ , and TNF-α and an increase in IL-5 expression
in a concentration-dependent manner, with 500 μg/mL
HM EVs and Digestion Survival (0.5 g/L) of EVs producing significant changes in all 4 of
Three studies provided evidence for gastric and pancreatic these cytokines and 50 μg/mL (0.05 g/L) of EVs resulted in a
digestion survival of miRNA and protein cargos within EVs statistically significant increase only in IL-5. In the case of the
in vitro, supporting the hypothesis that intact EV cargos changes seen with 0.5 g EVs/L, it is unclear if this corresponds
are delivered to intestinal cells where they may elicit their to physiologically relevant concentrations of EVs. In another
functions (22–24). Liao et al. (23) and Kahn et al. (22) study, Liao et al. (23) found that exosome concentrations
demonstrated that EVs can survive gastric and pancreatic in 8 breast-milk samples ranged from 0.06 g/L to 0.31 g/L
digestion while preserving their miRNA and protein cargos. of EVs. As such, the increased IL-5 production by PBMCs

64 O’Reilly et al.
incubated with 0.05 g/L of EVs is probably physiologically and placebo group or those with atopic dermatitis versus no
relevant, but cytokine changes after incubation with 0.5 g/L atopic dermatitis.
of EVs are unlikely to occur physiologically. Additionally, Although both studies demonstrated primarily negative
regulatory T-cell (Treg) analysis with allogeneic adult PBMCs results, failing to identify a strong effect of HM-derived
alone demonstrated an increase in forkhead box P3+ EVs on atopy and allergy (39, 44), the outcomes are
(FOXP3+ ) cells, a key transcription factor involved in the thought provoking. This may be due to the modest effect
expression of the Treg and anti-inflammatory phenotype (8, size of EVs in atopy and allergy given what is known of
58) in the presence of EVs. Nevertheless, small sample size the multifactorial pathogenesis of these conditions to date

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along with filtration technique using 0.45-μm pores that (61). Currently available information does not conclusively
could allow cell debris passage into the supernatant could determine if EVs are beneficial in protection from infections,
have yielded unrealistic EV concentrations, so the inferred allergic outcomes, or other immunological diseases. A large,
physiological relevance remains questionable. Additionally, multicenter prospective study would likely be required to
these findings represent the effects observed when PBMCs achieve the desired power to show the individual role of
are incubated with EVs in concentrations found within breast breast-milk EVs on the outcome of an atopic phenotype.
milk. The HM EV concentration in an infant’s circulation
would be substantially lower and therefore it is uncertain HM EVs and HIV
whether there would be sufficient concentrations to exert Three papers assessing the role of HM EVs in HIV trans-
similar effects on infant PBMCs. mission were identified; however, they yielded contradictory
Two large cohorts of patients were examined, 1 set from conclusions (34–36). Näslund et al. (34) demonstrated
the Assessment of Lifestyle and Allergic Disease During that EVs derived from monocyte-derived dendritic cells
INfancy (ALADDIN) study and the other from the Probiotics (MDDCs) of healthy donors, but not EVs derived from their
in the Prevention of Allergy among Children in Trondheim plasma, inhibited HIV-1 infection if they were preincubated
(ProPACT) trial, both of which examined atopy and allergic with HM EVs. Furthermore, co-culture of preincubated
phenotypes in the context of breastfeeding and breast-milk– MDDCs with HM exosomes and CD4− T cells showed
derived EVs (39, 44). This concept has good mechanistic prevention of HIV transfer from MDDCs to CD4+ T cells.
basis given what has been described in other included The authors suggested that HM-derived EVs may provide
literature of HM EVs inducing a Treg phenotype. protection against vertical transmission of HIV infection
Torregrosa Paredes et al. (44) explored the following (34). However, Sims et al. (35) found an enhanced HIV infec-
2 questions using samples from the ALADDIN cohort: 1) tion when treated with HM-derived EVs and demonstrated
does maternal lifestyle and sensitization affect HM EV ex- that HIV utilized exosomes entering into human immune
pression profile and 2) does the HM EV profile change from cells. In a follow-up study, Sims et al. (36) found that treating
“early” to “mature” milk? They found that EV phenotype cells with a CD9 antibody prevented augmentation of HIV-
changed in “early” (day 3–8) versus “mature” (2 mo) milk 1 entry into cells in the presence of HM EVs, indicating this
with high HLA-DR and low HLA-A,B,C expression in early process is tetraspanin dependent.
samples versus mature samples. Maternal lifestyle was found Possible reasons for the contradictory data could be
to be associated with EV expression in mothers following differences in measurement of infection—that is, the use of
a lifestyle characterized by the authors as “anthroposophic” virus-produced luciferase versus protein markers and the
(characterized by home deliveries, organic diet, and reduced direct inoculation of T-cell–like culture in the study by the
use of medications). Women characterized by an anthro- Sims group versus the use of MDDC co-culture in the paper
posophic lifestyle had significantly lower mucin (MUC-1) by the Näslund group. Similarly, differences in the individual
expression on their HLA-DR–enriched milk exosomes. The milk samples collected, with Näslund et al. (34) recruiting
identification of MUC-1 associated with HM EVs (8, 44) is a set of volunteers versus samples collected from remnants
particularly interesting given these proteins were previously after feeding in the neonatal intensive care unit by Sims et al.’s
described to prevent certain strains of viral and bacterial group. Additionally, Sims et al. (36) demonstrated that the
gastroenteritis (59, 60). This also reflects epidemiological increased infection of cells was abrogated when tetraspanins
data that show lower rates of gastroenteritis in breastfed in- (namely CD9) were blocked using an antibody. While this
fants (7). Finally, Torregrosa Paredes et al. (44) also reported gives a mechanistic basis for the role of EVs generally in HIV
an association between the immunological composition of transmission, the role of HM EVs in vertical transmission
EVs in mature milk and allergic sensitization at 2 y of age, of HIV has not yet been conclusively answered to date.
although these results should be interpreted with caution due Moreover, the aforementioned studies have all used HM from
to the small sample size (44). In the second study, Simpson HIV-negative donors.
et al. (39) used samples from ProPACT, a randomized Extremely limited data on HM EV glycome, the collection
controlled trial studying the effect of maternal probiotic of carbohydrates on the membrane surface, exist. They come
use on HM miRNAs and their potential role in atopic from a study investigating a single sample and from only
dermatitis development prevention. Small RNA-seq of the 1 individual (41). While establishing that the glycoprotein
EV-enriched HM samples did not demonstrate statistically coating of biofluid and cell culture–derived EVs was a
significant differences between the probiotic-treated group mixture of conserved moieties and parent cell–derived

Breast-milk EVs in child health 65

carbohydrates, they were unable to establish a role for these NEC. Wang et al. (37) extended the in vitro studies by
protein–carbohydrate complexes in human health, nor did treating an NEC rat model with term and preterm EVs and
they compare the HM EVs with a relevant “parent cell” (41). demonstrated greater proliferation of intestinal mucosa and
Glycosylation patterns also aid in the recognition and reduced severity of NEC with preterm EVs. They described
uptake of EVs by target cells (62). Wolf et al. (63) used differences in the expression of peptides between HM EVs
bovine-milk exosomes to demonstrate that, in human and from mothers who delivered at term versus preterm with
rat intestinal cells, glycosylation of the EVs and the cells 47 peptides upregulated and 23 downregulated. Finally, the
were responsible for recognition, endocytosis, and even- authors performed pathway analysis using Gene Ontology

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tual accumulation of the cargo miRNA within cells. This (GO) term enrichment to identify the putative protein
highlights the role of the EV glycome in targeted delivery domains responsible for this protective effect, suggesting that
of bioactive components. Moreover, identifying HM EV lactotransferrin and lactadherin proteins present in EVs are
glycosylation patterns might help clarify the contradictory likely involved in gut barrier function (37).
data regarding the impact of EVs on HIV infectivity. Since The identified studies established that intestinal cells in an
HIV contains heavily glycosylated membrane proteins (e.g., in vitro model could internalize EVs. They also established
gp120), EV glycome profile could elucidate their role in HIV that miRNA cargo is intact through this process (22–24).
particle penetration into target cells. Näslund et al. (34) also This suggests that EVs can effectively deliver miRNA cargo
demonstrated that preincubation of MDDCs with plasma- to infant gut allowing for maternal to infant transmission
derived exosomes did not have an effect on HIV transfer by of regulatory signals including those involved in gut and
MDDCs to CD4+ T cells, although the effect was observed immune system development during infancy. Additionally,
with HM-derived EVs “in play.” both Martin et al. (26) and Wang et al. (37) demonstrated
that breast-milk–derived EVs were effective either in an
HM EVs, Preterm Infants, and NEC in vitro or animal model of NEC in preventing cell death
Kahn et al. (22) examined the changes in HM EV miRNA and stimulating intestinal cell proliferation. The different
in infants delivered preterm. They collected 20 milk samples methodologies utilized by both papers, as well as their focus
that included 5 extremely preterm (<28 wk of gestation) on different cargos, suggests breast-milk EVs may be one of
and 5 preterm (28–32 wk of gestation) and 10 “early term” the components in breast milk leading to protection from
milk samples (>32 wk of gestation). They demonstrated that NEC (26). The absence of functional EVs in infant formula
miRNA content in preterm milk and early term milk was may explain the epidemiological observation that children
mostly identical and was not altered after in vitro digestion who receive donor breast milk instead of formula are less
in either group; however, they detected changes in preterm likely to develop NEC (45, 64). Future studies are needed
HM miRNA profile that were restricted to low abundance to determine if other breast-milk components (i.e., HMOs,
RNA reads. The most abundant RNA reads identified in both IgA) and EVs have synergistic roles in their protective effect
preterm and term populations are miRNA-22-3p, miRNA- on NEC (64, 65) and immune function during the infancy
148a-3p, and miRNA-146a, which are predominantly associ- period.
ated with immune function alongside cellular differentiation.
Additionally, immunofluorescence demonstrated internal-
ization of EVs by human intestinal epithelial crypt-like cells EVs’ Role as Therapeutics
(22). Moreover, miRNAs in both preterm and term and An additional rationale for examining HM EVs is for the
digested and undigested milk were similar, which indicates a possible therapeutic use of exogenously delivered EVs. This
high degree of miRNA content preservation within EVs. The is already a fertile ground for research with multiple neonatal
aforementioned findings suggest that both preterm and term conditions targeted using mesenchymal stem cell (MSC)–
milk may elicit immunomodulatory effects, either locally in derived EVs as “cell free” regenerative medicine. MSC-based
the gut or systemically through circulation with the help of therapies have also found application in BPD, a chronic lung
miRNA cargos within EVs in HM. disease that develops in preterm infants resulting from their
Martin et al. (26) utilized a different approach for isolated immature lung development, as well as from environmental
HM EVs, which were characterized by electron microscopy factors such as infection, invasive ventilation, and liberal
and nanoparticle tracking analysis. These EVs were assessed supplemental oxygen. This has resulted in a number of
for their ability to prevent cell death of intestinal cell phase I clinical trials (66–68). However, experimental animal
line 6 (IEC-6) cells treated with hydrogen peroxide, which models suggest that MSC cell-free media may ameliorate
constitutes a model of NEC. The researchers observed disease to a similar extent as the MSC-based treatment
reduced IEC-6 cell death due to oxidative stress after (69, 70). Numerous mechanisms have been postulated for
subjecting them to 200 μM H2 O2 in the presence of purified the MSC-derived exosome effect, including TNF-stimulated
HM-derived EVs. This study provides initial mechanistic gene 6 (TSG6) or through vascular endothelial growth factor
evidence for alleviation of oxidative stress by EVs in HM, (VEGF) signaling (71–73). The use of pluripotent stem cell
which is a key pathophysiological mechanism of necrosis. lines in newborns comes with a risk of tumorigenesis, which
However, the in vitro model cannot truly represent the wide would be avoided using MSC cell-free media or EVs, and
variety of variables that contribute to the development of there is some additional animal-derived evidence that EVs

66 O’Reilly et al.
 Downloaded from https://academic.oup.com/advances/article/12/1/59/5896686 by Royal Library Copenhagen University user on 24 November 2021
FIGURE 1 Summary of the role of EVs: HM EVs contain cargos (i.e., miRNA, proteins, metabolites, and glycopeptides). These EV cargos
likely have a biological impact on neighboring cells and may impact health outcomes in children. EV, extracellular vesicle; HM, human milk.

may provide other benefits over cell therapy in the treatment Overall, the data on HM EVs at present do not support
of BPD (70). making definitive statements regarding the role of EVs
As mentioned above, EVs may be involved in the protec- in immune function during infancy and long-term health
tive effect of HM against NEC. HM-derived EVs could be a outcomes, such as cardiovascular, endocrine, and autoim-
therapeutic option for neonates whose mothers are unable to mune disease development (78, 79). Studies determining the
breastfeed and where receiving donor-expressed breast milk mechanistic role of EVs on health outcomes in infancy and
is difficult. MSCs derived from various sources (amniotic childhood are needed, with a particular focus on elucidating
fluid, bone marrow) have been shown to enact protective the functions of breast-milk EVs, which suggest future
effects in NEC in rat models and that these effects are at directions in HM research.
least partially driven by EV-related mechanisms (74–76). EVs
have also been suggested as possible therapies for hypoxic- Acknowledgments
ischemic encephalopathy, retinopathy of prematurity, and The authors’ responsibilities were as follows—DO and DD:
spina bifida, which represent a substantial burden of disease performed the literature search; DO, NVA, NAN, AAE,
in the neonatal population (77). It may be hypothesized that LP, MRS, LY, and DM: prepared the initial draft of the
HM EVs may also be associated with the beneficial effects manuscript; JG: provided expertise with regard to the aspects
seen in the management of above mentioned conditions. of the reviewed topic; DO, MRS, and DM: developed the
Nonetheless, the mechanisms, efficacy, and feasibility of EVs initial design; and all authors: read and approved the final
in therapeutics need further investigation. manuscript.

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