Keywords
breast milk - COVID-19 - microbiota - neonate - SARS-CoV-2 - bacterial colonization
- pregnancy
Palavras-chave
leite materno - COVID-19 - microbiota - neonato - SARS-CoV-2 - colonização bacteriana
- gestação
Introduction
There is increasing evidence supporting the benefits of breastfeeding, both for starting
it as soon as possible and for maintaining it exclusively during the first stages
of life. Even though most of the evidence consists of observational and comparative
studies, supporting findings are steadily increasing in number and variety. Despite
this, many concerns about the safety of breastfeeding have been raised due to the
current COVID-19 pandemic. This has occurred even though the presence of the virus
in breast milk has rarely been reported, and a route of infection has not been successfully
demonstrated.[1]
[2]
[3] Therefore, the present article is a literature review concerning the microbiota
of breast milk and the influence of epigenetics in the susceptibility to COVID-19.
Methods
According to the typology presented by Grant et al.,[4] the present article is a literature review including published materials that provide
an examination of recent or current literature. The process for the present review
adhered to the Search, Appraisal, Synthesis, and Analysis (SALSA) framework[4]: the completeness of searching was determined by scope constraints, there was no
formal assessment of the quality of the studies, and a narrative and textual synthesis,
as well as a thematic and descriptive analysis, were used.
Results and Discussion
In a rapid systematic review including eight studies about pregnant women who were
reverse transcription-polymerase chain reaction (RT-PCR)-positive for SARS-CoV-2 and
presented with symptoms of the disease, no viral traits were found in breast milk
nor in newborns.[5] Another systematic review evaluated the published evidence regarding the presence
of SARS-CoV-2 in human milk. Only one study found some evidence of vertical transmission
of the virus from mother to infant through breastfeeding, and only one other study
attempted to find it directly in the milk, with negative results.[6] In addition, Dong et al.[7] found immunoglobulin G (IgG) and immunoglobulin A (IgA) in breast milk from an RT-PCR-positive
mother, but other body fluids such as urine, vaginal secretion, and sweat were negative
for the virus. Her baby was also negative when tested. Therefore, the authors suggested
that breast milk plays a protective role against viral infection.[6] However, SARS-CoV-2-specific IgG levels in the newborn do not stay elevated for
long periods (only for approximately a month a half), indicating a potential risk
of being infected after this period.[6] The only reported cases of vertically transmitted infection from the mother to her
newborn are related to intrauterine transmission.[8]
[9] In contrast, evidence suggests specific protection against SARS-CoV-2 inherited
by the newborn through breast milk.[10] Therefore, breastfeeding should be maintained and highly recommended even during
the pandemic, considering that the benefits outweigh the risks.[8]
[9]
[10]
In addition to the extensively reported nutritional contribution of breast milk to
newborns, a significant reduction in neonatal morbidity and mortality has been observed,
especially in premature newborns.[8]
[9]
[10]
[11] This is most likely a result of the contribution of active and passive immunity.[12] Besides that, there is growing evidence on the influence that breast milk exerts
on the formation of the microbiota of the neonate (intestinal, pulmonary, and other
parts of the body), which extends the positive effects throughout the lifetime of
the individual.
Detailed information on the active and passive immune contribution of breast milk
has been continuously reported. Breast milk provides immune cells (leukocytes, neutrophils,
eosinophils, monocytes, T and B lymphocytes, natural killer [NK] cells, immature granulocytes),
IgA and IgG, free fatty acids, monoglycerides, proteins such as lactoferrin, oligosaccharides,
and others.[12] All these components contribute to active immunity, stimulating the development
of the immune system and of the intestinal mucosa. Immunoglobulin A and IgG in breast
milk play a crucial role in direct and cross-protection against various pathogens.[13] Breastfeeding babies have shown a lower risk of developing asthma, inflammatory
bowel disease, obesity, and diabetes, and that they have an improved humoral response
to vaccination compared with formula-fed infants. The role of breast milk in the development
of the infant microbiota is increasingly being acknowledged and has become relevant
during the current pandemic. A prospective cohort study by CHILD-Canada collected
information of 3,500 pregnant mothers and their children since 2009 and reported that
breastfeeding positively influences the gut microbiota of the newborn.[14] Since this study, many others have published relevant information that has broadened
our current understanding of this field and have suggested that healthy microbiota
can be an additional tool against COVID-19.[15]
Studies performed in Ecuador, as in many other developing countries, are primarily
observational. For instance, Román Collazo et al.[16] suggest that breastfeeding for < 6 months may be associated with an increased risk
of overweight and obesity in infants from Cuenca. Another study performed in Quito
concluded that the levels of the immunoregulatory cytokine TNF in the breast milk
of adolescent mothers were higher than those in adult mothers.[17] Thompson et al.[18] associated differences in the microbiota of the newborn with the birth route (cesarean
section compared with vaginal delivery) in mothers from the Galapagos Islands. Reports
suggesting a high probability of congenital infection of COVID-19 are scarce. One
was reported in Toronto, Canada,[1] another in Tehran, Iran,[19] and one in San Luis Potosí, Mexico.[20] This last one reported negative results for SARS-CoV-2 in umbilical cord blood samples.
Additionally, the virus or antibodies against it have been found in few samples of
the placenta, amniotic fluid, and newborn blood.[11]
The World Health Organization (WHO) states that 3 out of 46 mothers who had breast
milk samples tested were positive for viral particles by RT-PCR but not for the live
virus.[21] Among the three infants whose mothers were positive, only one was positive for SARS-CoV-2,
but transmission through respiratory droplets or aerosols is the most plausible pathway
of infection. In addition to the congenital infection reported in Toronto,[1] there is another in Ulm, Sweden,[14] that reported the complete virus in breast milk. The infant described in the Toronto
study was born infected.[1] In the case from Ulm, the mother and her son had already been home for several days
when both began to show symptoms of COVID-19, and the virus was found in the maternal
milk for a few days. Thus, neither of the two cases showed a clear breast milk infection
route. On the other hand, reactive IgA against SARS-CoV-2 in breast milk implies that
breastfeeding promotes an additional defense against this disease.[11] However, it is unknown how the microbiota of the breast milk of COVID-19-positive
mothers influences the neonates. This issue needs further investigation.
Breastfeeding and the Immune System
Breastfeeding and the Immune System
During pregnancy, the embryo develops the first phases of the immune system, such
as macrophages, T and B lymphocytes, neutrophils, interleukins, cytokines etc. This
process is partially caused by the tolerance that both mother and embryo have. However,
the system of the baby is poorly developed and has a weakened or nonexistent response
compared with the adult immune system. For this reason, the neonate relies on the
mother to increase its immune defense capacity after birth. Antibodies are actively
transported from the mother to the embryo through the placenta, and the neonate is
born with higher IgG than the mother, which serves as protection during the first
months.[22] Similarly, during vaginal delivery, the neonate is exposed to 23 Bacteroidetes and Actinobacteria taxa that stimulate proinflammatory cytokines such as TNF and interleukin-18 (IL-18)
by producing lipopolysaccharides.[23]
After birth, breast milk is needed in order to continue this process. The colostrum
provides chemokines, cytokines, immunoglobulins, growth hormone, and a variety of
immune cells.[24] Because the lymphocyte populations found in breast milk are memory cells that have
already faced pathogens, they will carry this information to the infant; therefore,
directly providing protection.[25]
[26] In addition, the migration of maternal immune cells to the gastrointestinal mucous
tissue of the neonate is suggested as another protective mechanism. The literature
indicates that this process is more likely to occur during the early stages of lactation
due to the high leukocyte concentrations found in this period, contributing to the
development and to the correct maturation of the immune system in the intestinal mucosa.[26]
Human milk oligosaccharides are indigestible by humans, but they work as prebiotics
to stimulate the growth of bacterial species such as Bifidobacterium. These bacteria modulate the intestinal immune response and inhibit the adherence
of pathogens to the intestinal epithelium, preventing pathogenic invasion and colonization
from bacteria like Campylobacter jejuni, Helicobacter pylori, Entamoeba histolytica, and enterotoxigenic Escherichia coli.[27] Oligosaccharides may also contribute to an increase of short-chain fatty acids and
essential molecules for signaling pathways in the gastrointestinal tract.[25]
[27] Additionally, breast milk contains extracellular vesicles that are high in mRNA
and miRNA, which are essential for early metabolic regulation. This can enrich the
metabolic processes of biosynthesis, glycolysis, gluconeogenesis, and fatty acid biosynthesis
to enhance the functioning of the immune system and improve the establishment of healthy
bacterial communities.[25]
The exact reason for the low number of COVID-19 cases in children is not clear. One
of the main theories involves the different expressions of the ACE 2 enzyme, which
increases with age and is used by the virus to enter a cell. Another theory is the
passive immunity from the mother, such as type III interferon, which has antiviral
activity, demonstrated in a study on the Zika virus.[28] This immunity could act against SARS-CoV-2 without presenting the cytokinetic storm
associated with COVID-19-positive patients with the worst clinical diagnosis.
Microbiota and SARS-CoV-2
Microbiota and SARS-CoV-2
The relation between a healthy microbiota and a functional immune system is a theme
that demands investigation in the current pandemic. A Chinese study reported that
lung microbiota from COVID-19-infected patients showed alterations similar to those
observed in patients with community-acquired pneumonia,[29] such as the predominance of pathogenic species and a reduction of Lactobacillus and Bifidobacterium. This is an indication of dysbiosis in the lung microbiota of COVID-19 patients.
Gou et al.[30] studied the intestinal microbiota with proteomic biomarkers, finding an extremely
close correlation with inflammatory cytokines that could suggest an individual predisposition
to present with severe symptoms of COVID-19.
On the other hand, it has been reported that the administration of Lactobacillus isolated from breast milk to infants between 6 and 12 months old reduces the incidence
of respiratory and gastrointestinal tract infections by between 30 and 40%.[31]
[32] Di Pierro[33] states that the species Streptococcus salivarius K12, a predominant commensal in the oral, pulmonary, and upper respiratory tract,
has been safely used as a probiotic for 20 years now and has been correlated with
a significant reduction in upper respiratory tract viral infections in children and
adults.
SARS-CoV-2 RNA has been detected in fecal samples from children who recovered from
COVID-19,[34] suggesting that the virus interacts with elements of the gut microbiota or, at least,
with bacterial species of the digestive tract. A recent study including 15 acute/severe
COVID-19 patients in Hong Kong through March 2020 found an increased abundance compared
with controls of opportunistic intestinal pathogens known to cause bacteremia, such
as Clostridium hathewayi, Actinomyces viscous, and Bacteroides nordii, regardless of gender, age, comorbidities, and use of antibiotics.[35] In contrast, beneficial bacteria, such as Fecalibacterium prausnitzii, Lachnospiraceae bacterium, Eubacterium rectole, Ruminococcus
obeum, and Dorea formicigenerans, were greatly reduced in these cases. The researchers further concluded that there
were microbiome profiles correlated with worse COVID-19 symptoms: bacteria of the
genus Coprobacillus, known to increase ACE 2 expression in mice gut, Clostridium ramosum and Clostridium hathewayi. Alistipes onderdonkii, which is involved in the tryptophan-serotonin metabolism, is essential for gut homeostasis,
and Faecalibacterium prausnitzii produces butyrate, being associated with a better prognosis. These findings support
the need to assess the strengthening of microbiota as an additional tool for treating
and preventing COVID-19.[36]
COVID-19 Epigenetics
In the current COVID-19 pandemic, the fact that a large percentage of infected people
do not show any symptoms and that some show very mild symptoms, along with the wide
variation of symptoms of those who manifest them,[37] has been a topic for conjecture. One plausible explanation can be found on epigenetics.
Epigenetics is the study of heritable changes in the expression of the genome or phenotype,
mainly caused by DNA methylation, phosphorylation, and acetylation to modify histones,
noncoding RNA, ubiquitination, and other means.[38] The variety of organs affected by this virus in the human organism (lungs, brain,
heart, kidneys, digestive system, vascular system, among others) raises many questions
that could be answered by epigenetics.[39]
[40]
In the past two and a half decades, one of the research topics in epigenetics was
understanding the molecular mechanisms by which RNA viruses antagonize the signaling
and detection components that regulate the induction of innate immune and antiviral
defense programs in the host to an infection. Recently, increasing evidence supports
the hypothesis that viruses, including lytic RNA viruses that replicate in the cytoplasm,
have evolved to regulate the epigenome of the host cell and control the innate immune
system in order to promote robust virus replication and pathogenesis.[41] Coronaviruses have been a field of research for > 15 years. A study on the 2003
outbreak of Severe Acute Respiratory Syndrome (SARS) caused by the SARS-CoV virus
identified that the silencing of the TICAM2 gene increases the susceptibility to SARS-CoV in mice.[42] This gene encodes a protein that helps activate Toll-like receptors (TLRs) involved
in innate immunity, the first line of defense against pathogens.
Methylation and COVID-19
For the virus to evolve, one of two pathways must be used: direct fusion with the
host cell membrane or induction of host cells fusion. Both mechanisms facilitate viral
endocytosis, invasion of neighboring cells, and evasion of the innate antiviral immune
system.[43] The union of the virus with the cell forms a syncytium, a mechanism typical of coronaviruses
and of many other viruses.[44]
[45] Syncytia formation normally occurs during the development of the mammalian placenta,
in which syncytin genes are involved and produce syncytin-1 and -2 derived from two
human endogenous retroviruses.[46] Additionally, syncytia formation leads to the creation of multinucleated giant cells
in the placenta, making this tissue impermeable and generating mother-child immune
tolerance. For this reason, syncytin genes, which are hypomethylated, are functionally
active in the mammalian placenta. In other tissues, they are hypermethylated and,
therefore, silenced. The activation of these genes in other tissues has been associated
with various diseases, such as schizophrenia, multiple sclerosis, and type-1 diabetes.[47]
[48] For example, the Epstein-Barr virus and cytomegalovirus are known to demethylate
the syncytin-1 and -2 genes of the host, increasing gene transcription and causing
syncytia formation in tissues where those genes are normally hypermethylated and silenced.[29]
[31] Syncytia formation by SARS-CoV-2 is faster than that of SARS-CoV in 2002. Additionally,
it is known that syncytia formation is highly responsible for the virulence factor
and for the induction of a cytokine storm.[45]
[49]
Epigenetic Mechanisms in Susceptibility to SARS-CoV-2 and Severity of the Disease
Epigenetic Mechanisms in Susceptibility to SARS-CoV-2 and Severity of the Disease
Two recent studies[50]
[51] identified the importance of the gene encoding angiotensin-converting enzyme 2 (ACE2)
methylation pattern and determined that its expression rate is under epigenetic control.[52] Corley et al.[50] showed that the activity of the ACE2 gene, which is located on the X chromosome (Xp22.2), is associated with age and gender
based on the methylation pattern of various CpG promoter islands. This gene is mainly
present in the endothelium and the epithelium of many organs, such as the lungs and
upper airways, the intestines, the liver, the kidneys, the heart, the eyes, and central
nervous and vascular systems.[53] It was shown that the methylation rate in lung epithelial cells was the lowest compared
with in all other tissues, suggesting that the lungs have the highest rate of ACE2 transcription and expression.[53] It was also shown that age correlates with hypomethylation in lung tissue, which
provides a partial explanation for aging being set as a significant risk factor for
death by COVID-19. The opposite happens in children, in whom the ACE2 gene in the lungs and other tissues is hypermethylated and, therefore, silenced.[54] This suggests that certain subgroups of patients with known epigenetic features
are more susceptible to SARS-CoV-2, such as those with systemic lupus erythematosus
(SLE), in whom a strong overexpression of the lung protein ACE2 seems to be related
with hypomethylation.[55] Pique-Regi et al.[56] showed that the placenta expresses negligible amounts of both ACE2 and TMPRSS2,
which could explain the scarcity of reported infections between mother and child in
utero.
Breast Milk as an Epigenetic Regulator
Breast Milk as an Epigenetic Regulator
Breast milk has been considered an epigenetic regulator because of the ribonucleic
acids derived from exosomes (miRNA), which can modulate the action of the DNA methyltransferase
enzymes (DNMT).[57] These miRNAs are transferred by extracellular vesicles derived from the epithelial
cells of the mammary gland and are inserted into somatic cells by endocytosis mechanisms.
Breast milk also contains long-sequence noncoding RNAs known to be involved in regulating
gene expression at the post-transcriptional level. Thus, its effect is not only restricted
to DNA methylation. It is considered that consumption of human milk provides the appropriate
epigenetic signals to the genetic programming of the newborn and is perhaps one of
the mechanisms that protects against various respiratory conditions.[57] Regulatory exosomes of the immune function have been successfully isolated from
breast milk. These miRNAs are known to increase the number of FoxP3 cells, also known
as T cells (Tregs). Ribonucleic acid derived from exosomes binds to the enzymes DNMT1
and DNMT3b, which are associated with the FoxP3 locus on CD4 + T cells to mediate
its expression. On the other hand, breast milk exosomes have also been associated
with the function of the NR4A receptor family and with the expression of the nuclear
factor NFKB, which can also regulate genes associated with the immune response by
modulating DNMT. This combined evidence suggests that the microbiota of human milk
may shape immune function through DNA methylation and other epigenetic changes.[57]
[58] During the COVID-19 pandemic, it would be promising to study the ability of miRNAs
in human milk to epigenetically regulate the expression of various genes associated
with the antiviral immune response, such as HLA, IFN, and, most importantly, the ACE2 receptor.
Epidrugs against SARS-CoV-2
Epidrugs against SARS-CoV-2
Epigenetic regulation can be performed with natural interventions. An in-vitro study
suggested using vitamin D and quercetin to improve the symptoms of COVID-19,[59] which would be done by inhibiting the expression of ACE2. Another study proposed that the average vitamin D level in European countries is
inversely correlated with the COVID-19 death rate, but this study has several flaws
in its statistical approach, some recognized by the authors themselves.[60]
[61] Thus, it should be carefully considered before openly recommending vitamin D to
reduce the risk and symptoms of COVID-19. Another candidate for epigenetic silencing
of the ACE2 and interferon genes is curcumin.[62] Curcumin is a potent activator of DNA methyltransferases in viable clinical doses
and has ferritin-reducing effects.[63]
[64]
[65] This becomes important when considering that high ferritin values in patients with
COVID-19 are associated with greater severity of the disease.[66] Another so-called epidrug with proven methylation capability is sulforaphane from
broccoli.[67] All of these substances are natural, over-the-counter medicines and may help reduce
the severity and susceptibility of the disease. Pathways such as HIF-1 signaling,
autophagy, RIG-I signaling, toll receptor signaling, fatty acid oxidation/degradation,
Il-17 signaling, and others have been found to be sequestered or suppressed by viral
proteins leading to improved viral survival.[68] Furthermore, pathways such as relaxing signaling in the lungs suggest that aberration
by viral proteins could lead to the pulmonary pathophysiology found in COVID-19. The
epigenetic profile of the host tissue could allow the discovery of risk factors for
COVID-19 related to age and gender.
Genetic Susceptibility
Genetic susceptibility refers to the increased likelihood of developing a particular
disease based on an individual's genetic makeup. Genetic population studies show important
variations in protein-coding between different ethnic groups, making some of them
more susceptible to diseases. This might also help understand why most children are
unaffected by COVID-19, while a few still develop serious symptoms.[69] The ACE2 gene encodes a protein of 805 amino acids that exhibits observable variabilities
between different ethnic groups,[70]
[71]
[72] an interesting topic in this pandemic. There are three genetic patterns related
to predisposition to COVID-19.[73] The first one involves common variants at multiple loci that have a weak effect
with a unique contribution. However, they can show a significant effect through additive
contribution, increasing the risk and severity of infection. Second, moderately rare
variants in limited genes usually have strong effects and, therefore, would be highly
penetrating and even dominant. These variants could be the basis for severe forms
in young patients without any other medical conditions. Lastly, gene-environment interactions
could define the predisposition of the host. The genetic variants related to the immune
system that involve the previous strain of coronavirus may have a role in the susceptibility
to infection, since SARS-CoV-2 has a genetic identity of 80% with the previous virus.
Previous studies have supported the role of human leukocyte antigen (HLA) in the susceptibility
and severity of various viral infections.[74] Therefore, the genetic variability of the three main genes of the histocompatibility
complex (MHC) class I (HLA A, B and C) could also be involved in the susceptibility
and severity of SARS-CoV-2. Similarly, a synonymous variant in the IFN-induced transmembrane
protein 3 gene has been reported to cause severe clinical outcomes in patients infected
with the H7N9 and H1N1 influenza viruses. Likewise, it should be noted that the Mannose-Binding Lectin, CD147, CCL2, and interleukin-12 genes should also be considered regarding the susceptibility to COVID-19.[75]
Recommendations Regarding Breastfeeding and COVID-19
Recommendations Regarding Breastfeeding and COVID-19
It should be emphasized that, until now, we know with certainty that there is no vertical
transmission from a SARS-CoV-2 infected mother to her child and that breastmilk acts
as a protective element against this viral agent.[76] The WHO, The International Federation of Gynecology and Obstetrics, and The American
and Spanish Academies of Pediatrics highlight the considerable benefits of breastfeeding
both for the mother and the child, even during critical conditions like the actual
COVID-19 pandemic.[77] In June 2020, the protocol “Recommendations for health professionals from Human
Milk banks regarding the attention of pregnant women, mothers in puerperium and breastfeeding
mothers, premature or low-weight newborns with or without risk of infection of SARS-CoV-2
COVID-19” was published in Ecuador. In this document, breastfeeding and the support
that milk banks can provide to mothers are promoted for those who might not feel safe
breastfeeding or might not be in optimal conditions to do so. The permanent use of
personal protective equipment, handwashing for at least 20 seconds, wearing a face
mask, and keeping a two-meter safe distance between individuals are essential measures
to avoid the spread of the disease to and from mother, child, and the health professionals
taking care of them.[78]
Conclusion
The reviewed studies show evidence of a beneficial effect of breastfeeding and highlight
its importance on the current pandemic due to the immune reinforcement that it provides.
Breastfed individuals showed better clinical response due to the influence of breastfeeding
on microbiota and to its nutritional and immune contribution compared with those who
were not breastfed. Even though a few studies found SARS-CoV-2 fragments and one publication
found the full virus in breast milk, a route of infection through it has not been
established. This information needs to be updated as new evidence is published. On
the other hand, the relation between a healthy microbiota, especially derived from
breast milk, and a positive response to COVID-19 might suggest novel therapeutic options
by strengthening the microbiota. The genetic susceptibility of certain individuals
could be modified by little known epigenetic factors in which the microbiota could
play a key role through the interaction with the genome of the individual and with
SARS-CoV-2. Further experimental research needs to be performed in order to improve
the clinical guidelines in SARS-CoV-2-positive patients.
