DISCUSSION
The influence of early life nutrition on the development of the immune
response has not previously been studied in the first few weeks of life.
This is an important question as epidemiological data suggest that
breastfeeding is associated with long-term health benefits, such as a
lower incidence of allergy, asthma, diabetes, celiac disease and
multiple sclerosis19-22. This mechanism may be
imprinted early after birth when the immune system faces the dual
challenge of establishing inflammatory capacity against pathogens whilst
developing tolerance towards harmless antigens.
A striking finding was that Tregs expand substantially in the first 3
weeks of life and this expansion was more profound in breastfed babies
in contrast to those receiving formula feed. Tregs of breastfed neonates
also display an activated phenotype with increased expression of HLA-DR,
a marker of increased suppressive activity23. On the
other hand, changes in the profile of pro-inflammatory cytokine
production were also observed at this early stage of life. IL-8
production, a major phenotypic attribute within cord blood, was largely
maintained but a striking feature was the increase in intensity of IL-17
production by both CD4+ and CD8+ cells by 3 weeks of age. This is likely
to reflect recognition of bacterial antigen during establishment of the
microbiome and may be balanced by the coordinated Treg expansion during
this period. An increase in serum IL-17 has been shown at 4 weeks and is
likely explained by these observations5. Most of these
features were independent of nutrition although IFN-γ production by T
cells was lower in exclusively breastfed neonates. Our observation that
CD8+ cells are less abundant and have reduced cytotoxic capacity in
neonates compared to adults confirms previous
results24,25.
One of the most interesting findings was that T cells from breastfed
neonates display reduced proliferative responses and produce
substantially lower levels of Th1 cytokines when challenged with
maternal cells. This was specific to maternal antigens and was not
present against unrelated PBMC and did not result from an intrinsic
reduction of cytokine producing capacity. As such this reflects the
development of immunological tolerance against non-inherited maternal
antigens (NIMA) in exclusively breastfed neonates. Importantly, we were
also able to show that NIMA-specific tolerance was mediated by Tregs,
and is therefore linked to the expansion of this population in breastfed
neonates.
An additional observation was that neonatal PBMC at 3 weeks of age
triggered stronger immune responses from maternal PBMC compared to cord
blood cells. This may reflect maturation of antigen presentation
function by 3 weeks of age, although this was not assessed in this
study. This also indicates that the fetal immune system may contribute
to suppression of maternal immune recognition during pregnancy by
maintaining a tolerogenic phenotype prior to parturition.
It is interesting to speculate on potential mechanisms by which
breastfeeding can promote NIMA-specific tolerance in neonates. Our
observations likely reflect immune tolerance to gastrointestinal
presentation of maternal cells within breastmilk26.
Transplacental passage of cells during pregnancy leads to reciprocal
microchimerism that can persist for many years. Furthermore, this
’microchiome’ of maternal cells supports fertility in female offspring
by promoting immune tolerance to NIMA during next-generation
pregnancies27. Beneficial effects are also seen when
NIMA are shared between donors and recipients of allogeneic renal or
hemopoietic stem cell transplantation. Importantly, the establishment of
NIMA-specific tolerance has been shown to be dependent on breastfeeding
and nutritional history is also a determinant of NIMA-associated
transplant outcome 28,29. Our findings show that
breastfeeding directly promotes the development of Tregs that suppress
recognition of maternal tissue, thus likely supporting maternal
microchimerism and potentially conferring lifelong benefits in relation
to fertility and immune protection against infectious agents and
cancer27,30.
A further recent observation is that breastfeeding, through the transfer
of human milk oligosaccharides, exerts important prebiotic and
immunomodulatory effects including the development of tolerogenic
dendritic cells which prime Tregs31,32.
We were also interested to assess how nutrition could impact on the
formation of the early microbiome and how this might correlate with
immune function. Microbial composition was broadly comparable in
breastfed and formula-fed neonates and this is likely to reflect the
fact that all babies in our cohort were delivered by caesarean section.
Dysbiosis of the microbiota has been found to occur following delivery
by caesarean section and in infants who are not
breastfed33,34. Nevertheless, although it may take
several months for nutrition to markedly influence microbiome
composition35 subtle differences in microbial
diversity were already apparent at 3 weeks of life. In line with
previously published results36, we observed that the
gut microbiome of breastfed neonates is more abundant in short chain
fatty acid (SCFA) producing bacterial genera, such as Gemella andVeillonella . SCFAs, in particular propionate and butyrate, play
an important role in promoting Treg differentiation and proliferation
via the inhibition of histone deacetylases11. This
notion is supported by a link between the presence of Veillonellaand the proportion of Tregs at 3 weeks of age in our network modelling
analysis.
We selected elective caesarean deliveries for our study as labor is
known to promote pro-inflammatory changes37. Further
studies will therefore be needed to establish the relative contribution
of mode of delivery and nutritional history on the development of
NIMA-specific tolerance in the neonate. A further limitation of this
work is the relatively low number of neonates in the examined feeding
groups. Nevertheless, we established a unique and homogenous cohort of
healthy neonates who were sampled at 3 weeks of age exclusively for the
purposes of this study.
In summary, we demonstrate that the neonatal immune system undergoes
substantial maturation in the first 3 weeks of life with an increase in
IL-17 production in T cells and a balanced increase in the Treg
population. Moreover, breastfed neonates show a specific and Treg
dependent reduction in proliferative T cell responses to NIMA,
associated with a reduction in inflammatory cytokine production. These
findings add to our understanding of mechanisms by which early life
nutrition can determine long term health outcomes38.