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Immunological programming by breast milk creates an anti-inflammatory
cytokine milieu in breast-fed infants compared to formula-fed infants
Essi Kainonen*, Samuli Rautava and Erika Isolauri
Department of Paediatrics, Turku University Hospital, Kiinamyllynkatu 4-8, Turku 20520, Finland
(Submitted 26 September 2011 – Final revision received 10 May 2012 – Accepted 28 June 2012 – First published online 30 October 2012)
Abstract
Breast milk provides important maturational stimuli to an infant’s developing immune system. However, data concerning the role of
breast-feeding in reducing the risk of allergic disease remain contradictory. Previous studies have centred on comparative analyses of
breast milk and formula compositions. We chose a slightly different angle, whereby we focused on the effects of the chosen diet on
the infant himself, comparing the immune development of formula-fed and breast-fed children. The objective of the present study was
to determine how the mode of feeding affects infant immunology. Altogether, eighteen formula-fed infants with limited breast-feeding
for , 3 months and twenty-nine infants who were exclusively breast-fed for . 3 months were included in the study. Concentrations of
interferon g, TNF-a IL-10, IL-5, IL-4 and IL-2 were measured simultaneously from the same serum sample through use of a multiplexed
flow cytometric assay at the ages of 1, 3, 6 and 12 months. Transforming growth factor b2 (TGF-b2) was measured using ELISA at the
same time points. Serum TNF-a and IL-2 concentrations were significantly higher in formula-fed than in breast-fed infants during the
first year of life (ANOVA, P¼0·002). The serum concentrations of TGF-b were significantly lower in formula-fed than in breast-fed infants
throughout the first year of life (ANOVA, P#0·0001). Exclusive breast-feeding promotes an anti-inflammatory cytokine milieu, which
is maintained throughout infancy. Such an immunological environment limits hyper-responsiveness and promotes tolerisation, possibly
prohibiting the onset of allergic disease.
Key words: Infants: Breast milk: Immunology: Allergy: Transforming growth factor b: Tolerisation: Cytokines
Breast milk has undergone hundreds of thousands of years
of product development during the evolution of our species.
It can, with confidence, be claimed to be the ideal form of
nutrition for the human infant during the first months of life
and the ideal supplement to solid nutrition during the infant’s
first years. In addition to providing developmentally appro-
priate nutrition, breast milk contains a multitude of immuno-
logically active components. In the same manner that breast
milk has evolved to fit the needs of our infants, it can be
assumed that our infants have evolved to develop in the
presence of breast milk. Despite being well-equipped for
extra-uterine life at the time of birth, the newborn infant
also has several shortcomings, and the most profound are
those faced by the immature immune system. The infant
undergoes a transition from the sterile environment experi-
enced in utero to one that is filled with microbes, and he
must also begin to feed. It is, thus, essential that his immune
system learns to differentiate between hostile and innocuous
antigens.
Breast milk may help infants in these essential adaptations.
It provides infants with direct anti-pathogenic effects via
maternal microbe-specific Ig and various other antimicrobial
substances
(1 – 3)
, and has an extensively documented ability
to ward off infective disease, such as infantile diarrhoea,
in developing countries
(4 – 6)
, but also common causes of
pathogen-related infant morbidity in western settings
(7 – 10)
.
There is also an impressive amount of data to suggest that
its recipients are protected from common disruptions of the
immune system such as type 1 diabetes
(11)
, inflammatory
bowel disease
(12)
, coeliac disease
(13)
and atopic diseases,
including allergies and asthma
(10,14 – 21)
. Breast-feeding pro-
motes gut colonisation with beneficial bifidobacteria in
infants
(1,22 – 24)
, and this finding may be linked with many
of the advantages associated with breast-feeding.
Nevertheless, results on the potency of breast milk in
reducing the risk of atopic disease remain unclear and con-
tradictory, with evidence for both decreased
(14 – 21,25)
and
increased
(26 – 31)
risk of atopic disease in breast-fed children,
and no proven dose–response pattern. The effects of breast-
feeding, or the lack of it, on infant immune physiology have
thus remained elusive.
In the present study, we determined blood plasma concen-
trations of key cytokines and circulatory antibody-secreting
cells in exclusively breast-fed infants, and compared them with
* Corresponding author: E. Kainonen, fax þ 358 2 323 1460, email esskai@utu.fi
Abbreviation: TGF-b2, transforming growth factor b2.
British Journal of Nutrition (2013), 109, 1962–1970 doi:10.1017/S0007114512004229
q The Authors 2012
British Journal of Nutrition
those of infants who had experienced early termination of
breast-feeding and had been primarily fed with formula. The
present study aimed to shed light on whether differing immune
physiology is evident in breast-fed and formula-fed infants.
Materials and methods
Design
The infants in the study were selected from a prospective
study (n 98) that had followed them up from birth to the
age of 12 months. The majority of the participants were
genetically at high risk for atopy, with the mother or other
first-degree relatives with atopic disease. This family history
was self-reported. A case–control design was chosen, as it is
not feasible to conduct a randomised study on breast-feeding.
The inclusion criteria for the formula-fed group were the
complete cessation of breast-feeding before 3 months of
age and consequent exclusive formula feeding at that age.
Infants who were exclusively breast-fed at 3 months of age
were chosen as controls. Infants with chronic disease were
excluded from the study. Altogether, eighteen formula-fed
and twenty-nine exclusively breast-fed infants fulfilled the
criteria of the study. The present study was conducted accord-
ing to the guidelines laid down in the Declaration of Helsinki,
and all procedures involving human patients were approved
by the Turku University Central Hospital Ethics Committee.
Written informed consent was obtained from all patients.
Determining plasma cytokine concentrations using
cytometric bead assays and ELISA
Venous blood samples were drawn, heparinised and stored at
2 808C for later analysis at the ages of 1, 3, 6 and 12 months.
The samples of formula-fed infants were drawn between
September 2000 and February 2002 and the samples of
breast-fed infants between May 2000 and August 2002 in
almost parallel follow-up periods. The concentrations of the
cytokines interferon g, TNF-a, IL-10, IL-5, IL-4 and IL-2 were
measured simultaneously from the same blood plasma
sample through use of a multiplexed bead-based flow cyto-
metric assay between August and September 2003, and all
samples had been stored at the same facility in similar con-
ditions. A commercial Human Th1/Th2 Cytokine Kit, BD
Cytometric Bead Array, a FACSCalibur flow cytometer and
BD CBA Software (BD Immunocytometry Systems and BD
Biosciences Pharmingen) were used for the analysis according
to the manufacturer’s specifications. The detection limits for
these cytokines according to the manufacturer were 7·1 pg/ml
for interferon g, 2·6 pg/ml for IL-2, 2·6 pg/ml for IL-4, 2·4 pg/ml
for IL-5, 2·8 pg/ml for IL-10 and 2·8 pg/ml for TNF-a.
Cytometric bead assay analyses were carried out simul-
taneously during August and September 2003. The plasma
concentrations of transforming growth factor b2 (TGF-b2)
were measured in duplicate using commercial sandwich
ELISA specific for the molecule (R&D Systems). Activation
of latent TGF-b2 and all determinations were carried out
according to the manufacturer’s instructions.
Determining the number of Ig-secreting cells
The total number of Ig-secreting cells and the number
of cells producing antibodies directed specifically against
the cows’ milk allergen, casein, were measured using the
enzyme-linked immunospot assay, as detailed in previous
work (Isolauri et al), from blood samples obtained at the
ages of 3, 6 and 12 months. On the day of sample collection,
peripheral blood mononuclear cells were isolated using Ficoll-
Paque gradient centrifugation. Isolated cells were washed
three times in Hanks balanced salt solution and suspended
in Roswell Park Memorial Institute 1640 medium containing
Table 1. Clinical characteristics of participants
(Mean values and ranges; number of participants and percentages)
Formula-fed (n 18) Breast-fed (n 29)
Mean Range Mean Range P
Boys 0·53
n 714
%3848
Gestational age (weeks) 40·1 36·7–42·3 39·2 37·0 –42·0 0·051
Birth weight (g) 3485 2580–4260 3544 2730– 4890 0·68
Caesarian section 0·12
n 53
%2810
Duration of exclusive breast feeding (months) 0·5 0·0–1·5 4·1 3·0–6·0 ,0·0001
Overall duration of breast feeding (months) 1·4 0·0–2·8 9·9 4·2–12·0 , 0·0001
Family history of AD
First-degree relative AD
n 14 29 0·04
%77100
Maternal AD
n 12 29 0·009
%67100
AD, atopic disease.
Immunological programming by breast milk 1963
British Journal of Nutrition
Table 2. Serum cytokine concentrations in formula-fed and breast-fed infants
(Geometric means and 95 % confidence intervals)
Age
(months)
All participants
P (ANOVA
for
repeated
measures)
Only atopic mothers
P (ANOVA
for
repeated
measures) P
Formula-fed (n 18) Breast-fed (n 29) Formula-fed (n 12) Breast-fed (n 29)
Geometric
mean 95 % CI
Geometric
mean 95 % CI P
Geometric
mean 95 % CI
Geometric
mean 95 % CI
Serum TGF-b2
(pg/ml)
1 89 68, 116 300 325, 356 , 0·0001 , 0·0001 84 61, 117 340 325, 356 ,0·0001 ,0·0001
3 82 53, 129 406 376, 438 , 0·0001 98 57, 169 406 376, 438 ,0·0001
6 150 84, 268 285 216, 376 0·039 132 63, 277 285 216, 376 0·029
12 35 18, 71 362 280, 469 , 0·0001 45 20, 104 362 280, 469 , 0·0001
Serum IFN-g
(pg/ml)
1 25·7 7·8, 82·3 26 16·1, 41·8 0·10 11·9 3·0, 45·9 26 16·1, 41·8 0·36
3 40·7 14·9, 111 12 9·6, 15·1 43·6 12·1, 157·7 12 9·6, 15·1
6 71·7 27·6, 187 15·4 9·6, 24·5 59·3 19·4, 181·6 15·4 9·6, 24·5
12 27·8 13·2, 58·2 17·5 12·5, 24·4 20 9·7, 41·4 17·5 12·5, 24·4
Serum TNF-a
(pg/ml)
1 1·6 1·1, 2·2 1·1 0·8, 1·3 0·002 0·039 1·6 1·0, 2·3 1 0·8, 1·3 0·005 0·076
3 1·5 1·0, 2·3 1 0·7, 1·2 0·1 1·6 0·9, 2·8 1 0·7, 1·2 0·094
6 3·1 1·4, 6·4 1 0·8, 1·2 0·001 3 1·2, 7·1 1 0·8, 1·2 0·002
12 2·8 2·0, 4·5 1 0·8, 1·3 0·0001 2·6 1·9, 3·6 1 0·8, 1·3 , 0·0001
Serum IL-10
(pg/ml)
1 6·5 5·8, 7·2 5·7 4·9, 6·6 0·05 6 5·2, 6·9 5·7 4·9, 6·6 0·12
3 8·3 5·8, 11·8 6·5 5·8, 7·3 8·6 5·2, 14·1 6·5 5·8, 7·3
6 21·6 9·2, 50·3 6·3 4·9, 8·1 19·9 7·3, 54·5 6·3 4·9, 8·1
12 11 7·2, 16·9 8 7·0, 9·1 8·4 6·3, 11·1 8 7·0, 9·1
Serum IL-5
(pg/ml)
1 2·5 1·9, 3·2 2·6 2·1, 3·3 0·84 2·3 1·6, 3·3 2·6 2·1, 3·3 0·44
3 2·1 1·5, 2·8 2 1·7, 2·4 2·3 1·5, 3·5 2 1·7, 2·4
6 4·9 2·6, 9·1 3·2 2·5, 4·0 4·1 2·1, 7·8 3·2 2·5, 4·0
12 3 2·0, 4·5 2·6 2·1, 3·2 2·3 1·6, 3·1 2·6 2·1, 3·2
Serum IL-4
(pg/ml)
1 4·8 2·4, 9·8 2·1 1·7, 2·5 0·28 4·5 1·9, 10·5 2·1 1·7, 2·5 0·067
3 2·5 1·5, 4·3 2·1 1·6, 2·9 3·1 1·7, 5·8 2·1 1·6, 2·9
6 5·1 2·5, 10·2 2·5 1·9, 3·2 4·8 1·7, 13·3 2·5 1·9, 3·2
12 3·7 2·2, 60·1 2·8 2·3, 3·5 2·6 1·5, 4·6 2·8 2·5, 3·5
Serum IL-2
(pg/ml)
1 2·2 1·5, 3·2 1 0·8, 1·2 0·0001 0·0003 2·6 1·7, 4·0 1 0·8, 1·2 0·0001 0·0002
3 3 1·9, 4·7 1·1 0·8, 1·4 0·001 3·5 1·8, 6·5 1·1 0·8, 1·4 0·002
6 4·4 1·9, 9·7 1 0·7, 1·1 , 0·0001 3·6 1·5, 8·8 1 0·7, 1·1 0·0003
12 2·6 1·6, 4·3 1·3 1·0, 1·7 0·014 2 1·2, 3·3 1·3 1·0, 1·7 0·12
TGF-b2, transforming growth factor b2; IFN-g, interferon g.
E. Kainonen et al.1964
British Journal of Nutrition
10 % fetal calf serum and adjusted to a concentration of
1–2 £ 10
6
cells/ml.
To determine the total number of Ig-secreting cells, the
wells were coated for 2 h at 378C with rabbit anti-human
IgA, IgG and IgM (Dako A/S) diluted in the ratio 1:100 in
PBS (pH 7·4), and to detect the number of cells secreting
casein-specific antibodies, the coating was performed with
b-casein (20 mg/ml; Sigma Chemical Company). Uncoated
binding sites were blocked with 1 % bovine serum albumin
(Boehringer Mannheim) in PBS (pH 7·4) for 30 min at 378C.
After washing, the lymphocyte suspension was incubated on
antigen-coated flat-bottomed ninety-six-well microtitre plates
(Immunoplate RI, A/S Nunc) at 378C for 2 h. The detection
of antibodies secreted during that time was performed with
alkaline phosphatase-conjugated goat antiserum to human
IgA, IgG and IgM (Sigma Chemical Company), diluted in 1 %
bovine serum albumin–PBS (pH 7·4) and incubated overnight
at room temperature, followed by a substrate agarose overlay
and observation of the coloured spots.
Statistical evaluation
The clinical data are described as means with ranges. The
differences in clinical characteristics between the study
groups were assessed using Student’s t test and the
x
2
test.
The results are expressed as geometric means with 95 % CI.
The differences between serum cytokine concentrations
in the breast-fed and formula-fed infants during the course
of the follow-up were assessed using ANOVA for repeated
measures after logarithmic transformation. If the ANOVA
suggested a difference between groups, further analyses at
each time point were conducted using Student’s t test after
logarithmic transformation. A P value of less than 0·05 was
considered statistically significant.
Results
Clinical data
The clinical characteristics of the participants are shown in
Table 1. The study groups were comparable with regard to
gestational age, sex, birth weight and the mode of delivery,
while differing on the mode of feeding according to the
study design. Due to the design of the original prospective
cohort, the family history of atopy differed between the for-
mula-fed and breast-fed groups. A total of fourteen of the
eighteen (77 %) formula-fed infants had a first-degree relative
with atopic disease compared to all of the twenty-nine (100 %)
breast-fed infants (P ¼ 0·04). A total of twelve of the eighteen
(67 %) formula-fed infants had a mother with clinical atopic
disease compared to all of the twenty-nine (100 %) in the
breast-fed group (P ¼ 0·009). The groups are, thus, signifi-
cantly different regarding family history of atopy. However,
the cytokine results observed persist and remain significant
even when only the participants with atopic mothers are eval-
uated (i.e. when the six formula-fed participants without an
atopic mother are excluded from the evaluation), suggesting
that the differences witnessed in cytokine concentrations are
not due to the differences in family history of atopy (Table 2).
Differing plasma cytokine concentrations in formula-fed
and breast-fed infants
ANOVA for repeated measures showed a significant difference
in TNF-a concentrations (P ¼ 0·0019), with elevated serum
TNF-a concentrations in formula-fed infants compared to
their breast-fed counterparts (Fig. 1). Serum IL-2 concen-
trations (P ¼ 0·001) were also significantly higher in
formula-fed than in breast-fed infants, with significant differ-
ences seen throughout the first year of life (Fig. 2). A contrary
situation was evident with TGF-b2. The concentrations of
TGF-b2(P# 0·0001) were significantly lower in formula-fed
than in breast-fed infants throughout the first year of life,
with up to ten-fold differences seen (Fig. 3).
No statistically significant differences were evident between
the serum concentrations of interferon g (P ¼ 0·10), IL-10
(P ¼ 0·05), IL-5 (P ¼ 0·84) or IL-4 (P ¼ 0·28) (Table 2)
Differing numbers of Ig-secreting cells in formula-fed and
breast-fed infants
The different levels of cows’ milk antigen exposure
between the two study groups were well-reflected in the
7000
6000
5000
4000
3000
2000
1000
0
1 month 3 months 6 months 12 months
TNF-α concentration (pg/ml)
Fig. 1. Serum TNF-a concentrations in formula-fed ( ) and breast-fed infants ( ). ANOVA for repeated measures showed a significant difference in TNF-a con-
centrations (P ¼ 0·0019). Statistically significant differences were evident for 1 month (P ¼ 0·039), 3 months (P¼ 0·010), 6 months (P, 0·0011) and 12 months
(P¼ 0·039).
Immunological programming by breast milk 1965
British Journal of Nutrition
enzyme-linked immunospot assay results for casein-specific
Ig. The numbers of casein-specific IgA- and IgG-secreting
cells were significantly greater in formula-fed than in breast-
fed children at 3 and 6 months of age, but not at a later age
(Table 3). In the infants receiving formula, the highest
numbers of casein-specific Ig-secreting cells were seen at
3 months, and the total number of secreting cells decreased
with age. The opposite was true in the breast-fed group,
where low amounts of casein-specific Ig-secreting cells were
seen at 3 months, and the number of total secreting cells
increased with age so that by age 12 months, no significant
difference remained between the groups.
Discussion
The gold standard and optimum goal of early infant nutrition
should be the healthy, breast-fed infant. Defining correct
nutrition by detailing the precise composition of what the
infant feeds on is an approach that is prone to erroneous con-
clusions. The composition of breast milk varies from one indi-
vidual to another, and can also change in each lactating
woman due to diet, parity, season and stage of lactation.
The biologically active components of breast milk are diverse
and show even more variation than the nutritive components.
The dynamics of how cytokines and other biologically active
breast milk components affect each individual infant are
impossible to predict. Thus, we must study and compare the
physiology, in the case of the present study, the immune
physiology of infants under different modes of feeding in
order to assess the nutritive and physiological appropriateness
of each diet.
One of the most striking differences between breast-fed
and formula-fed infants was evident in the serum concen-
trations of the TGF-b isoform, TGF-b2, with breast-fed infants
exhibiting significantly higher levels of this anti-inflammatory
cytokine. TGF-b2 has a wide variety of effects extending
from regulation of cell proliferation and differentiation to
modulation of immune responses
(32 – 35)
. It also appears to
contribute to mucosal barrier function by inducing IgA pro-
duction
(36)
. Several studies have indicated the importance of
TGF-b2 in the process of tolerisation
(37,38)
. Higher serum
concentrations of the potentially pro-inflammatory cytokines
TNF-a and IL-2 were seen in formula-fed than in breast-fed
infants. TNF-a is a pleiotropic cytokine with wide-ranging
pro-inflammatory effects, and its ability to disrupt mucosal
barrier function is well-acknowledged
(39,40)
. Elevated concen-
trations of TNF-a prevailed in formula-fed infants throughout
the first year of life. The present results thus suggest that early
12 000
10 000
8000
6000
4000
2000
0
1 month 3 months 6 months 12 months
IL-2 concentration (pg/ml)
Fig. 2. Serum IL-2 concentrations in formula-fed ( ) and breast-fed infants ( ). ANOVA for repeated measures showed a significant difference in IL-2 concen-
trations (P# 0·001). Statistically significant differences were evident for 1 month (P¼ 0·0003), 3 months (P¼ 0·001), 6 months (P¼ 0·0001) and 12 months
(P# 0·014).
600
500
400
300
200
100
0
1 month 3 months 6 months 12 months
TGF-β concentration (pg/ml)
Fig. 3. Serum transforming growth factor b2 (TGF-b2) concentrations in formula-fed ( ) and breast-fed infants ( ). ANOVA for repeated measures showed
a significant difference in TGF-b2 concentrations (P# 0·0001). Statistically significant differences were evident for 1 month (P# 0·0001), 3 months (P# 0·0001),
6 months (P¼ 0·039) and 12 months (P# 0·0001).
E. Kainonen et al.1966
British Journal of Nutrition
Table 3. Numbers of Ig-secreting (Ig-scr.) cells in formula-fed and breast-fed infants (enzyme-linked immunospot assay)
(Geometric means and 95 % confidence intervals)
Age
(months)
All participants
P
(ANOVA
for
repeated
measures) P
Only atopic mothers
P
(ANOVA
for
repeated
measures) P
Formula-fed (n 18) Breast-fed (n 29) Formula-fed (n 12) Breast-fed (n 29)
Geometric
mean 95 % CI
Geometric
mean 95 % CI
Geometric
mean 95 % CI
Geometric
mean 95 % CI
Total Ig-scr.
cells/10
6
cells
3 1425 1079, 1883 1329 1048, 1684 0·18 1628 1102, 2405 1329 1048, 1684 0·34
6 1873 1400, 2564 1782 1330, 2388 1542 1121, 2121 1782 1330, 2388
12 2320 1604, 3354 2352 1635, 3383 2267 1472, 3491 2352 1635, 3383
Total Ig-scr.
cells/10
4
cells
3 1402 1114, 1765 1359 1051, 1757 0·77 1513 1092, 2098 1359 1051, 1757 0·52
6 1748 1192, 2564 1734 1223, 2460 1191 884, 1604 1734 1223, 2460
12 2417 1692, 3452 1981 1385, 2833 2339 1592, 3436 1981 1385, 2833
Total IgM-scr.
cells/10
6
cells
3 610 458, 796 422 332, 600 0·63 707 483, 1032 422 332, 600 0·98
6 743 464, 1189 693 519, 868 533 300, 948 693 519, 868
12 971 586, 1611 1166 763, 1781 786 450, 1374 1166 763, 1781
Casein-specific
IgA-scr.
cells/10
6
cells
3 3·2 1·8, 5·7 1 0·8, 1·3 0·0005 0·0004 2·6 1·2, 5·4 1 0·8, 1·3 0·01 0·008
6 2·5 1·4, 4·4 1·1 0·8, 1·5 0·015 2·4 1·2, 4·9 1·1 0·8, 1·5 0·041
12 2·3 1·4, 3·8 1·3 0·9, 1·9 0·1 2·2 1·2, 4·1 1·3 0·9, 1·9 0·19
Casein-specific
IgG-scr.
cells/10
6
cells
3 5·4 3·1, 9·4 0·9 0·8, 1·0 , 0·0001 , 0·0001 5·5 3·0, 10·0 0·9 0·8, 1·0 , 0·0001 , 0·0001
6 3·3 1·8, 6·1 1·1 0·8, 1·4 0·001 4·3 2·1, 8·7 1·1 0·8, 1·4 0·0002
12 2·6 1·3, 5·2 1·5 1·0, 2·2 0·17 2·2 1·0, 4·6 1·5 1·0, 2·2 0·39
Casein-specific
IgM-scr.
cells/10
6
cells
3 4·1 2·4, 7·0 1·9 1·1, 3·1 0·24 4·9 2·4, 9·8 1·9 1·1, 3·1 0·38
6 3·3 1·5, 7·5 2·2 1·5, 3·4 3·3 1·1, 6·1 2·2 1·5, 3·4
12 2·9 1·3, 6·6 2·6 1·5, 4·6 2·2 1·0, 4·9 2·6 1·5, 4·6
Immunological programming by breast milk 1967
British Journal of Nutrition
consumption of formula is associated with pro-inflammatory
immune responsiveness during the first year of life. In con-
trast, exclusive breast-feeding promotes an anti-inflammatory
cytokine milieu, which is maintained throughout infancy.
There are several possible explanatory models for the
observed results. We propose it is likely that the differences
seen between breast-fed and formula-fed infants could
be due to direct immunoregulatory effects of breast milk
TGF-b2, the effects of breast-feeding on infant gut microbiota,
functional immune cells and other immunoactive substances
contained in breast milk.
It is likely that breast-fed infants have ingested more exo-
genic TGF-b2 than the infants fed with formula. In a mouse
model, enterally given TGF-b emerged as systemic, immuno-
logically active TGF-b in nursing mouse pups
(41)
. In another
animal experiment, enterally given TGF-b was shown to
induce endogenous TGF-b production
(42)
. We, and others,
have shown in previous studies that the concentrations of
breast milk cytokines not only mirror the clinical immunologi-
cal status of the mother, but are also reflected in the cytokine
concentrations and clinical manifestations of their child
(43 – 45)
.
For example, high breast milk TGF-b2 concentrations are
associates with reduced risk of atopic disease in the
infant
(46)
. Serum cytokine levels not only reflect the present
state of immune activity, but also have the potential to guide
the development and polarisation process of various
immune cells, yielding long-lasting, perhaps even permanent,
effects on the individual’s immune function.
Indeed, the elevated serum TGF-b2 in breast-fed infants
was seen to persist throughout the first year of life, despite
reversion to a similar diet as the formula-fed group due to
the consumption of solid foods and cows’ milk, as well as
the cessation of breast-feeding. In light of the present finding,
it seems that the consumption of breast milk may not only
have direct immunoregulatory effects, but also the potential
to guide the infant’s own immune system towards the pro-
duction of regulatory T cells and healthy immune function.
The persistent anti-inflammatory effects of breast-feeding
may also be due to the effects of breast milk on infant gut
microbiota. In addition to active microbes
(47 – 49)
and mol-
ecules operative in microbial recognition
(50)
, breast milk
contains an abundance of complex oligosaccharides that the
microbial populations in the developing intestine utilise
(51 – 53)
.
These oligosaccharides are believed to facilitate development
of a healthy, infant intestinal microbiota rich in bifidobacteria.
Recent genomic analyses suggest an intricate co-evolution
among the human host, milk glycans and the microbes they
enrich, as human milk oligosaccharides appear to very specifi-
cally favour colonisation with certain microbial species that
have previously been shown to be beneficial to health
(54)
,
further underlining the unique role of breast milk in the
immunological education of the neonate.
The present results showed a similar phenomenon regard-
ing casein-specific Ig-secreting cells, as others focusing on
antibody levels have done previously
(55 – 58)
. The higher
levels witnessed in formula-fed infants may simply be a reflec-
tion of the higher antigen exposure that the formula-fed
infants are subjected to, and, consequently, antibody levels
in themselves should not be regarded as an indication of
permanently altered immune development. The witnessed
enzyme-linked immunospot assay results validate the present
study group division and show that formula-fed infants are
exposed to foreign antigens at an earlier age and to a greater
extent than breast-fed infants. This phenomenon in itself
could also contribute to the immunological differences
witnessed between the groups, but conclusions on causality
cannot be drawn from the present study. It is interesting
to note that differences in serum cytokine concentrations
outlast the differences witnessed in the numbers of cows’
milk-specific Ig-secreting cells, again suggesting a type of
immune programming by breast milk.
The breast-fed infants in the present study manifested high
levels of TGF-b2 and low levels of pro-inflammatory cytokines
throughout the first year of life and exceeding the duration
of breast-feeding. Such an immunological environment limits
IgE production and hyper-responsiveness and promotes
tolerisation, possibly prohibiting the onset of allergic disease.
In the pursuit for improved forms of alternative infant nutri-
tion, manufacturers should not necessarily solely endeavour
to mimic the composition of breast milk, but rather aim to
achieve similar physiological effects in the infant.
Acknowledgements
We would like to thank Mika Korkeama
¨
ki and Heikki
Arvilommi for their contributions towards the present study.
Funding for the present study was received from the Academy
of Finland and the Finnish Cultural Foundation Varsinais-
Suomi regional fund. The authors are not aware of any conflict
of interest. All authors contributed intellectually to the manu-
script to the extent that they can defend the contents.
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