of June 13, 2013.
This information is current as
Modulated by Human Milk
Responses Are Specifically and Differentially
Recognition: TLR-Mediated Innate Immune
Modulation of Neonatal Microbial
Thornton and Mario O. Labéta
Raby, Michael Affolter, Karine Vidal, Catherine A.
Emmanuel LeBouder, Julia E. Rey-Nores, Anne-Catherine
2006; 176:3742-3752; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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The Journal of Immunology
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Modulation of Neonatal Microbial Recognition: TLR-Mediated
Innate Immune Responses Are Specifically and Differentially
Modulated by Human Milk1
Emmanuel LeBouder,* Julia E. Rey-Nores,†Anne-Catherine Raby,* Michael Affolter,‡
Karine Vidal,§Catherine A. Thornton,¶and Mario O. Labe ´ta2*
The mechanisms controlling innate microbial recognition in the neonatal gut are still to be fully understood. We have sought
specific regulatory mechanisms operating in human breast milk relating to TLR-mediated microbial recognition. In this study, we
report a specific and differential modulatory effect of early samples (days 1–5) of breast milk on ligand-induced cell stimulation
via TLRs. Although a negative modulation was exerted on TLR2 and TLR3-mediated responses, those via TLR4 and TLR5 were
enhanced. This effect was observed in human adult and fetal intestinal epithelial cell lines, monocytes, dendritic cells, and PBMC
as well as neonatal blood. In the latter case, milk compensated for the low capacity of neonatal plasma to support responses to LPS.
Cell stimulation via the IL-1R or TNFR was not modulated by milk. This, together with the differential effect on TLR activation,
suggested that the primary effect of milk is exerted upstream of signaling proximal to TLR ligand recognition. The analysis of
TLR4-mediated gene expression, used as a model system, showed that milk modulated TLR-related genes differently, including
those coding for signal intermediates and regulators. A proteinaceous milk component of >80 kDa was found to be responsible
for the effect on TLR4. Notably, infant milk formulations did not reproduce the modulatory activity of breast milk. Together, these
findings reveal an unrecognized function of human milk, namely, its capacity to influence neonatal microbial recognition by
modulating TLR-mediated responses specifically and differentially. This in turn suggests the existence of novel mechanisms
regulating TLR activation. The Journal of Immunology, 2006, 176: 3742–3752.
dominance of LPS-producing Gram-negative bacteria may con-
tribute to the pathogenesis of infections and a variety of inflam-
matory conditions. However, the epithelial layer, together with the
intraepithelial and lamina propria immunocompetent cells, coor-
dinate adequate local innate and adaptive immune responses to the
microbial challenge. Such regulated recognition of microorgan-
isms in the neonatal gut is crucial to the maintenance of gut ho-
Notably, a lower incidence of gastrointestinal infections and in-
flammatory conditions as well as allergic diseases in breast-fed
newborns has long been reported (5–13). Over the years, protec-
tion by breast-feeding has been variously ascribed to a number of
mechanisms, and to milk components such as maternal immuno-
competent cells, Igs, antimicrobial peptides, oligosaccharides,
uring bacterial colonization of the newborn’s gut, a vast
microbial inoculum is brought into acute contact with
the hitherto sterile neonatal intestine. The initial pre-
growth factors, cytokines, lysozyme, lactoferrin, complement, and
nutrients (8, 14). However, we have sought additional and specific
mechanisms related to the recognition of microbes in the gut, be-
cause microbial recognition appears as a common factor directly or
indirectly involved in the pathological conditions that are claimed
to be reduced by breastfeeding. Such mechanisms remain to be
The activity of the mammalian TLR family is crucial to an im-
mediate and efficient innate recognition of an array of microor-
ganisms and their cell-wall components (15). Thirteen mammalian
TLRs have so far been identified (TLR1–TLR13) and the ligand
specificity described for most of them (16–18). Efficient microbial
a coreceptor, CD14, which is expressed as a cell surface molecule
(membrane-bound CD14; mCD14)3mainly in myeloid cells, and also
in serum as a soluble (s) coreceptor (sCD14). Through its coreceptor
activity, CD14 enhances cellular responses to most microbial com-
ponents activating via TLR2 or TLR4 (19–24).
TLRs are expressed preferentially in tissues that are in constant
contact with microorganisms, such as the lung and the gastro-
intestinal tract, as well as in immunologically important cell
types such as blood leukocytes and dendritic cells (DC)
(25–27). The latter are also distributed along the intestinal
epithelium and can sample pathogens directly into the gut
lumen by extending dendrites outside the epithelium (28).
Recent work highlighted the crucial role that TLRs play in the
gut by controlling intestinal epithelial homeostasis and protect-
ing it from direct injury (4, 29, 30).
*Infection and Immunity, Department of Medical Biochemistry and Immunology,
Cardiff University, College of Medicine, Cardiff, United Kingdom;†School of Ap-
plied Sciences, University of Wales Institute, Cardiff, United Kingdom;‡Department
of Bioanalytical Science, Nestle ´ Research Center, Lausanne, Switzerland;§Depart-
ment of Nutrition and Health, Nestle ´ Research Center, Lausanne, Switzerland; and
¶Newborn Immunity, School of Medicine, University of Wales Swansea, United
Received for publication September 1, 2005. Accepted for publication December
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Wellcome Trust of Great Britain.
2Address correspondence and reprint requests to Dr. Mario O. Labe ´ta, Infection and
Immunity, Department of Medical Biochemistry and Immunology, Cardiff Univer-
sity, College of Medicine, Henry Wellcome Research Building, Heath Park, Cardiff
CF14 4XX, United Kingdom. E-mail address: email@example.com
3Abbreviations used in this paper: mCD14, membrane-bound CD14; s, soluble; DC,
dendritic cell; poly(I:C), polyinosinic-polycytidylic acid; IEC, intestinal epithelial
cell; LBP, LPS binding protein.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
by guest on June 13, 2013
TLR engagement triggers the recruitment and activation of a
number of signal adaptors and intermediates. This leads to tran-
scription factor activation and the induction of genes that code for
a variety of proinflammatory and immunoregulatory cytokines,
chemokines, and costimulatory molecules (31). This process re-
sults in a prompt and efficient response to the microbial challenge.
However, the excessive release of some of these proinflammatory
molecules may lead to serious inflammatory conditions, which
may result in tissue damage and septic shock. Therefore, TLR
triggering has to be tightly regulated (32, 33). This is of particular
importance during bacterial colonization of the newborn’s gut, be-
cause only a finely controlled recognition of microorganisms will
result in the maintenance of neonatal gut homeostasis.
We have therefore sought TLR-specific mechanisms operating
in milk that may modulate microbial recognition in the gut, thereby
contributing to the beneficial effects of breast-feeding. We recently
reported the presence in human milk, but not in infant milk for-
mulations, of high concentrations of the coreceptor sCD14 (34).
We have proposed that milk sCD14 may enable efficient TLR
activation during bacterial colonization of the neonatal gut, and
suggested that milk sCD14-mediated TLR triggering might be
tightly regulated (34). We have therefore looked for regulatory
mechanisms. More recently, we described the existence of sol-
uble forms of TLR2 (sTLR2) capable of regulating cell activa-
tion via cell surface TLR2, and naturally expressed in human
milk as well as in plasma (35). Interestingly, sTLR2, like
sCD14, was not detected in infant milk formulations. These
findings prompted us to test in this study whether additional
regulatory mechanisms operating in milk are involved in mod-
ulating TLR-mediated microbial recognition.
Materials and Methods
Ultra-pure LPS (Escherichia coli O111:B4 strain), bacterial peptidoglycan,
heat-killed Listeria monocytogenes, bacterial flagellin, and polyinosinic-
polycytidylic acid (poly(I:C)) were purchased from InvivoGen. The syn-
thetic bacterial lipopeptide Pam3-Cys-Ser-(Lys)4HCl (Pam3Cys) was ob-
tained from EMC Microcollections. Heat-killed E. coli (NLTC 1048) was
provided by Dr. S. Jackson (Department of Medical Microbiology, Cardiff
University, College of Medicine, Cardiff, U.K.). IL-1? and TNF-? were
obtained from R&D Systems. Human milk-derived sCD14 was isolated
and purified as described previously (34). The anti-human CD14 (clone
MY4, IgG2b), and CD86-PE conjugate (clone IT2.2, IgG2b) mAbs were
obtained from Beckman Coulter, and BD Pharmingen, respectively. The
anti-TLR2 mAb (clone TLR2.1, IgG2a) was obtained from eBioscience.
The anti-TLR4 mAb (clone HTA125, IgG2a) was provided by K. Miyake
(Saga Medical School, Saga, Japan). The anti-TLR3 (clone 40C1285.6,
IgG1) and TLR5 (clone 85B152.5, IgG2a) mAbs were donated by Immu-
noKontact (AMS Biotech, Oxon, U.K.). The isotype-matched controls
IgG2b, IgG1- and IgG2b-PE conjugate were obtained from Diaclone. GM-
CSF (Leukine, Sargramostim) was obtained from Berlex Laboratories.
IL-13 was donated by Sanofi-Aventis. Proteinase K was obtained from
Roche Diagnostics. All chemicals were reagent grade.
Milk, neonatal (cord) blood, cells, and cell activation
Human milk from clinically healthy mothers of term infants (38–39 wk
gestation) was obtained after written consent. Any relevant pathological
condition (e.g., mastitis) was an exclusion criterion. Milk samples contain-
ing ?6 pg/ml LPS (Limulus amebocyte lysate test) and/or capable of in-
ducing detectable amounts (above background) of IL-8 or IL-6 (ELISA) by
Mono Mac-6 monocytes were excluded from this study. Cell-free milk
aliquots were kept at ?80°C until use. Three different commercially avail-
able infant milk formulations were prepared just before using, following
the manufacturer’s instructions. Newborn cord blood—from infants born at
39? wk of gestation (with the exception of one at 37 wk 4 days)—was
collected following local Health Authority Ethical Committee approval
from the umbilical cord immediately after vaginal birth or after cesarean
section delivery of the placenta. The blood was anticoagulated (129 mM
sodium citrate), and the hemocytes were washed (600 ? g for 6 min at
room temperature) with phenol red-free RPMI 1640 medium (Invitrogen
Life Technologies) and resuspended in autologous plasma, adult (AB)
plasma, or milk, as indicated in Results, in preparation for the experiments.
The adult intestinal epithelial cell (IEC) lines HT-29, SW-620, Caco-2, and
T-84 were obtained from the American Type Culture Collection. The non-
malignant fetal IEC line H-4 was developed from a 20-wk-old normal fetal
small intestine (36) (provided by Dr. A. Walker, Massachusetts General
Hospital, Department of Pediatrics, Harvard Medical School, Boston, MA).
The adult IEC lines HT-29, T-84, and Caco-2 were cultured in DMEM
medium (Invitrogen Life Technologies) supplemented with 10% FBS (de-
fined FBS; HyClone), 2 mM glutamine, and further supplemented with 1%
nonessential amino acids for Caco-2. The H-4 cell line was cultured in
DMEM medium supplemented with 10% FBS (HyClone), 2 mM glu-
tamine, 1 mM sodium pyruvate, 10 mM HEPES, 1% nonessential amino
acids, and 10 ?g/ml insulin (Invitrogen Life Technologies). The human
monocytic cell line Mono Mac-6 (provided by H. W. L. Ziegler-Heitbrock,
Department of Immunology, Leicester University, Leicester, U.K.) was
cultured in RPMI 1640 medium supplemented with 10% FBS (HyClone),
2 mM glutamine, 1 mM pyruvate, 1% nonessential amino acids, and 10
?g/ml insulin (all obtained from Invitrogen Life Technologies). PBMC
were obtained after Ficoll density-gradient centrifugation of buffy coats
from heparinized blood of healthy donors. Monocyte-derived DCs were
obtained following culture of monocyte preparations from PBMC (2 h at
37°C adherence) for at least 6 days in phenol red-free RPMI 1640 supple-
mented with 10% FBS (HyClone), 2 mM glutamine, 2 mM sodium pyru-
vate, 1% nonessential amino acids, GM-CSF (800 IU/ml), and IL-13 (100
ng/ml). At day 3, fresh medium containing the cytokines was added to the
cultures. The purity of the DC preparations was always ?95%, as evalu-
ated by FACS with anti-CD3, -CD19, -CD14, -CD11c, -CD40, -CD80, and
-CD83 mAbs. The expression level of the particular TLRs tested in the
cells shown or referred to in Results was determined by FACS (data not
shown). Specifically, a relatively moderate level of expression of TLR4
was detected in adult PBMC, a low level in cord blood and H-4 cells, a
very low level in HT-29 cells, Mono Mac-6 monocytes, and DCs, and
undetectable expression (inferred, as indicated by the cell stimulation as-
says shown in Results, and Northern blots) in Caco-2 and T-84 cells. The
TLR5 expression in Mono Mac-6 monocytes was low. TLR2 expression
was moderate in Mono Mac-6 cells, and the TLR3 expression in DCs was
relatively high. For cell stimulation experiments, the adult IEC lines, the
Mono Mac-6 monocytes, PBMC (2 ? 105cells) as well as the fetal IEC
line H-4 (1 ? 105cells) and DCs (1 ? 104cells), were cultured in 96-well
plates in phenol red-free RPMI 1640/1 mM glutamine medium without
serum in the absence or presence of the indicated concentrations of sCD14
or milk, and stimulated with different concentrations of TLR2, TLR3,
TLR4, or TLR5 ligands, IL-1? or TNF-?, as indicated in Results. At the
indicated time points, culture supernatants were collected and tested for
IL-6, IL-8, TNF-?, and IP-10 by ELISA (Duoset; R&D Systems).
Immunofluorescence and FACS analysis
Detection of cell surface CD86 in DCs with the anti-CD86-PE mAb was
performed by immunofluorescence, followed by FACS analysis, as de-
scribed previously (37). In this study, DCs were preincubated for 10 min
with 20% normal rabbit serum before washing and staining.
Gene microarray analysis
For gene expression profile analyses, quadruplicate cultures of Mono
Mac-6 monocytes (5 ? 106cells) in serum-free and phenol red-free RPMI
1640/1 mM glutamine medium were supplemented or not with 0.125%
human milk (n ? 2; collected at day 2 and 3 postpartum). The concentra-
tion of sCD14 in the cultures was normalized by supplementing the cul-
tures without milk with purified milk-derived sCD14 at the same concen-
tration as that in the 0.125% milk sample used. The cultures were
stimulated with 5 ng/ml LPS for 2, 4, and 16 h. At each time point, the
culture supernatants were collected for cytokine determinations (IL-6,
IL-8, TNF-?, and IP-10), and total RNA was extracted from the cells in
preparation for the gene array analysis by using the RNeasy kit (Qiagen)
following the manufacturer’s instructions. Comparative analyses of gene
expression between cells stimulated in the presence and absence of milk
were performed by using a TLR-focused, cDNA-based microarray (GE-
Arrays; SuperArray Bioscience) following the manufacturer’s instructions.
One hundred one cDNA fragments (300–600 bp), corresponding to TLR-
related (96 cDNAs; see Table I) and control (5 cDNAs: pUC18, GAPDH,
cyclophilin A, RPL13A, and ?-actin) genes, and printed in a tetra-spot
format on nylon membranes, were hybridized with biotin-labeled cDNA
probes prepared from the RNAs (3 ?g) extracted from the experimental
3743The Journal of Immunology
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samples. The specifically hybridized probes were detected as spots in the
microarrays by using a chemiluminescent detection system (SuperArray
Bioscience). Data were collected by scanning the x-ray films, and pixel
levels were analyzed with the ScanAlyze software (SuperArray Bio-
science). Expression values of transcripts scoring as present after back-
ground subtraction and normalization, according to an internal program
algorithm, were compared. Transcripts with a ratio of normalized expres-
sion levels between the two experimental conditions (with or without hu-
man milk) altered by ?2-fold (average of three independent experiments)
were considered modulated.
In preparation for the cell stimulation experiments described (see Fig. 6),
0.125% milk samples were either: 1) boiled (5 min), 2) subjected to pro-
tease digestion (proteinase K, 0.5 U/ml milk, 30 min, 37°C), 3) ultrafiltered
(Ultra-free MC centrifugal filters, PL-10 10-kDa cutoff membrane; Milli-
pore), or 4) the milk sample was fractionated by using a restricted access
media column (provided by Tosoh Biosciences) consisting of size-exclu-
sion (exclusion limit 80 kDa) and anion-exchange chromatographic media.
Up to 4 ml of defatted and casein-depleted milk were loaded onto the
column and fractionated as described recently (38). The flow-through and
Ratio LPS ? HM/LPS ? sCD14 (fold change)
2 h4 h16 h
aThe gene expression profile of cells stimulated with LPS in the absence
(?sCD14) and presence of human milk (?HM) was compared by performing a TLR-
focused microarray analysis as described in Materials and Methods. The ratio be-
tween the expression values of transcripts in the two experimental conditions after
background subtraction and normalization is shown for each gene at the indicated
time points poststimulation. Transcripts with ratios altered by ?2-fold were consid-
ered not affected (?).
Table I. Effect of human milk on gene expression by LPS-stimulated
Mono Mac-6 monocytesa
Ratio LPS ? HM/LPS ? sCD14 (fold change)
2 h4 h16 h
C-type lectin M
3744 MODULATION OF TLR-MEDIATED RESPONSES BY HUMAN MILK
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eluted fractions were dialyzed (PBS), and their sCD14 concentrations nor-
malized by addition of purified sCD14 before cell stimulation experiments.
Enhancing effect of human breast milk on TLR4- and
TLR5-mediated cellular responses
To test for additional TLR-specific regulatory mechanisms oper-
ating in milk, we first focused on TLR4, and evaluated the role that
milk sCD14 plays in IEC stimulation via this receptor. IEC stim-
ulation via TLR4 strictly depended on milk sCD14, because an
anti-CD14 mAb (MY4) blocked the milk-mediated stimulation of
the human (mCD14?) IEC lines HT-29 (Fig. 1) and SW620 (data
not shown) induced by LPS or whole Gram-negative bacteria.
These results confirmed our previous findings (34). We found,
however, that milk sCD14 was necessary, but not sufficient, for
efficient TLR4-mediated IEC stimulation, because purified milk-
derived sCD14 did not reproduce the effect of whole milk, even
when sCD14 was used at concentrations up to fifty times higher
than those in the milk samples (Fig. 2A). This enhancing effect of
milk was observed in the adult IEC line HT-29, the nonmalignant
IEC line of fetal origin H-4 (mCD14?), human monocytes (Mono
Mac-6), and DCs (Fig. 2A), as well as in PBMC and the adult IEC
lines Caco-2 and T-84 (data not shown). The production of IL-8,
IL-6, TNF-?, and IP-10 was found to be affected (Fig. 2A). The
effect of milk, however, was not restricted to the LPS-induced
production of cytokines, because costimulatory molecule (CD86)
expression in DCs was also found to be enhanced (Fig. 2B). The
enhancing effect was not observed after a week postpartum (Fig.
2C) and, notably, was not reproduced by using infant milk formu-
lations, even when they were supplemented with sCD14 (Fig. 2D).
This effect of milk was not limited to stimulation via TLR4. In fact,
cell stimulation via TLR5 by the TLR5 ligand bacterial flagellin
was found also to be enhanced (Fig. 2E).
Negative effect of human milk on cell stimulation via TLR2
The enhancing effect of milk posed the question of whether milk
exerts only a positive regulation on TLR-mediated cell stimulation.
In contrast to the enhancing effect on TLR4 and TLR5, in the
presence of milk cell stimulation induced via TLR2 by a number
of TLR2-specific ligands was found significantly reduced (Fig.
3A), as compared with that in the presence of the coreceptor
sCD14 (used at the same concentration as in the milk sample) or
to stimulation in the absence of any supplement (cells alone) at
high ligand concentration. A similarly negative effect of milk was
observed when DCs were stimulated via TLR3 with the viral
dsRNA mimic TLR3 ligand poly(I:C) (Fig. 3B). Interestingly,
poly(I:C)-induced CD86 expression in DCs was not affected by
milk (Fig. 3C). This was also in contrast to the positive effect
observed when TLR4 was tested (Fig. 2B). The inhibitory effect
was not reproduced by using infant milk formulations (Fig. 3D),
like the enhancing effect on TLR4 (Fig. 2C).
Cell stimulation via the IL-1R or TNFR is not modulated by
We asked whether the regulatory activity of human milk was re-
stricted to TLRs, or whether other receptors were also affected. To
address this issue, we tested the effect of milk on cell stimulation
via the IL-1R, which shares with TLRs the (MyD88-dependent)
signaling pathway, because both receptors have a conserved region
in their intracytoplasmic domain, known as the Toll/IL-1R do-
main, which is crucial for signaling (31). In addition, the effect of
milk on signaling via a TLR nonrelated receptor, the TNFR, was
tested. Fig. 4 shows that cell stimulation via either the IL-1R or
TNFR was not affected by milk. This finding, together with the
capacity of anti-CD14 mAb to block milk-mediated cell stimula-
tion (Figs. 1 and 2A), and the lack of effect of milk on cells alone
(Figs. 1, 2, and 3), indicated that the modulatory effect is exerted
in a specific manner on the ligand-induced TLR-mediated
Human milk modulates newborn hemocyte sensitivity to
We then asked whether the modulatory effect of milk observed in
a number of cell types and lines as well as in adult PBMC can be
reproduced in normal newborn immunocompetent cells. It has also
recently been demonstrated that newborn (cord) plasma confers
greatly reduced sensitivity to TLR4-mediated leukocyte stimula-
tion, as compared with adult plasma (39). Therefore, we also asked
whether milk behaves differently from neonatal plasma. To ad-
dress these issues, we used whole neonatal (cord) blood as a min-
imally perturbed ex vivo model system, and compared the capacity
of newborn plasma and milk to mediate cell stimulation induced
via TLR4. Washed newborn hemocytes were resuspended in 100%
autologous plasma, 100% adult (AB) plasma, or 1–2% human
natants of the IEC line HT-29 (2 ? 105cells) cultured for 16 h in the absence or presence of human milk (HM, day 2) and stimulated with 1 ?g/ml
ultrapure E. coli LPS or varying numbers of whole E. coli. The anti-CD14 mAb MY4 (IgG2b, 10 ?g/ml), but not its isotype-matched control, blocked
LPS and E. coli stimulation. Results are mean ? SD of triplicate cultures of one experiment representative of five performed with milk samples
collected at days 1–5 postpartum. The differences in cytokine release between MY4- and isotype control-treated cultures were compared using the
Student’s t test; ???, p ? 0.0001.
IEC stimulation by LPS or Gram-negative bacteria depends on milk sCD14. IL-8 production (ELISA) was tested in culture super-
3745 The Journal of Immunology
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105cells), the nonmalignant fetal IEC line H-4 (1 ? 105cells), Mono Mac-6 (MM6) monocytes (2 ? 105cells), and DCs (1 ? 104cells) were stimulated
for 16 h with the indicated concentrations of ultrapure E. coli LPS (A) or flagellin (E) in the absence or presence of 0.125% human breast milk (HM) or
purified milk-derived sCD14 (6 ?g/ml). IL-8, IL-6, TNF-?, and IP-10 concentrations in the culture supernatants were determined by ELISA. Results are
mean ? SD of triplicate cultures of one experiment representative of five (A) or three (E). B, Fluorescence profiles of CD86 expression in DCs stimulated
for 16 h with 2.5 ng/ml LPS in the absence or presence of human milk or purified milk-derived sCD14 as described for A and E. The shaded
profile corresponds to the staining with the PE-conjugated isotype-matched control Ab. Results shown are representative of four experiments.
Human milk, but not infant milk formulations, enhances TLR4- and TLR5-mediated responses. A and E, The adult IEC line HT-29 (2 ?
3746 MODULATION OF TLR-MEDIATED RESPONSES BY HUMAN MILK
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milk. Notably, just a 2% concentration of milk was sufficient toen-
hance IL-8 and TNF-? release by newborn hemocytes in response
to LPS, as compared with neonatal plasma (Fig. 5). This finding
indicated that newborn peripheral immunocompetent cells are also
susceptible to regulation by milk, and that milk can compensate for
the low capacity of neonatal plasma to support responses to LPS.
C, IL-8 released by HT-29 cells stimulated as in A with 1 ?g/ml LPS in the absence or presence of 0.125% human milk (collected at day 2, 7, or 15
postpartum) or purified milk sCD14 (1 ?g/ml). Results are representative of three experiments. D, IL-8 concentrations in culture supernatants of HT-29
cells stimulated as in A with 1 ?g/ml LPS in the absence or presence of 0.125% human milk, 0.125% commercially available infant milk formulations (n ?
3), or purified milk-derived sCD14 (1 ?g/ml). Results are from one experiment (?SD) representative of three. Experiments in A, B, D, and E were
performed with milk samples collected at day 1, 2 (shown), 3, and 5 postpartum. The sCD14 concentration in the 0.125% human milk samples (n ? 3)
used in the experiments shown in A–E was 122 ? 5 ng/ml (day 2), and in C, day 7 and 15, 61 ? 12 ng/ml and 47 ? 16 ng/ml, respectively. The differences
in cytokine release between human milk and sCD14 supplemented cultures were significant: ?, p ? 0.005; ??, p ? 0.001; ???, p ? 0.0001.
105cells) or DCs (1 ? 104cells) were stimulated for 16 h with the indicated concentrations of the TLR2-specific ligands (MM6) Pam3-Cys-Ser-(Lys)4(Pam3),
in the absence or presence of 0.125% human breast milk (HM) or 125 ng/ml purified milk-derived sCD14 (TLR2 stimulation). IL-8 levels in the culture
supernatants were determined by ELISA. C, Fluorescence profiles of CD86 expression in DCs (1 ? 104cells) stimulated or not with 80 ?g/ml poly(I:C) for 16 h
in the absence or presence of 0.125% human milk. The shaded profile corresponds to the staining with the PE-conjugated isotype-matched control Ab. D, IL-8
levels in the culture supernatants of Mono Mac-6 cells stimulated as in A with 5 ?g/ml PGN in the absence or presence of 0.125% human milk, 0.125% infant
milk formulations (n ? 3), or 125 ng/ml purified milk-derived sCD14. Results shown in A to D are from one experiment (?SD in A, B, and D) representative
of four (A–C) or three (D) performed with milk collected at day 1, 2 (shown), 3, and 5 postpartum. The sCD14 concentration in the 0.125% human milk samples
(n ? 3) used in the experiments shown in A–D was 122 ? 5 ng/ml (day 2). The differences in IL-8 release between human milk and purified sCD14 supplemented
cultures or cells (poly(I:C) stimulation) were significant: ??, p ? 0.001; ???, p ? 0.0001.
Negative effect of human milk, but not infant milk formulations, on cell stimulation via TLR2 and TLR3. A and B, Mono Mac-6 monocytes (2 ?
3747The Journal of Immunology
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Differential modulatory effect of milk on the LPS-induced TLR-
related gene expression
To evaluate the extent of milk’s regulatory effect and obtain an insight
into the underlying mechanism, we used a gene array approach. We
focused on TLR4, and used the TLR4-mediated (Mono Mac-6)
monocyte stimulation by LPS as a model system. We asked whether
the enhancing effect observed on the LPS-induced cytokine and co-
milk on the ligand-induced TLR4-mediated gene expression. To ad-
dress this issue, we performed a comparative, TLR-focused, microar-
ray analysis of the expression of 96 TLR-signaling-related genes by
monocytes stimulated with LPS in the presence, as opposed to the
absence, of human milk (preliminary gene array studies and the re-
sults shown in Figs. 1, 2, and 3 indicated that milk has no effect on
nonstimulated cells). To normalize the concentration of sCD14, cell
cultures stimulated in the absence of milk were supplemented with
sCD14. Primary and secondary transcriptional changes in gene ex-
pression were evaluated by conducting the analysis at relatively early
(2 and 4 h) and late (16 h) time points poststimulation.
Eighty four percent of the genes (81 genes) were found affected
(?2-fold) by milk upon LPS stimulation (Table I). Eighty three
percent of the genes affected (67 genes) were up-modulated 2 h
poststimulation (Table I, early up-modulation category). These
early up-modulations were accompanied or not by later effects.
Notably, however, a total of 34 genes (42%) were found down-
modulated, including 10 genes after 2 h or 4 h (Table I, early
down-modulation category), 3 after 16 h (Table I, late modulations
category), and a further 21 preceded by earlier modulations (Table
I, early up-modulation category). The relative down-modulation of
16 of these genes was observed at 4 h, and was preceded by an
earlier (2 h) up-modulation (Table I, early up-modulation cate-
gory). This reflects, most likely, the effect of milk on the expres-
sion kinetics of these genes (i.e., faster kinetics), rather than an
absolute negative effect on their expression levels at 4 h.
Early up-modulations affected many genes coding for TLR sig-
nal intermediates, e.g., ECSIT, IL-1R-associated kinase family
members, and isoforms of p38MAPK. The transcriptional factor
IFN regulatory factor-3 and those of the NF-?B family, I-REL
(REL-B), KBF1 (p105/p50), NFKB2 (p100/p52), NFKB3 (REL-
A), proinflammatory mediators and cytokines (IFN-?1, IFN-?, IL-
1?, IL-1?, IL-2, IL-6, IL-8, IL-12?, IL-12?, IP-10, TNF-?, TNF-
?), TLRs (TLR2, TLR4, TLR5, TLR7, TLR8, TLR9), the TLR4
accessory molecule MD2, as well as the apoptosis intermediate
and NF-?B inducer FLICE (caspase-8) were also found up-mod-
ulated. Some TLR signal intermediates were, however, down-
modulated early in the presence of milk, e.g., JIP3, MKK3, TAK1,
TIRAP (MAL) as well as the apoptosis intermediate Fas-associated
via death domain protein. Notably, down-modulation of mRNA
levels (2 h) also strongly affected the transcript coding for TLR3.
Late (16 h, positive or negative) effects of milk, accompanied or
not by earlier modulations, were observed in ?31% of the genes
affected (25 genes). These late effects most likely depended on
protein synthesis induced in the initial wave of gene induction.
Interestingly, 64% of the late-modulated genes (16 genes) were
subjected to down-regulation. Late modulations not accompanied
by earlier effects were observed in only four genes (Table I, late
modulations), and were negative, with the notable exception of the
relatively strong up-regulation of the gene coding for the TLR-
negative regulator molecule SIGIRR (TIR8).
The validity of the microarray analysis was confirmed by the
correlation between the relatively high concentrations of IL-8,
IL-6, TNF-?, and IP-10 detected in the cultures supplemented with
milk and the early up-modulation of the corresponding transcripts
(Fig. 2A and Table I) as well as by performing Northern blots for
RNA analysis of prototypical examples of early down-modulated
(Fas-associated death domain protein) and late-modulated (IKK1)
genes (data not shown).
A milk protein component is responsible for the modulatory
effect on LPS-induced cell stimulation
To obtain an insight into the molecular nature of the milk compo-
nent responsible for the modulatory effect of milk on LPS-induced
cell activation, the milk samples were subjected to heat, protease
digestion, and size fractionation. Fig. 6 shows that boiling as well
as proteinase K digestion of the milk samples before the experi-
ments abrogated the enhancing effect (Fig. 6, A and B). Further-
more, the enhancing effect of milk was reproduced by using the
retentate, but not the filtrate following size fractionation of the
samples by ultrafiltration through a 10-kDa cutoff membrane (Fig.
milk. IL-8 levels (ELISA) in culture supernatants of HT-29 cells (2 ? 105
cells) stimulated for 16 h with the indicated concentrations of IL-1? or
TNF-? in the absence or presence of 0.125% human milk (day 2). Results
are mean ? SD of triplicate cultures of one experiment representative of
Cell stimulation via the IL-1R or TNFR is not affected by
milk. Newborns’ cord blood (n ? 4) was washed, and 200 ?l of hemocyte
aliquots were resuspended in 100% autologous or heterologous plasma, 1%
or 2% human milk, and stimulated for 16 h with 5 ng/ml LPS. IL-8 and
TNF-? concentrations in the culture supernatants were determined by
ELISA. Results are from one experiment (?SD) representative of four
performed with milk samples collected at day 2 (shown), 3, and 5 post-
partum. The concentration of sCD14 in the 2% milk sample and 100%
autologous plasma used in the experiment shown was 1.26 ?g/ml and 1.65
?g/ml, respectively. The differences in cytokine release between hemo-
cytes resuspended in autologous plasma and human milk were significant;
???, p ? 0.0001.
Newborn hemocyte sensitivity to LPS is modulated by
3748 MODULATION OF TLR-MEDIATED RESPONSES BY HUMAN MILK
by guest on June 13, 2013
6C). The milk samples were fractionated further by using a com-
bination of anion-exchange and size-exclusion (exclusion limit, 80
kDa) chromatography in a single column. The sCD14 concentra-
tion in the flow-through and eluted fractions was normalized by the
addition of purified milk-derived sCD14, and the capacity of both
fractions to mediate HT-29 cell stimulation by LPS was tested.
Fig. 6D shows that the enhancing effect of milk was reproduced
by using the flow-through, but not the eluted fraction, and the
supernatants of HT-29 cells stimulated, as described in Fig. 2A, with 1 ?g/ml LPS in the absence or presence of human milk or purified milk-derived sCD14
(1 ?g/ml). For some experiments (D), the anti-CD14 mAb MY4 (IgG2b) or its isotype-matched control (10 ?g/ml) was added to the cultures. Before the
experiments, the 0.125% milk preparations were as follows: A, boiled (ø); B, proteinase K treated (Prot. K); C, ultrafiltered (10-kDa cutoff membrane). In
D, the milk sample was fractionated by using restricted access media chromatography as described in Materials and Methods. The sCD14 concentration
in the flow-through and eluted fractions was normalized by adding purified sCD14 before cell stimulation experiments. The sCD14 concentration in the
0.125% milk samples (day 2) used in A–C was 122 ? 5 ng/ml (n ? 3). Results are mean (?SD) of triplicate cultures of two independent experiments (D)
or of one experiment representative of three (A and B) or two (C).
A milk protein component is responsible for the enhancing effect on LPS-induced cell stimulation. A–D, IL-8 concentrations in culture
3749The Journal of Immunology
by guest on June 13, 2013
combination of both fractions restored the original effect of the
unfractionated milk. Together, these results indicated that a
proteinaceous milk component(s) of ?80 kDa is responsible for
the TLR4-enhancing effect.
This study reveals an unrecognized function of human milk,
namely, its capacity to influence neonatal microbial recognition by
modulating TLR-mediated responses specifically and differen-
tially. This in turn suggests the existence of novel mechanisms
regulating TLR activation.
The use of individual TLR ligands, mostly corresponding to or
representing main microbial components expressed and released
by a variety of bacteria colonizing the neonatal gut (except for
heat-killed L. monocytogenes and poly(I:C)), allowed us to dissect
and analyze the response of the TLR tested separately.
Following ligand-induced cell stimulation, the modulatory ef-
fects exerted by human milk on TLR2 and TLR3, and those on
TLR4 and TLR5 were found to be opposite. Although a negative
modulation was exerted on TLR2- and TLR3-mediated responses,
those via TLR4 and TLR5 were enhanced. These effects were ob-
served only when relatively early milk samples (days 1–5 post-
partum) were tested. The fact that milk did not affect IL-1R sig-
naling (Fig. 4), and that it affected TLR2 and TLR3 differently
from TLR4 and TLR5, indicates that the primary effect of milk is
exerted upstream of the signaling pathway, most probably proxi-
mal to ligand recognition.
As regards the enhancing effect on TLR4-mediated responses,
the mRNA level for TLR4 was found up-modulated (2-fold) early
(2 h) in the presence of milk (Table I). Increased TLR4 expression
may contribute to the enhancing effect. However, it cannot explain
the modulatory effect of milk on genes that were found affected as
early as the one for TLR4, nor how TLR4 was up-modulated early
(2 h) in the first place. The potency of the receptor, rather than
receptor numbers, seems to be affected, because the sensitivity to
low doses of ligand was increased preferentially (Fig. 2A; HT-29),
whereas flow cytometric analysis did not show variations in TLR-4
cell surface expression (data not shown). Notably, the mRNA level
for the accessory molecule MD2 was strongly up-regulated in the
presence of milk at a relatively early time point (Table I). It is well
documented that this secreted molecule is crucial to LPS recogni-
tion and the function of the LPS receptor complex (29, 40, 41). It
is thus possible that an increased level of MD2 results in selective
(up- or down-) modulation or recruitment of signal intermediates
following LPS triggering of the receptor. This in turn may influ-
ence the quality and extent of the biological response. However,
this cannot explain the effect of milk on genes that were found
affected as early as the one for MD2 (Table I). It could be argued
that MD2 present in milk contributes to this effect. This would be
consistent with the proteinaceous nature of the TLR4-enhancing
milk component (Fig. 6). Although this possibility cannot be ex-
cluded, we failed to detect MD2 in a number of milk samples (our
unpublished observations). The presence of the LPS binding pro-
tein (LBP) in milk would also explain the enhancing effect of milk
on cytokine and costimulatory molecule expression, because
LBP—at low concentrations—is known to facilitate the interaction
of LPS with CD14, thereby increasing cell sensitivity to LPS sub-
stantially (42). However, this is unlikely because of the following:
1) we have reported that the concentration of LBP in milk is ex-
tremely low, at the limit of detection (34), and at the concentra-
tions of milk used in this study (0.1–0.2%), milk LBP was unde-
tectable; 2) we also found that preincubating milk with anti-LBP
Ab does not affect IEC sensitivity to LPS (43); 3) supplementing
milk samples with highly purified LBP (0.5 ?g/ml) does not in-
crease the enhancing capacity (our unpublished observations); and
4) the Mrof LBP (60, 000) is not consistent with that estimated for
the TLR4-enhancing milk component (?80,000; Fig. 6D). We also
tested for the possible involvement of GM-CSF, because this
growth factor was shown to up-modulate TLR-mediated responses
(44, 45). However, in our hands, GM-CSF (100 or 200 IU/ml)
either alone or as a milk supplement did not affect Mono Mac-6
sensitivity to LPS (our unpublished observations).
Further experimentation will be necessary to determine the na-
ture of the primary event resulting in the modulatory effect of milk
on TLR4 signaling. Similarly, the primary events responsible for
the positive effect on TLR5 and negative effects on TLR2 and
TLR3 remain to be elucidated. With regard to the negative effect,
milk markedly inhibited the TLR3-mediated poly(I:C)-induced cy-
tokine production by DCs without affecting costimulatory mole-
cule expression (Fig. 3, B and C), suggesting that the MyD88-
dependent (cytokine production), but not the independent pathway,
which controls costimulatory molecule expression and is the main
signaling pathway for TLR3 (31), was affected. This was in
marked contrast to the effect of milk on TLR4, because here both
cytokine production and costimulatory molecule expression were
affected, and in a positive manner (Fig. 2, A and B). Similar to
TLR3, the negative regulation of TLR2 appears to result from an
effect on the MyD88-dependent pathway, because this is the only
signaling pathway used by this receptor. At present, we are eval-
uating the extent of the milk sTLR2’s contribution to this negative
regulation. Interestingly, TLR5 was positively affected by milk
despite only using the MyD88-dependent signaling pathway, like
TLR2. These findings support the claim that the primary event(s)
affecting TLRs is exerted upstream of signaling.
The individual expression of TLRs in the initial cultures of the
cells tested is unlikely to contribute to the differences observed,
because the different capacity of milk, sCD14, and nonsupple-
mented cultures (cells alone) to mediate ligand-induced cell stim-
ulation were observed in the same cell line by testing and com-
paring parallel cultures of the same cell line preparation.
Moreover, the modulatory effect of milk was observed in cells
irrespective of the expression level of the TLR involved, e.g., the
TLR4-enhancing effect was observed in cells with moderate
(PBMC), low or very low (H-4, cord blood, Mono Mac-6, HT-29,
DCs) as well as undetectable—inferred—(Caco-2 and T-84 cells)
The TLR4-mediated cellular response was used as a model sys-
tem in comparative, TLR-focused, gene array analysis to deter-
mine the extent of modulatory activity, and to obtain an insight
into the underlying mechanism. The majority of the TLR-related
genes tested were up-modulated by milk upon cellular stimulation
by LPS. However, a substantial number of genes were down-mod-
ulated or not affected, and others were only modulated late as a
result of secondary transcriptional events (Table I). These findings
indicated that milk behaves as a general TLR modulator, and not
as an enhancer of a discrete number of genes. Notably, among the
genes up-modulated early were those coding for TLR signal in-
termediates, including three of the four isoforms of p38MAPK,
which are involved in the MyD88-dependent and -independent sig-
naling pathways of gene induction, and the transcriptional factor
IFN regulatory factor-3, which is crucial to the MyD88-independent
not only cytokine production (Fig. 2A), which is controlled by the
MyD88-dependent and -independent signaling pathways, but also
IP-10 and costimulatory molecule expression (Fig. 2, A and B), which
follows a MyD88-independent signaling pathway (31). Transcripts
coding for family members of the transcription factor NF-?B and the
protease FLICE (caspase-8) were also found up-modulated early
3750MODULATION OF TLR-MEDIATED RESPONSES BY HUMAN MILK
by guest on June 13, 2013
(Table I). Interestingly, caspase-8, although involved in cellular apo-
ptosis, has recently been shown to be required for the efficient nuclear
translocation of the transcription factor NF-?B following TLR4 stim-
milk, because NF-?B plays a pivotal role in LPS-mediated gene
induction (31). The gene array analysis also identified transcripts
coding for molecules influencing T cell activation and responses that
were up-modulated by milk, including IL-1?, IL-1?, IL-12? (IL-
12p35), and IL-12? (IL-12p40). It will thus be important to evaluate
the effect that this modulation may have on the adaptive immune
response. A late (16 h), but relatively strong up-modulation of the
transcript coding for SIGIRR was observed. This cell surface mole-
cule is known to negatively regulate TLR4 signaling (47). The
putative overexpression of SIGIRR, together with that of other TLR-
namely IL-1R-associated kinase-M and TOLLIP (Table I), may be
part of a negative feedback to control the extent of the milk’s
No correlation between the milk-induced enhanced expression
of CD86 and the level of the corresponding mRNA was observed
(Fig. 2B and Table I). It is possible that the (up) regulation of
CD86 was exerted at the protein synthesis or posttranslational lev-
els. Alternatively, after LPS stimulation, milk may have induced
mobilization to the cell surface of an internal pool of this molecule.
Overall, the data support the notion that the primary effect of
milk is exerted upstream of the signaling pathway. In this regard,
recent studies have demonstrated that different cell surface mole-
cules associating with TLRs impart specificity to TLR-mediated
microbial recognition (48–50). Furthermore, it has been shown
that, following LPS stimulation, TLR4, MD2, and a number of
additional cell surface molecules are recruited to lipid rafts, and
that the composition and stoichiometry of this receptor cluster may
dictate the type of signal transduced (51). We therefore hypothe-
size that milk component(s) may induce or facilitate the differential
association of cell surface molecules with TLRs. These differential
molecular associations may affect the capacity of TLRs to recog-
nize or respond to ligands and constitute the primary events in
milk-mediated TLR signaling. This would be consistent with doc-
umented observations, indicating that the reactivity pattern of gut
immunocompetent cells is influenced by the local environment
(52–54). Our findings thus suggest that a human breast milk-, but
not an infant milk formulation-rich environment may affect the
reactivity pattern of gut immunocompetent cells by modulating
TLR reactivity specifically and differentially.
The biological reason for the opposite modulatory effect of milk
on TLR2 (negative) and TLR4 (positive), the two main receptors
for microbial components, is not clear. It is possible that following
birth, the opposite modulation by milk promotes the efficient rec-
ognition of and response to potentially harmful LPS-producing
Gram-negative bacteria via TLR4, whereas allowing the establish-
ment of bifidobacteria (Gram-positive) as the predominant intes-
tinal microbiota through a low TLR2 reactivity. Consistent with
this possibility is the observation that breast-fed, but not formula-
fed, full-term infants have a preferred intestine microbial flora with
predominance of the probiotic bifidobacteria (55). More experi-
mentation will, however, be required to determine whether the
modulatory effects on TLR-mediated immune responses described
in this study contribute to the reported protective effects of breast-
feeding against necrotizing enterocolitis, an inflammatory condi-
tion affecting premature infants. Testing the effect of preterm milk
samples may help to clarify this issue.
The modulatory effects of human milk seem to extend to neo-
natal peripheral leukocytes (Fig. 5), suggesting that if the milk
component(s) ultimately responsible for modulating TLR re-
sponses reach the periphery, they would influence neonatal innate
immune responses to systemic infections. This study paves the
way for the identification of such milk components.
The findings reported in this study indicate that, in addition to
sCD14 and sTLR2, other TLR modulatory molecules are present
in milk and involved in novel mechanisms regulating the extent
and quality of TLR activation. The data increases our understand-
ing of how breast milk components contribute to adequate inter-
actions between microorganisms and immunocompetent cells. The
identification of these molecules will advance our knowledge of
the mechanisms controlling innate immune responses and may in-
form the design of improved infant milk formulations, as well as
being of relevance to gut inflammatory conditions where optimiz-
ing TLR-mediated tissue responses may be of therapeutic
We thank H. W. L. Ziegler-Heitbrock (Department of Immunology, Leic-
ester University, Leicester, U.K.) and B. P. Morgan (Department of Med-
ical Biochemistry and Immunology, Cardiff University, College of Medi-
cine, Cardiff, U.K.) for helpful discussions and critical reading of this
M.A. and K.V. are currently employees of Nestle Research Center, Lau-
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