Lactobacillus rhamnosus (LGG) Regulates IL-10 Signaling
in the Developing Murine Colon through Upregulation of
the IL-10R2 Receptor Subunit
Julie Mirpuri1,2*, Ilya Sotnikov1, Loren Myers1, Timothy L. Denning1,3, Felix Yarovinsky4,
Charles A. Parkos3, Patricia W. Denning1, Nancy A. Louis1*
1Department of Pediatrics, Division of Neonatal-Perinatal Medicine, Emory University, Atlanta, Georgia, United States of America, 2Department of Pediatrics, University of
Texas Southwestern, Dallas, Texas, United States of America, 3Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta,
Georgia, United States of America, 4Department of Immunology, University of Texas Southwestern, Dallas, Texas, United States of America
The intestinal microflora is critical for normal development, with aberrant colonization increasing the risk for necrotizing
enterocolitis (NEC). In contrast, probiotic bacteria have been shown to decrease its incidence. Multiple pro- and anti-
inflammatory cytokines have been identified as markers of intestinal inflammation, both in human patients with NEC and in
models of immature intestine. Specifically, IL-10 signaling attenuates intestinal responses to gut dysbiosis, and disruption of
this pathway exacerbates inflammation in murine models of NEC. However, the effects of probiotics on IL-10 and its
signaling pathway, remain poorly defined. Real-time PCR profiling revealed developmental regulation of MIP-2, TNF-a, IL-12,
IL-10 and the IL-10R2 subunit of the IL-10 receptor in immature murine colon, while the expression of IL-6 and IL-18 was
independent of postnatal age. Enteral administration of the probiotic Lactobacillus rhamnosus GG (LGG) down-regulated the
expression of TNF-a and MIP-2 and yet failed to alter IL-10 mRNA and protein expression. LGG did however induce mRNA
expression of the IL-10R2 subunit of the IL-10 receptor. IL-10 receptor activation has been associated with signal transducer
and activator of transcription (STAT) 3-dependent induction of members of the suppressors of cytokine signaling (SOCS)
family. In 2 week-old mice, LGG also induced STAT3 phosphorylation, increased colonic expression of SOCS-3, and
attenuated colonic production of MIP-2 and TNF-a. These LGG-dependent changes in phosphoSTAT3, SOCS3, MIP-2 and
TNF-a were all inhibited by antibody-mediated blockade of the IL-10 receptor. Thus LGG decreased baseline
proinflammatory cytokine expression in the developing colon through upregulation of IL-10 receptor-mediated signaling,
most likely due to the combined induction of phospho-STAT3 and SOCS3. Furthermore, LGG-dependent increases in IL-10R2
were associated with reductions in TNF-a, MIP-2 and disease severity in a murine model of intestinal injury in the immature
Citation: Mirpuri J, Sotnikov I, Myers L, Denning TL, Yarovinsky F, et al. (2012) Lactobacillus rhamnosus (LGG) Regulates IL-10 Signaling in the Developing Murine
Colon through Upregulation of the IL-10R2 Receptor Subunit. PLoS ONE 7(12): e51955. doi:10.1371/journal.pone.0051955
Editor: Josef Neu, University of Florida, United States of America
Received August 17, 2012; Accepted November 7, 2012; Published December 18, 2012
Copyright: ? 2012 Mirpuri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants DK62007 (N.A.L), K12 HD 068369 (J.M.), R03DK076613 & R01HD059122 (P.W.D.);
AA01787001 & AI083554 (T.L.D); DK79392 (C.A.P.); DK 063399 (C.A.P., N.A.L, P.W.D., and T.L.D.). Additional support came from a Career Development Award from
the Crohn’s and Colitis Foundation of America, an Emory Egleston Children’s Research Center seed grant (T.L.D.) and a Children’s Medical Center Foundation Grant
(J.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Patricia Denning is a co-author and is also a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all PLOS
ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org (JM); email@example.com (NAL)
Exaggerated proinflammatory responses and deficient inflam-
matory resolution in the developing intestine are implicated in the
pathogenesis of intestinal diseases such as necrotizing enterocolitis
(NEC) [1,2]. NEC has been linked to aberrant mucosal responses
to bacterial colonization both in the intestine of premature infants
[3,4,5] and in experimental models of NEC-like inflammation .
Promotion of normal colonization through the oral administration
of commensal or probiotic flora has been shown to attenuate
intestinal inflammation [7,8] and promote barrier in experimental
models of the developing intestine [9,10] and NEC .
Additionally, meta-analyses of several clinical trials have shown
an association between probiotic administration and reduced
incidence of NEC in preterm infants [12,13].
While the specific mechanisms by which protection against
NEC remain to be elucidated, it is easy to speculate that the effects
of LGG on cytokine production are relevant to either the onset or
evolution of disease. Furthermore, NEC in the premature infant is
recognized to most commonly occur within a developmental
window, peaking at 31 weeks post conception, independent of
gestational age at birth. Therefore, the effects of probiotics on
cytokine and chemokine production and inflammatory signaling in
the context of developmental changes in the immature gut are of
particular interest. Specifically, increases in the expression of TNF-
a [14,15,16,17], MIP-2  IL-6 [19,20], IL-12 , and IL-18
 have all been implicated as marking or exacerbating
inflammation in models of NEC-like inflammation. With the
exception of IL-18, the expression of all of these mediators is in
turn negatively regulated through activation of the IL-10 pathway
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[22,23,24,25,26,27,28]. Furthermore, while its expression does not
appear to be IL-10-dependent, IL-18-mediated cytokine pro-
duction can be antagonized by IL-10 . Thus IL-10-dependent
suppression of cytokine signaling could be a final common
pathway protecting against these inflammatory mediators during
NEC-like inflammation in the developing intestine.
We have independently shown that commensal strains of
Escherichia coli exert protective effects in the developing colon
through the induction of the type I interferon IFNaA , and
that the anti-inflammatory effects of IFNaA are dependent on IL-
10 production in adult models of colitis . Additionally, animal
models identify interleukin-10 (IL-10) as a critical regulator of
mucosal inflammation in response to colonizing flora . IL-10
deficient mice spontaneously develop a chronic colitis in response
to colonization with commensal flora, with initial pathologic
changes reported after the second week of life . These mice are
also more susceptible to NEC-like inflammation in experimental
models [34,35,36]. Therefore, alterations in IL-10 signaling may
be important for the protective effects of probiotics in the
The vulnerability to NEC-like inflammation seen in IL102/2
mice is attenuated when IL-10 deficient pups are fed by wild type
foster mothers, arguing for a protective role for maternal milk-
derived IL-10 . Breast milk feeding is also known to reduce the
incidence of NEC in preterm infants . Interestingly, human
breast milk is rich in IL-10  and IL-10 is present in amniotic
fluid at concentrations which increase throughout gestation .
Thus, it is possible that breast milk may serve as an important
source of exogenous IL-10, protecting the immature intestine
following premature birth.
IL-10 mediates its effects through binding to a heterotetrameric
cell surface receptor, consisting of two heterodimers of the IL-
10R1 and IL-10R2 protein subunits . While IL-10R1 is
sufficient for IL-10 binding , the IL-10R2 subunit has been
shown to be critical for IL-10 receptor-mediated signaling
responses in several cell types [42,43]. Specifically, cytokine
binding to the IL-10 receptor results in phosphorylation of tyrosine
residues within members of the STAT family, through activation
of Janus Kinase-1 (JAK1) and Tyrosine Kinase-2 (TYK2) 
followed by phospho-STAT3-dependent upregulation of the
expression of the SOCS genes, including SOCS3 . We
hypothesized that the IL-10 signaling pathway regulates in-
flammation during colonization of the immature intestine, and
contributes to the protective effects of probiotics such as LGG.
Here, we characterize the baseline mRNA expression of IL-10
and its receptor subunits as well as that of the proinflammatory
mediators MIP-2, TNF-a, IL-6, the p40 subunit of IL-12 (IL-
12p40), and IL-18 in developing murine colon. The mRNA
expression of MIP-2 and TNF-a was highest during the first week
of life, and then subsequently declined, while that of IL-12p40,
also initially high, declined later, after the second week of life. IL-6
and IL-18 expression was independent of postnatal age. In
contrast, IL-10 production increased over the first three weeks of
life. When compared to adult animals, 2 week-old mice showed
higher colonic expression of MIP-2, TNF-a, and IL-12p40 and
diminished IL-10. We thus sought to determine whether LGG
might alter the baseline inflammatory tone of the developing colon
of 2 week-old mice through induction of IL-10 or its receptor.
Examination of both cytokine expression and the IL-10 signaling
pathway revealed that enteral feeding with LGG attenuated the
expression of proinflammatory cytokines and triggered phosphor-
ylation of STAT3 and expression of SOCS3 through the IL-10
receptor, implying a role for IL-10 signaling. Finally, in a murine
model of NEC-like intestinal inflammation, LGG was protective
against injury induced by the combined injection of platelet
activating factor (PAF) and lipopolysaccharide (LPS). While IL-10
expression was induced by intestinal injury alone with or without
LGG, LGG-mediated protective effects were associated with
increases in the expression of both IL-10R2 and SOCS3. Thus,
LGG appears to mediate its effects on both the baseline
inflammatory potential of murine colon and colonic responses to
injury through induction of the IL-10R2 receptor subunit.
The Baseline Colonic Expression of Cytokines and IL-10
Receptor Subunits is Developmentally Regulated Prior to
Enterocytes from preterm human infants mount increased
intestinal IL-8 responses to proinflammatory stimuli . How-
ever, little is known about cytokine production in the developing
whole intestine exposed solely to normal commensal colonization.
Therefore, we further characterized the baseline inflammatory
phenotype of the developing murine intestine. Real-time PCR was
employed to profile cytokine mRNA expression in the colon of 1, 2
and 3 week-old mice. The proinflammatory cytokines MIP-2 and
TNF-a were highest during the first week of life, with mRNA
expression subsequently declining over the second and third weeks
of life (Figure 1A, *p,0.05). IL-12p40 was also high during the
first week, yet remained elevated, not falling until the third week of
life while baseline mRNA expression of IL-6 and IL-18 remained
unchanged, both prior to weaning and into adulthood (Figure
S1A). In contrast, colonic IL-10 mRNA expression (Figure 1B)
and cytokine production (Figure 1C) increased with postnatal age.
Interestingly, while the mRNA expression of IL-10R1 showed no
developmental variation, baseline colonic mRNA expression of IL-
10 R2 declined significantly by the third week, coincident with the
substantial increases in IL-10 cytokine expression after the second
week of life (Figure 1B).
2 Week-old Mice have Higher Colonic Levels of
Proinflammatory and Lower Levels of Anti-inflammatory
Mediators than Adults
Given these findings, we next compared the colonic cytokine
expression at 2 weeks of life with that of adult mice. The colonic
mRNA expression of MIP-2, TNF-a, and IL-12p40 (Figure 2A)
was decreased in adult mice relative to 2 week-old pups. IL-12p40,
but not IL-18 or IL-6, was also increased relative to adults (Figure
S1B). In contrast, colonic mRNA expression of both IL-10 and the
IL-10R2 receptor subunit (Figure 2B) was decreased at 2 weeks of
age, relative to adults. Expression levels of the IL-10R1 receptor
subunit were comparable in 2 week-old and adult mice. These
changes in MIP-2, TNF-a and IL-10 expression were confirmed at
the protein level (Figure 2C) in the supernatants from colonic
explants cultured for 24 hours. We can conclude from these
experiments that the colons of conventionally raised 2 week-old
mice have enhanced expression of proinflammatory cytokines and
a reduction in elements of the IL-10 signaling pathway, relative to
LGG Increases IL-10R2 Receptor Subunit Expression and
Reduces MIP-2 and TNF-a Expression in the Colon of 2
Commensal and probiotic flora signal in part through TLR9-
dependent induction of type I interferons such as IFNaA [46,47],
which regulates inflammation through induction of IL-10 in adult
models of colitis . However, the specific effects of probiotics on
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IL-10 signaling remain to be defined in the developing intestine. In
order to examine the effects of probiotics on IL-10 and its
receptor, 2 week-old mice were gavage-fed with LGG or vehicle,
and sacrificed after 6 hours. Analyses of the mRNA expression of
IL-10 and the IL-10 receptor subunits revealed that IL-10 was
unchanged following LGG exposure (Figure 3A). In contrast, IL-
10R2 mRNA was induced by 6.862.1-fold, relative to vehicle-fed
controls (Figure 3A). We can conclude that induction of IL-10R2
may contribute to downstream LGG-dependent attenuation of
inflammation in the developing colon. To determine if the effects
of LGG on IL-10 signaling required viable bacteria, we gavage fed
2 week-old mice with heat-inactivated LGG or vehicle and
performed similar analysis. We found that heat-killed LGG did
not result in enhanced expression of IL-10R2 (Figure S2A).
Therefore live LGG is required for the induction of the IL-10R2
subunit. Coincident with changes in IL-10R2 expression, LGG
feeding of 2 week-old mice resulted in a downstream decrease in
the mRNA expression of MIP-2 and TNF-a at 24 hours
(Figure 3B). Thus MIP-2 and TNF-a were selected as markers
on the mRNA level of anti-inflammatory properties of LGG. In
contrast, IL12p40 was unchanged following this time interval
while both IL-6 and IL-18 were increased in response to enteral
LGG (Figure S2B), making these mediators less likely to be strong
contributors to any anti-inflammatory effects of IL-10 signaling in
the developing intestine.
LGG Triggers IL-10 Receptor-dependent Phosphorylation
of STAT3 and Induces SOCS3 in the Colon of 2 Week-old
The binding of IL-10 to its receptor triggers phosphorylation-
dependent activation of the transcription factor STAT3. Activated
STAT3 then upregulates the gene expression of members of the
SOCS family and downregulates the expression of proinflamma-
tory cytokines, such as TNF-a and MIP-2, shown above to be
inhibited by LGG. The net effect of these changes is IL-10
dependent suppression of inflammation . In order to assess
whether amplification of the IL-10R2 subunit resulted in altered
phosphosignaling, the effect of enteral administration of LGG on
phosphorylated STAT3 was determined. In colons harvested from
mice 6 hours after feeding with LGG or vehicle alone, western blot
analysis revealed a statistically significant, 8-fold increase in the
ratio of phospho-STAT3 to total STAT3 protein in LGG-fed mice
(Figure 4A). To confirm the specific contributions of the IL-10
receptor to LGG-dependent changes in STAT3 phosphorylation, 2
week-old mice were treated by intraperitoneal injection with either
an IL-10 receptor blocking monoclonal Ab [49,50] or an isotype-
matched control, then gavage-fed with either LGG or vehicle.
When mice were sacrificed 6 hours later, pretreatment with the
IL-10 receptor blocking antibody attenuated the effect of LGG on
STAT3 phosphorylation, relative to LGG-fed mice pretreated with
the isotype control antibody (Figure 4A). Additionally, after
antibody-mediated blockade of the IL-10 receptor, the ratio of
phospho-STAT3 to total STAT3 was no longer significantly
different in LGG-fed mice, relative to vehicle-fed controls.
Similarly, when mice were sacrificed 24 hours after feeding with
LGG, the IL-10 receptor-blocking antibody also attenuated the
LGG-associated induction of SOCS3 (Figure 4B). Thus, LGG-
mediated changes in STAT3 activation and downstream SOCS3
production proceed through the IL-10 receptor and most likely
depend in part on induction of IL-10R2 expression.
LGG-mediated Suppression of MIP-2 and TNF-
a Expression in the Developing Murine Colon is
Dependent on the IL-10 Receptor
We next examined whether changes in IL-10 signaling were
responsible for the LGG-associated reduction of colonic MIP-2 and
TNF-a. Cytokine production was analyzed from colonic explant
cultures of 2 week-old mice treated with either an IL-10 receptor
blocking Ab or an isotype-matched control Ab. 24 hours later,
Figure 1. The baseline colonic expression of cytokines and IL-
10 receptor subunits is developmentally regulated prior to
weaning. Cytokine and IL-10 receptor subunit expression was analyzed
by real time PCR in whole colon from 1, 2 and 3 week-old C57BL/6J
mice. The data represent mean fold change 6 SEM, relative to 1 week-
old mice (A, B). C. IL-10 cytokine levels in supernatants of whole colon
incubated for 24 hours were assayed by ELISA and depicted as mean
concentration 6 SEM (*p,0.05, n=527 mice per age group).
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mice were sacrificed and colonic explants were prepared. Super-
natants were collected and assessed by ELISA for MIP-2
(Figure 5A) and TNF-a (Figure 5B). Antibody-blockade of the
IL-10 receptor prevented LGG-mediated decreases in the expres-
sion of MIP-2 (Figure 5A) and TNF-a (Figure 5B). Taken together,
these findings indicate that LGG-mediated reduction in inflamma-
Figure 2. 2 week-old mice have higher colonic levels of proinflammatory and lower levels of anti-inflammatory mediators than
adults. Mice were raised under conventional conditions and sacrificed at 2 weeks or as adults (6–8 weeks of life) and the colon excised and
processed for analysis of mRNA (A) of the proinflammatory cytokines MIP-2 and TNF-a. mRNA expression data is depicted as mean fold change 6
SEM, relative to adult mice. B. Colonic mRNA expression of IL-10 and its receptor subunits is depicted for 2 week-old mice, relative to adult controls. C.
MIP-2, TNF-a and IL-10 cytokine levels of colonic supernatants determined by ELISA are depicted as mean concentration 6 SEM. (*p,0.05, **p,0.01,
n=527 per age group).
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tory cytokines depends, at least in part, on the induction of IL-
LGG Protects against Intestinal Injury Induced by PAF and
In order to investigate if enhanced IL-10 signaling as a result of
IL-10R2 induction by LGG would be protective against injury in
the developing intestine, we used a previously characterized model
of NEC-like inflammation in which gut mucosal injury is induced
in 2 week-old mice by intraperitoneal administration of PAF and
LPS [51,52,53]. 24 hours prior to treatment with PAF/LPS, mice
were gavage-fed 108CFU LGG or an equal volume of vehicle. The
severity of intestinal injury was scored based on blinded histologic
analysis, and the effect of cytokine expression and SOCS3
induction was measured by qRT-PCR. Pre-treatment with LGG
significantly protected mice from intestinal injury, with average
injury scores of 861 in non-LGG and 561 in LGG pre-treated
mouse pups (Figure 6A and 6B). LGG treatment also attenuated
the injury-associated induction of pro-inflammatory cytokines
MIP-2 and TNF-a (Figure 6B). Interestingly, the anti-inflamma-
tory cytokine IL-10 was induced by injection with PAF/LPS
alone, independent of LGG (Figure 6C). In contrast, the expression
of IL-10R2 was essentially unchanged in response to PAF/LPS,
yet was markedly increased in the protected LGG-treated group.
Furthermore, SOCS3 was also found to be upregulated in colon of
LGG pretreated mice that were protected from NEC-like intestinal
injury (Figure 6C). Expression of the IL-10R1 was not changed by
either PAF/LPS injection alone or by LGG (data not shown).
Taken together we can conclude that the protective effects of LGG
in NEC-like intestinal injury appear more specifically related to its
induction of the IL-10R2 subunit and downstream SOCS3.
In these studies, we examine the effects of LGG on both baseline
cytokine levels and on cytokine signaling responses to intestinal
injury in the developing murine intestine. In initial studies, we
showed that even in the absence of exogenous inflammatory
stimuli, the baseline expression of key cytokines mediating both
promotion and resolution of intestinal inflammation in models of
NEC-like injury are developmentally regulated during the post-
natal period. Initial profiling identified TNF-a, MIP-2, and IL-
12p40, and IL-10 as developmentally regulated prior to weaning
in the immature intestine. While expression of proinflammatory
Figure 3. LGG increases IL-10R2 receptor subunit expression and reduces MIP-2 and TNF-a expression in the colon of 2 week-old
mice. 2 week-old mice were gavage fed 108CFUs of LGG or equal volume of HBSS, then sacrificed 6 hours later. A. The colon was excised and the
mRNA expression of IL-10, IL-10R1, and IL-10R2 was analyzed by real-time PCR. B. Colonic mRNA expression for MIP-2 and TNF-a was measured at 24
hours after LGG or vehicle control. Data are depicted as mean 6 SEM (n=627 mice per condition).
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cytokines, MIP-2, TNF-a and IL-12p40 was highest during the
first two weeks of life, then declined, IL-10 expression was initially
low and increased between two and three weeks of life. Therefore,
2 week-old mice had increased basal expression of proinflamma-
tory cytokines, at a time when IL-10 expression remained low.
Enteral feeding with the probiotic LGG was not associated with
significant changes in IL-10 production. However, LGG did
upregulate the R2 subunit of the IL-10 receptor, as well as IL-10
receptor-dependent phosphorylation of STAT3, and downstream
expression of SOCS3. Furthermore, LGG-dependent changes in
IL-10 receptor expression were associated with a reduction in the
expression of the proinflammatory cytokines MIP-2 and TNF-
a and protection from intestinal injury. These results suggest that
LGG may increase sensitivity to IL-10, and implicate IL-10
signaling as a key pathway mediating the anti-inflammatory effects
of LGG. Of interest, IL-10 is increased in models of NEC and in
patients with severe or surgical NEC  we found that IL-10 was
in fact increased after intestinal injury in both LGG- and vehicle-
treated mice. Thus, increases in IL-10 cytokine production might
be a response to counterbalance the pro-inflammatory response of
NEC/intestinal injury. Alternatively, increased IL-10 production
by itself may play a pathophysiological role by inhibiting T-cell
effector function . However, in the PAF/LPS model of
intestinal injury, the protective effects of LGG were more
specifically associated with changes in IL-10R2 expression,
implying that this protective mechanism, based on receptor rather
Figure 4. LGG triggers IL-10 receptor-dependent phosphorylation of STAT3 and induces SOCS3 in the colon of 2 week-old mice. 2
week-old mice were treated by intraperitoneal injection with either 200 mg IL-10R Ab or an isotype-matched control immediately prior to gavage
feeding with 108CFUs LGG or vehicle alone. 6 hours after treatment, the mice were sacrificed and the colon was processed for western blot analysis
of phospho- and total STAT3 (A). Similar analysis was performed for SOCS3 at 24 hours after treatment (B). Densitometric analysis was performed and
the mean ratio 6 SEM of detected phosphorylated STAT3 to total STAT3 was plotted for each condition (A). (n=3 separate experiments, 5 mice per
condition, *p,0.05, **p,0.01).
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than cytokine expression has the potential to be regulated in a cell-
type specific manner. Therefore, the role of the IL-10 receptor in
this background may be crucial for enhancement of effective
downstream anti-inflammatory responses, and in our studies LGG-
dependent induction of IL-10R2 and SOCS3 was associated with
protection from intestinal injury.
Imbalances in inflammatory signaling have been implicated in
the pathogenesis of NEC [1,56,57]. Walker and colleagues have
reported that fetal intestinal cells mount an exaggerated proin-
flammatory cytokine response to inflammatory stimuli and
bacterial antigens . Additional evidence argues that decreased
capacity for IL-10-dependent signaling may contribute to in-
creased inflammatory responses in the immature intestine.
Specifically, IL-10 is present in human breast milk, and Fituch
et al. have demonstrated that premature infants who developed
NEC despite receiving maternal milk feedings had mothers with
lower concentrations of IL-10 in their breast milk .
The findings in this study demonstrating developmental
regulation of IL-10 and its receptor in the murine intestine are
intriguing given the experimental evidence implicating protective
roles for the IL-10 pathway both during colonization of the
developing intestine and in infants with NEC. Our study is focused
on the effect on the developing colon since as the site of maximal
bacterial colonization, however, it should be noted that probiotic
effects may differ in large and small intestine, both at baseline and
under conditions of inflammation such as in NEC. The effect of
LGG may also not be limited to epithelial cells but may also involve
other populations of lamina propria cells. We can speculate,
however, that lower levels of IL-10 production early in intestinal
development may predispose the immature intestine to increased
or sustained inflammatory responses to luminal bacteria. Thus,
part of the protective influence of maternal milk may reside in its
provision of exogenous IL-10 [37,38]. Furthermore, probiotic
induction of the IL-10 receptor may offer some protection by
increasing the capacity for IL-10 receptor signaling in response to
IL-10 present in maternal milk.
The IL-10 pathway appears to have the potential for regulation
through two temporally distinct mechanisms in the developing
intestine, initially by flora-dependent receptor induction, followed
later by increased baseline capacity for IL-10 secretion. Our
findings indicate the potential for probiotic-dependent induction of
the IL-10 receptor promoting activation of the IL-10 signaling
pathway prior to the induction of a robust cytokine response. This
early receptor-based mechanism of augmenting sensitivity to IL-10
through induction of IL-10R2 may represent a cell-type specific
mode of regulation of cytokine signaling in the developing
Probiotics provide protective effects in animal models of NEC
[59,60,61] and in initial human trials [62,63,64]. However, the
safety and long-term consequences of probiotic administration
have yet to be established. Furthermore, probiotics have been
shown in case reports to cause sepsis in immunocompromised
patients including premature infants [65,66,67]. Our study
identifies a potential mechanism by which probiotic bacteria
protect the developing intestine through increased potential for IL-
10 receptor signaling in response to low levels of endogenous
cytokine as well as exogenous IL-10 derived from maternal milk.
While safety concerns remain, the availability of probiotic bacteria
which have been genetically engineered to secrete human IL-10
 offers a candidate alternative therapy for the prevention of
NEC, particularly in cases where maternal milk is not available.
SOCS3-signaling still needs to be carefully evaluated in future
clinical trials with probiotics as well, due to its additional role in
negatively regulating fetal liver hematopoiesis by attenuating
erythropoietin signaling . However, specific targeting of
probiotic-dependent signaling pathways such as IL-10 receptor-
mediated activation of SOCS3 may provide pharmacologic
alternatives to the administration of live bacteria to our most
LGG (ATCC, Manassas, VA) was prepared overnight in
Lactobacillus broth at 37uC as per ATCC guidelines. LGG cultures
Figure 5. LGG mediated suppression of MIP-2 and TNF-
a expression in the developing murine colon is dependent on
the IL-10 receptor. 2 week-old mice were treated by intraperitoneal
injection with either 200 mg IL-10R Ab, or an isotype-matched control
Ab, immediately prior to gavage feeding with 108CFUs LGG or vehicle
alone. 24 hours after treatment, colonic supernatants were collected
and assayed for MIP-2 (A) and TNF-a (B) by ELISA. Data are depicted as
mean 6 SEM (n=3 separate experiments, for a total of 7 mice per
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were washed, concentrated in HBSS to 109CFU/ml and gavage-
fed to 2-week old mice at a dose of 108CFUs. LGG was heat-killed
by heating 1010CFUs for 20 mins at 80uC .
All animal experiments were conducted according to protocols
approved by the Institutional Animal Care and Use Committees at
Emory University and the University of Texas Southwestern.
C57BL/6J mice were bred and maintained within the animal
facilities at Emory University or the University of Texas
For probiotic experiments, 2 week-old mice were orally gavage-
fed 0.1 ml of HBSS, with or without LGG at a dose of 108CFUs
then sacrificed after 6 hours or 24 hours. For experiments lasting
longer than 6 hours, mice were returned to their mother for the
indicated time interval. Then, mice were sacrificed by CO2
inhalation followed by cervical dislocation. Whole colons were
isolated and immediately frozen in TRIzol for RNA isolation.
Excised mouse colons were homogenized in TRIzol (Invitrogen
Life Technologies) then subjected to phenol-chloroform extraction
according to the manufacturer’s protocol and as previously
described . RNA was digested with DNaseI (Ambion, Austin,
TX) to remove contamination with genomic DNA, then cDNA
was synthesized by reverse transcription using oligo (dT12-18)
primers and superscript II reverse transcriptase (Invitrogen,
Carlsbad, CA). Real time PCR was performed using a MyIQ
real-time PCR machine and SYBR Green supermix (Biorad,
Primer sequences were as follows:
sense, 59-CTCTCAAGGGCGGTCAAAAAGTT-39 and anti-
sense, 59-TCAGACAGCGAGGCACATCAGGTA-39; Murine
anti-sense, 59-TGGGCTACAGGCTTGTCACT-39; Murine IL-
10 sense, 59-ATGCTGCCTGCTCTTACTGACTG-3, and anti-
sense, 59-CCCAAGTAACCCTTAAAGTCCTGC-39; Murine
IL-6 sense, 59-CTGATGCTGGTGACAACCAC-39, and anti-
12p40 sense, 59-ACAGCACCAGCTTCTTCATCAG-39, anti-
sense 59- TCTTCAAAGGCTTCATCTGCAA-39; Murine IL-18
sense, 59-GACAGCCTGTGTTCGAGGAT-39, anti-sense 59-
TGGATCCATTTCCTCAAAGG-39; Mouse IL-10R1 sense, 59-
AGG CAG AGG CAG CAG GCC CAG CAG AAT GCT-39,
antisense, 59-TGG AGC CTG GCT AGC TGG TCA CAG
TAG GTC-39; Mouse IL-10R2 (CRF2-4) sense, 59-GCC AGC
TCT AGG AAT GAT TC-39, antisense, 59-AAT GTT CTT
CAA GGT CCA C-39; Mouse SOCS3 sense, 59-ATTTGCCT-
CAATCACTTTTAT-39, antisense, 59-ACTGGGATTTGGTT-
GAGTTT-39. Data were analyzed by the DDCtthreshold cycle
method with normalization for starting template performed using
the housekeeping gene SRP14 as previously described .
Western Immunoblot Analysis
Frozen whole colon tissues were homogenized in 0.5 ml of lysis
buffer containing protease inhibitors (10% mammalian tissue
protease inhibitor mixture and 1 mM PMSF, Sigma-Aldrich, St.
Louis, MO). The homogenates were centrifuged and the super-
natants were removed for detection of protein concentration.
Equal amounts of protein were resolved by polyacrylamide gel
electrophoresis and subjected to electrophoretic transfer to
activated polyvinylidene fluoride membranes (BioRad, Hercules,
CA). Membranes were blocked with 0.5% skim milk (BioRad,
Hercules, CA) and probed with rabbit anti-mouse antibody
specific for SOCS3 (Cell Signaling Technology, Beverly, MA)
for 10 minutes, washed three times in phosphate-buffered saline
containing 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO) and
bound antibody was detected by probing with species-specific
peroxidase conjugated secondary antibodies followed by visuali-
zation using BM chemiluminescence substrate (Roche, Indiana-
polis, IN). All washes and antibody incubation were performed
using the SNAP-ID Protein Detection system (Millipore, Billerica,
In vivo Blockade of the IL-10R Receptor
C57BL/6 mice were treated by intraperitoneal injection with
200 mg per mouse of a rat anti-mouse IL-10R (CD210) mono-
clonal antibody specific for the IL-10 binding domain of the IL-10
receptor (clone 1B1.3a, BD Pharmingen, San Diego, CA), as
previously described . Control mice were injected with a rat
IgG1isotype control antibody (clone R3-34, BD Pharmingen, San
Diego, CA). Mice were then fed either vehicle control or LGG at
a dose of 108CFUs, by gavage. Mice were sacrificed 6 hours after
treatment for analysis of expression of total and phosphoSTAT3.
Detection of STAT3 Phosphorylation
The colonic mucosal layer was homogenized in a lysis buffer
containing 50 mM Tris (pH 8), 0.5% NP-40, 1 mM EDTA,
150 mM NaCL, 10% glycerol, 1 mM sodium vanadate, 50 mM
sodium fluoride, 10 mM sodium pyrophosphate, 1 mM phenyl-
methylsulfonyl fluoride, and 10% protease inhibitor cocktail
(Roche, Indianapolis, IN) . The lysates (15 mg total protein/
lane) were resolved by SDS-PAGE, and specific proteins were
detected by immunoblotting using an ECL detection system
(Roche, Indianapolis, IN). Phosphorylation of the tyrosine 705
residue, critical for activation of both the a and b isoforms of
STAT3, was detected by using anti-phospho-STAT3 mAb (Cell
Signaling Technology, Beverly, MA). After stripping the mem-
brane of anti-phospho-STAT-3 specific antibody, the membranes
were probed again using anti-STAT3 Ab (Cell Signaling
Technology, Beverly, Massachusetts). The amount of detected
protein was quantified by densitometric analysis, and the ratio of
phosphorylated STAT3 to total STAT3 was determined following
correction for background signal intensity.
Each colon sample was weighed, cut longitudinally, washed
extensively with PBS and incubated overnight in cell culture media
(RPMI +10% FBS). MIP-2, TNF-a and IL-10 concentrations in
Figure 6. LGG protects against intestinal injury induced by PAF and LPS. 2 week-old mice were gavage-fed either LGG or PBS and then
exposed to PAF/LPS 24 hours later. 2 hours after PAF/LPS exposure, mice were then sacrificed and colon was harvested for histologic analysis
following H&E staining. PAF/LPS-treated mice that were pre-treated with LGG had lower intestinal injury scores than those pre-treated with vehicle
alone (A), representative images are shown. Inflammatory cytokines MIP-2 and TNF-a were also measured by qRT-PCR after PAF/LPS and found to be
decreased in the mice pre-treated with LGG (B). IL-10R2 and SOCS3 were significantly induced in the LGG pre-treated mice after PAF/LPS compared to
vehicle-fed mice (C), while IL-10 was unchanged. Data are depicted as mean 6 SEM (n=527 mice per condition, *p,0.05, **p,0.01).
IL-10 Signaling and Lactobacillus GG
PLOS ONE | www.plosone.org9December 2012 | Volume 7 | Issue 12 | e51955
the supernatants were determined with ELISA kits from
eBioscience accordingto the
(n=6 mice per condition).
PAF/LPS Model of Intestinal Injury
Mucosal injury was induced in the intestines of 12–14 day
mouse pups by intraperitoneal administration of PAF (50 ug/kg)
and LPS (1 mg/kg) [51,52,53]. In all experiments, mice were
sacrificed 2 hours after PAF and LPS administration and mucosal
injury was graded in a blinded-fashion using a scale as follows: For
crypt integrity: 0, normal; 1, irregular crypts; 2, mild crypt loss; 3,
severe crypt loss; 4, complete crypt loss with an intact epithelial cell
layer; 5, complete loss of crypts and surface epithelium (,10 crypt
width); and 6, complete loss of crypts and surface epithelium (.10
crypts). For infiltration of inflammatory cells into the mucosa: 0,
normal; 1, mild; 2, modest; and 3, severe. For infiltration of the
submucosa: 0, normal; 1, mild; 2, modest; and 3, severe. For
infiltration of the muscle: 0, normal; 1, mild; 2, modest; and 3,
severe. To determine whether or not LGG was protective against
PAF/LPS-induced intestinal injury, mice were gaavage fed either
LGG or vehicle-control 24 hours prior to IP injection with PAF/
Quantification and Statistical Analysis
Statistical differences were analyzed by ANOVA and t-test
using Prism 5 for Mac OS X, version 5.0 a, 1992–2008 GraphPad
Software, Inc (San Diego, CA). Values are expressed as mean 6
SEM, with statistical significance identified as a p value ,0.05.
found to be important in NEC. The baseline colonic mRNA
expression of IL-12p40, IL-18 and IL-6 was analyzed by qRT-
PCR. IL-12p40 was increased in 1 and 2 week-old mice compared
to 3 week-old (A) or adult mice (B). The data represent mean fold
change 6 SEM, relative to 1 week-old mice (A, B). IL-18 and IL-6
were not developmentally regulated. Data is depicted as mean fold
change 6 SEM (*p,0.05, n=527 mice per age group).
Developmental profiling of other cytokines
was analyzed in whole colon by qRT-PCR 6 hours after
gavage feeding. Heat-killed LGG had no effect on IL-10, IL-
10R1 or IL-10R2 (A). IL-12, IL-18 and IL-6 were analyzed 24
hours after gavage of live LGG (B). Data are depicted as mean 6
SEM (n=527 mice per condition, *p,0.05).
Effect of heat-killed LGG on IL-10 signaling
Conceived and designed the experiments: JM IS NAL PWD FY.
Performed the experiments: JM IS LM. Analyzed the data: JM NAL
PWD TLD. Contributed reagents/materials/analysis tools: JM CAP TLD
NAL FY. Wrote the paper: JM NAL PWD TLD CAP.
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