TLR9 Activation Coupled to IL-10 Deficiency Induces Adverse
Jessica E. Thaxton,* Roberto Romero,†and Surendra Sharma2*
Pregnancy outcome is severely compromised by intrauterine infections and inflammation. Although the pregnant uterine micro-
environment is replete with innate immune cells and TLR expression, the mechanisms that facilitate adverse effects of their
activation are largely unknown. In this study, we mimic the activation of TLR9 with its pathogenic ligand hypomethylated CpG
and demonstrate that IL-10 proficiency protects against CpG-induced pregnancy complications. We show that fetal resorption and
preterm birth are rapidly induced in IL-10?/?mice by low doses of CpG (?25 ?g/mouse) when injected i.p. on gestational day
6 or gestational day 14, respectively. In contrast, wild-type mice failed to experience such effects at comparable doses, but
pups born at term displayed craniofacial/limb defects in response to higher doses (?400 ?g/mouse). Pregnancy complications
in IL-10?/?mice were associated with unexpected and robust TLR9-triggered activation and amplification of uterine neu-
trophil and macrophage subpopulations followed by their migration to the placental zone. Furthermore, a dramatic increase
in serum levels of mouse KC and TNF-? production by uterine F4/80?cells, but not uterine NK or Gr-1?CD11b?cells, was
observed. Depletion of F4/80?macrophages or neutralization of TNF-? rescued pregnancy to term. Our results have im-
portant implications for IL-10-mediated “uterine tolerance” against CpG-driven innate immune activation.
Immunology, 2009, 183: 1144–1154.
innate immune system at the maternal-fetal interface in response to
normal pregnancy intrauterine milieu or inflammatory stimuli has
attracted an abundance of recent interest. Although a vigorous
uterine immune system juxtaposes the fetal tissue predominated by
innate sentinels, NK cells and macrophages, it does not pose any
intolerance to normal fetal development and survival of invading
trophoblasts (3–5). We and others have proposed that uterine NK
(uNK)3cells produce angiogenic and pregnancy-compatible fac-
tors and are involved in local endovascular processes and regula-
tion of trophoblast invasion (3, 6–8). On the other hand, in a
mouse model, we have demonstrated that uNK cells become an-
tagonistic to pregnancy in response to bacterial products (9, 10).
Thus, the underlying mechanisms of diverse pathogen-mediated
The Journal of
common link for a significant proportion of early and
late pregnancy maladies lies in intrauterine infections
and inflammation (1, 2). In this regard, the role of the
inflammatory events that may trigger cytotoxic activation of ma-
ternal immune cells and trophoblasts require further exploration.
A group of innate immune sentinel receptors known as TLRs are
present not only on uterine leukocytes, but also on trophoblast
cells, thus implying an active cross-talk between the placenta and
local immunity (11–13). TLRs have evolved to recognize specific
pathogen-associated molecular patterns enabling them to serve as
the first line of defense in the innate immune system (14, 15).
Although TLRs are ancient receptors without memory require-
ments for cells that express them, they harbor the ability to trans-
duce both negative and positive signals depending on interactions
with their immediate microenvironment (16). Moreover, patho-
genic load and gene-environment interactions are likely to be over-
riding factors in the outcome of inflammation-induced positive
versus negative cascades. Our recent studies provide support to
this hypothesis in that very low doses of LPS cause adverse preg-
nancy outcomes in IL-10?/?mice compared with their wild-type
(WT) counterparts (9, 10).
Systemic or intrauterine bacterial and viral infectious agents,
and their breakdown products, are likely to lead to the excessive
presence of pathogenic hypomethylated CpG DNA motifs
which are recognized by TLR9 (17, 18). Few studies have fo-
cused on the role of TLR9-mediated immune activation during
pregnancy. Using C57BL/6 mice, it has been shown that high
doses of CpG cause deformities in pups born to treated dams
(19). On the other hand, CpG has been used as an adjuvant to
reduce negative fetal outcomes induced by Listeria monocyto-
genes in BALB/c pregnant dams (20). In other settings, it has
been shown that direct injection of CpG DNA in neuroblasto-
mas induces complete tumor rejection in mice and elicits long-
term Th1-driven immunity (21). In addition, antitumor effects
of CpG have been demonstrated in different intracranial models
of syngeneic glioma (22, 23). These observations imply that
CpG motifs are capable of triggering strong and polarized im-
mune responses which may be beneficial or harmful depending
on the intrinsic microenvironment.
*Department of Pediatrics, Women and Infants Hospital of Rhode Island, Warren
Alpert Medical School of Brown University, Providence, RI 02905; and†Perinatol-
ogy Research Branch, Eunice Kennedy Shriver National Institute of Child Health and
Human Development, National Institutes of Health/Department of Health and Human
Services, Detroit, MI 48201
Received for publication March 11, 2009. Accepted for publication May 21, 2009.
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 in part by a grant from National Institutes of Health,
National Center for Research Resources (P20RR018728), The Intramural Division of
the Eunice Kennedy Shriver National Institute of Child Health and Human Develop-
ment, National Institutes of Health, and Subcontract WSU05056 under National In-
stitute of Child Health and Human Development Contract N01-HD-2-3342. J.E.T.
was supported by a Superfund Basic Research Program Award (P42ES013660) from
the National Institute of Environmental Health Sciences.
2Address correspondence and reprint requests to Dr. Surendra Sharma, Department
of Pediatrics, Women and Infants Hospital, 101 Dudley Street, Providence, RI 02905.
E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: uNK, uterine NK; ODN, oligodeoxynucleotide;
UMGC, uterine mononuclear and granular cells; DHR, dihydrorhodamine 123; gd,
gestational day; ROS, reactive oxygen species; mKC, mouse KC.
The Journal of Immunology
Given the proposed widespread use of CpG as a treatment and
vaccine tool among the general population, including pregnant in-
dividuals, it is important to determine whether overactivation of
the immune system in response to nonteratogenic doses of CpG
can lead to negative pregnancy outcomes (20, 24–28). In this
study, we examined the role of the CpG-TLR9 axis in a mouse
model of pregnancy with a focus on the protective role of preg-
nancy-compatible cytokines such as IL-10. CpG-induced TLR9
activation has been associated mainly with stimulation of systemic
immunity. Our observations allow us to elucidate a link between
CpG-mediated activation of innate immune responses and IL-10
deficiency at the maternal-fetal interface that leads to adverse preg-
Materials and Methods
Mice used in this study, C57BL/6 and C57BL/6 IL-10?/?, were obtained
from The Jackson Laboratory. All mice were housed in a specific patho-
gen-free facility supervised by the Central Research Department of Rhode
Island Hospital. All protocols were approved by the Lifespan Animal Wel-
fare Committee and conducted according to its guidelines. Mice of 8–10
wks of age were mated and each experimental group contained at least
three mice. The day of vaginal plug appearance was designated gestational
day (gd) 0.
In vivo treatment of pregnant mice
WT and IL-10?/?mice received i.p. injections of CpG oligodeoxynucle-
otide (ODN 1826; InvivoGen) at doses of 15, 25, 100, 300, or 400 ?g on
gd6 or gd14. For IL-10?/?mice, suitable doses were 15 or 25 ?g/dam
since higher doses caused maternal demise. Cellular depletions were per-
formed with CpG injection on gd6 or gd14. One hundred microliters of
anti-asialo GM1 (Wako) or nonimmune rabbit serum (Antibodies) was
administered on gd4, 6, and 9 or gd9, 11, and 14 for NK cell depletion.
Two hundred fifty micrograms of anti-Gr-1 (RB6-8C5; BD Biosciences) or
isotype Ab (rat IgG1; BD Biosciences) was administered at gd5. Two
hundred fifty micrograms of anti-F4/80 (BM8; eBioscience) or isotype Ab
(rat IgG2a, ?; eBioscience) was given on gd5 and 7 or gd13 and 15. Com-
petitive antagonist experiments were performed in IL-10?/?mice and i.p.
injections of 100 ?g or 50 ?g of antagonist ODN (ODN 2088; InvivoGen)
were given with 25 ?g of CpG ODN on gd6. Control experiments were
performed using 50 or 100 ?g of antagonist ODN alone or 50 or 100 ?g
of antagonist ODN plus 25 ?g of CpG ODN on gd6. Monoclonal anti-
TNF-? Ab (Gr81-2626; BD Pharmingen) was administered i.p. at 250 ?g
on gd5 and gd7 with CpG ODN injection on gd6 or on gd13 and gd15 with
CpG ODN injection on gd14.
Uterine mononuclear and granular cells (UMGC) were obtained via minc-
ing and mechanical dispersion of whole gd 8–9 or gd15 uteroplacental
tissue in RPMI 1640 supplemented with 10% FBS, penicillin/streptomycin,
and L-glutamine. Single-cell suspensions from uterine horns were run
through a 100-?m cell strainer and subjected to density gradient separation
using Fico-Lite LM (Atlanta Biologicals). Experiments were performed
on the three layers obtained from Ficoll gradient separation to determine in
which layer granulocytes accumulated. It was found that granulocytes col-
lect directly below the monocyte layer and both layers were harvested
together for all experiments.
Abs to NK1.1 (NKR-PIC), CD3 (145-2C11), CD45 (30-F11), F4/80
(BM8), Gr-1(RB6-8C5), CD11b (M1/79), and Ly6G (1A8) were purchased
(BD Biosciences). Isolated UMGC were washed in PBS and resuspended
in PBS containing 2% FBS (stain buffer). Combinations of Abs were
added for extracellular staining for 30 min at 4°C, rinsed with stain buffer,
and acquired via flow cytometry (FACSCanto; BD Biosciences). Fluoro-
chrome-conjugated isotype-matched Abs were used as controls. Abs to
TNF-? (MP6-XT22) and IFN-? (XMG1.2) were purchased for intracellu-
lar staining (BD Biosciences). UMGC were washed with stain buffer and
incubated in 96-well plates for 4–6 h with Brefeldin A (BD Biosciences),
PMA (Calbiochem), and ionomycin (Calbiochem). Cells were washed
twice with stain buffer and stained extracellularly as described above. For
intracellular staining, UMGC were washed with Perm Wash (BD Bio-
sciences) and fixed with Cytofix/Cytoperm (BD Biosciences) for 25 min at
4°C and incubated with Abs for 30 min at room temperature. Cells were
washed and analyzed via flow cytometry.
Intracellular reactive oxygen species (ROS) production was assessed
with dihydrorhodamine 123 (DHR; Sigma-Aldrich) by flow cytometry.
This nonfluorescent dye becomes fluorescent upon oxidation to rhodamine
by ROS produced during the respiratory burst. Directly after UMGC were
prepared, DHR (10 ?mol/ml) was added simultaneously with Abs Gr-1,
CD45, and CD11b or with irrelevant isotype Abs, and the mixture was
incubated at 37°C for 15 min. UMGC were washed and immediately pro-
cessed by flow cytometry.
TNF-?, IL-12, IFN-?, mouse KC (mKC), MIP-1?, and MIP-2 were mea-
sured in serum. Blood samples were collected via cardiac puncture into
1-ml tubes, allowed to clot for 30 min at room temperature, spun at 8000
rpm for 20 min at 4°C, and supernatants were collected and frozen for
further analysis. TNF-?, IL-12, IFN-?, mKC, MIP-1?, and MIP-2 were
assayed using Quantikine ELISA kits (R&D Systems) and experiments
were performed according to the manufacturer’s instructions. Separate se-
rum samples were collected from each experimental treatment group
(n ? 9).
Fetoplacental units from varying experimental conditions were removed
from uterine horns and placed in 4% buffered formalin or snap frozen
in a cassette with OCT. Samples in the former treatment were paraffin
embedded 24 h after fixation. Staining for mKC (KC Rabbit Polyclonal
Ab; BioVision) was performed on paraffin-embedded tissue from gd9-
or gd15-treated and control-matched samples as previously described
(29). F4/80?(MCA497R rat and mouse Ag; Serotec) cells were de-
tected using paraffin-embedded tissue as described previously (30).
Gr-1 (purified rat anti-mouse Ly6G and Ly6C; BD Biosciences) stain-
ing was performed on frozen gd9 and gd15 tissue cut into 10-?m sec-
tions as described previously (31).
Statistical significance of pregnancy outcomes was examined using the
one-way ANOVA method. All experiments where flow cytometry plots
were analyzed for n ? 3 or more animals per condition and significance
was assessed via the t test. Data are expressed as means ? SD. A p ? 0.05
was considered to be statistically significant.
CpG ODN treatment induces fetal resorption and preterm birth
We studied the effect of CpG ODN treatment on pregnancy in
either a fetal resorption or preterm birth model using C57BL/6
mice. The CpG ODN motif, CpG 1826, used in these studies has
been widely used to trigger B or T cell-specific immunity (32, 33).
To assess the ability of CpG ODN to induce fetal resorption, we
injected i.p. varying doses of CpG ODN in IL-10?/?or WT mice
on gd6. We initially tested a dose range from 5 to 400 ?g/dam and
found that the 25-?g dose was optimal in IL-10?/?mice to induce
complete resorption of uterine horns. At the dose of 100 ?g/dam
or higher, we observed maternal wasting in IL-10?/?mice. As Fig.
1A demonstrates, fetal resorption was consistently observed in IL-
10?/?mice in numerous matings (n ? 37) at the 25-?g/dam dose.
In contrast, no negative pregnancy effects were observed at this
dose in WT mice (n ? 8) as they maintained pregnancy to term
(?20 days) and delivered healthy pups.
Next, we aimed to find a dose of CpG ODN that caused a neg-
ative pregnancy outcome in WT mice in response to gd6 admin-
istration. A range of doses up to 250 ?g/dam did not exert any
adverse consequences on pregnancy outcome or gestational length.
However, at a dose of 400 ?g/dam, a significant number of pups
(43%) were born at term with cranial and distal limb malforma-
tions (Fig. 1C). These results are consistent with previously pub-
lished data demonstrating similar deformities to pups born to dams
treated with 300 ?g of CpG ODN (19).
1145The Journal of Immunology
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1154TLR9-MEDIATED PREGNANCY COMPLICATIONS