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: email@example.com
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
We have previously shown that LPS administered to IL-10?/?
and WT mice on gd14 induces preterm birth on gd17, albeit at
20-fold higher doses in WT mice (10). For preterm birth outcomes
in response to CpG ODN administration, a similar approach was
used. CpG ODN was administered i.p. on gd14 and mice were
checked twice daily for preterm birth of newborns. As shown in
Fig. 1B, IL-10?/?mice (n ? 18) consistently delivered stillborn
pups at a dose of 15 ?g/dam within 24–48 h of injection. Lower
doses of CpG ODN failed to induce preterm delivery. Similar to
fetal resorption observations, WT mice (n ? 4) failed to expe-
rience any negative consequences of CpG ODN administration
at a comparable or higher dose in the range of 300 ?g/dam (n ?
8), except for deformities at higher doses similar to that shown
in Fig. 1C.
Regarding fetal resorption in IL-10?/?mice, we noted that ef-
fects were rapid with severe uteroplacental pathology at 25 ?g of
CpG ODN (Fig. 1A). To characterize the kinetics and general pa-
thology in IL-10?/?mice in response to CpG ODN administra-
tion, we visually assessed fetal resorption from gd7 through gd9.
Fig. 2A demonstrates fetal resorption at 25 ?g of CpG ODN as
observed in uterine horns harvested as early as gd7 and the pla-
cental pathology appeared to increase in severity on gd8 and gd9.
In contrast, control ODN-treated mice did not experience fetal
CpG ODN-mediated effects on pregnancy outcome are TLR9
CpG ODN 1826 is a promiscuous molecule which may signal not
just through TLR9, but also through an extracellular receptor (34,
35). Thus, we assessed whether negative pregnancy outcomes in
IL-10?/?mice were due to direct TLR9 stimulation. To address
this issue, we used CpG ODN 2088 (antagonist ODN), an ODN
sequence which binds intracellular TLR9 with high affinity but
does not induce downstream stimulation of the TLR pathway (36–
38). Antagonist ODN injected on gd6 did not induce any pathology
in placental units harvested on gd9 (Fig. 2B). Importantly, mice
treated with antagonist ODN when allowed to deliver gave birth to
healthy litters at term. Antagonist ODN, when used with patho-
genic CpG ODN at a 4:1 or 2:1 ratio, blocked fetal resorption
compared with CpG ODN alone as demonstrated by normal pla-
cental units (Fig. 2B). If allowed to deliver, antagonist ODN plus
CpG ODN-treated mice gave birth to healthy pups. These results
demonstrate that antagonist ODN was able to successfully blunt
the TLR9 receptor-mediated signaling and displace the binding of
CpG ODN at a dose ratio as low as 2:1 as demonstrated by lack of
resorption on gd9.
CpG ODN administration in IL-10?/?mice results in
amplification and placental migration of uterine macrophages
In normal pregnancy, uNK cells, macrophages, and low numbers
of T cells, but not Gr-1?CD11b?cells, are normally detected in
sorption and preterm birth in WT or IL-10?/?mice. CpG or control ODN
was injected i.p. in WT or IL-10?/?mice after visualization of a vaginal
plug on gd0. A, Fetal resorption in uterine horns from IL-10?/?or WT
mice was assessed visually on gd9 and mice were injected with CpG or
control ODN on gd6. Twenty-five micrograms of CpG ODN induced full
resorption of uterine horns in IL-10?/?mice (n ? 37), whereas control
ODN failed to elicit this pathology (n ? 20). WT mice did not respond to
25 ?g of CpG ODN nor control ODN. B, gd14 i.p. injection of 15 ?g of
CpG ODN induced preterm birth of stillborn pups within 24–48 h after
injection in IL-10?/?mice (n ? 15). Control ODN-treated IL-10?/?mice
did not experience preterm delivery (n ? 4). WT mice at this dose did not
experience any preterm birth in the control ODN condition (n ? 4) or CpG
ODN condition (n ? 4). C, In WT mice, a dose of 400 ?g of CpG ODN
given i.p. on gd6 caused cranial-facial and distal limb malformations, de-
noted by ?, in 43% of pups born at term (n ? 12). Similarly, a dose of 300
?g of CpG ODN in WT mice given i.p. on gd14 resulted in pups born at
term with similar malformations (n ? 8). WT mice treated with control
ODN at these doses did not display teratogenic effects.
Dose-dependent CpG ODN-mediated induction of fetal re-
curs with rapid kinetics and is TLR9 dependent. A, Representative uterine
horns of control and CpG ODN-treated IL-10?/?mice harvested on gd7,
gd8, and gd9 represent multiple experiments of kinetic evaluation on the
insult of placental pathologies. B, Representative uterine horns harvested
on gd9 from IL-10?/?mice treated on gd6 with antagonist ODN 2088 (50
?g; top panel), antagonist ODN (50 ?g) plus stimulatory CpG ODN (25
?g; middle panel), and CpG ODN alone (bottom panel) are shown. Data
demonstrate antagonistic binding of antagonist ODN to TLR9 prevented
fetal resorption (n ? 4 animals/treatment).
CpG ODN-induced fetal resorption in IL-10?/?mice oc-
1146 TLR9-MEDIATED PREGNANCY COMPLICATIONS
the pregnant mouse uterus by gd7 (4, 39, 40). These cells remain
localized to the maternal side of uteroplacental tissue within the
mesometrial triangle and decidua basalis. However, it is not yet
clear whether these uterine immune cells acquire a cytotoxic phe-
notype and migrate to the placental zone to cause local damage. In
this regard, we have recently shown for the first time that uNK
cells are indeed activated in response to LPS administration and
cause apoptosis in the placental region (10). It is possible that
severe and rapid pathology in CpG ODN-treated mice (Figs. 1 and
2A) may be programmed by the mechanisms of cellular activation
and placental injury.
There is abundant evidence in nonpregnant mice that CpG ODN
treatment leads to activation of dendritic cells (41). We prepared
single-cell suspensions of CD45?uterine mononuclear and gran-
ular cells as described in Materials and Methods and utilized flow
cytometry to probe for the presence of CD11c?(dendritic cells),
CD11b?Gr-1?(neutrophils), CD11b?F4/80?(macrophages), or
NK1.1?CD3?(uNK cells) in IL-10?/?and WT mice treated or
untreated with CpG ODN. In the case of fetal resorption, we
harvested uterine tissue and spleen on gd8 and 9, whereas for
the preterm birth setting, we harvested uterine tissue and spleen
24 h after CpG ODN injection on gd15. Surprisingly, we did not
suspensions of UMGC populations were isolated from IL10?/?(A and C) or WT (B and D) mice on gd9 or gd15 after treatment with control or CpG ODN.
Cells were gated on CD45?populations for CD11b?Gr-1?(A and B) and CD11b?F4/80?(C and D); data shown are representative of multiple experiments
using four animals per condition. A, IL-10?/?mice showed a significant increase in the CD11b?Gr-1?population on gd9 and gd15, p ? 0.05. B, WT mice
under similar conditions did not show any significant changes in this population. C, IL-10?/?mice showed a significant increase in the CD11b?F4/80?
cell population on gd9 and gd15, p ? 0.05, compared with control ODN-treated mice. D, WT mice under similar conditions did not show any significant
changes in this population.
CD11b?Gr-1?and CD11b?F4/80?uterine cell populations amplify in response to CpG ODN in IL-10?/?, but not WT mice. Single-cell
1147The Journal of Immunology
see any changes in the dendritic cell populations of IL-10?/?or
WT mice treated with 25 ?g of CpG ODN on gd6 (data not
Interestingly, analysis of gd8 and 9 uterine mononuclear and
granular cells from CpG ODN-treated IL-10?/?mice showed a
significant increase in the CD11b?Gr-1?cellular population com-
pared with vehicle-treated mice (9.3 ? 2.5 to 28.8 ? 4.4%; Fig.
3A). Similarly, we observed an increase in the CD11b?GR1?cel-
lular population in IL-10?/?mice under preterm birth conditions
when analyzed on gd15 (6 ? 1.4 to 23.7 ? 3.5%; Fig. 3A). A
simultaneous increase occurred in CD11b?F4/80?populations.
When analyzed on gd8 and 9, this population increased from
11.1 ? 0.9% to 35.0 ? 4.0% and a similar increase occurred in the
preterm birth condition, 9.5 ? 3.1% to 25 ? 2.6% (Fig. 3C). At the
doses used to induce fetal resorption or preterm birth in IL-10?/?
mice, we did not observe any significant changes in either of these
cellular populations in WT mice (Fig. 3, B and D).
Analysis of uNK cells revealed a slight increase in NK1.1?
CD3?cells in CD45?mononuclear and granular cells from gd8
and 9 and gd15 tissues from IL-10?/?but not WT mice. However,
depletion of uNK cells using anti-asialo GM1 Ab proved ineffec-
tive in rescuing fetal resorption or preterm birth in IL-10?/?mice
treated with CpG ODN (data not shown). Thus, we focused on the
role of macrophages and neutrophils in adverse pregnancy out-
comes because these cells were highly amplified in response to
CpG ODN treatment.
mKC is significantly induced in CpG ODN-treated IL-10?/?
We addressed the possibility that a suitable chemokine was in-
duced in response to CpG ODN treatment in IL-10?/?mice that
led to the unscheduled recruitment and amplification of granulo-
cyte and monocyte (CD11b?Gr-1?, or CD11b?F4/80?) cellular
populations. Initial screening was done for serum levels of MIP-
1?, MIP-2, and mKC using chemokine-specific ELISAs. No sig-
nificant changes were observed for MIP-1? or MIP-2 in serum
samples from control ODN or CpG ODN-treated mice (data not
shown). However, mKC, the mouse homologue of human IL-8 and
a known chemoattractant of macrophages and neutrophils (42, 43),
was markedly increased in samples from CpG ODN-treated IL-
10?/?but not WT mice. The mKC levels in sera collected on gd9
from IL-10?/?mice averaged 94.3 ? 42.5 pg/ml in control ODN-
treated mice and increased to an average of 319.1 ? 85.7 pg/ml in
CpG ODN-treated mice, demonstrating a significant difference
(Fig. 4A). Under identical conditions, no significant differences
were observed in WT mice (Fig. 4A). Sera collected from gd15
IL-10?/?mice showed larger increases in mKC levels from an
average of 63.4 ? 7.4 pg/ml in control animals (n ? 9) to an
average of 1242.9 ? 311.2 pg/ml in CpG ODN-treated animals
(n ? 9) with no significant changes in WT mice (Fig. 4B). IL-8 has
been shown to be intrinsically produced at physiological levels by
human uNK cells and it has been proposed that this chemokine
mice on gd9 (A) or gd15 (B). Data represent average values from nine different serum samples. IL-10?/?mice (f) showed a significant increase in mKC
levels above control ODN-treated mice (3) on gd9 (A) and gd15 (B); ?, p ? 0.05. C and D, mKC was evaluated by immunohistochemistry done on
uteroplacental tissue collected from control or CpG ODN-treated IL-10?/?mice. A marked increase in mKC protein was observed in CpG ODN-treated
uterine tissue compared with control ODN-treated tissue on gd9 (C) and gd15 (D). M, Demarcates the mesometrial triangle, whereas P indicates the
CpG ODN treatment leads to elevated production of mKC in IL-10?/?mice. mKC was measured in sera collected from IL-10?/?or WT
1148TLR9-MEDIATED PREGNANCY COMPLICATIONS