Objective
Intrauterine infection such as by Escherichia coli and Ureaplasma spp induce placental inflammation and are one of the leading causes of preterm birth. Here we evaluated hydroxylated fullerene (C 60 [OH] 44 ) for its in vitro antiinflammatory and antioxidant effects against host cellular responses to the ureaplasma toll-like receptor 2 (TLR2) ligand, UPM-1. In addition, we investigated the preventative effects of C 60 (OH) 44 in vivo in a mouse preterm birth model that used UPM-1.
Study Design
TLR2-overexpressing cell lines and the primary cultures of mouse peritoneal macrophages were pretreated with C 60 (OH) 44 . After UPM-1 addition to the cell lines, the activation of the nuclear factor kappa-light chain-enhancer of activated B cells (NF-kappaB) signaling cascade and the production of reactive oxygen species were monitored. The levels of expression of inflammatory cytokines of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and the production of reactive oxygen species were quantified after stimulation with UPM-1. The in vivo preventative effects of C 60 (OH) 44 on mice preterm birth were evaluated by analyzing the preterm birth rates and fetal survival rates in the preterm birth mouse model with placental histological analyses.
Results
Pretreatment with C 60 (OH) 44 significantly suppressed UPM-1-induced NF-kappaB activation and reactive oxygen species production in TLR2-overexpressing cell lines. In the primary culture of mouse peritoneal macrophages, UPM-1-induced production of reactive oxygen species and the expression of inflammatory cytokines such as IL-6, IL-1β, and TNF-α were significantly reduced by pretreatment with C 60 (OH) 44 . In the UPM-1-induced preterm birth mouse model, the preterm birth rate decreased from 72.7% to 18.2% after an injection of C 60 (OH) 44 . Placental examinations of the group injected with C 60 (OH) 44 reduced the damage of the spongiotrophoblast layer and reduced infiltration of neutrophils.
Conclusion
C 60 (OH) 44 was effective as a preventative agent of preterm birth in mice.
The World Health Organization has defined preterm birth as birth that occurs prior to the 37th gestational week. The estimated number of preterm births worldwide was 15 million in 2010 (11.1%). Preterm birth is a risk factor in more than 50% of all incidents of perinatal mortality and infantile respiratory failure, and it results in long-term neurologic morbidity.
Babies born before 28 gestational weeks have a risk of disease during childhood and a risk of cerebrovascular and ischemic heart disease in early adulthood. The development of perinatal care has raised the survival rate of infants with extremely low birthweights, which resulted in the recent observation that females born preterm have an increased risk of reproductive difficulties. Considerable efforts have been focused on the control of preterm birth; however, the preterm birth rate is still increasing.
It is widely accepted that intrauterine bacterial infections are one of the most important causes of preterm birth. Lipopolysaccharide (LPS) from Escherichia coli , which is a ligand of the toll-like receptor (TLR) 4, activates downstream inflammatory responses. LPS has often been used in mammalian models of preterm birth.
There is growing evidence that Ureaplasma spp, belonging to the family Mycoplasmataceae , has been a common causative bacteria of chorioamnionitis (CAM) and, consequently, preterm birth. Patients with threatened preterm birth and intact membranes who test positive on polymerase chain reaction (PCR) for U urealyticum in the amniotic fluid are at risk of going into preterm birth with adverse perinatal outcomes.
We analyzed 151 preterm and stillbirth placentas, and 63 (42%) of them were positive for Ureaplasma spp. Although Ureaplasma spp has been considered to be involved in the pathobiology of preterm birth for more than half a century, the mechanism of its involvement remains unclear. Synthesized diacylated 21 N-terminal amino acids of the ureaplasmal outer membrane lipoprotein (UPM-1) activates the signaling cascade of nuclear factor-kappa–light-chain enhancer of activated B cells (NF-kappaB) through TLR2. UPM-1 has been shown to induce preterm birth and intrauterine fetal death in C3H/HeN mice.
Fullerene, a nanomaterial with radical-scavenging activity, is a molecule that comprises carbon in the form of a hollow sphere. These observations suggest fullerene is an antioxidant agent. In our previous study, we demonstrated injections of the 70 nm silica nanoparticle restricted fetal growth and induced abortion in pregnant mice. However, fullerene did not cause any pregnancy-associated complications at the dose used in our study.
In this study, we aimed to clarify the antiinflammatory and antioxidant effects of water-soluble hydroxylated fullerene (C 60 [OH] 44 ) on TLR2-mediated inflammation in vitro. We found that C 60 (OH) 44 suppressed the inflammation and the production of reactive oxygen species in cultured cells. Furthermore, C 60 (OH) 44 reduced preterm birth and fetal loss in our UPM-1–induced preterm mouse model, suggesting C 60 (OH) 44 has potential as a therapeutic agent for preterm birth in mice.
Materials and Methods
Cell culture and C 60 (OH) 44 cytotoxicity
Hydroxylated fullerene [C 60 (OH) 44 ], which has an average molecular formula of C 60 (OH) 44 ·8H 2 O, was synthesized with hydrogen peroxide, according to a previously reported method.
HeLa and HEK293T cells lines were purchased from RIKEN (Saitama, Japan). The cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Co LLC, St. Louis, MO) containing 10% fetal calf serum, 100 U/mL of penicillin G, and 100 mg/mL of streptomycin under the condition of 5% CO 2 in humid air at 37°C. Both cell lines (6.0 × 10 3 ) were cultured overnight and exposed to various concentrations of C 60 (OH) 44 (5, 10, 25, or 50 μM).
Cell viability was assayed with a cell-counting kit-8 (Dojindo Laboratories, Kumamoto, Japan) at different time points (24, 48, or 72 hours). In brief, we added 10 μL of the cell-counting kit-8 reagent at different time points and incubated for 2 hours. The optical density at a wavelength of 450 nm (OD 450 ) as absorbance was calculated with a microplate reader (Bio-Rad model 3550; Bio-Rad Laboratories, Hercules, CA). Cell viability was calculated as follows: [{(OD 450 ) of sample − (OD 450 ) of blank}/{(OD 450 ) of control − (OD 450 ) of blank}] × 100(%).
NF-kappaB reporter assay
An NF-kappaB reporter assay was conducted as previously described. In brief, after an overnight culture of HeLa cells or HEK293T cells (2.4 × 10 4 ), 0.3 μg of pNF-kappaB-luciferase (Agilent Technologies, Santa Clara, CA), 0.1 μg of pFLAG-TLR2 and 0.1 μg of pRL-tk (Promega Corp, Madison, WI) were cotransfected with the lipofection reagent, FuGENE 6 (Promega Corp). After 48 hours of transfection, we added 0–50 μM of C 60 (OH) 44 . One hour after adding the reagents, the transfected cells were stimulated with 27.2 nM of UPM-1. After a further 8 hour exposure to UPM-1, the activated cells were lysed and a luciferase assay was performed with a dual-luciferase reporter assay system (Promega Corp) and quantified with a Luminescence Reader BLR-301 (Hitachi Aloka Medical, Ltd, Tokyo, Japan).
Flow cytometry for the detection of reactive oxygen species production in HEK293T cells
After the overnight culture of HEK293T cells (2.4 × 10 4 ), the cells were transfected with 0.4 μg of pF-TLR2 with FuGENE 6 (Promega Corp). Forty-eight hours after transfection, 50 μM of C 60 (OH) 44 was added to the cells. One hour after adding the fullerene, the cells were stimulated with 27.2 nM of UPM-1. Eight hours after stimulation, 5 μM of the cellROX green reagent (Life Technologies, Carlsbad, CA) was added to the culture medium, and the relative fluorescence units (RFUs) of every 10,000 cells were measured with BD FACScalibur HG (Becton, Dickinson and Co, Franklin Lakes, NJ). The mean fluorescence was calculated with CellQuest Pro software (Becton, Dickson and Co).
Reactive oxygen species production of mouse peritoneal macrophages
C57BL/6J mice were from Japan SLC, Inc (Hamamatsu, Japan). The experimental protocols of the animal studies were approved by the animal experiment committee of Osaka Medical Center and the Research Institute for Maternal and Child Health.
Peritoneal macrophages were collected according to the method of Zhang et al with a slight modification. In brief, 1–3 mL of 3% sterilized fluid thioglycollate medium II (Eiken Chemical Co, Ltd, Tokyo, Japan) was injected into the intraperitoneal cavities of 8–13 week old nonpregnant female mice.
Four days later, the refluxed intraperitoneal fluid was collected, and macrophages were kept in the RPMI 1640 medium (Sigma-Aldrich Co) containing 10% fetal calf serum, 100 U/mL of penicillin G, and 100 mg/mL of streptomycin. The next day, 1 hour prior to the stimulation of UPM-1 and LPS ( E coli O55:B5; Sigma-Aldrich Co), we replaced the RPMI 1640 medium with a medium containing 50 μmol/L of C 60 (OH) 44 , and 8 hours after stimulation with 36.0 nM of UPM-1 and 1 μg/mL of LPS, we added 5 μmol/L of the CellROX green reagent (Life Technologies).
Reactive oxygen species ( ROS) production was expressed in RFUs by using a fluorescence microscope (ECLIPSE TE-2000E; Nikon Corp, Tokyo, Japan) that was equipped with EM-CCD iXon+ (Andor Technology, Ltd, Belfast, UK), and the data were analyzed using Andor iQ2 software (Andor Technology, Ltd). At least 450 macrophages were quantified in each group.
Reverse transcription–polymerase chain reaction
We performed quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate the inflammatory cytokines. Mouse peritoneal macrophages (1.95 × 10 6 ) were collected and stimulated as in the ROS production assay. After 8 hours of UPM-1 stimulation, total ribonucleic acid (RNA) was extracted with an SV RNA isolation kit (Promega Corp). First-strand complementary deoxyribonucleic acid was synthesized from 100 to 200 ng of RNA with random primers with a PrimeScript first-strand complementary deoxyribonucleic acid synthesis kit (Takara Bio Inc, Otsu, Japan).
qRT-PCR was performed to determine the levels of mouse interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α with a QuantiTect SYBR Green PCR kit (QIAGEN Benelux B.V., Venlo, The Netherlands) on a Chromo 4 real-time PCR system (Bio-Rad Laboratories, Inc). The primers are listed in Table 1 . qRT-PCR was performed as follows: 40 cycles of 95°C for 20 seconds, 57°C for 30 seconds, and 72°C for 30 seconds.
| Gene | Sequence | Product size, bp |
|---|---|---|
| Mouse IL-6 | 5′-tccagttgccttcttgggac-3′ | 331 |
| 5′-gtactccagaagaccagagg-3′ | ||
| Mouse IL-1β | 5′-ctccatgagctttgtacaagg-3′ | 245 |
| 5′-tgctgatgtaccagttgggg-3′ | ||
| Mouse TNF-α | 5′-gcatgatccgcgacgtggaa-3′ | 304 |
| 5′-agatccatgccgttggccag-3′ |
Preterm birth mouse model
We used our previously reported preterm birth mouse model. In brief, C3H/HeN mice (Japan SLC, Inc) were pair mated. We designated the day the vaginal plug was confirmed as day 0 of gestation. On day 14, a minilaparotomy was performed under isoflurane anesthesia. The uterus was exposed by incision to visualize all of the gestational sacs. The number of sacs was counted. Twenty microliters of UPM-1 (750 ng/μL, 0.27 mM; 5.4 nmol/body) or distilled water in the control group was injected into the uterus between the 2 lower gestational sacs on the right side of the uterus. Four hours after UPM-1 stimulation, 100 μg (68 nmol) of C 60 (OH) 44 in 100 μL of phosphate-buffered saline (pH 7.4) was injected into the tail vein.
Forty-eight hours after exposure of the uterus to UPM-1, a cesarean delivery was performed, and the gestational sacs were checked. Preterm birth was detected when we could see at least 1 baby in the cage within 48 hours of the injection of UPM-1 or when we could not detect 1 gestational sac in the uterus when the cesarean delivery was performed. The number of live or dead pups in the uterus was recorded. Intrauterine fetal death was detected if white discoloration, markedly smaller fetal size, or maceration changes were observed.
Histological evaluations
The collected placentas were embedded in paraffin and stained with hematoxylin and eosin and a myeloid cell–specific esterase/naphthol AS-D chloroacetate esterase (3-hydroxy-2-naphthoic-o-toluidide chloroacetate) staining kit (Muto Pure Chemicals Co, Ltd, Tokyo, Japan). A light microscope (Olympus BX51; Olympus Corp, Tokyo, Japan) was used. The ratio of the spongiotrophoblast layer area to the total placental area was calculated according to previous reports. In brief, the spongiotrophoblast layer and total placental areas were assessed with hematoxylin and eosin–stained sections and quantitatively analyzed with NIS-Elements Documentation, version 3.13 (Nikon Corp).
Statistical analysis
PASW statistics 18 (IBM Corp, Armonk, NY) was used for the statistical analyses. Differences were considered statistically significant when P < .05. The data are expressed as mean ± SE for the cell viability analysis, NF-kappaB reporter assay.
To determine the dose dependency of C 60 (OH) 44 against NF-kappaB activation, we conducted Pearson’s regression analysis of the NF-kappaB reporter assay. For the quantification of ROS production, the mean fold of RFUs of the group of interest per RFU of the control group were calculated from 3 independent experiments of the flow cytometry analyses of every 10,000 cells or fluorescence microscopic observations of at least 450 cells.
Tukey’s post hoc tests were performed with 1-way analyses of variance (ANOVAs) on the data from the previously mentioned experiments. For qRT-PCR of inflammatory cytokines of mouse peritoneal macrophages, a nonparametric statistical analysis with the Kruskal-Wallis method was conducted from 3 independent experiments.
Fisher exact tests were applied to the data for the preterm birth. The fetal survival rate per dam was compared with 1-way ANOVAs with the Tukey-Kramer post hoc test.
In the histological examination, the ratios of the spongiotrophoblast layer to the placenta were analyzed by 1-way ANOVAs with Tukey’s post hoc tests (6 placentas per group).
Results
C 60 (OH) 44 pretreatment inhibited activation of the NF-kappaB signal cascade
C 60 (OH) 44 (0–50 μM) was added to the culture media of both cell lines. There was no significant difference in cell viability in both cell lines ( Figure 1 , A and B), which suggested the treatment with C 60 (OH) 44 did not result in cellular toxicity at any of the concentrations or time points tested.
To investigate the inhibitory effects of C 60 (OH) 44 pretreatment on inflammatory responses, we monitored NF-kappaB activation by using luciferase as a reporter in both cell lines. UPM-1–activated NF-kappaB signals were diminished by pretreatment with 50 μM of C 60 (OH) 44 to 49.4% in HeLa cells ( P < .01; Figure 1 , C) and 75.0% in HEK293T cells ( P < .01; Figure 1 , D).
We conducted a Pearson’s regression analysis to determine whether C 60 (OH) 44 regulates NF-kappaB activation in a dose-dependent manner. In HeLa cells, it had a weak negative correlation in a dose-dependent manner (r = −0.442; P = .066). In HEK293T cells, it had a relatively strong negative correlation (r = −0.617; P < .01).
In vitro antioxidant activity and proinflammatory cytokine messenger RNA levels were suppressed by pretreatment with C 60 (OH) 44
In HEK293T cells ( Figure 2 , A), UPM-1 activated ROS production by 1.83-fold, whereas 50 μM of C 60 (OH) 44 suppressed it to background levels ( P < .01).
We examined the influence of C 60 (OH) 44 on ROS production in a primary culture of mouse peritoneal macrophages. Because the collected mouse macrophages adhered tightly to the culture plate dishes and the harvested cell count was relatively limited, we used a fluorescence microscopic analysis instead of a flow cytometric analysis. As shown in Figure 2 , B, the fluorescence of ROS in each mouse macrophage was visualized as a green dot. The mean RFUs were measured. As in the HEK293T cells stimulated with UPM-1, UPM-1 increased the activation of ROS production by 1.58-fold in mouse macrophages. Pretreatment with 50 μM of C 60 (OH) 44 significantly blocked the UPM-1–induced ROS production, which was at background levels ( P < .01; Figure 2 , B and C).
LPS (1.0 μg/mL), a TLR4 ligand, also increased ROS production by 1.99-fold compared with the control, whereas pretreatment with 50 μmol/L of C 60 (OH) 44 significantly blocked LPS-induced ROS production to the background level ( P < .01; Figure 2 , D and E).
We quantified the expression levels of the proinflammatory cytokine messenger RNA (mRNA) after stimulation with UPM-1 ( Figure 3 ). Fifty micromoles of C 60 (OH) 44 significantly inhibited IL-6 by 75% ( P = .027), IL-1β by 72% ( P = .050), and TNF-α by 84.1% ( P = .039).
