Placenta
Volume 31, Issue 5 , Pages 387-391, May 2010

Maternal obesity up-regulates inflammatory signaling pathways and enhances cytokine expression in the mid-gestation sheep placenta

  • M.J. Zhu

      Affiliations

    • Center for the Study of Fetal Programming, Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA
    • Corresponding Author InformationCorresponding author. Tel.: +1 307 766 3140; fax: +1 307 766 2355.
    • ,
  • M. Du

      Affiliations

    • Center for the Study of Fetal Programming, Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA
    • ,
  • P.W. Nathanielsz

      Affiliations

    • Center for the Study of Fetal Programming, Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA
    • Center for Pregnancy and Newborn Research, University of Texas, Health Sciences Center, San Antonio, TX 78229, USA
    • ,
  • S.P. Ford

      Affiliations

    • Center for the Study of Fetal Programming, Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA

Accepted 3 February 2010. published online 25 February 2010.

Article Outline

Abstract 

Obesity in pregnant women is a growing public health concern. The placenta is a source of cytokines which can induce maternal gestational insulin resistance and alter nutrient transport to the fetus. Obesity induces placental inflammation at term, but the impact of obesity on placental inflammation earlier in pregnancy has not been defined. Using sheep as an experimental model, we hypothesized that maternal obesity (MO) would induce inflammation in the cotyledonary (COT) tissue of the placentome by mid-gestation. Nonpregnant ewes were randomly assigned to a control (C, 100% of NRC recommendations) or obese (OB, 150% of NRC) group from 60 days before conception to 75 day of gestation (dG), when ewes were necropsied and placental COT tissue collected for analyses. Free fatty acids content, triglyceride and cholesterol content were higher (P < 0.05) in the fetal plasma of OB compared to C ewes on day 75. MO increased mRNA levels of toll-like receptor (TLR) 2 (P < 0.05) and TLR4 (P = 0.06), macrophage markers cluster of differentiation (CD)11b (P = 0.06), CD14 and CD68 (P < 0.05), and proinflammatory cytokines tumor necrosis factor (TNF)α (P < 0.01), interleukin (IL)-6 (P < 0.05), IL-8(P < 0.01) and IL-18 (P = 0.06), in COT tissue. Inflammatory c-Jun N-terminal kinase (JNK)/c-Jun and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways were up-regulated (P < 0.05) in COT of OB ewes. In conclusion, MO enhanced the placental inflammatory response in OB ewes at mid-gestation, possibly as a result of increased TLR4 and free fatty acids.

Keywords: Inflammation, Obesity, Placenta, Sheep, Toll-like receptors

 

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1. Introduction 

In the USA, pre-pregnancy obesity in women increased from 13.0% in 1993–1994 to 22.0% in 2002–2003, a net increase of 69.3% [16]. Recent evidence suggests that high pre-pregnancy BMI and maternal diabetes are associated with macrosomia, newborn adiposity and complications for offspring in later life [3], [8], [20], [21], [23], [33]. Mechanisms linking maternal obesity (MO) to the increased incidence of obesity and metabolic diseases in offspring remain poorly defined.

Obesity leads to low-grade inflammation in adipose tissue [9], [37], [40]. Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling and the c-Jun N-terminal kinase (JNK) pathway are the primary signaling pathways of the inflammatory response [2], which further enhances expression of proinflammatory cytokines, producing a vicious cycle [15]. Toll-like receptor (TLR) 4 is a major factor that drives the inflammatory NF-κB pathway [43] and activates the JNK pathway [24]. A crucial recent finding demonstrated that free fatty acids are ligands for TLR4 receptor [26], [31]. Therefore, excessive fatty acids in the fetal circulation in the setting of MO would tend to activate TLR4 signaling, resulting in inflammation of fetal tissues.

The placenta mediates nutrient transport from the maternal to fetal circulation, and is also an important immune organ, producing a number of cytokines [10]. A recent study reported that obesity induced inflammation in the placenta of obese women at term [5]. However, whether MO induces inflammation in the placenta earlier in gestation has not been assessed. Mid-gestation is a critical stage for fetal development, and inflammation during this stage is expected to impact placental development and function as well as the development of fetuses [42]. Since human tissue is difficult to obtain at mid-gestation from otherwise normal and overweight women, we have developed an animal model of MO in sheep, a species commonly studied in pregnancy. We hypothesized that MO increases lipid concentration in the fetal circulation, in association with increased expression of inflammatory signaling molecules in placental COT tissue at mid-gestation.

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2. Materials and methods 

2.1. Care and use of animals 

All animal procedures were approved by the University of Wyoming Animal Care and Use Committee. From 60 days before conception to day 75 of gestation, 3–4 yr old multiparous Rambouillet/Columbia ewes of similar weight and body condition score were placed in individual pens and fed either a highly palatable diet at 100% (control, C; n = 10) of National Research Council (NRC) recommendations [25] or 150% (obesogenic, OB; n = 10) of NRC recommendations on a metabolic body weight basis (BW0.75) as previously reported [47]. Ewes were weighed at weekly intervals so that individual diets could be adjusted for weight gain, and body condition scores were obtained at monthly intervals to evaluate changes in fatness. A body condition score of 1 (emaciated) to 9 (obese) was assigned by two trained observers after palpation of the transverse and vertical processes of the lumbar vertebrae (L2 through L5) and the region around the tail head as previously described [29].

2.2. Tissue collection 

On the day of necropsy, ewes were sedated with an i.v. infusion of Ketamine (22.2 mg/kg body wt), and general anesthesia induced with isofluorane inhalation anesthetic (0.5–2.5%). Jugular venous blood samples were collected from five twin bearing ewes in each dietary group while under anesthesia. Maternal blood was collected into a chilled tube (EDTA, Sigma) and plasma frozen at −80 °C until utilized for analysis. Following midventral laparotomy, the gravid uterus was located and opened, and the umbilical cord of each fetus was isolated. Umbilical venous blood was collected from each fetus via venipuncture and plasma were collected and stored as described for maternal blood. Ewes were then exsanguinated while under general anesthesia and the gravid uterus immediately recovered and opened from base to tip. COT tissue was obtained from several type A placentomes of similar size associated with the placental membranes of each conceptus and located within 10 cm of each umbilical cord attachment site and frozen in liquid nitrogen and stored at −80 °C for Western blot and real-time reverse transcript (RT)-PCR analysis as previously described [46]. All placentomes present in each uterus of both C and OB ewes in this study were classified as type A using the criteria previously described [39]. In each dietary group, six female fetuses and four male fetuses are used. Since there is no significant difference between female and male fetuses on day 75 gestation, data from male and female fetuses were pooled together for analyses.

2.3. Colorimetric total free fatty acid (FAA) assay 

Fetal plasma total FFA content was analyzed colorimetrically following the published method by Tinnikov and Boonstra [38]. Briefly, 10 μL EDTA-plasma was added to glass vials containing 1ml of CHN (chloroform-heptane (4:3, v/v), 2% methanol). Then, 50 mg 120 °C oven activated silicic acid was added to each tube to eliminate phospholipid interference. After centrifugation at 2000 × g for 3 min, the upper 0.8 mL subsample was transferred to another vial containing 0.4 mL of Cu-TEA solution (50mM Cu(NO3)2, 100 mM triethanolamine, 35 mM NaOH, 33% NaCl, pH8.1), vortexed for 5 min and centrifuged for 3 min at 2000 × g. The 0.6 mL of upper phase was carefully transferred into a new vial, which was evaporated under air in a fume hood. Then 200 μL of 100% ethanol was added to each tube, incubated at 37 °C for 15 min, and then vortexed for 5 min. Finally, 0.4% diphenylcarbazide solution was added for colorimetric determination of copper at 550 nm. Total FAA concentration was calculated based on the standard curve. Each sample was analyzed in duplicate and mean values were reported [38]. All sample were analyzed in a single assay and intra-assay coefficient of variation < 2.0%.

2.4. Lipid analyses 

A Boehringer Mannhiem/Hitachi 912 analyzer was used to analyze fetal plasma for cholesterol and triglyceride (Roch Diagnostics, Indianapolis, IN) as previously described [19]. The lipid analyses were conducted by Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia.

2.5. Antibodies 

Antibodies against phos-SAPK/JNK (Thr183/Tyr185) (Cat # 9251), SAPK/JNK (Cat # 9252), phos-c-Jun (Ser63) (Cat # 9261), phos-c-Jun (Ser73) (Cat # 9164), c-Jun (60A8) (Cat #9165), phos-IKKα/β (Ser176/180) (Cat # 2697), IKKβ (Cat # 2678), phos-NF-kB p65 (Ser536) (Cat # 3033) and NF-kB p65 (Cat# 4764) were purchased from Cell Signaling (Danvers, MA). Anti-β-tubulin(Cat #T4026) antibody was purchased from Sigma (Saint Louis, Missouri).

2.6. Western blotting analysis 

Western blotting analyses were used to analyze JNK, c-Jun, and IKK and NF-κB total protein and their phosphorylated protein content, and conducted by procedures previously published from our laboratory [44], [45]. Briefly, protein extractions were separated by 5–15% SDS-PAGE gels and transferred to nitrocellulose membranes. Following transfer, membranes were blocked with 5% nonfat milk powder in TBST (50 mM Tris–HCl, pH7.6, 150 mM NaCl, 0.05% Tween-20) for 2 h. Blocked membranes were then incubated overnight at 4 °C with the primary antibodies (1:1000 dilution in TBST with 5% BSA). Following three time of TBST washing, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature. After three 15 min washes, membranes were visualized using ECL™ Western blotting detection reagents (Amersham Bioscience) and exposure to film (MR, Kodak, Rochester, NY). The density of bands was quantified by using an Imager Scanner II (Amersham Bioscience) and ImageQuant TL software (Amersham Bioscience). Band density among different blots was normalized according to the density of a reference sample. Band density was also normalized according to the β-tubulin content [44], [45].

2.7. Real-time reverse transcript PCR (RT-PCR) analysis 

Real-time RT-PCR was used to analyze the mRNA expression of Cluster of differentiation (CD)11b, CD14, CD68, Interleukin (IL)-6, IL-8, IL-18, TLR2, TLR4, and tumor necrosis factor (TNF)-α. Total RNA was extracted using Trizol®Reagent (Invitrogen, Carlsbad, CA), treated with DNase I (Qiagen, Valencia, CA) and cleanup with RNeasy Mini kit (Qiagen, Valencia, CA). cDNA was synthesized with SuperScriptTM III first-strand synthesis for RT-PCR kit (Invitrogen, Carlsbad, CA). Real-time quantitative RT-PCR (qRT-PCR) was conducted on a Bio-Rad iQ5 machine. Primers used in this study were synthesized by Invitrogen. β-tubulin was used as the housekeeping gene. Primer information for CD11b, CD14, CD68, IL-6, IL-8, IL-18, TLR2, TLR4, TNF-α and β-tubulin were listed in Supplementary Table 1. SYBR Green Master Mix (Bio-Rad Laboratories, Hercules, CA) was used in all PCR reaction (20μl total volume). The final primer concentration is 200nM for each gene. The amplification efficiency was 0.90–0.99. The qRT-PCR conditions was 95 °C, 3min; 35 cycles of 95 °C for 10 s and 58 °C for 30 s. At the end of each run, dissociation melt curves were obtained.

2.8. Statistics 

Data were analyzed as a complete randomized design using GLM (General Linear Model of Statistical Analysis System) [30]. Means separation was performed using LSMEANS. Means ± SEM were considered different when P < 0.05, and a trend was indicated when P < 0.10.

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3. Results 

At d75 of gestation, OB ewes fed the obesogenic diet were heavier than ewes fed the control diet (98.5 ± 3.1 vs 73.9 ± 4.20 kg; P < 0.01) and fatter as indicated by their body condition scores (7.9 ± 0.2 vs 5.0 ± 0.5; P < 0.01). At necropsy, fetal body weight was greater in OB than C ewes (234.4 ± 6.61 vs 185.7 ± 6.89 g; P < 0.05). Free fatty acid content, triglyceride and cholesterol concentrations were higher in fetal blood of OB compared to the C animals (P < 0.05; Table 1).

Table 1. Total free fatty acids, triglyceride and cholesterol content in the fetal plasma on 75 dG of control (C) and obese (OB) ewes.
COBP value
Free fatty acid (mM)0.49 ± 0.030.61 ± 0.030.008
Cholesterol (mg/dl)34.0 ± 2.442.0 ± 2.40.050
Triglyceride (mg/dl)38.8 ± 1.848.0 ± 1.80.006

Mean ± SEM; n = 10 in each group.

Maternal obesity increased COT mRNA levels of both TLR2 (P < 0.05) and TLR4 (P = 0.06), key mediators of the innate immune response compared to COT of C ewes (Fig. 1). In addition, monocyte activation marker CD11b (P = 0.06) and macrophage differentiation and maturation markers CD14 (P < 0.05) and CD68 (P < 0.05) were all increased in COT of OB ewes when compared to C ewes (Fig. 2A). Consistent with these results, OB ewes exhibited enhanced mRNA expression of the proinflammatory cytokines, TNF-α (P < 0.01), IL-6 (P < 0.05), IL-8 (P < 0.01) and IL-18 (P = 0.06) in COT tissue compared to C ewes (Fig. 2B).

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  • Fig. 1 

    Toll-like receptor 2 and 4 mRNA expressions in COT tissue of control (C) and obese (OB) ewes on 75 dG ■: C ewes; □: OB ewes. Mean ± SEM; *: P < 0.05; †: P = 0.06; n = 10 in each group.

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  • Fig. 2 

    Macrophage markers CD11b, CD14 and CD68 and proinflammatory cytokines TNF-α, IL-6, IL-8 and IL-18 mRNA expressions in COT tissue of control (C) and obese (OB) ewes on 75 dG ■: C ewes; □: OB ewes. Mean ± SEM; **: P < 0.01; *: P < 0.05; †: P = 0.06; n = 10 in each group.

Western blotting further indicated that phos-JNK (Thr183/Tyr185) (P < 0.05) and its downstream target phos-c-Jun at Ser63 (P < 0.05) and Ser73 (P < 0.01) sites were up-regulated (P < 0.05) in the OB group versus the C group (Fig. 3A). In addition, both phos-IκB kinase α/β (Ser176/180) and its downstream component NF-κB, phos-p65 (Ser536) were also (P < 0.05) up-regulated in COT of OB versus C ewes (Fig. 3B).

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  • Fig. 3 

    JNK and NF-κB inflammatory signaling pathway protein content and phosphorylation in COT tissue of control (C) and obese (OB) ewes on 75 dG ■: C ewes; □: OB ewes. Mean ± SEM; **: P < 0.01; *: P < 0.05, n = 10 in each group.

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4. Discussion 

Placental uptake of free fatty acids from the maternal circulation provides substrate for both placental metabolism and delivery to the fetus [32], [41]. Placental tissue expresses lipoprotein lipase activity [6] as well as phospholipase A2 [27] and intracellular lipase activities [4]. Once released into fetal circulation, fatty acids that have been transported across the placenta are taken up by the fetal liver, esterified and released back into the circulation as triglycerides. In this study, we report that free fatty acids, cholesterol and triglycerides were elevated in the fetal blood of OB compared to C ewes and fetuses. Such an increase in free fatty acid and lipid content in fetal circulation is due to enhanced uptake of fatty acids by the placenta, which could be resulted from both higher lipid content in maternal circulation due to MO and higher efficiency of cross-placental fatty acid transport. Indeed, a linear correlation has been reported between maternal and fetal plasma triglycerides in the rat [11]. This relationship may have important implications for fetal growth, because a direct relationship between maternal triglycerides and newborn weight has been found in human pregnancy [17], [18].

Excessive adipose tissue associated with obesity induces chronic low-grade inflammation in peripheral tissues as shown in studies linking insulin resistance, obesity and Type 2 Diabetes [35]. Obesity in pregnant women induces an exaggerated inflammatory response in the placenta at term [5], consistent with our observations described here at mid-gestation in obese pregnant ewes. However, the cause of the placental inflammation associated with MO has not been clearly defined. We hypothesized that TLR4 has a crucial role in mediating this inflammation.

TLRs function as pattern-recognition receptors in mammals and play an important role in the recognition of microbial components [1]. TLR4 acts as the receptor for lipopolysaccharide (LPS) and TLR2 as the receptor for lipoproteins in bacterial cell membranes [26]. When LPS binds to TLR4 and its co-receptor, CD14 (cluster of differentiation 14), the adaptor protein MyD88 (myeloid differentiation factor-88) is recruited to the TLR4 receptor leading to auto-phosphorylation of IL-1R-associated kinase (IRAK) and activation of NF-κB signaling [26].

Recently, free fatty acids have been identified as ligands of TLR4 [31], [34], linking obesity, TLR4 and inflammation (Fig. 4). We observed that the TLR4 was up-regulated in COT of OB ewes compared to C ewes at mid-gestation. In addition, the elevated free fatty acid concentrations in fetal blood were detected. These free fatty acids should be mainly derived from maternal FFA which goes to the maternal portion of the placentome, transported into the fetal portion of the placentome and integrated into the whole fetal circulation. Enhanced TLR4 activation in combination with increased free fatty acid concentrations is likely to activate inflammatory signaling. In support of this mechanism, both NF-κB and JNK signaling were up-regulated in OB COT, a finding that is in agreement with previous reports that obesity activates inflammatory signaling pathways, mainly NF-κB and JNK pathways in the ovary [7]. As a transcription factor, NF-κB activates target genes, many of which are inflammatory cytokines, including TNF-α, a cytokine known to induce systemic inflammation [5], [47]. In this study, the expression of inflammatory cytokines was higher in COT of OB ewes compared to C ewes, consistent with the presence of enhanced inflammatory signaling. In addition, expression of the macrophage markers, CD11b, CD14 and CD68 were also increased in OB COT, consistent with a previous report that obesity stimulates macrophage infiltration in the placenta at term [5] and is also in agreement with the up-regulation of inflammatory signaling pathways in OB COT.

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  • Fig. 4 

    A hypothetical model for pathways linking maternal obesity to inflammation in placenta. Maternal obesity increases free fatty acids in fetal circulation, which activates TLR4 signaling and inflammatory signaling pathways, NF-κB and JNK pathways. As a result, the expression of inflammatory cytokines was increased, which further induces inflammation forming a vicious circle. TLR4: Toll-like receptor 4; NF-κB: Nuclear factor kappa B; JNK: Jun N-terminal kinase; AP-1: Activator protein 1.

Maternal obesity is known to increase the incidence of fetal overgrowth and adiposity, and is associated with the development of preeclampsia and associated fetal death [22], [36]. Inflammation is likely to be a key contributor to such adverse changes, as it is known to impair fetal brain and nervous system development [12]. Since the placenta is a major source of certain inflammatory cytokines, such as TNF-α, in the fetal circulation [28], placental inflammation may induce systemic fetal inflammation, thereby negatively affecting fetal development. Hence, maternal obesity may lead to fetal inflammatory responses via the mechanism of placental inflammation (Fig. 4). If left uncontrolled placental inflammation may lead to the impairment of overall placental function such as increased free fatty acid delivery in fetal circulation as observed in this study, which is expected to alter fetal growth and development. Indeed, in our previous study using the same group of sheep, a higher concentration of TNF-α was detected in OB fetal circulation when compared to Con sheep [47]. It is reported that proinflammatory cytokines IL-6 and TNF-α stimulate the activity of amino acid transporter system A, but not system L, in cultured human primary trophoblast cells [13]. Up-regulation of placental glucose transporter-1 and amino acid transporter constitute a mechanism linking maternal high-fat diet and obesity to fetal overgrowth [14]. In this study, we observed that cross-placenta fatty acid transportation is enhanced, and further studies to assess the consequence of these inflammatory processes and lipid changes on the placental function, especially fatty acid transportation, throughout gestation are warranted.

In conclusion, MO up-regulates inflammatory NF-κB and JNK signaling pathways and enhanced the expression of cytokines. The content of TLR4 was higher in OB compared to C placenta. As a receptor activated by fatty acids, the up-regulation of TLR4 may provide a potential link between MO and placental inflammation.

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Acknowledgements 

The authors would like to thank Dr. Myrna Miller and Mr. Ryan Gustafson for assistance with animal care and tissue collection. This project was supported by National Research Initiative Competitive Grant no. 2008-35203-19084 and 2009-65203-05716 from the USDA Cooperative State Research, Education and Extension Service and by University of Wyoming INBRE P20 RR016474.

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Appendix. Supplementary material 

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PII: S0143-4004(10)00055-X

doi:10.1016/j.placenta.2010.02.002

Placenta
Volume 31, Issue 5 , Pages 387-391, May 2010

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