Gestational restricted- and over-feeding promote maternal and offspring inflammatory responses that are distinct and dependent on diet in sheep

Gestational restricted- and over-feeding promote maternal and offspring inflammatory responses... Abstract Inflammation may be a mechanism of maternal programming because it has the capacity to alter the maternal environment and can persist postnatally in offspring tissues. This study evaluated the effects of restricted- and over-feeding on maternal and offspring inflammatory gene expression using reverse transcription (RT)-PCR arrays. Pregnant ewes were fed 60% (Restricted), 100% (Control), or 140% (Over) of National Research Council requirements beginning on day 30.2 ± 0.2 of gestation. Maternal (n = 8–9 ewes per diet) circulating nonesterified fatty acid (NEFA) and expression of 84 inflammatory genes were evaluated at five stages during gestation. Offspring (n = 6 per diet per age) inflammatory gene expression was evaluated in the circulation and liver at day 135 of gestation and birth. Throughout gestation, circulating NEFA increased in Restricted mothers but not Over. Expression of different proinflammatory mediators increased in Over and Restricted mothers, but was diet-dependent. Maternal diet altered offspring systemic and hepatic expression of genes involved in chemotaxis at late gestation and cytokine production at birth, but the offspring response was distinct from the maternal. In the perinatal offspring, maternal nutrient restriction increased hepatic chemokine (CC motif) ligand 16 and tumor necrosis factor expression. Alternately, maternal overnutrition increased offspring systemic expression of factors induced by hypoxia, whereas expression of factors regulating hepatocyte proliferation and differentiation were altered in the liver. Maternal nutrient restriction and overnutrition may differentially predispose offspring to liver dysfunction through an altered hepatic inflammatory microenvironment that contributes to immune and metabolic disturbances postnatally. Introduction Maternal programming is defined as changes to the maternal or intrauterine environment that alter fetal development and can permanently impair tissue function to predispose offspring to chronic diseases postnatally [1]. Maternal nutrition is one factor that can cause programming of offspring through altered macro- or micronutrient ingestion during gestation. Both maternal nutrient restriction and overnutrition result in offspring who exhibit poor postnatal growth, reduced muscle mass, increased adiposity, disrupted metabolism, and altered innate immunity [2–4]. Further, offspring born to nutrient restricted and overnourished mothers are at increased risk to develop chronic metabolic diseases during adulthood, including obesity, type 2 diabetes, atherosclerosis, and hepatic steatosis [1]. Chronic inflammation is common to the pathogenesis of these metabolic diseases [5], and thus may be a persistent consequence of poor maternal nutrition during gestation [6, 7]. Although the phenotype of offspring exposed to poor maternal nutrition during gestation is similar in response to maternal restriction and overnutrition, the mechanisms of each diet are distinct [4, 8]. Furthermore, the prevalence of over- and restricted nutrition is concerning in women of reproductive age (20–39 years). In the USA, 58.5% of women in this age group are overweight (body mass index [BMI] > 24) and 31.8% are obese (BMI > 29; [9]). Likewise, up to 28% of women aged 20–39 years are underweight (BMI 16–18.5) in developing nations [10]. Therefore, it is necessary to understand the effects of both maternal nutrient excess and restriction on offspring to improve outcomes of fetal programming worldwide. Inflammation has been proposed as a mechanism contributing to fetal programming because it has the capacity to alter the maternal environment and is known to persist postnatally in metabolically important tissues of offspring exposed to adverse intrauterine environments [11, 12]. In the maternal environment, low-grade, chronic inflammation is present in healthy pregnancies to facilitate maternal tolerance to the fetus, which exhibits paternal antigens [13, 14]. This involves timely coordination of the maternal endocrine and innate immune system throughout gestation and the onset of parturition [13, 14]. Failure of the maternal innate immune system to tolerate the fetus and elicit a chronic but controlled inflammatory response can result in pregnancy complications such as early embryonic loss, abortion, or preterm labor [13, 14]. This is usually provoked by a source of inflammation that is independent of pregnancy, including stress, infection, or diet [12]. For example, excessive intake and metabolism of macronutrients is associated with low-grade, chronic inflammation in nonpregnant women [5], and this inflammation is exaggerated in overweight and obese pregnant women [15]. When coupled with pregnancy, dietary-induced inflammation has the potential to negatively impact pregnancy success, maternal health, and fetal development. Diet-associated inflammation in the mother has the potential to impact the fetus through direct transfer of immune modulators across the placenta or by modulating transport of nutrients and oxygen to alter the intrauterine environment [12]. Epidemiological evidence demonstrates that offspring born to both over- or underweight women exhibit an increased inflammatory score as infants [16]. Additionally, animal studies have demonstrated that sheep offspring exposed to maternal nutrient restriction or overnutrition during gestation exhibit increased tissue-specific inflammation postnatally in muscle, cardiac, intestine, and liver tissues, indicating that inflammation caused by poor maternal nutrition may have persistent underlying effects [17–21]. The fetal liver may have an integral role in priming the postnatal innate immune and metabolic systems [22]. During gestation, the liver is a major hematopoietic organ responsible for differentiating resident and peripheral macrophages, and therefore the fetal hepatic microenvironment has the potential to prime cells of the innate immune system to favor a pro- or anti-inflammatory phenotype postnatally [22–24]. Postnatally, the liver is a vital endocrine and metabolic organ whose function is related to the immune cells and cytokines present in the hepatic tissue [25]. Thus, it is necessary to investigate how the liver of offspring may be affected by the maternal diet during gestation, as well as mechanisms by which inflammation may contribute to altered postnatal hepatic microenvironment and function. This study describes changes in maternal systemic gene expression of inflammatory mediators throughout gestation and in response to diet, and reports maternal nonesterified fatty acid (NEFA) concentrations as a potential link between gestational diet and maternal inflammation. Additionally, this study investigates offspring systemic inflammation in response to both maternal nutrient restriction and overnutrition, allowing comparisons between maternal and fetal inflammation, and between diets, in one study. Finally, we report offspring hepatic changes in inflammatory gene expression which may predispose offspring to postnatal metabolic and liver dysfunction. Materials and Methods Animals and experimental design All animal procedures were approved by the University of Connecticut's Institutional Animal Care and Use Committee. Multiparous western white-faced ewes were estrous synchronized, bred by live cover to one of four related Dorset rams, and confirmed pregnant at day 28.5 ± 0.4 of gestation using transabdominal ultrasound as previously described [26]. The ewes used for this study were a subset of a larger flock and collaborative experiment detailed in Pillai et al. [27]. All ewes were bred and maternal and offspring samples were collected in the same breeding season (e.g., all samples are from the same breeding cohort). Briefly, on individual housing at day 20 of gestation, ewes were transitioned onto a complete pelleted feed to meet 100% of the requirements of a pregnant ewe carrying twins as recommended by the National Research Council (NRC [28]). At day 30.2 ± 0.2 of gestation, pregnant ewes were randomly assigned to one of three experimental diets that met 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC total digestible nutrient requirements (TDN [28]). Offspring from ewes fed Control, Restricted, and Over diets are referred to as CON, RES, and OVER, respectively. Body weights of ewes were recorded weekly, allowing rations to be adjusted for individual changes in body weight. Six weeks before parturition, rations were adjusted based on NRC recommendations to meet the TDN requirement for a late gestation ewe expecting twins (28.5% increase of TDN), while maintaining feeding levels of 60%, 100% and 140% [28]. Daily requirements were provided in two rations per day and ewes remained on diet until parturition (gestation length 147.4 ± 1.9 days). Body condition scoring was also performed weekly on ewes by the same trained observers using a scale of 0–5 to evaluate maternal adiposity (0 = extremely emaciated and 5 = extremely obese [29]). This method of assessing adiposity is highly correlated (r = 0.94) with carcass fat content determined by chemical analysis, with a body condition score of 3 (28.7% adiposity) being ideal [29]. Maternal sample collection Ewe blood collection was performed in the fasted state, before morning feeding. Whole blood was collected via jugular venipuncture at days 23 ± 1.2, 45 ± 1.4, 90 ± 1.4, and 135 ± 1.6 of gestation for RNA and serum, at day 142 ± 3.3 of gestation for serum only, and within 24 h of parturition (birth) for RNA only (Figure 1). For RNA processing, 3 mL of whole blood were transferred into a Tempus Blood RNA Tube (Applied Biosystems, Foster City, CA) containing RNA stabilizer and shaken vigorously to precipitate RNA from whole blood. Tempus tubes were stored on ice until returned to the laboratory and then stored at –20°C until processed. For serum, 5 mL of whole blood was transferred into a nonheparinized tube and processed as previously described [30]. Figure 1. View largeDownload slide Experimental design and sample collection. Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation (gest) through parturition [28]. Whole blood was collected for RT-PCR arrays (black) or serum (gray) analyses from pregnant ewes and offspring at the time points indicated. Liver was collected from offspring at necropsy for RT-PCR array and ELISA assays at day 135 of gestation (n = 6 per diet; three females and three males in CON and RES, four females and two males in OVER) or within 24 h of birth (n = 6 per diet; three females and three males in CON and OVER, four females and two males in RES). All ewes were bred and maternal and offspring samples were collected in the same breeding season (e.g., all samples are from the same breeding cohort). Figure 1. View largeDownload slide Experimental design and sample collection. Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation (gest) through parturition [28]. Whole blood was collected for RT-PCR arrays (black) or serum (gray) analyses from pregnant ewes and offspring at the time points indicated. Liver was collected from offspring at necropsy for RT-PCR array and ELISA assays at day 135 of gestation (n = 6 per diet; three females and three males in CON and RES, four females and two males in OVER) or within 24 h of birth (n = 6 per diet; three females and three males in CON and OVER, four females and two males in RES). All ewes were bred and maternal and offspring samples were collected in the same breeding season (e.g., all samples are from the same breeding cohort). Offspring sample collection Following parturition, lambs (n = 6 lambs per diet [three females and three males in CON and OVER, four females and two males in RES]) from Control, Restricted, or Over ewes (n = 3–5 ewes per diet) nursed from their dam for up to 24 h to receive colostrum. Within 24 h of parturition, whole blood was obtained from live lambs via jugular venipuncture and processed for RNA or serum as described above (Figure 1). Lambs were subsequently euthanized with an i.v. overdose of Beuthanasia-D Special (390 ng/mL sodium pentobarbital and 50 mg/mL phenytoin based on body weight; Merck Animal Health, Summit, NJ) and exsanguinated. Liver tissue was collected, snap-frozen in liquid nitrogen, and stored at –80°C. Another group of ewes (n = 3–5 ewes per diet) from the same experiment [27] were euthanized at day 135 of gestation to acquire fetus(es) for sampling (Figure 1). Ewes were euthanized with an i.v. overdose of Beuthanasia-D Special (Merck Animal Health) and exsanguinated. Subsequently, a midline abdominal incision was performed on the ewe to remove the fetus(es). Following euthanasia, whole blood (3 mL) was collected via cardiac puncture for RNA, and liver was dissected from six fetuses per diet (three females and three males in CON and RES, four females, and two males in OVER). Samples were processed and stored as described for lambs. RNA isolation Isolation of RNA from whole blood was performed using the Perfect Pure RNA Blood kit according to the manufacturer's protocol (5 Prime, Inc., Gaithersburg, MD). The GeneJET Cleanup and Concentration Micro Kit (Thermo Scientific, Lafayette, CO) was used to concentrate the eluted RNA into 10 μL of RNase Free water. Hepatic RNA isolation was performed using 100 mg of tissue homogenized using a standard bead beating method in TRIzol Reagent (Invitrogen, Carlsbad, CA [31]). Quantity and quality of RNA was determined using a NanoDrop spectrophotometer (Thermo Scientific) and Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA), respectively. Real-time reverse transcription-PCR array Genomic DNA elimination was performed on each 500 ng sample of RNA and reverse transcribed using the RT2 First Strand Kit (SABiosciences). Quantitative real-time reverse transcription (RT)-PCR was performed using the Inflammatory Cytokines & Receptors RT2 Profiler PCR Array (Catalog no. PABT-011Z; SABiosciences, Germantown, MD). This array measures the expression of 84 genes mediating the inflammatory response, and has been previously validated in sheep [32]. One cDNA sample was analyzed per PCR array with RT2 SYBR Green Master Mix (SABiosciences). Cycling conditions were one cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min, performed using the ABI 7900 HT Fast Real-time PCR machine (Applied Biosystems). Cycle threshold (CT) values were obtained, and the ΔΔCT method was used to determine changes in gene expression [33]. Beta-actin (ACTB) was chosen as the housekeeping gene for all samples, as the CT values were not different between diet or stage of gestation in ewes (P > 0.21), and were not different between maternal diet or gender in offspring (P > 0.13). NEFA and ELISA assays NEFA concentrations were quantified from ewe and lamb serum using the acyl-CoA synthetase-acyl-CoA oxidase method (NEFA-HR, Wako Pure Chemical Industries, Dallas, TX; detection limit: 0.01–4.00 mEq/L). Lamb serum CXCL8 was quantified using a sheep-specific enzyme-linked immunosorbent assay (ELISA; Genorise Scientific, Inc., Glen Mills, PA; detection limit: 12–800 pg/mL). Hepatic protein was isolated by homogenizing 100 mg tissue using standard bead beating method and centrifugation to collect the supernatant. Total protein was quantified using the Quick Start Bradford Protein Assay (BioRad, Hercules, CA) to normalize subsequent assays. Offspring hepatic c-reactive protein (CRP) was determined with a sheep-specific ELISA using undiluted samples per the manufacturer's protocol (MyBioSource, San Diego, CA; detection range 0.25–8 μg/mL). Hepatic bone morphogenetic protein 2 (BMP2), hepatocyte growth factor (HGF), transforming growth factor beta 1 (TGFB1), and tumor necrosis factor (TNF) were quantified from offspring birth samples diluted 1:10 using sheep-specific ELISAs (Genorise Scientific, Inc.; detection limits: 62–4000 pg BMP2/mL; 94–6000 pg HGF/mL; 31–2000 pg TGFbeta/mL; 35–2400 pg TNF/mL). The assay sensitivities were 8 pg BMP2/mL, 0.9 pg CXCL8/mL, 0.1 μg CRP/mL, 18 pg HGF/mL, 5 pg TGFB/mL, and 7 pg TNF/mL. The concentration of each factor was measured within a single assay, and the intra-assay coefficient of variation from duplicate samples ranged from 6.13% to 23.2%. Data analyses The power calculations performed for this study were based on anticipated differences in maternal and offspring body weight in response to dietary treatment, with 90% power to detect significance at 5%. We anticipated that a 15% difference in maternal BW (16.5 kg) would be achieved at parturition in response to dietary treatment during gestation, with an animal variability of 8 kg, indicating that seven ewes per diet were needed for maternal measurements. Six offspring per diet were required based on an anticipated 20% difference in body weight (996 g) and animal variability of 480 g. Offspring gender could not be controlled for, and therefore the study did not have the power to investigate effects of gender within a diet. All data analyses were performed using the MIXED procedure of Statistical Analysis System version 9.4 (SAS Institute, Cary, NC). Ewe data were analyzed as a completely randomized design. Percent change in body weight from day 30 of gestation, percent change in BCS from day 30 of gestation, and NEFA concentrations were analyzed with repeated measures. The model included fixed effects of diet, day of gestation, and their interaction, with the subject defined as ewe. Covariate structures were chosen based on the lowest Akaike Iteration Criterion value for each dependent variable [34]. Compound symmetry was chosen for percent change BCS, autoregressive was chosen for percent change in body weight, and unstructured was chosen for NEFA concentration. Data are presented as least squares mean (lsmean) ± SE. Due to insufficient RNA quantity from ewe blood (<500 ng), PCR arrays could not be run for all days of gestation within each ewe. Thus, ewe array data were analyzed as a cross-sectional study with fixed effects of diet, day of gestation, and their interaction. Heat maps of ewe inflammatory gene expression were generated in the gplots package in R version 3.3.1. Ewe gene expression data are expressed relative to Control at day 23 in the presence of an interaction, relative to Control in the presence of a main effect of diet, or relative to day 23 in the presence of a main effect of gestation. Offspring data were analyzed as a completely randomized design. Offspring body weight and CRP were analyzed with the fixed effects of maternal diet, gender, and stage of gestation, and interaction of maternal diet or gender by stage of gestation. For the blood and liver PCR arrays, the housekeeping gene ACTB cycled differently at 135 days of gestation and birth (P < 0.0001). Thus, offspring array data from each time point were analyzed separately, with fixed effects of maternal diet and gender only. Offspring gene data are expressed relative to CON or females. Lamb NEFA, and TNF, TGFB1, BMP2, and HGF ELISA data were analyzed at birth only, and thus the model included fixed effects of maternal diet and gender only. Data are expressed as lsmean ± SE. Significance is discussed when P ≤ 0.05. Results Maternal body weight, BCS, and circulating NEFA concentration There was a significant interaction of diet by day of gestation (Figure 2) on ewe body weight (P < 0.0001), BCS (P = 0.001), and circulating NEFA concentrations (P < 0.0001). Upon the start of the dietary treatment at day 30 of gestation, the body weight of pregnant ewes did not differ between diet groups (78.2 ± 2.4, 81.2 ± 2.2, 75.7 ± 2.4 kg for Control, Restricted, and Over, respectively; P ≥ 0.09). Between days 79 and 135 of gestation, the percent change in body weight from day 30 of gestation was different between Control, Restricted, and Over ewes (P ≤ 0.05; Figure 2A). That is, the body weight of Control and Over ewes increased by 19.4% and 22.7%, respectively, between day 79 and 135, whereas the body weight of Restricted ewes was reduced by 3.5% at day 79, and increased by 5.8% at day 135 of gestation (Figure 2A). Five days before parturition (day 142), the percent change in body weight from day 30 was greater in Over (31.4 ± 5.3%) and Control (26.6 ± 2.1%) ewes than in Restricted ewes (5.9 ± 2.4%; P < 0.0001; Figure 2A). Similar to ewe body weight, ewe BCS did not differ between diets at day 30 of gestation (3.07 ± 0.04, 3.00 ± 0.04, 2.99 ± 0.04 for Control, Restricted, and Over, respectively; P ≥ 0.85). However, by day 142 of gestation, the percent change in BCS from day 30 was greater in Over (5.7 ± 1.4%) and less in Restricted (–5.7 ± 0.83%) ewes, compared with Control (0.6 ± 2.6%; P ≤ 0.02; Figure 2B). Circulating NEFA concentrations were not different in ewes before beginning of experimental diets (P ≥ 0.71; Figure 2C). Throughout dietary treatment, Restricted ewes had greater (P < 0.0001) circulating NEFA concentrations compared with Control and Over, which did not differ (P ≤ 0.55; Figure 2C). Figure 2. View largeDownload slide Change in maternal body weight (A), body condition score (B), and circulating NEFA concentration (C). Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation (gest) through parturition [28]. Data are presented as lsmean ± SE. Within a day of gestation, pairwise comparisons between diets are denoted when P ≤ 0.05 by ‡ (Restricted vs. Over, with Control intermediate), † (Restricted vs. Control and Over), or * (Restricted vs. Control vs. Over). Figure 2. View largeDownload slide Change in maternal body weight (A), body condition score (B), and circulating NEFA concentration (C). Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation (gest) through parturition [28]. Data are presented as lsmean ± SE. Within a day of gestation, pairwise comparisons between diets are denoted when P ≤ 0.05 by ‡ (Restricted vs. Over, with Control intermediate), † (Restricted vs. Control and Over), or * (Restricted vs. Control vs. Over). Maternal systemic inflammatory gene expression Analysis of inflammatory related gene expression in pregnant ewes indicated that there was an interaction of diet by day of gestation on the expression of interleukin (IL) 6 receptor (IL6R) and platelet factor 4 (PF4) in the circulation of pregnant ewes (P ≤ 0.04; Figure 3; Supplementary Table S1). Compared with day 23 of gestation, the expression of IL6R did not change as gestation advanced in Control ewes (P > 0.05). Expression of IL6R increased at day 135 of gestation in Restricted compared with Control and Over ewes and within 24 h of birth in Over compared with Restricted ewes (Figure 3A; P ≤ 0.05). As gestation advanced, expression of PF4 did not change in Control ewes, compared with day 23. (Figure 3B: P ≤ 0.05). At day 45 of gestation, PF4 gene expression in Over ewes was greater than Control, whereas Restricted was greater than Control at day 90 of gestation (P ≤ 0.05). Figure 3. View large Download slide Maternal systemic inflammatory gene expression is altered by gestational diet and stage of gestation (gest). Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC TDN requirements from day 30.2 ± 0.2 of gestation through parturition [28]. An interaction of diet by day of gestation was observed for the mRNA expression of Interleukin 6 receptor and Platelet factor 4. Within a day of gestation, pairwise comparisons between diets are denoted when P ≤ 0.05 by † (Restricted vs. Control and Over), ‡ (Restricted vs. Over, with Control intermediate), § (Over vs. Control and Restricted), or # (Restricted vs. Control, with Over intermediate). Relative expression values are available in Supplementary Table S1. Figure 3. View large Download slide Maternal systemic inflammatory gene expression is altered by gestational diet and stage of gestation (gest). Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC TDN requirements from day 30.2 ± 0.2 of gestation through parturition [28]. An interaction of diet by day of gestation was observed for the mRNA expression of Interleukin 6 receptor and Platelet factor 4. Within a day of gestation, pairwise comparisons between diets are denoted when P ≤ 0.05 by † (Restricted vs. Control and Over), ‡ (Restricted vs. Over, with Control intermediate), § (Over vs. Control and Restricted), or # (Restricted vs. Control, with Over intermediate). Relative expression values are available in Supplementary Table S1. Thirteen genes exhibited a main effect of diet and are reported as a heat map in Figure 4 and as relative expression in Supplementary Table S2. Regardless of the stage of gestation, the expression of C-X-C motif chemokine ligand 9 (CXCL9), IL4, IL7, lymphotoxin beta (LTB), TNF super family member 10 (TNFSF10), and IL10 receptor subunit alpha (IL10RA) increased in Over ewes compared with Control (P ≤ 0.05). In Restricted ewes, expression of BMP2, C-X-C motif chemokine receptor 1 (CXCR1), IL1B, and TNFSF13B increased compared with Control (P ≤ 0.05). Expression of C-C motif chemokine ligand 4 (CCL4), lymphotoxin alpha (LTA), and TNFSF11 was greater in Over ewes than Restricted (P = 0.001), but neither differed from Control (P ≥ 0.09). Figure 4. View large Download slide Maternal systemic inflammatory gene expression is altered by gestational diet. Pregnant ewes were assigned to 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirement from day 30.2 ± 0.2 of gestation through parturition [28]. A main effect of diet (P ≤ 0.05) was observed for the expression of 13 inflammatory genes. Within a gene, red indicates the least expression and blue indicates the greatest expression. Relative expression values are available in Supplementary Table S2. Abbreviations: Bone morphogenetic protein 2 (BMP2); C-C motif chemokine ligand 4 (CCL4); C-X-C motif chemokine ligand 9(CXCL9); C-X-C motif chemokine receptor 1 (CXCR1); Interleukin 1 beta (IL1B), Interleukin (IL)-4, 7; Interleukin 10 receptor subunit alpha (IL10RA); lymphotoxin alpha (LTA); lymphotoxin beta (LTB); TNF super factor family member (TNFSF)-10, 11, 13B. Figure 4. View large Download slide Maternal systemic inflammatory gene expression is altered by gestational diet. Pregnant ewes were assigned to 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirement from day 30.2 ± 0.2 of gestation through parturition [28]. A main effect of diet (P ≤ 0.05) was observed for the expression of 13 inflammatory genes. Within a gene, red indicates the least expression and blue indicates the greatest expression. Relative expression values are available in Supplementary Table S2. Abbreviations: Bone morphogenetic protein 2 (BMP2); C-C motif chemokine ligand 4 (CCL4); C-X-C motif chemokine ligand 9(CXCL9); C-X-C motif chemokine receptor 1 (CXCR1); Interleukin 1 beta (IL1B), Interleukin (IL)-4, 7; Interleukin 10 receptor subunit alpha (IL10RA); lymphotoxin alpha (LTA); lymphotoxin beta (LTB); TNF super factor family member (TNFSF)-10, 11, 13B. Forty-one genes exhibited a main effect of day of gestation, reported as a heat map in Figure 5 and as relative expression in Supplementary Table S3. Genes with a main effect of gestation exhibited different expression patterns throughout gestation. Compared with day 23 of gestation, expression of CCL2 and IL2RB increased at day 45, 90, and 135 of gestation (P ≤ 0.04). Expression of secreted phosphoprotein 1 (SPP1) peaked at day 45 of gestation (P ≤ 0.04), whereas IL17B was greatest at day 135 of gestation (P ≤ 0.05). Expression of C-C motif chemokine receptor 6 (CCR6) increased at day 45 and 90 of gestation (P ≤ 0.03), whereas colony stimulating factor 3 (CSF3) increased at day 90 and 135 of gestation (P ≤ 0.05). Compared with day 23 of gestation, CXCR1 expression decreased at day 45 and increased at day 135 of gestation and birth (P ≤ 0.05). At day 90 of gestation and after parturition, IL5 expression was reduced (P ≤ 0.02). Between day 23, 45, and 90 of gestation, expression of CCL4, CXCL9, C-X3-C motif chemokine receptor 1 (CX3CR1), CD40 ligand (CD40LG), Fas ligand (FASLG), and TNFSF13B did not differ, but all were reduced at day 135 and 24 h after parturition (P ≤ 0.05). Expression of aminoacyl tRNA synthase complex interacting multifunctional protein 1 (AIMP1), CCL3, CCL5, CCL22, macrophage migration inhibitory factor (MIF), CCR1, CCR3, CCR5, CCR8, CXCR3, CSF1, interferon gamma (IFNG), IL7, IL13, IL16, IL6 signal transducer (IL6ST), IL9R, LTA, LTB, TNF and TNFSF11 did not change during gestation (P > 0.06), but exhibited reduced expression 24 h after parturition (P ≤ 0.05). Expression of CXCL8 decreased only at day 45 of gestation (P ≤ 0.03), whereas CXCL10, IL4, IL15, nicotinamide phosphoribosyltransferase (NAMPT), and TNFSF10 continued to decline from day 45 of gestation through parturition (P ≤ 0.05). Figure 5. View large Download slide Maternal systemic inflammatory gene expression is altered by stage of gestation. Pregnant ewes were assigned to 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation through parturition [28]. A main effect of gestation (P ≤ 0.05) was observed for the expression of 41 inflammatory genes. Within a gene, red indicates the least expression and blue indicates the greatest expression. Relative expression values are available in Supplementary Table S3. Abbreviations: Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 (AIMP1); CD40 ligand (CD40LG); C-C motif chemokine ligand (CCL)-2, 3, 4, 5, 22; C-X-C motif chemokine ligand (CXCL)-8, 9, 10; C-C motif chemokine receptor (CCR)-1, 3, 5, 6, 8; C-X-C motif chemokine receptor (CXCR)- 1, 3; C-X3-C motif chemokine receptor 1 (CX3CR1); Colony stimulating factor (CSF)-1, 3; Fas ligand (FASLG); Interferon gamma (IFNG); Interleukin (IL)-4, 5, 7, 13, 15, 16, 17B; Interleukin receptors (IL2RB; IL6R; IL9R); Interleukin 6 signaling transducer (IL6ST); Lymphotoxin alpha (LTA); Lymphotoxin beta (LTB); Macrophage migration inhibitory factor (MIF); Nicotinamide phosphoribosyltransferase (NAMPT); Secreted phosphoprotein 1 (SPP1); Tumor necrosis factor (TNF); TNF factor super family member (TNFSF)-10, 11, 13B. Figure 5. View large Download slide Maternal systemic inflammatory gene expression is altered by stage of gestation. Pregnant ewes were assigned to 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation through parturition [28]. A main effect of gestation (P ≤ 0.05) was observed for the expression of 41 inflammatory genes. Within a gene, red indicates the least expression and blue indicates the greatest expression. Relative expression values are available in Supplementary Table S3. Abbreviations: Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 (AIMP1); CD40 ligand (CD40LG); C-C motif chemokine ligand (CCL)-2, 3, 4, 5, 22; C-X-C motif chemokine ligand (CXCL)-8, 9, 10; C-C motif chemokine receptor (CCR)-1, 3, 5, 6, 8; C-X-C motif chemokine receptor (CXCR)- 1, 3; C-X3-C motif chemokine receptor 1 (CX3CR1); Colony stimulating factor (CSF)-1, 3; Fas ligand (FASLG); Interferon gamma (IFNG); Interleukin (IL)-4, 5, 7, 13, 15, 16, 17B; Interleukin receptors (IL2RB; IL6R; IL9R); Interleukin 6 signaling transducer (IL6ST); Lymphotoxin alpha (LTA); Lymphotoxin beta (LTB); Macrophage migration inhibitory factor (MIF); Nicotinamide phosphoribosyltransferase (NAMPT); Secreted phosphoprotein 1 (SPP1); Tumor necrosis factor (TNF); TNF factor super family member (TNFSF)-10, 11, 13B. Offspring body weight and NEFA concentrations A main effect of maternal diet was observed on offspring body weight in that RES offspring weighed 15.4% and 7.7% less than CON and OVER, respectively (CON: 5007.8 ± 187.6 g, RES: 4236.5 ± 188.5 g, OVER: 4591.8 ± 188.6 g; P = 0.02). Offspring body weight did not differ with stage of gestation (P = 0.611) or gender (P = 0.09). No differences were observed in the lamb serum NEFA concentration between maternal diets (CON: 872.3 ± 109.7 μmol/L, RES: 803.9 ± 112.0 μmol/L, OVER: 1138.3 ± 134.3 μmol/L; P = 0.18) or males and females (924.2 ± 91.6 and 952.1 ± 102.4, μmol/L, respectively; P = 0.84). Offspring circulation In offspring circulation at day 135 of gestation, there were main effects of maternal diet for five genes (P ≤ 0.05; Figure 6A) and gender for six genes (P ≤ 0.05; Table 1). Systemic CCL22 expression was reduced in RES compared with CON (P ≤ 0.05). Systemic expression of CXCL12, CXCR1, and IL1A were reduced in OVER and RES compared with CON (P ≤ 0.05). Systemic expression of MIF was reduced in OVER compared with CON (P ≤ 0.05). At day 135 of gestation, systemic CCL22, CXCL1, CXCL9, CXCL12, IL4, and IL15 expression was reduced in male fetuses compared with females, regardless of maternal diet (P ≤ 0.05; Table 1). Figure 6. View largeDownload slide Main effect of maternal diet (P ≤ 0.05) on the systemic mRNA expression of offspring euthanized at day 135 of gestation (A) or within 24 h of birth (B), and CXCL8 protein concentration at birth (C). Offspring (n = 6 per diet at each stage) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. At each stage of gestation, there were 10 female and 8 male offspring. Within a stage of gestation, mRNA expression is expressed relative to CON. Within a gene, treatments with different letters differ (P ≤ 0.05). Abbreviations: C-C motif chemokine ligand 22 (CCL22); C-X-C motif chemokine ligand (CXCL)-8, 12; C-X-C motif chemokine receptor 1 (CXCR1); interleukin 1 alpha (IL1A); lymphotoxin beta (LTB); macrophage migration inhibitory factor (MIF); platelet factor 4 (PF4); TNF super family member 13 (TNFSF13); vascular endothelial growth factor A (VEGFA). Figure 6. View largeDownload slide Main effect of maternal diet (P ≤ 0.05) on the systemic mRNA expression of offspring euthanized at day 135 of gestation (A) or within 24 h of birth (B), and CXCL8 protein concentration at birth (C). Offspring (n = 6 per diet at each stage) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. At each stage of gestation, there were 10 female and 8 male offspring. Within a stage of gestation, mRNA expression is expressed relative to CON. Within a gene, treatments with different letters differ (P ≤ 0.05). Abbreviations: C-C motif chemokine ligand 22 (CCL22); C-X-C motif chemokine ligand (CXCL)-8, 12; C-X-C motif chemokine receptor 1 (CXCR1); interleukin 1 alpha (IL1A); lymphotoxin beta (LTB); macrophage migration inhibitory factor (MIF); platelet factor 4 (PF4); TNF super family member 13 (TNFSF13); vascular endothelial growth factor A (VEGFA). Table 1. Effect of gender on gene expression of offspring systemic inflammatory mediators.   Gender    Gene1  Female (n = 10)  Male (n = 8)  SEM  P-value  D 135  CCL22  1.52  0.53  0.30  0.03  CXCL1  1.37  0.40  0.20  0.24  CXCL9  2.07  0.43  0.56  0.02  CXCL12  2.22  0.59  0.68  0.45  IL4  1.10  0.49  0.14  0.002  Birth  CXCR1  1.11  0.63  0.11  0.02  TNFSF13  1.23  2.55  0.36  0.002    Gender    Gene1  Female (n = 10)  Male (n = 8)  SEM  P-value  D 135  CCL22  1.52  0.53  0.30  0.03  CXCL1  1.37  0.40  0.20  0.24  CXCL9  2.07  0.43  0.56  0.02  CXCL12  2.22  0.59  0.68  0.45  IL4  1.10  0.49  0.14  0.002  Birth  CXCR1  1.11  0.63  0.11  0.02  TNFSF13  1.23  2.55  0.36  0.002  1 Expression relative to females. Abbreviations: C-C- motif chemokine ligand 22 (CCL22); C-X-C motif chemokine ligand (CXCL)-1, 9, 12; C-X-C motif chemokine receptor 1 (CXCR1); Interleukin 4 (IL4), 15; TNF super family member (TNFSF)-13. View Large In offspring circulation at birth, there were main effects of maternal diet for five genes (P ≤ 0.05; Figure 6B) and gender for two genes (P ≤ 0.05; Table 1). Systemic CXCL8 increased in RES and OVER compared with CON (P ≤ 0.05). The systemic protein concentration of CXCL8 at birth was 47% and 57% greater in OVER compared with CON and RES, respectively (P ≤ 0.02; Figure 6C), with no effect of gender (P = 0.82). Systemic LTB was reduced in RES compared with CON and OVER (P ≤ 0.05). Systemic PF4, TNFSF13, and VEGFA were increased in OVER compared with CON and RES (P ≤ 0.05). Compared with females, systemic CXCR1 expression was reduced, whereas TNFSF13 expression increased in male lambs at birth (P ≤ 0.05; Table 1). Offspring liver In offspring liver at day 135 of gestation, there were main effects of maternal diet for three genes (P ≤ 0.05; Figure 7A) and gender for seven genes (P ≤ 0.05; Table 2). Hepatic CCL16 expression increased in RES compared with CON (P ≤ 0.05). Hepatic LTB was reduced in OVER compared with CON and RES (P ≤ 0.05). Hepatic TNFSF11 was increased in OVER compared with RES, with CON intermediate (P ≤ 0.05; Table 2). Compared with females, hepatic expression of Complement C5 (C5), CCL3, CCR1, CCR6, and IL2RG was reduced in male fetuses (P ≤ 0.05). Oppositely, hepatic TNFSF14 was increased in male fetuses compared with females (P ≤ 0.05; Table 2). Figure 7. View largeDownload slide Main effect of maternal diet (P ≤ 0.05) on the hepatic mRNA expression of offspring euthanized at day 135 of gestation (A) or within 24 h of birth (B). Offspring (n = 6 per diet at each stage) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. At each stage of gestation there were 10 female and 8 male offspring. Within a stage of gestation, mRNA expression is expressed relative to CON. Within a gene, treatments with different letters differ (P ≤ 0.05). Abbreviations: bone morphogenetic protein 2 (BMP2); C-C motif chemokine ligand 16 (CCL16); C-X-C motif chemokine ligand (CXCL)-10, 12; lymphotoxin beta (LTB); tumor necrosis factor (TNF); TNF super family member 11 (TNFSF11). Figure 7. View largeDownload slide Main effect of maternal diet (P ≤ 0.05) on the hepatic mRNA expression of offspring euthanized at day 135 of gestation (A) or within 24 h of birth (B). Offspring (n = 6 per diet at each stage) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. At each stage of gestation there were 10 female and 8 male offspring. Within a stage of gestation, mRNA expression is expressed relative to CON. Within a gene, treatments with different letters differ (P ≤ 0.05). Abbreviations: bone morphogenetic protein 2 (BMP2); C-C motif chemokine ligand 16 (CCL16); C-X-C motif chemokine ligand (CXCL)-10, 12; lymphotoxin beta (LTB); tumor necrosis factor (TNF); TNF super family member 11 (TNFSF11). Table 2. Effect of gender on hepatic gene expression of inflammatory mediators in offspring.   Gender      Gene1  Female (n = 10)  Male (n = 8)  SEM  P-value  D 135  C5  1.84  0.42  0.35  0.03  CCL3  1.12  0.50  0.18  0.01  CCR1  3.04  0.27  0.72  0.01  CCR6  6.36  0.19  1.54  0.04  IL2RG  1.04  0.68  0.12  0.03  TNFSF14  1.23  2.94  0.43    Birth  CCL1  1.52  6.80  0.96  0.0005  CXCL9  1.35  0.33  0.22  0.004  CXCL10  1.50  0.46  0.26  0.04  IL1R1  1.43  0.48  0.27  0.03  IL5  1.12  0.61  0.16  0.02  IL7  1.14  0.52  0.14  0.01  IL15  1.08  0.61  0.11  0.01  MIF  1.11  3.59  0.70  0.05  TNFSF10  1.05  0.36  0.24  0.01    Gender      Gene1  Female (n = 10)  Male (n = 8)  SEM  P-value  D 135  C5  1.84  0.42  0.35  0.03  CCL3  1.12  0.50  0.18  0.01  CCR1  3.04  0.27  0.72  0.01  CCR6  6.36  0.19  1.54  0.04  IL2RG  1.04  0.68  0.12  0.03  TNFSF14  1.23  2.94  0.43    Birth  CCL1  1.52  6.80  0.96  0.0005  CXCL9  1.35  0.33  0.22  0.004  CXCL10  1.50  0.46  0.26  0.04  IL1R1  1.43  0.48  0.27  0.03  IL5  1.12  0.61  0.16  0.02  IL7  1.14  0.52  0.14  0.01  IL15  1.08  0.61  0.11  0.01  MIF  1.11  3.59  0.70  0.05  TNFSF10  1.05  0.36  0.24  0.01  1Expression relative to females. Abbreviations: Complement C5 (C5); C-C motif chemokine ligand (CCL)-1, 3; C-C motif chemokine receptor (CCR)-1, 6; C-X-C motif chemokine ligand (CXCL)-9, 10; Interleukin (IL)-2RG, 5, 7, 15; Interleukin 1 Receptor 1 (IL1R1); Macrophage migration inhibitory factor (MIF); TNF super family member 14 (TNFSF14). View Large In offspring liver at birth, there were main effects of maternal diet for four genes (P ≤ 0.05; Figure 7B) and gender for eight genes (P ≤ 0.05; Table 2). Hepatic BMP2 and CXCL12 expression was reduced in OVER compared with CON and RES, whereas CXCL10 was reduced in OVER compared with RES with CON intermediate (P ≤ 0.05). Hepatic TNF expression increased in RES compared with CON and OVER (P ≤ 0.05). Compared with females, hepatic expression of CCL1, CXCL9, CXCL10, IL1R1, IL5, IL7, IL15, and TNFSF10 was reduced in males, but MIF expression was greater (P ≤ 0.05; Table 2). To confirm the PCR array results and further understand the effects of maternal nutrient restriction and overnutrition during gestation on offspring liver development, the protein concentrations of TNF, TGFB1, BMP2, and HGF were evaluated in offspring liver at birth (Figure 8). Maternal diet did not alter the protein concentration of TNF (P = 0.32; Figure 8A) or TGFB1 (P = 0.07; Figure 8B). Consistent with mRNA expression, the protein concentration of BMP2 was reduced by 20% in OVER compared with RES (P = 0.004; Figure 8C), whereas the protein concentration of HGF was increased by 18.5% in RES and 16.1% in OVER compared with CON (P ≤ 0.02; Figure 8D). There was no effect of gender on the hepatic protein concentration of TNF, TGFB1, BMP2, or HGF at birth (P ≥ 0.09). Figure 8. View largeDownload slide Main effect of maternal diet on the hepatic protein concentration of tumor necrosis factor (TNF; P = 0.32), transforming growth factor beta 1 (TGFB1; P = 0.07), bone morphogenetic protein 2 (BMP2; P = 0.01), and hepatocyte growth factor (HGF; P = 0.02) in offspring at birth. Offspring (n = 6 per diet) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. There were 10 females and 8 males at birth. Within a graph, treatments with different letters differ (P < 0.05). Figure 8. View largeDownload slide Main effect of maternal diet on the hepatic protein concentration of tumor necrosis factor (TNF; P = 0.32), transforming growth factor beta 1 (TGFB1; P = 0.07), bone morphogenetic protein 2 (BMP2; P = 0.01), and hepatocyte growth factor (HGF; P = 0.02) in offspring at birth. Offspring (n = 6 per diet) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. There were 10 females and 8 males at birth. Within a graph, treatments with different letters differ (P < 0.05). No interactions (P ≥ 0.07) or main effects were observed in the concentration of CRP in offspring liver between day 135 of gestation and birth (5.02 ± 0.81 and 5.67 ± 0.88 μg, respectively; P = 0.59), males and females (4.41 ± 0.91 and 6.28 ± 0.77 μg, respectively; P = 0.13), or maternal diets (CON: 4.65 ± 1.03 μg, RES: 6.18 ± 1.03 μg, OVER: 5.20 ± 1.06 μg; P = 0.57). Discussion This study evaluated inflammatory gene expression in both the mother and fetus to begin to understand inflammation as a potential fetal programming mechanism. Our findings indicate that maternal inflammatory gene expression is altered by poor nutrition during gestation, but the expression of inflammatory mediators differs during restricted- or overnutrition. Further, our data indicate that the offspring inflammatory response to maternal nutrient restriction or overnutrition is distinct from the inflammatory response of the mother, indicating that maternal inflammation may program fetal development through indirect mechanisms. Finally, we report alterations in proinflammatory cytokine, chemokine, and growth factor expression in the liver of offspring, which may be responsible for predisposition to hepatic dysfunction later in life. In addition to evaluating the maternal and fetal inflammatory response to gestational diet, this study also reports changes to maternal inflammation at multiple stages of gestation, in the absence of dietary effects. Previous literature evaluating inflammation throughout gestation has only evaluated one stage of gestation, or performed longitudinal analyses of few inflammatory mediators [15, 35, 36]. Our study is unique in that inflammatory changes are reported at multiple stages of gestation in the same cohort of females, with the mRNA abundance of 84 inflammatory genes evaluated, providing a more comprehensive analysis. These data have implications in understanding how the maternal innate immune system responds during gestation. Importantly, this information is reported in sheep, a common biomedical model for human pregnancy due to the similar fetal to maternal biomass ratio, delivery of precocial offspring, and continuity of fetal programming consequences [37]. In women, healthy pregnancies are associated with a chronic, low-grade inflammatory response which is pertinent to pregnancy recognition, maintenance, and parturition [13, 14]. It has been proposed that CCL2, CCL3, CCL4, and CCL5 are relevant chemokines during pregnancy due to their role in mediating leukocyte chemotaxis between the systemic and local intrauterine environment [38]. This is supported by our data in sheep, in which CCL2 increased 3- to 4-fold between day 23 of gestation and parturition. Further, the expression of CCL3, CCL4, and CCL5 was maintained during early and midgestation and decreased during late-gestation or postpartum, indicating a greater role in pregnancy maintenance than parturition. In our study, the mRNA expression of TNF and CXCL8 was greatest after parturition, consistent with longitudinal reports in women in which the serum concentrations of TNF and CXCL8 were greatest postpartum [15, 35]. Our data also demonstrate that the maternal immune response involves the expression of multiple cytokine, chemokine, and TNFSF ligands that previously may have not been considered during gestation. Although additional research and protein expression is necessary to investigate the roles and relationships of each inflammatory mediator reported, our data support the integral role of the innate immune system during pregnancy in sheep which is consistent with reports in women. While chronic, low-grade inflammation is associated with healthy pregnancies [13, 14], improper activation of the innate immune system due to maternal stressors may have negative consequences on the success of the pregnancy and development of the fetus during gestation [13, 14]. In our study, proinflammatory gene expression was observed in both restricted and overnourished mothers, yet there was no evidence of disrupted pregnancy success as no early fetal losses occurred after dietary treatment began [26], and the gestation length of ewes was not different based on diet [27]. However, the gene expression of proinflammatory mediators may indirectly affect fetal development by modulating fetal nutrient availability through altered placental transport [12]. Although our study did not investigate the placental tissues, we did observe increased systemic PF4 during early- and midgestation of both Restricted and Over pregnancies. In sheep, placental development is rapid during early to midgestation, and complete by day 90 [39]. Increased PF4 may have a role in altered angiogenesis and vascularization during this timeframe, potentially having negative consequences on the formation and transport capacity of blood to and across the placenta to affect fetal development [40, 41]. Previous models of maternal overnutrition have associated reduced uterine and umbilical blood flow in ewes with reduced fetal nutrient delivery [40–42]. Additionally, in ewes who were nutrient restricted during the first 40% of gestation, placental vasoconstriction contributed to fetal intrauterine growth restriction [42]. In our model, OVER lambs at birth exhibited increased systemic gene expression of PF4 and VEGFA, which promote cell survival and adaptation to hypoxic conditions. Thus, future research may investigate the role of PF4 on placental structure and transport, as well as the presence of a hypoxic intrauterine environment, during overnutrition. Nutrient restriction and overnutrition resulted in differing maternal metabolic and inflammatory phenotypes, indicating that inflammation affects maternal health and fetal development differently based on nutrient status (Figure 9). Nutrient restricted ewes demonstrated depletion of nutrient reserves by the gradual loss of body condition [29] and increased circulating NEFA concentrations as gestation advanced. Additionally, RES offspring weighed less at both day 135 of gestation and birth. This maternal–fetal phenotype is consistent with previous pregnant sheep models of limited maternal nutrient intake that are associated with slowed fetal growth in late gestation [43–45]. Mobilization of NEFAs may have contributed to inflammation in Restricted mothers because sustained increases of NEFA are known to activate the innate immune system through toll-like receptors (TLR [46]), a network of transmembrane receptors that typically respond to pathogen or danger-associated molecular patterns. Specifically, stimulation of TLR4 by NEFA causes a proinflammatory signaling cascade through the activation of master inflammatory axes, nuclear factor kappa B (NFκB), and mitogen-activated kinase [46, 47]. In our model, Restricted ewes exhibited a proinflammatory response during gestation via increased mRNA expression of cytokines IL1B and TNFSF13B, and decreased mRNA expression of anti-inflammatory cytokine IL4 and chemokine CCL4. The cytokines IL1B and TNFSF13B activate both canonical and noncanonical inflammatory signaling pathways, and act in a positive feedback loop to mediate a chronic inflammatory environment and depress anti-inflammatory mediators [48]. Over ewes did not exhibit increased circulating NEFA concentrations. However, the BCS of Over ewes increased as gestation advanced, indicating increased accumulation of maternal adipose tissue [29]. Therefore, the maternal inflammation observed in response to overnutrition was not related to increased circulating NEFA. Rather, increased adiposity may have contributed to inflammation in Over ewes. During prolonged overnutrition, TNF production antagonizes peroxisome proliferator-activated receptor gamma, inhibiting adipocyte differentiation and resulting in adipocyte hypertrophy instead of hyperplasia [49]. Consequently, engorged adipocytes promote local and systemic inflammation through local hypoxia and necrosis, resulting in macrophage infiltration and the production of adipokines that travel peripherally to induce systemic inflammation [49]. The systemic mRNA expression of TNFSF ligands and lymphotoxins in Over ewes supports an environment that would promote adipocyte hypertrophy and hypoxia and contribute to systemic inflammation [49]. Figure 9. View largeDownload slide Maternal inflammation is promoted by nutrient restriction and may be linked to increased nonesterified fatty acid (NEFA) concentrations, whereas inflammation related to overnutrition does not appear to be linked to NEFA concentrations in our study. The offspring inflammatory response to maternal nutrient restriction and overnutrition is distinct from the maternal response. Inflammation may predispose offspring to liver dysfunction via increased tumor necrosis factor (TNF) in response to maternal nutrient restriction or increased hepatocyte growth factor (HGF) and reduced bone morphogenetic protein 2 (BMP2) in response to maternal overnutrition. Figure 9. View largeDownload slide Maternal inflammation is promoted by nutrient restriction and may be linked to increased nonesterified fatty acid (NEFA) concentrations, whereas inflammation related to overnutrition does not appear to be linked to NEFA concentrations in our study. The offspring inflammatory response to maternal nutrient restriction and overnutrition is distinct from the maternal response. Inflammation may predispose offspring to liver dysfunction via increased tumor necrosis factor (TNF) in response to maternal nutrient restriction or increased hepatocyte growth factor (HGF) and reduced bone morphogenetic protein 2 (BMP2) in response to maternal overnutrition. To investigate the offspring inflammatory response to maternal restricted- and overnutrition, our study focused on the perinatal period because T and B cells of the innate immune system are mature by late gestation, and differentiation of monocytes into macrophages is ongoing [6, 23]. Postnatally, the systemic inflammatory response is a critical component of the innate immune system because it is responsible for providing the first-line of defense in response to an immune challenge. At day 135 of gestation, both RES and OVER lambs exhibited reduced chemokine expression in circulation (Figure 9), which may be indicative of a depressed innate immune system that would impair health and survival in neonates. In support of this, previous studies have demonstrated that the ability of neonate offspring to elicit an immune response to lipopolysaccharide challenge was diminished as a result of maternal nutrient restriction during gestation [3]. During gestation, the fetal liver receives the first exposure of nutrients and oxygen from the maternal circulation for secondary distribution to the brain, heart, and peripheral tissues [50]. Thus, the fetal liver is susceptible to altered delivery of metabolites and cytokines from the maternal circulation that would change the hepatic microenvironment. In the present study, there was increased expression of hepatic CCL16 which is primarily expressed in the liver in response to TLR stimulation [51]. Thus, exposure of the fetal liver to altered nutrients from the mother has the potential to alter CCL16 expression and subsequent the hepatic microenvironment in a way that primes hematopoietic cells to favor an M1, proinflammatory phenotype, postnatally. This would pose potential consequences for the innate immune system of the offspring. At the onset of parturition and the glucocorticoid surge, the fetal liver partakes in glucose metabolism rather than hematopoiesis, and is responsible for performing gluconeogenesis and maintaining glucose homeostasis postnatally [22]. However, the activity of macrophages, cytokines, and metabolites continues to regulate liver function. Cytokines produced by the liver, such as TNF, are established to play a role in the pathogenesis of liver dysregulation and metabolic disease by disrupting insulin signaling and promoting fibrosis [24, 52]. In our study, RES offspring exhibited a more than 4-fold increase of TNF expression, but the lack of change in TGFB1 protein concentration suggests that maternal nutrient restriction does not have immediate effects on liver fibrosis. However, the effects of maternal nutrient restriction are not typically observed until maturity because of the lengthy pathogenesis of liver disease. For example, 1-year-old lambs born to restricted-fed mothers exhibited hepatic steatosis and reduced oxidative metabolism when challenged with nutrient excess at weaning [18]. Further, at 6 years of age, offspring born to restricted-fed mothers had increased hepatic glycogen and lipid content and reduced insulin sensitivity [53]. Thus, birth may be too early to observe liver dysfunction, but the persistence of increased hepatic TNF expression would likely play a role in the pathogenesis of liver disease that originated during fetal life (Figure 9). The potential mechanism by which maternal overnutrition may impact offspring liver function differs from nutrient restriction. Liver development is regulated by multiple factors such as HGF, which promotes cellular proliferation and expansion, and BMP2, which negatively regulates proliferation to induce differentiation [54, 55]. In our study, OVER offspring exhibited increased HGF and decreased BMP2, characteristic of hepatocyte proliferation rather than differentiating to acquire the complex metabolic capabilities necessary for normal postnatal liver function. This finding suggests that the liver of OVER offspring may not be as prepared to function independent of the mother postnatally. Thus, the effect of maternal overnutrition on offspring postnatal liver function may be a result of adjusting to the mismatched intrauterine and postnatal environment, whereas the effect of maternal nutrient restriction resulted in the production of cytokines that will promote liver metabolism to store nutrients for survival (Figure 9). In conclusion, we demonstrate that maternal nutrient restriction and overnutrition during gestation promote a proinflammatory environment in both the mother and offspring, yet the inflammatory responses of each are distinct. During nutrient restriction, maternal inflammation is linked with mobilization of NEFAs, whereas overnutrition is not. We propose that maternal inflammation provoked by diet indirectly promotes systemic and hepatic inflammation in the perinatal offspring because changes to inflammatory expression in the mother and offspring were not similar. Further, maternal nutrient restriction and overnutrition differentially alter the offspring hepatic microenvironment in ways that would favor liver dysfunction postnatally. Our study also identified gender differences in offspring inflammatory gene expression, with an overall observation of increased inflammatory mRNA expression in females. Although this study was not powered to discriminate gender differences within a diet, gender is an important consideration for future research in the maternal programming field, considering that baseline gender differences exist. Future research regarding the effect of maternal inflammation on placental transport and integrity is necessary to completely understand how maternal inflammation alters the intrauterine environment to program the fetus, as well as to create strategies that mitigate the long-term consequences of poor maternal nutrition on offspring postnatally. Supplementary data Supplementary data are available at BIOLRE online. Supplementary Table S1. Interaction of diet and stage of gestation on the gene expression of systemic inflammatory mediators of pregnant ewes. Supplementary Table S2. Main effect of diet on gene expression of systemic inflammatory mediators of pregnant ewes. Supplementary Table S3. Main effect of stage of gestation on systemic inflammatory mediators of pregnant ewes, expressed relative to day 23 of gestation. Acknowledgments The authors thank V. Delaire and the UConn Livestock staff for their technical assistance, and T. Hoagland and the University of Connecticut Animal Science undergraduate students for animal care during the duration of this experiment. The authors also thank Zoetis (Florham Park, NJ) for their kind donation of the controlled intravaginal release devices used for estrous synchronization of ewes during breeding. Authors’ contributions: AKJ conceived of study, participated in design and coordination, acquired and analyzed data, drafted manuscript; MLH participated in design and coordination, acquired data, critically evaluated manuscript; SMP participated in design and coordination, acquired data; KKM participated in design and coordination, acquired data, critically evaluated manuscript; SAZ conceived of study, participated in design and coordination, critically evaluated manuscript; KEG conceived of study, participated in design and coordination, critically evaluated manuscript; SAR conceived of study, participated in design and coordination, critically evaluated manuscript. All authors read and approved final manuscript. Footnotes † Grant Support: This work was supported by the University of Connecticut Research Excellence Program (SAR) and USDA-NIFA Project 2013-01919 (SAZ). Notes Conference Presentation: Presented in part at the Joint Annual Meeting of the American Society of the Animal Science and American Dairy Science Society, 19–23 July 2016, Salt Lake City, Utah and the Annual Meeting and Trade Show of the American Society of Animal Science and Canadian Society of Animal Science, 8–12 July 2017, Baltimore, Maryland. Edited by Dr. Romana Nowak, PhD, University of Illinois Urbana-Champaign. References 1. Nathanielsz PW, Poston L, Taylor PD. In utero exposure to maternal obesity and diabetes: animal models that identify and characterize implications for future health. Obstet Gynecol Clin North Am  2007; 34: 201– 212. Google Scholar CrossRef Search ADS PubMed  2. Wu G, Bazer FW, Wallace JM, Spencer TE. BOARD-INVITED REVIEW: intrauterine growth retardation: implications for the animal sciences. J Anim Sci  2006; 84: 2316– 2337. Google Scholar CrossRef Search ADS PubMed  3. 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Early maternal undernutrition programs increased feed intake, altered glucose metabolism and insulin secretion, and liver function in aged female offspring. Am J Physiol Regul Integr Comp Physiol  2012; 302: R795– R804. Google Scholar CrossRef Search ADS PubMed  54. Xu C-P, Ji W-M, van den Brink GR, Peppelenbosch MP. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver. World J Gastroenterol  2006; 12: 7621– 7625. Google Scholar CrossRef Search ADS PubMed  55. Shin D, Monga SPS. Cellular and molecular basis of liver development. Compr Physiol  2013; 3: 799– 815. Google Scholar PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biology of Reproduction Oxford University Press

Gestational restricted- and over-feeding promote maternal and offspring inflammatory responses that are distinct and dependent on diet in sheep

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Abstract

Abstract Inflammation may be a mechanism of maternal programming because it has the capacity to alter the maternal environment and can persist postnatally in offspring tissues. This study evaluated the effects of restricted- and over-feeding on maternal and offspring inflammatory gene expression using reverse transcription (RT)-PCR arrays. Pregnant ewes were fed 60% (Restricted), 100% (Control), or 140% (Over) of National Research Council requirements beginning on day 30.2 ± 0.2 of gestation. Maternal (n = 8–9 ewes per diet) circulating nonesterified fatty acid (NEFA) and expression of 84 inflammatory genes were evaluated at five stages during gestation. Offspring (n = 6 per diet per age) inflammatory gene expression was evaluated in the circulation and liver at day 135 of gestation and birth. Throughout gestation, circulating NEFA increased in Restricted mothers but not Over. Expression of different proinflammatory mediators increased in Over and Restricted mothers, but was diet-dependent. Maternal diet altered offspring systemic and hepatic expression of genes involved in chemotaxis at late gestation and cytokine production at birth, but the offspring response was distinct from the maternal. In the perinatal offspring, maternal nutrient restriction increased hepatic chemokine (CC motif) ligand 16 and tumor necrosis factor expression. Alternately, maternal overnutrition increased offspring systemic expression of factors induced by hypoxia, whereas expression of factors regulating hepatocyte proliferation and differentiation were altered in the liver. Maternal nutrient restriction and overnutrition may differentially predispose offspring to liver dysfunction through an altered hepatic inflammatory microenvironment that contributes to immune and metabolic disturbances postnatally. Introduction Maternal programming is defined as changes to the maternal or intrauterine environment that alter fetal development and can permanently impair tissue function to predispose offspring to chronic diseases postnatally [1]. Maternal nutrition is one factor that can cause programming of offspring through altered macro- or micronutrient ingestion during gestation. Both maternal nutrient restriction and overnutrition result in offspring who exhibit poor postnatal growth, reduced muscle mass, increased adiposity, disrupted metabolism, and altered innate immunity [2–4]. Further, offspring born to nutrient restricted and overnourished mothers are at increased risk to develop chronic metabolic diseases during adulthood, including obesity, type 2 diabetes, atherosclerosis, and hepatic steatosis [1]. Chronic inflammation is common to the pathogenesis of these metabolic diseases [5], and thus may be a persistent consequence of poor maternal nutrition during gestation [6, 7]. Although the phenotype of offspring exposed to poor maternal nutrition during gestation is similar in response to maternal restriction and overnutrition, the mechanisms of each diet are distinct [4, 8]. Furthermore, the prevalence of over- and restricted nutrition is concerning in women of reproductive age (20–39 years). In the USA, 58.5% of women in this age group are overweight (body mass index [BMI] > 24) and 31.8% are obese (BMI > 29; [9]). Likewise, up to 28% of women aged 20–39 years are underweight (BMI 16–18.5) in developing nations [10]. Therefore, it is necessary to understand the effects of both maternal nutrient excess and restriction on offspring to improve outcomes of fetal programming worldwide. Inflammation has been proposed as a mechanism contributing to fetal programming because it has the capacity to alter the maternal environment and is known to persist postnatally in metabolically important tissues of offspring exposed to adverse intrauterine environments [11, 12]. In the maternal environment, low-grade, chronic inflammation is present in healthy pregnancies to facilitate maternal tolerance to the fetus, which exhibits paternal antigens [13, 14]. This involves timely coordination of the maternal endocrine and innate immune system throughout gestation and the onset of parturition [13, 14]. Failure of the maternal innate immune system to tolerate the fetus and elicit a chronic but controlled inflammatory response can result in pregnancy complications such as early embryonic loss, abortion, or preterm labor [13, 14]. This is usually provoked by a source of inflammation that is independent of pregnancy, including stress, infection, or diet [12]. For example, excessive intake and metabolism of macronutrients is associated with low-grade, chronic inflammation in nonpregnant women [5], and this inflammation is exaggerated in overweight and obese pregnant women [15]. When coupled with pregnancy, dietary-induced inflammation has the potential to negatively impact pregnancy success, maternal health, and fetal development. Diet-associated inflammation in the mother has the potential to impact the fetus through direct transfer of immune modulators across the placenta or by modulating transport of nutrients and oxygen to alter the intrauterine environment [12]. Epidemiological evidence demonstrates that offspring born to both over- or underweight women exhibit an increased inflammatory score as infants [16]. Additionally, animal studies have demonstrated that sheep offspring exposed to maternal nutrient restriction or overnutrition during gestation exhibit increased tissue-specific inflammation postnatally in muscle, cardiac, intestine, and liver tissues, indicating that inflammation caused by poor maternal nutrition may have persistent underlying effects [17–21]. The fetal liver may have an integral role in priming the postnatal innate immune and metabolic systems [22]. During gestation, the liver is a major hematopoietic organ responsible for differentiating resident and peripheral macrophages, and therefore the fetal hepatic microenvironment has the potential to prime cells of the innate immune system to favor a pro- or anti-inflammatory phenotype postnatally [22–24]. Postnatally, the liver is a vital endocrine and metabolic organ whose function is related to the immune cells and cytokines present in the hepatic tissue [25]. Thus, it is necessary to investigate how the liver of offspring may be affected by the maternal diet during gestation, as well as mechanisms by which inflammation may contribute to altered postnatal hepatic microenvironment and function. This study describes changes in maternal systemic gene expression of inflammatory mediators throughout gestation and in response to diet, and reports maternal nonesterified fatty acid (NEFA) concentrations as a potential link between gestational diet and maternal inflammation. Additionally, this study investigates offspring systemic inflammation in response to both maternal nutrient restriction and overnutrition, allowing comparisons between maternal and fetal inflammation, and between diets, in one study. Finally, we report offspring hepatic changes in inflammatory gene expression which may predispose offspring to postnatal metabolic and liver dysfunction. Materials and Methods Animals and experimental design All animal procedures were approved by the University of Connecticut's Institutional Animal Care and Use Committee. Multiparous western white-faced ewes were estrous synchronized, bred by live cover to one of four related Dorset rams, and confirmed pregnant at day 28.5 ± 0.4 of gestation using transabdominal ultrasound as previously described [26]. The ewes used for this study were a subset of a larger flock and collaborative experiment detailed in Pillai et al. [27]. All ewes were bred and maternal and offspring samples were collected in the same breeding season (e.g., all samples are from the same breeding cohort). Briefly, on individual housing at day 20 of gestation, ewes were transitioned onto a complete pelleted feed to meet 100% of the requirements of a pregnant ewe carrying twins as recommended by the National Research Council (NRC [28]). At day 30.2 ± 0.2 of gestation, pregnant ewes were randomly assigned to one of three experimental diets that met 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC total digestible nutrient requirements (TDN [28]). Offspring from ewes fed Control, Restricted, and Over diets are referred to as CON, RES, and OVER, respectively. Body weights of ewes were recorded weekly, allowing rations to be adjusted for individual changes in body weight. Six weeks before parturition, rations were adjusted based on NRC recommendations to meet the TDN requirement for a late gestation ewe expecting twins (28.5% increase of TDN), while maintaining feeding levels of 60%, 100% and 140% [28]. Daily requirements were provided in two rations per day and ewes remained on diet until parturition (gestation length 147.4 ± 1.9 days). Body condition scoring was also performed weekly on ewes by the same trained observers using a scale of 0–5 to evaluate maternal adiposity (0 = extremely emaciated and 5 = extremely obese [29]). This method of assessing adiposity is highly correlated (r = 0.94) with carcass fat content determined by chemical analysis, with a body condition score of 3 (28.7% adiposity) being ideal [29]. Maternal sample collection Ewe blood collection was performed in the fasted state, before morning feeding. Whole blood was collected via jugular venipuncture at days 23 ± 1.2, 45 ± 1.4, 90 ± 1.4, and 135 ± 1.6 of gestation for RNA and serum, at day 142 ± 3.3 of gestation for serum only, and within 24 h of parturition (birth) for RNA only (Figure 1). For RNA processing, 3 mL of whole blood were transferred into a Tempus Blood RNA Tube (Applied Biosystems, Foster City, CA) containing RNA stabilizer and shaken vigorously to precipitate RNA from whole blood. Tempus tubes were stored on ice until returned to the laboratory and then stored at –20°C until processed. For serum, 5 mL of whole blood was transferred into a nonheparinized tube and processed as previously described [30]. Figure 1. View largeDownload slide Experimental design and sample collection. Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation (gest) through parturition [28]. Whole blood was collected for RT-PCR arrays (black) or serum (gray) analyses from pregnant ewes and offspring at the time points indicated. Liver was collected from offspring at necropsy for RT-PCR array and ELISA assays at day 135 of gestation (n = 6 per diet; three females and three males in CON and RES, four females and two males in OVER) or within 24 h of birth (n = 6 per diet; three females and three males in CON and OVER, four females and two males in RES). All ewes were bred and maternal and offspring samples were collected in the same breeding season (e.g., all samples are from the same breeding cohort). Figure 1. View largeDownload slide Experimental design and sample collection. Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation (gest) through parturition [28]. Whole blood was collected for RT-PCR arrays (black) or serum (gray) analyses from pregnant ewes and offspring at the time points indicated. Liver was collected from offspring at necropsy for RT-PCR array and ELISA assays at day 135 of gestation (n = 6 per diet; three females and three males in CON and RES, four females and two males in OVER) or within 24 h of birth (n = 6 per diet; three females and three males in CON and OVER, four females and two males in RES). All ewes were bred and maternal and offspring samples were collected in the same breeding season (e.g., all samples are from the same breeding cohort). Offspring sample collection Following parturition, lambs (n = 6 lambs per diet [three females and three males in CON and OVER, four females and two males in RES]) from Control, Restricted, or Over ewes (n = 3–5 ewes per diet) nursed from their dam for up to 24 h to receive colostrum. Within 24 h of parturition, whole blood was obtained from live lambs via jugular venipuncture and processed for RNA or serum as described above (Figure 1). Lambs were subsequently euthanized with an i.v. overdose of Beuthanasia-D Special (390 ng/mL sodium pentobarbital and 50 mg/mL phenytoin based on body weight; Merck Animal Health, Summit, NJ) and exsanguinated. Liver tissue was collected, snap-frozen in liquid nitrogen, and stored at –80°C. Another group of ewes (n = 3–5 ewes per diet) from the same experiment [27] were euthanized at day 135 of gestation to acquire fetus(es) for sampling (Figure 1). Ewes were euthanized with an i.v. overdose of Beuthanasia-D Special (Merck Animal Health) and exsanguinated. Subsequently, a midline abdominal incision was performed on the ewe to remove the fetus(es). Following euthanasia, whole blood (3 mL) was collected via cardiac puncture for RNA, and liver was dissected from six fetuses per diet (three females and three males in CON and RES, four females, and two males in OVER). Samples were processed and stored as described for lambs. RNA isolation Isolation of RNA from whole blood was performed using the Perfect Pure RNA Blood kit according to the manufacturer's protocol (5 Prime, Inc., Gaithersburg, MD). The GeneJET Cleanup and Concentration Micro Kit (Thermo Scientific, Lafayette, CO) was used to concentrate the eluted RNA into 10 μL of RNase Free water. Hepatic RNA isolation was performed using 100 mg of tissue homogenized using a standard bead beating method in TRIzol Reagent (Invitrogen, Carlsbad, CA [31]). Quantity and quality of RNA was determined using a NanoDrop spectrophotometer (Thermo Scientific) and Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA), respectively. Real-time reverse transcription-PCR array Genomic DNA elimination was performed on each 500 ng sample of RNA and reverse transcribed using the RT2 First Strand Kit (SABiosciences). Quantitative real-time reverse transcription (RT)-PCR was performed using the Inflammatory Cytokines & Receptors RT2 Profiler PCR Array (Catalog no. PABT-011Z; SABiosciences, Germantown, MD). This array measures the expression of 84 genes mediating the inflammatory response, and has been previously validated in sheep [32]. One cDNA sample was analyzed per PCR array with RT2 SYBR Green Master Mix (SABiosciences). Cycling conditions were one cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min, performed using the ABI 7900 HT Fast Real-time PCR machine (Applied Biosystems). Cycle threshold (CT) values were obtained, and the ΔΔCT method was used to determine changes in gene expression [33]. Beta-actin (ACTB) was chosen as the housekeeping gene for all samples, as the CT values were not different between diet or stage of gestation in ewes (P > 0.21), and were not different between maternal diet or gender in offspring (P > 0.13). NEFA and ELISA assays NEFA concentrations were quantified from ewe and lamb serum using the acyl-CoA synthetase-acyl-CoA oxidase method (NEFA-HR, Wako Pure Chemical Industries, Dallas, TX; detection limit: 0.01–4.00 mEq/L). Lamb serum CXCL8 was quantified using a sheep-specific enzyme-linked immunosorbent assay (ELISA; Genorise Scientific, Inc., Glen Mills, PA; detection limit: 12–800 pg/mL). Hepatic protein was isolated by homogenizing 100 mg tissue using standard bead beating method and centrifugation to collect the supernatant. Total protein was quantified using the Quick Start Bradford Protein Assay (BioRad, Hercules, CA) to normalize subsequent assays. Offspring hepatic c-reactive protein (CRP) was determined with a sheep-specific ELISA using undiluted samples per the manufacturer's protocol (MyBioSource, San Diego, CA; detection range 0.25–8 μg/mL). Hepatic bone morphogenetic protein 2 (BMP2), hepatocyte growth factor (HGF), transforming growth factor beta 1 (TGFB1), and tumor necrosis factor (TNF) were quantified from offspring birth samples diluted 1:10 using sheep-specific ELISAs (Genorise Scientific, Inc.; detection limits: 62–4000 pg BMP2/mL; 94–6000 pg HGF/mL; 31–2000 pg TGFbeta/mL; 35–2400 pg TNF/mL). The assay sensitivities were 8 pg BMP2/mL, 0.9 pg CXCL8/mL, 0.1 μg CRP/mL, 18 pg HGF/mL, 5 pg TGFB/mL, and 7 pg TNF/mL. The concentration of each factor was measured within a single assay, and the intra-assay coefficient of variation from duplicate samples ranged from 6.13% to 23.2%. Data analyses The power calculations performed for this study were based on anticipated differences in maternal and offspring body weight in response to dietary treatment, with 90% power to detect significance at 5%. We anticipated that a 15% difference in maternal BW (16.5 kg) would be achieved at parturition in response to dietary treatment during gestation, with an animal variability of 8 kg, indicating that seven ewes per diet were needed for maternal measurements. Six offspring per diet were required based on an anticipated 20% difference in body weight (996 g) and animal variability of 480 g. Offspring gender could not be controlled for, and therefore the study did not have the power to investigate effects of gender within a diet. All data analyses were performed using the MIXED procedure of Statistical Analysis System version 9.4 (SAS Institute, Cary, NC). Ewe data were analyzed as a completely randomized design. Percent change in body weight from day 30 of gestation, percent change in BCS from day 30 of gestation, and NEFA concentrations were analyzed with repeated measures. The model included fixed effects of diet, day of gestation, and their interaction, with the subject defined as ewe. Covariate structures were chosen based on the lowest Akaike Iteration Criterion value for each dependent variable [34]. Compound symmetry was chosen for percent change BCS, autoregressive was chosen for percent change in body weight, and unstructured was chosen for NEFA concentration. Data are presented as least squares mean (lsmean) ± SE. Due to insufficient RNA quantity from ewe blood (<500 ng), PCR arrays could not be run for all days of gestation within each ewe. Thus, ewe array data were analyzed as a cross-sectional study with fixed effects of diet, day of gestation, and their interaction. Heat maps of ewe inflammatory gene expression were generated in the gplots package in R version 3.3.1. Ewe gene expression data are expressed relative to Control at day 23 in the presence of an interaction, relative to Control in the presence of a main effect of diet, or relative to day 23 in the presence of a main effect of gestation. Offspring data were analyzed as a completely randomized design. Offspring body weight and CRP were analyzed with the fixed effects of maternal diet, gender, and stage of gestation, and interaction of maternal diet or gender by stage of gestation. For the blood and liver PCR arrays, the housekeeping gene ACTB cycled differently at 135 days of gestation and birth (P < 0.0001). Thus, offspring array data from each time point were analyzed separately, with fixed effects of maternal diet and gender only. Offspring gene data are expressed relative to CON or females. Lamb NEFA, and TNF, TGFB1, BMP2, and HGF ELISA data were analyzed at birth only, and thus the model included fixed effects of maternal diet and gender only. Data are expressed as lsmean ± SE. Significance is discussed when P ≤ 0.05. Results Maternal body weight, BCS, and circulating NEFA concentration There was a significant interaction of diet by day of gestation (Figure 2) on ewe body weight (P < 0.0001), BCS (P = 0.001), and circulating NEFA concentrations (P < 0.0001). Upon the start of the dietary treatment at day 30 of gestation, the body weight of pregnant ewes did not differ between diet groups (78.2 ± 2.4, 81.2 ± 2.2, 75.7 ± 2.4 kg for Control, Restricted, and Over, respectively; P ≥ 0.09). Between days 79 and 135 of gestation, the percent change in body weight from day 30 of gestation was different between Control, Restricted, and Over ewes (P ≤ 0.05; Figure 2A). That is, the body weight of Control and Over ewes increased by 19.4% and 22.7%, respectively, between day 79 and 135, whereas the body weight of Restricted ewes was reduced by 3.5% at day 79, and increased by 5.8% at day 135 of gestation (Figure 2A). Five days before parturition (day 142), the percent change in body weight from day 30 was greater in Over (31.4 ± 5.3%) and Control (26.6 ± 2.1%) ewes than in Restricted ewes (5.9 ± 2.4%; P < 0.0001; Figure 2A). Similar to ewe body weight, ewe BCS did not differ between diets at day 30 of gestation (3.07 ± 0.04, 3.00 ± 0.04, 2.99 ± 0.04 for Control, Restricted, and Over, respectively; P ≥ 0.85). However, by day 142 of gestation, the percent change in BCS from day 30 was greater in Over (5.7 ± 1.4%) and less in Restricted (–5.7 ± 0.83%) ewes, compared with Control (0.6 ± 2.6%; P ≤ 0.02; Figure 2B). Circulating NEFA concentrations were not different in ewes before beginning of experimental diets (P ≥ 0.71; Figure 2C). Throughout dietary treatment, Restricted ewes had greater (P < 0.0001) circulating NEFA concentrations compared with Control and Over, which did not differ (P ≤ 0.55; Figure 2C). Figure 2. View largeDownload slide Change in maternal body weight (A), body condition score (B), and circulating NEFA concentration (C). Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation (gest) through parturition [28]. Data are presented as lsmean ± SE. Within a day of gestation, pairwise comparisons between diets are denoted when P ≤ 0.05 by ‡ (Restricted vs. Over, with Control intermediate), † (Restricted vs. Control and Over), or * (Restricted vs. Control vs. Over). Figure 2. View largeDownload slide Change in maternal body weight (A), body condition score (B), and circulating NEFA concentration (C). Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation (gest) through parturition [28]. Data are presented as lsmean ± SE. Within a day of gestation, pairwise comparisons between diets are denoted when P ≤ 0.05 by ‡ (Restricted vs. Over, with Control intermediate), † (Restricted vs. Control and Over), or * (Restricted vs. Control vs. Over). Maternal systemic inflammatory gene expression Analysis of inflammatory related gene expression in pregnant ewes indicated that there was an interaction of diet by day of gestation on the expression of interleukin (IL) 6 receptor (IL6R) and platelet factor 4 (PF4) in the circulation of pregnant ewes (P ≤ 0.04; Figure 3; Supplementary Table S1). Compared with day 23 of gestation, the expression of IL6R did not change as gestation advanced in Control ewes (P > 0.05). Expression of IL6R increased at day 135 of gestation in Restricted compared with Control and Over ewes and within 24 h of birth in Over compared with Restricted ewes (Figure 3A; P ≤ 0.05). As gestation advanced, expression of PF4 did not change in Control ewes, compared with day 23. (Figure 3B: P ≤ 0.05). At day 45 of gestation, PF4 gene expression in Over ewes was greater than Control, whereas Restricted was greater than Control at day 90 of gestation (P ≤ 0.05). Figure 3. View large Download slide Maternal systemic inflammatory gene expression is altered by gestational diet and stage of gestation (gest). Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC TDN requirements from day 30.2 ± 0.2 of gestation through parturition [28]. An interaction of diet by day of gestation was observed for the mRNA expression of Interleukin 6 receptor and Platelet factor 4. Within a day of gestation, pairwise comparisons between diets are denoted when P ≤ 0.05 by † (Restricted vs. Control and Over), ‡ (Restricted vs. Over, with Control intermediate), § (Over vs. Control and Restricted), or # (Restricted vs. Control, with Over intermediate). Relative expression values are available in Supplementary Table S1. Figure 3. View large Download slide Maternal systemic inflammatory gene expression is altered by gestational diet and stage of gestation (gest). Pregnant ewes were individually fed 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC TDN requirements from day 30.2 ± 0.2 of gestation through parturition [28]. An interaction of diet by day of gestation was observed for the mRNA expression of Interleukin 6 receptor and Platelet factor 4. Within a day of gestation, pairwise comparisons between diets are denoted when P ≤ 0.05 by † (Restricted vs. Control and Over), ‡ (Restricted vs. Over, with Control intermediate), § (Over vs. Control and Restricted), or # (Restricted vs. Control, with Over intermediate). Relative expression values are available in Supplementary Table S1. Thirteen genes exhibited a main effect of diet and are reported as a heat map in Figure 4 and as relative expression in Supplementary Table S2. Regardless of the stage of gestation, the expression of C-X-C motif chemokine ligand 9 (CXCL9), IL4, IL7, lymphotoxin beta (LTB), TNF super family member 10 (TNFSF10), and IL10 receptor subunit alpha (IL10RA) increased in Over ewes compared with Control (P ≤ 0.05). In Restricted ewes, expression of BMP2, C-X-C motif chemokine receptor 1 (CXCR1), IL1B, and TNFSF13B increased compared with Control (P ≤ 0.05). Expression of C-C motif chemokine ligand 4 (CCL4), lymphotoxin alpha (LTA), and TNFSF11 was greater in Over ewes than Restricted (P = 0.001), but neither differed from Control (P ≥ 0.09). Figure 4. View large Download slide Maternal systemic inflammatory gene expression is altered by gestational diet. Pregnant ewes were assigned to 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirement from day 30.2 ± 0.2 of gestation through parturition [28]. A main effect of diet (P ≤ 0.05) was observed for the expression of 13 inflammatory genes. Within a gene, red indicates the least expression and blue indicates the greatest expression. Relative expression values are available in Supplementary Table S2. Abbreviations: Bone morphogenetic protein 2 (BMP2); C-C motif chemokine ligand 4 (CCL4); C-X-C motif chemokine ligand 9(CXCL9); C-X-C motif chemokine receptor 1 (CXCR1); Interleukin 1 beta (IL1B), Interleukin (IL)-4, 7; Interleukin 10 receptor subunit alpha (IL10RA); lymphotoxin alpha (LTA); lymphotoxin beta (LTB); TNF super factor family member (TNFSF)-10, 11, 13B. Figure 4. View large Download slide Maternal systemic inflammatory gene expression is altered by gestational diet. Pregnant ewes were assigned to 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirement from day 30.2 ± 0.2 of gestation through parturition [28]. A main effect of diet (P ≤ 0.05) was observed for the expression of 13 inflammatory genes. Within a gene, red indicates the least expression and blue indicates the greatest expression. Relative expression values are available in Supplementary Table S2. Abbreviations: Bone morphogenetic protein 2 (BMP2); C-C motif chemokine ligand 4 (CCL4); C-X-C motif chemokine ligand 9(CXCL9); C-X-C motif chemokine receptor 1 (CXCR1); Interleukin 1 beta (IL1B), Interleukin (IL)-4, 7; Interleukin 10 receptor subunit alpha (IL10RA); lymphotoxin alpha (LTA); lymphotoxin beta (LTB); TNF super factor family member (TNFSF)-10, 11, 13B. Forty-one genes exhibited a main effect of day of gestation, reported as a heat map in Figure 5 and as relative expression in Supplementary Table S3. Genes with a main effect of gestation exhibited different expression patterns throughout gestation. Compared with day 23 of gestation, expression of CCL2 and IL2RB increased at day 45, 90, and 135 of gestation (P ≤ 0.04). Expression of secreted phosphoprotein 1 (SPP1) peaked at day 45 of gestation (P ≤ 0.04), whereas IL17B was greatest at day 135 of gestation (P ≤ 0.05). Expression of C-C motif chemokine receptor 6 (CCR6) increased at day 45 and 90 of gestation (P ≤ 0.03), whereas colony stimulating factor 3 (CSF3) increased at day 90 and 135 of gestation (P ≤ 0.05). Compared with day 23 of gestation, CXCR1 expression decreased at day 45 and increased at day 135 of gestation and birth (P ≤ 0.05). At day 90 of gestation and after parturition, IL5 expression was reduced (P ≤ 0.02). Between day 23, 45, and 90 of gestation, expression of CCL4, CXCL9, C-X3-C motif chemokine receptor 1 (CX3CR1), CD40 ligand (CD40LG), Fas ligand (FASLG), and TNFSF13B did not differ, but all were reduced at day 135 and 24 h after parturition (P ≤ 0.05). Expression of aminoacyl tRNA synthase complex interacting multifunctional protein 1 (AIMP1), CCL3, CCL5, CCL22, macrophage migration inhibitory factor (MIF), CCR1, CCR3, CCR5, CCR8, CXCR3, CSF1, interferon gamma (IFNG), IL7, IL13, IL16, IL6 signal transducer (IL6ST), IL9R, LTA, LTB, TNF and TNFSF11 did not change during gestation (P > 0.06), but exhibited reduced expression 24 h after parturition (P ≤ 0.05). Expression of CXCL8 decreased only at day 45 of gestation (P ≤ 0.03), whereas CXCL10, IL4, IL15, nicotinamide phosphoribosyltransferase (NAMPT), and TNFSF10 continued to decline from day 45 of gestation through parturition (P ≤ 0.05). Figure 5. View large Download slide Maternal systemic inflammatory gene expression is altered by stage of gestation. Pregnant ewes were assigned to 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation through parturition [28]. A main effect of gestation (P ≤ 0.05) was observed for the expression of 41 inflammatory genes. Within a gene, red indicates the least expression and blue indicates the greatest expression. Relative expression values are available in Supplementary Table S3. Abbreviations: Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 (AIMP1); CD40 ligand (CD40LG); C-C motif chemokine ligand (CCL)-2, 3, 4, 5, 22; C-X-C motif chemokine ligand (CXCL)-8, 9, 10; C-C motif chemokine receptor (CCR)-1, 3, 5, 6, 8; C-X-C motif chemokine receptor (CXCR)- 1, 3; C-X3-C motif chemokine receptor 1 (CX3CR1); Colony stimulating factor (CSF)-1, 3; Fas ligand (FASLG); Interferon gamma (IFNG); Interleukin (IL)-4, 5, 7, 13, 15, 16, 17B; Interleukin receptors (IL2RB; IL6R; IL9R); Interleukin 6 signaling transducer (IL6ST); Lymphotoxin alpha (LTA); Lymphotoxin beta (LTB); Macrophage migration inhibitory factor (MIF); Nicotinamide phosphoribosyltransferase (NAMPT); Secreted phosphoprotein 1 (SPP1); Tumor necrosis factor (TNF); TNF factor super family member (TNFSF)-10, 11, 13B. Figure 5. View large Download slide Maternal systemic inflammatory gene expression is altered by stage of gestation. Pregnant ewes were assigned to 100% (Control; n = 8), 60% (Restricted; n = 9), or 140% (Over; n = 8) of NRC requirements from day 30.2 ± 0.2 of gestation through parturition [28]. A main effect of gestation (P ≤ 0.05) was observed for the expression of 41 inflammatory genes. Within a gene, red indicates the least expression and blue indicates the greatest expression. Relative expression values are available in Supplementary Table S3. Abbreviations: Aminoacyl tRNA synthetase complex interacting multifunctional protein 1 (AIMP1); CD40 ligand (CD40LG); C-C motif chemokine ligand (CCL)-2, 3, 4, 5, 22; C-X-C motif chemokine ligand (CXCL)-8, 9, 10; C-C motif chemokine receptor (CCR)-1, 3, 5, 6, 8; C-X-C motif chemokine receptor (CXCR)- 1, 3; C-X3-C motif chemokine receptor 1 (CX3CR1); Colony stimulating factor (CSF)-1, 3; Fas ligand (FASLG); Interferon gamma (IFNG); Interleukin (IL)-4, 5, 7, 13, 15, 16, 17B; Interleukin receptors (IL2RB; IL6R; IL9R); Interleukin 6 signaling transducer (IL6ST); Lymphotoxin alpha (LTA); Lymphotoxin beta (LTB); Macrophage migration inhibitory factor (MIF); Nicotinamide phosphoribosyltransferase (NAMPT); Secreted phosphoprotein 1 (SPP1); Tumor necrosis factor (TNF); TNF factor super family member (TNFSF)-10, 11, 13B. Offspring body weight and NEFA concentrations A main effect of maternal diet was observed on offspring body weight in that RES offspring weighed 15.4% and 7.7% less than CON and OVER, respectively (CON: 5007.8 ± 187.6 g, RES: 4236.5 ± 188.5 g, OVER: 4591.8 ± 188.6 g; P = 0.02). Offspring body weight did not differ with stage of gestation (P = 0.611) or gender (P = 0.09). No differences were observed in the lamb serum NEFA concentration between maternal diets (CON: 872.3 ± 109.7 μmol/L, RES: 803.9 ± 112.0 μmol/L, OVER: 1138.3 ± 134.3 μmol/L; P = 0.18) or males and females (924.2 ± 91.6 and 952.1 ± 102.4, μmol/L, respectively; P = 0.84). Offspring circulation In offspring circulation at day 135 of gestation, there were main effects of maternal diet for five genes (P ≤ 0.05; Figure 6A) and gender for six genes (P ≤ 0.05; Table 1). Systemic CCL22 expression was reduced in RES compared with CON (P ≤ 0.05). Systemic expression of CXCL12, CXCR1, and IL1A were reduced in OVER and RES compared with CON (P ≤ 0.05). Systemic expression of MIF was reduced in OVER compared with CON (P ≤ 0.05). At day 135 of gestation, systemic CCL22, CXCL1, CXCL9, CXCL12, IL4, and IL15 expression was reduced in male fetuses compared with females, regardless of maternal diet (P ≤ 0.05; Table 1). Figure 6. View largeDownload slide Main effect of maternal diet (P ≤ 0.05) on the systemic mRNA expression of offspring euthanized at day 135 of gestation (A) or within 24 h of birth (B), and CXCL8 protein concentration at birth (C). Offspring (n = 6 per diet at each stage) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. At each stage of gestation, there were 10 female and 8 male offspring. Within a stage of gestation, mRNA expression is expressed relative to CON. Within a gene, treatments with different letters differ (P ≤ 0.05). Abbreviations: C-C motif chemokine ligand 22 (CCL22); C-X-C motif chemokine ligand (CXCL)-8, 12; C-X-C motif chemokine receptor 1 (CXCR1); interleukin 1 alpha (IL1A); lymphotoxin beta (LTB); macrophage migration inhibitory factor (MIF); platelet factor 4 (PF4); TNF super family member 13 (TNFSF13); vascular endothelial growth factor A (VEGFA). Figure 6. View largeDownload slide Main effect of maternal diet (P ≤ 0.05) on the systemic mRNA expression of offspring euthanized at day 135 of gestation (A) or within 24 h of birth (B), and CXCL8 protein concentration at birth (C). Offspring (n = 6 per diet at each stage) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. At each stage of gestation, there were 10 female and 8 male offspring. Within a stage of gestation, mRNA expression is expressed relative to CON. Within a gene, treatments with different letters differ (P ≤ 0.05). Abbreviations: C-C motif chemokine ligand 22 (CCL22); C-X-C motif chemokine ligand (CXCL)-8, 12; C-X-C motif chemokine receptor 1 (CXCR1); interleukin 1 alpha (IL1A); lymphotoxin beta (LTB); macrophage migration inhibitory factor (MIF); platelet factor 4 (PF4); TNF super family member 13 (TNFSF13); vascular endothelial growth factor A (VEGFA). Table 1. Effect of gender on gene expression of offspring systemic inflammatory mediators.   Gender    Gene1  Female (n = 10)  Male (n = 8)  SEM  P-value  D 135  CCL22  1.52  0.53  0.30  0.03  CXCL1  1.37  0.40  0.20  0.24  CXCL9  2.07  0.43  0.56  0.02  CXCL12  2.22  0.59  0.68  0.45  IL4  1.10  0.49  0.14  0.002  Birth  CXCR1  1.11  0.63  0.11  0.02  TNFSF13  1.23  2.55  0.36  0.002    Gender    Gene1  Female (n = 10)  Male (n = 8)  SEM  P-value  D 135  CCL22  1.52  0.53  0.30  0.03  CXCL1  1.37  0.40  0.20  0.24  CXCL9  2.07  0.43  0.56  0.02  CXCL12  2.22  0.59  0.68  0.45  IL4  1.10  0.49  0.14  0.002  Birth  CXCR1  1.11  0.63  0.11  0.02  TNFSF13  1.23  2.55  0.36  0.002  1 Expression relative to females. Abbreviations: C-C- motif chemokine ligand 22 (CCL22); C-X-C motif chemokine ligand (CXCL)-1, 9, 12; C-X-C motif chemokine receptor 1 (CXCR1); Interleukin 4 (IL4), 15; TNF super family member (TNFSF)-13. View Large In offspring circulation at birth, there were main effects of maternal diet for five genes (P ≤ 0.05; Figure 6B) and gender for two genes (P ≤ 0.05; Table 1). Systemic CXCL8 increased in RES and OVER compared with CON (P ≤ 0.05). The systemic protein concentration of CXCL8 at birth was 47% and 57% greater in OVER compared with CON and RES, respectively (P ≤ 0.02; Figure 6C), with no effect of gender (P = 0.82). Systemic LTB was reduced in RES compared with CON and OVER (P ≤ 0.05). Systemic PF4, TNFSF13, and VEGFA were increased in OVER compared with CON and RES (P ≤ 0.05). Compared with females, systemic CXCR1 expression was reduced, whereas TNFSF13 expression increased in male lambs at birth (P ≤ 0.05; Table 1). Offspring liver In offspring liver at day 135 of gestation, there were main effects of maternal diet for three genes (P ≤ 0.05; Figure 7A) and gender for seven genes (P ≤ 0.05; Table 2). Hepatic CCL16 expression increased in RES compared with CON (P ≤ 0.05). Hepatic LTB was reduced in OVER compared with CON and RES (P ≤ 0.05). Hepatic TNFSF11 was increased in OVER compared with RES, with CON intermediate (P ≤ 0.05; Table 2). Compared with females, hepatic expression of Complement C5 (C5), CCL3, CCR1, CCR6, and IL2RG was reduced in male fetuses (P ≤ 0.05). Oppositely, hepatic TNFSF14 was increased in male fetuses compared with females (P ≤ 0.05; Table 2). Figure 7. View largeDownload slide Main effect of maternal diet (P ≤ 0.05) on the hepatic mRNA expression of offspring euthanized at day 135 of gestation (A) or within 24 h of birth (B). Offspring (n = 6 per diet at each stage) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. At each stage of gestation there were 10 female and 8 male offspring. Within a stage of gestation, mRNA expression is expressed relative to CON. Within a gene, treatments with different letters differ (P ≤ 0.05). Abbreviations: bone morphogenetic protein 2 (BMP2); C-C motif chemokine ligand 16 (CCL16); C-X-C motif chemokine ligand (CXCL)-10, 12; lymphotoxin beta (LTB); tumor necrosis factor (TNF); TNF super family member 11 (TNFSF11). Figure 7. View largeDownload slide Main effect of maternal diet (P ≤ 0.05) on the hepatic mRNA expression of offspring euthanized at day 135 of gestation (A) or within 24 h of birth (B). Offspring (n = 6 per diet at each stage) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. At each stage of gestation there were 10 female and 8 male offspring. Within a stage of gestation, mRNA expression is expressed relative to CON. Within a gene, treatments with different letters differ (P ≤ 0.05). Abbreviations: bone morphogenetic protein 2 (BMP2); C-C motif chemokine ligand 16 (CCL16); C-X-C motif chemokine ligand (CXCL)-10, 12; lymphotoxin beta (LTB); tumor necrosis factor (TNF); TNF super family member 11 (TNFSF11). Table 2. Effect of gender on hepatic gene expression of inflammatory mediators in offspring.   Gender      Gene1  Female (n = 10)  Male (n = 8)  SEM  P-value  D 135  C5  1.84  0.42  0.35  0.03  CCL3  1.12  0.50  0.18  0.01  CCR1  3.04  0.27  0.72  0.01  CCR6  6.36  0.19  1.54  0.04  IL2RG  1.04  0.68  0.12  0.03  TNFSF14  1.23  2.94  0.43    Birth  CCL1  1.52  6.80  0.96  0.0005  CXCL9  1.35  0.33  0.22  0.004  CXCL10  1.50  0.46  0.26  0.04  IL1R1  1.43  0.48  0.27  0.03  IL5  1.12  0.61  0.16  0.02  IL7  1.14  0.52  0.14  0.01  IL15  1.08  0.61  0.11  0.01  MIF  1.11  3.59  0.70  0.05  TNFSF10  1.05  0.36  0.24  0.01    Gender      Gene1  Female (n = 10)  Male (n = 8)  SEM  P-value  D 135  C5  1.84  0.42  0.35  0.03  CCL3  1.12  0.50  0.18  0.01  CCR1  3.04  0.27  0.72  0.01  CCR6  6.36  0.19  1.54  0.04  IL2RG  1.04  0.68  0.12  0.03  TNFSF14  1.23  2.94  0.43    Birth  CCL1  1.52  6.80  0.96  0.0005  CXCL9  1.35  0.33  0.22  0.004  CXCL10  1.50  0.46  0.26  0.04  IL1R1  1.43  0.48  0.27  0.03  IL5  1.12  0.61  0.16  0.02  IL7  1.14  0.52  0.14  0.01  IL15  1.08  0.61  0.11  0.01  MIF  1.11  3.59  0.70  0.05  TNFSF10  1.05  0.36  0.24  0.01  1Expression relative to females. Abbreviations: Complement C5 (C5); C-C motif chemokine ligand (CCL)-1, 3; C-C motif chemokine receptor (CCR)-1, 6; C-X-C motif chemokine ligand (CXCL)-9, 10; Interleukin (IL)-2RG, 5, 7, 15; Interleukin 1 Receptor 1 (IL1R1); Macrophage migration inhibitory factor (MIF); TNF super family member 14 (TNFSF14). View Large In offspring liver at birth, there were main effects of maternal diet for four genes (P ≤ 0.05; Figure 7B) and gender for eight genes (P ≤ 0.05; Table 2). Hepatic BMP2 and CXCL12 expression was reduced in OVER compared with CON and RES, whereas CXCL10 was reduced in OVER compared with RES with CON intermediate (P ≤ 0.05). Hepatic TNF expression increased in RES compared with CON and OVER (P ≤ 0.05). Compared with females, hepatic expression of CCL1, CXCL9, CXCL10, IL1R1, IL5, IL7, IL15, and TNFSF10 was reduced in males, but MIF expression was greater (P ≤ 0.05; Table 2). To confirm the PCR array results and further understand the effects of maternal nutrient restriction and overnutrition during gestation on offspring liver development, the protein concentrations of TNF, TGFB1, BMP2, and HGF were evaluated in offspring liver at birth (Figure 8). Maternal diet did not alter the protein concentration of TNF (P = 0.32; Figure 8A) or TGFB1 (P = 0.07; Figure 8B). Consistent with mRNA expression, the protein concentration of BMP2 was reduced by 20% in OVER compared with RES (P = 0.004; Figure 8C), whereas the protein concentration of HGF was increased by 18.5% in RES and 16.1% in OVER compared with CON (P ≤ 0.02; Figure 8D). There was no effect of gender on the hepatic protein concentration of TNF, TGFB1, BMP2, or HGF at birth (P ≥ 0.09). Figure 8. View largeDownload slide Main effect of maternal diet on the hepatic protein concentration of tumor necrosis factor (TNF; P = 0.32), transforming growth factor beta 1 (TGFB1; P = 0.07), bone morphogenetic protein 2 (BMP2; P = 0.01), and hepatocyte growth factor (HGF; P = 0.02) in offspring at birth. Offspring (n = 6 per diet) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. There were 10 females and 8 males at birth. Within a graph, treatments with different letters differ (P < 0.05). Figure 8. View largeDownload slide Main effect of maternal diet on the hepatic protein concentration of tumor necrosis factor (TNF; P = 0.32), transforming growth factor beta 1 (TGFB1; P = 0.07), bone morphogenetic protein 2 (BMP2; P = 0.01), and hepatocyte growth factor (HGF; P = 0.02) in offspring at birth. Offspring (n = 6 per diet) from ewes fed 100%, 60%, or 140% of NRC requirements beginning on day 30.2 ± 0.2 through gestation are referred to as CON, RES, and OVER, respectively. There were 10 females and 8 males at birth. Within a graph, treatments with different letters differ (P < 0.05). No interactions (P ≥ 0.07) or main effects were observed in the concentration of CRP in offspring liver between day 135 of gestation and birth (5.02 ± 0.81 and 5.67 ± 0.88 μg, respectively; P = 0.59), males and females (4.41 ± 0.91 and 6.28 ± 0.77 μg, respectively; P = 0.13), or maternal diets (CON: 4.65 ± 1.03 μg, RES: 6.18 ± 1.03 μg, OVER: 5.20 ± 1.06 μg; P = 0.57). Discussion This study evaluated inflammatory gene expression in both the mother and fetus to begin to understand inflammation as a potential fetal programming mechanism. Our findings indicate that maternal inflammatory gene expression is altered by poor nutrition during gestation, but the expression of inflammatory mediators differs during restricted- or overnutrition. Further, our data indicate that the offspring inflammatory response to maternal nutrient restriction or overnutrition is distinct from the inflammatory response of the mother, indicating that maternal inflammation may program fetal development through indirect mechanisms. Finally, we report alterations in proinflammatory cytokine, chemokine, and growth factor expression in the liver of offspring, which may be responsible for predisposition to hepatic dysfunction later in life. In addition to evaluating the maternal and fetal inflammatory response to gestational diet, this study also reports changes to maternal inflammation at multiple stages of gestation, in the absence of dietary effects. Previous literature evaluating inflammation throughout gestation has only evaluated one stage of gestation, or performed longitudinal analyses of few inflammatory mediators [15, 35, 36]. Our study is unique in that inflammatory changes are reported at multiple stages of gestation in the same cohort of females, with the mRNA abundance of 84 inflammatory genes evaluated, providing a more comprehensive analysis. These data have implications in understanding how the maternal innate immune system responds during gestation. Importantly, this information is reported in sheep, a common biomedical model for human pregnancy due to the similar fetal to maternal biomass ratio, delivery of precocial offspring, and continuity of fetal programming consequences [37]. In women, healthy pregnancies are associated with a chronic, low-grade inflammatory response which is pertinent to pregnancy recognition, maintenance, and parturition [13, 14]. It has been proposed that CCL2, CCL3, CCL4, and CCL5 are relevant chemokines during pregnancy due to their role in mediating leukocyte chemotaxis between the systemic and local intrauterine environment [38]. This is supported by our data in sheep, in which CCL2 increased 3- to 4-fold between day 23 of gestation and parturition. Further, the expression of CCL3, CCL4, and CCL5 was maintained during early and midgestation and decreased during late-gestation or postpartum, indicating a greater role in pregnancy maintenance than parturition. In our study, the mRNA expression of TNF and CXCL8 was greatest after parturition, consistent with longitudinal reports in women in which the serum concentrations of TNF and CXCL8 were greatest postpartum [15, 35]. Our data also demonstrate that the maternal immune response involves the expression of multiple cytokine, chemokine, and TNFSF ligands that previously may have not been considered during gestation. Although additional research and protein expression is necessary to investigate the roles and relationships of each inflammatory mediator reported, our data support the integral role of the innate immune system during pregnancy in sheep which is consistent with reports in women. While chronic, low-grade inflammation is associated with healthy pregnancies [13, 14], improper activation of the innate immune system due to maternal stressors may have negative consequences on the success of the pregnancy and development of the fetus during gestation [13, 14]. In our study, proinflammatory gene expression was observed in both restricted and overnourished mothers, yet there was no evidence of disrupted pregnancy success as no early fetal losses occurred after dietary treatment began [26], and the gestation length of ewes was not different based on diet [27]. However, the gene expression of proinflammatory mediators may indirectly affect fetal development by modulating fetal nutrient availability through altered placental transport [12]. Although our study did not investigate the placental tissues, we did observe increased systemic PF4 during early- and midgestation of both Restricted and Over pregnancies. In sheep, placental development is rapid during early to midgestation, and complete by day 90 [39]. Increased PF4 may have a role in altered angiogenesis and vascularization during this timeframe, potentially having negative consequences on the formation and transport capacity of blood to and across the placenta to affect fetal development [40, 41]. Previous models of maternal overnutrition have associated reduced uterine and umbilical blood flow in ewes with reduced fetal nutrient delivery [40–42]. Additionally, in ewes who were nutrient restricted during the first 40% of gestation, placental vasoconstriction contributed to fetal intrauterine growth restriction [42]. In our model, OVER lambs at birth exhibited increased systemic gene expression of PF4 and VEGFA, which promote cell survival and adaptation to hypoxic conditions. Thus, future research may investigate the role of PF4 on placental structure and transport, as well as the presence of a hypoxic intrauterine environment, during overnutrition. Nutrient restriction and overnutrition resulted in differing maternal metabolic and inflammatory phenotypes, indicating that inflammation affects maternal health and fetal development differently based on nutrient status (Figure 9). Nutrient restricted ewes demonstrated depletion of nutrient reserves by the gradual loss of body condition [29] and increased circulating NEFA concentrations as gestation advanced. Additionally, RES offspring weighed less at both day 135 of gestation and birth. This maternal–fetal phenotype is consistent with previous pregnant sheep models of limited maternal nutrient intake that are associated with slowed fetal growth in late gestation [43–45]. Mobilization of NEFAs may have contributed to inflammation in Restricted mothers because sustained increases of NEFA are known to activate the innate immune system through toll-like receptors (TLR [46]), a network of transmembrane receptors that typically respond to pathogen or danger-associated molecular patterns. Specifically, stimulation of TLR4 by NEFA causes a proinflammatory signaling cascade through the activation of master inflammatory axes, nuclear factor kappa B (NFκB), and mitogen-activated kinase [46, 47]. In our model, Restricted ewes exhibited a proinflammatory response during gestation via increased mRNA expression of cytokines IL1B and TNFSF13B, and decreased mRNA expression of anti-inflammatory cytokine IL4 and chemokine CCL4. The cytokines IL1B and TNFSF13B activate both canonical and noncanonical inflammatory signaling pathways, and act in a positive feedback loop to mediate a chronic inflammatory environment and depress anti-inflammatory mediators [48]. Over ewes did not exhibit increased circulating NEFA concentrations. However, the BCS of Over ewes increased as gestation advanced, indicating increased accumulation of maternal adipose tissue [29]. Therefore, the maternal inflammation observed in response to overnutrition was not related to increased circulating NEFA. Rather, increased adiposity may have contributed to inflammation in Over ewes. During prolonged overnutrition, TNF production antagonizes peroxisome proliferator-activated receptor gamma, inhibiting adipocyte differentiation and resulting in adipocyte hypertrophy instead of hyperplasia [49]. Consequently, engorged adipocytes promote local and systemic inflammation through local hypoxia and necrosis, resulting in macrophage infiltration and the production of adipokines that travel peripherally to induce systemic inflammation [49]. The systemic mRNA expression of TNFSF ligands and lymphotoxins in Over ewes supports an environment that would promote adipocyte hypertrophy and hypoxia and contribute to systemic inflammation [49]. Figure 9. View largeDownload slide Maternal inflammation is promoted by nutrient restriction and may be linked to increased nonesterified fatty acid (NEFA) concentrations, whereas inflammation related to overnutrition does not appear to be linked to NEFA concentrations in our study. The offspring inflammatory response to maternal nutrient restriction and overnutrition is distinct from the maternal response. Inflammation may predispose offspring to liver dysfunction via increased tumor necrosis factor (TNF) in response to maternal nutrient restriction or increased hepatocyte growth factor (HGF) and reduced bone morphogenetic protein 2 (BMP2) in response to maternal overnutrition. Figure 9. View largeDownload slide Maternal inflammation is promoted by nutrient restriction and may be linked to increased nonesterified fatty acid (NEFA) concentrations, whereas inflammation related to overnutrition does not appear to be linked to NEFA concentrations in our study. The offspring inflammatory response to maternal nutrient restriction and overnutrition is distinct from the maternal response. Inflammation may predispose offspring to liver dysfunction via increased tumor necrosis factor (TNF) in response to maternal nutrient restriction or increased hepatocyte growth factor (HGF) and reduced bone morphogenetic protein 2 (BMP2) in response to maternal overnutrition. To investigate the offspring inflammatory response to maternal restricted- and overnutrition, our study focused on the perinatal period because T and B cells of the innate immune system are mature by late gestation, and differentiation of monocytes into macrophages is ongoing [6, 23]. Postnatally, the systemic inflammatory response is a critical component of the innate immune system because it is responsible for providing the first-line of defense in response to an immune challenge. At day 135 of gestation, both RES and OVER lambs exhibited reduced chemokine expression in circulation (Figure 9), which may be indicative of a depressed innate immune system that would impair health and survival in neonates. In support of this, previous studies have demonstrated that the ability of neonate offspring to elicit an immune response to lipopolysaccharide challenge was diminished as a result of maternal nutrient restriction during gestation [3]. During gestation, the fetal liver receives the first exposure of nutrients and oxygen from the maternal circulation for secondary distribution to the brain, heart, and peripheral tissues [50]. Thus, the fetal liver is susceptible to altered delivery of metabolites and cytokines from the maternal circulation that would change the hepatic microenvironment. In the present study, there was increased expression of hepatic CCL16 which is primarily expressed in the liver in response to TLR stimulation [51]. Thus, exposure of the fetal liver to altered nutrients from the mother has the potential to alter CCL16 expression and subsequent the hepatic microenvironment in a way that primes hematopoietic cells to favor an M1, proinflammatory phenotype, postnatally. This would pose potential consequences for the innate immune system of the offspring. At the onset of parturition and the glucocorticoid surge, the fetal liver partakes in glucose metabolism rather than hematopoiesis, and is responsible for performing gluconeogenesis and maintaining glucose homeostasis postnatally [22]. However, the activity of macrophages, cytokines, and metabolites continues to regulate liver function. Cytokines produced by the liver, such as TNF, are established to play a role in the pathogenesis of liver dysregulation and metabolic disease by disrupting insulin signaling and promoting fibrosis [24, 52]. In our study, RES offspring exhibited a more than 4-fold increase of TNF expression, but the lack of change in TGFB1 protein concentration suggests that maternal nutrient restriction does not have immediate effects on liver fibrosis. However, the effects of maternal nutrient restriction are not typically observed until maturity because of the lengthy pathogenesis of liver disease. For example, 1-year-old lambs born to restricted-fed mothers exhibited hepatic steatosis and reduced oxidative metabolism when challenged with nutrient excess at weaning [18]. Further, at 6 years of age, offspring born to restricted-fed mothers had increased hepatic glycogen and lipid content and reduced insulin sensitivity [53]. Thus, birth may be too early to observe liver dysfunction, but the persistence of increased hepatic TNF expression would likely play a role in the pathogenesis of liver disease that originated during fetal life (Figure 9). The potential mechanism by which maternal overnutrition may impact offspring liver function differs from nutrient restriction. Liver development is regulated by multiple factors such as HGF, which promotes cellular proliferation and expansion, and BMP2, which negatively regulates proliferation to induce differentiation [54, 55]. In our study, OVER offspring exhibited increased HGF and decreased BMP2, characteristic of hepatocyte proliferation rather than differentiating to acquire the complex metabolic capabilities necessary for normal postnatal liver function. This finding suggests that the liver of OVER offspring may not be as prepared to function independent of the mother postnatally. Thus, the effect of maternal overnutrition on offspring postnatal liver function may be a result of adjusting to the mismatched intrauterine and postnatal environment, whereas the effect of maternal nutrient restriction resulted in the production of cytokines that will promote liver metabolism to store nutrients for survival (Figure 9). In conclusion, we demonstrate that maternal nutrient restriction and overnutrition during gestation promote a proinflammatory environment in both the mother and offspring, yet the inflammatory responses of each are distinct. During nutrient restriction, maternal inflammation is linked with mobilization of NEFAs, whereas overnutrition is not. We propose that maternal inflammation provoked by diet indirectly promotes systemic and hepatic inflammation in the perinatal offspring because changes to inflammatory expression in the mother and offspring were not similar. Further, maternal nutrient restriction and overnutrition differentially alter the offspring hepatic microenvironment in ways that would favor liver dysfunction postnatally. Our study also identified gender differences in offspring inflammatory gene expression, with an overall observation of increased inflammatory mRNA expression in females. Although this study was not powered to discriminate gender differences within a diet, gender is an important consideration for future research in the maternal programming field, considering that baseline gender differences exist. Future research regarding the effect of maternal inflammation on placental transport and integrity is necessary to completely understand how maternal inflammation alters the intrauterine environment to program the fetus, as well as to create strategies that mitigate the long-term consequences of poor maternal nutrition on offspring postnatally. Supplementary data Supplementary data are available at BIOLRE online. Supplementary Table S1. Interaction of diet and stage of gestation on the gene expression of systemic inflammatory mediators of pregnant ewes. Supplementary Table S2. Main effect of diet on gene expression of systemic inflammatory mediators of pregnant ewes. Supplementary Table S3. Main effect of stage of gestation on systemic inflammatory mediators of pregnant ewes, expressed relative to day 23 of gestation. Acknowledgments The authors thank V. Delaire and the UConn Livestock staff for their technical assistance, and T. Hoagland and the University of Connecticut Animal Science undergraduate students for animal care during the duration of this experiment. The authors also thank Zoetis (Florham Park, NJ) for their kind donation of the controlled intravaginal release devices used for estrous synchronization of ewes during breeding. Authors’ contributions: AKJ conceived of study, participated in design and coordination, acquired and analyzed data, drafted manuscript; MLH participated in design and coordination, acquired data, critically evaluated manuscript; SMP participated in design and coordination, acquired data; KKM participated in design and coordination, acquired data, critically evaluated manuscript; SAZ conceived of study, participated in design and coordination, critically evaluated manuscript; KEG conceived of study, participated in design and coordination, critically evaluated manuscript; SAR conceived of study, participated in design and coordination, critically evaluated manuscript. All authors read and approved final manuscript. Footnotes † Grant Support: This work was supported by the University of Connecticut Research Excellence Program (SAR) and USDA-NIFA Project 2013-01919 (SAZ). 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Biology of ReproductionOxford University Press

Published: Feb 1, 2018

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