MATERNAL OBESITY MAY CONTRIBUTE TO INCREASED PLACENTAL AND FETAL INFLAMMATION Molecular indicators of stress as indicators of immune status AMANDA JONES April 22, 2015 MCB5255

Background Obesity Maternal obesity Pre-pregnancy obesity Gestational diabetes High prevalence of obesity in women of reproductive age

Causes: Maternal Age Pre-pregnancy obesity Pre-existing chronic medical conditions

Background Maternal Impact Complications Cesarean delivery Gestational diabetes Fetal Consequences Fetal programming- Thrifty Phenotype (Barker) Hypothesis Fetal stress- Intrauterine Growth Restriction (IUGR)

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Paper 1 Objective: Evaluate how the placenta may display exaggerate inflammation in response to maternal obesity.

Methods Lean mothers (n=15) Obese mothers (n=20) Pre-gravid BMI Singleton pregnancies Cesarean delivery Maternal blood draw Fluorescence activated cell sorter (FACS) analysis Umbilical blood draw Glucose, insulin, cytokine- plasma assay Placental Biopsy RNA extraction Primary cell isolation- FACS analysis Immunohistochemistry- MP’s

Results

Results: Plasma Assays

Results: Increased gene expression of maternal cytokines and monocytes in maternal PBMC’s

Results: Increased localization of macrophages in obese placental tissue Lean Obese CD68+CD14+

Results: Accumulation of monocyte heterogeneity in placenta of obese women

Results: Higher expression of macrophages in the placenta, along with increased inflammatory cytokine expression

Paper 1 Conclusion Evidence of macrophage accumulation in the placenta of OB mothers Macrophages in the placenta produce pro-inflammatory molecules Obesity exacerbates basal inflammatory state of pregnancy, and may influence the in utero environment for fetal development

Sheep versus Rat model Sheep Monotocous Comparable birth weight Similar maternal:fetal ratio Similar organogenesis and growth rate Lack of available antibodies, reagents, etc. Labor intensive Ruminants Rat Litter bearing High growth and metabolic rates Short gestation Cost effective Easier genetic manipulation

Paper 2 Objective: Evaluate effects of maternal obesity on adipogenesis, inflammatory signaling, and insulin pathways at late gestation when ovine fetal skeletal muscle matures

Methods Control Diet: 100% NRC requirement (n=6) Obesogenic Diet: 150% NRC requirement (n=6) 2 singleton, 4 twins = 3 M, 3F Necropsy Collection Fetal blood (umbillical vein) Glucose and Insulin assay Semitendenosus (St) muscle Histology mRNA extraction, RT-PCR Immunoblot Analysis -60d0d 135d Start Diet Conception Necropsy (148d) (Full Term)

Results: Obese model achieved

Results: Evidence of increased adipogenesis in OB offspring OB Offspring  # fat cells  fat cell size Fat cell infiltration  # muscle cells (G)  muscle cell diameter (H)  PPAR ϒ mRNA and protein CONOB

Results: Gene expression of TLR4 and co-receptors is higher in OB offspring

NFkB and JNK Pathway

Results: Phosphorylation of key NFkB components are increased

Results: Phosphorylation of key JNK components are increased

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Results: Disregulation of PKC, Akt and AMPK suggest insulin insensitivity and promoteds adipogenesis

Conclusion, Paper 1 Enhanced adipogenesis in OB offspring during late gestation Increased TLR4 expression may contribute to activation of NFkB and JNK pathway, leading to inflammation in the ST Increase of p-PKC and reduction of the insulin receptor and p-AKT indicate decreased insulin signaling Reduction in AMPK activity is indicative of lipogenesis and adipogenesis Obesity during gestation is associated with evidence of inflammation fetal muscle, indicative of fetal immune stress during important developmental stages

Grant Proposal Objective: Establish maternal and fetal systemic inflammatory profile Sheep model Control Overfed Restricted Isolate RNA from white blood cells PCR Array Bovine Inflammatory Cytokines and Receptors Maternal: Longitudinal analysis Fetal: pre- versus post- parturition

Grant Schematic 90d45d 30d0d 135d DietConception 148d Maternal Fetal