Inflammatory Determinants of Pregravid Obesity in Placenta and Peripheral Blood

Abstract

Pre-pregnancy (pregravid) obesity has been linked to several adverse health outcomes for both mother and offspring. Complications during pregnancy include increased risk for gestational diabetes, hypertension, preeclampsia, placental abruption, and difficulties during delivery. Several studies suggest that these negative outcomes are mediated by heightened systemic inflammation as well as changes in placental development and function. However, the molecular mechanisms by which pregravid obesity affects these processes are poorly understood. In this study, we aimed to address this question by carrying out a comprehensive analysis of the systemic maternal immune system coupled with placental gene expression and microbial profiling at term delivery (11 lean and 14 obese). Specifically, we examined the impact of pregravid obesity on circulating cytokines, chemokine, adipokines, and growth factors using multiplex Luminex assay. Innate and adaptive immune cell frequencies and their cytokine production in response to stimuli were measured using flow cytometry. Finally, changes in placental transcriptome and microbiome were profiled using RNA- and 16S-sequencing, respectively. Pregravid obesity is characterized by insulin and leptin resistance, high levels of circulating inflammatory markers IL-6 and CRP, in addition to chemokine IL-8 (p < 0.01). Moreover, pregravid obesity was associated with lower frequency of naïve CD4+ T-cells (p < 0.05), increased frequency of memory CD4+ T-cells (p < 0.01), and a shift towards Th2 cytokine production (p = 0.05). Myeloid cells from the obese cohort produced higher levels of pro-inflammatory cytokines but lower levels of chemokines following TLR stimulation (p < 0.05). Lastly, pregravid obesity is associated with increased abundance of Bacteroides and changes in the expression of genes important for nutrient transport and immunity (FDR < 0.05). Collectively, these data indicate that pregravid obesity is associated with heightened systemic inflammation and of dysregulated nutrient transport in the placenta and provide insight into the basis of fetal reprogramming.

Keywords: PBMC; RNA-Seq; cytokines and inflammation; inflammation; maternal obesity; microbiome; placenta.

FIGURE 1

Pregravid obesity alters plasma levels of inflammatory mediators. (A) Study design: circulating factors in plasma were measured using Luminex and ELISA while frequency and cytokine production by immune cell subsets were quantified using flow cytometry. (B) Plasma levels of leptin and lipocalin 2. (C) Plasma levels of EGF. (D) Circulating levels of canonical markers of inflammation-IL6 and CRP as well as chemokines CCL22, IL8, and CCL4. Significant differences between groups are indicated as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. All of the bar graphs show median values with interquartile ranges (IQR).

FIGURE 2

Pregravid obesity alters frequencies of circulating CD4+ T-cells and their responses to stimulation. (A) Changes in percentages of circulating naïve and memory CD4+ T-cells subsets measured by flow cytometry and (B) their homeostatic proliferation. (C) Changes in frequency of IL17 and IL4 secreting CD4+ T-cells following in vitro CD3/CD28 activation. Significant differences between groups are indicated as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. All of the bar graphs show median values with interquartile ranges (IQR).

FIGURE 3

Maternal obesity establishes a hyper-responsive phenotype in circulating monocytes. (A) Changes in the frequency of circulating CD14+ monocytes, CD56+ NK cells, and DCs (B) Relative frequencies of myeloid and plasmacytoid DCs. (C) Clustered heatmap of cytokine, chemokine, and growth factor responses following stimulation with TLR2 (Pam3CSK4), 3 (PolyI:C), 4 (LPS), 7 (Imiquimod), and 9 (ODN2216) agonists. Responses are reported as scaled post stimulatory concentrations in pg/mL. Significant differences between groups are indicated as follows: ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001. All of the bar graphs show median values with inter quartile ranges (IQR).

FIGURE 4

Obesity during pregnancy alters the placental transcriptome. (A) Principal Component Analysis of absolute gene expression of 500 most variable genes. (B) Functional enrichment of the 500 variable genes evaluated using DAVID. Numbers adjacent to the bar represent number of genes mapping to the gene ontology (GO) term. (C) Pie chart denoting number of differentially expressed genes (DEG) identified by edgeR with at least two-fold change in expression and FDR controlled at 5%. (D) Functional enrichment of 110 DEG using DAVID. Numbers adjacent to the bar represent number of DEGs mapping to the GO term (FDR < 5%). (E) Heatmap of genes mapping to disease terms “Immune System Diseases” and “Pregnancy Complications (FDR < 5%) (See Supplementary Figure 4A).

FIGURE 5

Pregravid obesity is not associated with structural changes in its placental microbiome. (A) Median relative abundance of microbial communities at the phylum level reported by QIIME. No significant differences were observed. (B) Differences in the relative abundance of Bacteroides detected by both edgeR and Linear Discriminant Analysis (LDA) (p < 0.01). (C) LDA of PICRUSt-predicted KEGG functions of microbial communities in the placentas from lean and obese subjects identified using LEfSe. Significant differences between groups are indicated as follows: ^∗∗p < 0.01.