Obesity in pregnancy stimulates macrophage accumulation and inflammation in the placenta

Abstract

Obesity and pregnancy are associated with a combination of insulin resistance and inflammatory changes which exacerbate in combination. Based on the similarity between the inflammatory transcriptomes of adipose tissue and placenta, we hypothesized that the placenta develops exaggerated inflammation in response to obesity. The aim of this study was to characterize placental inflammatory mediators and macrophage accumulation in relation to peripheral inflammation in obesity. Placental macrophages and maternal peripheral blood mononuclear cells (PBMC) from 20 obese and 15 lean women were functionally and phenotypically characterized using immunohistochemistry, flow cytometry and expression for macrophage markers and inflammatory cytokines. The number of resident CD68+ and CD14+ cells was increased 2-3 fold in the placenta of obese as compared to lean women. The macrophage population was characterized by a marked phenotypic heterogeneity with complex subsets of CD14+, CD68+ and CD11b+ (mac-1) cells and by an increased expression of the pro-inflammatory cytokines IL-1, TNF-alpha, IL-6. Placental inflammation was associated with an activation of PBMC gene expression with an increase in the monocyte differentiation and maturation markers CD14 and CD68 in maternal but not fetal PBMC. The inflammatory changes were associated with higher plasma concentrations of C-reactive protein and IL-6 in obese compared to lean women. In conclusion, the chronic inflammation state of pre-gravid obesity is extending to in utero life with accumulation of a heterogeneous macrophage population and pro-inflammatory mediators in the placenta. The resulting inflammatory milieu in which the fetus develops may have critical consequences for short and long term programming of obesity.

Figure 1. Systemic inflammatory status in obese pregnant women

Peripheral blood mononuclear cells (PBMC) were separated from maternal and umbilical venous plasma samples obtained at the time of elective cesarean delivery. Aliquots were processed for total RNA purification. Expression of inflammatory markers was assessed by real-time RT-PCR. Results normalized for actin are expressed as fold change in obese vs lean women. Results are given as mean ± SE of duplicate determinations with 19 obese and 15 lean women. *: p <0.001. White bars: lean, grey bars: obese.

Figure 2. Accumulation of CD68+ and CD14+ macrophages in placenta of obese women

Upper left panel: histochemical analysis of whole placental villous sections from a representative lean (pre-gravid BMI:22.5) woman shows rare CD68⁺ (A) and CD14⁺ (B) staining. By contrast, the sections from a representative obese (pre-gravid BMI: 31.7) woman show an increased number of dark brown CD68⁺ (C) and CD14⁺ (D) stained cells. Original magnification A-D: x20. Right panel: quantification of CD68⁺ and CD14⁺ macrophage number in placental sections. Mean ± SE of N = 10 placental sections. *: p < 0.001 obese vs lean. Lower panel: higher magnification showing localization of macrophages (thick arrows) in the villous stroma with no staining in the syncytiotrophoblast outlayer (maternal side) or vascular endothelium (fetal side). Original magnification (x60). Thin arrow: syncytiotrophoblast nuclei, #: fetal blood space, *: maternal blood space. Scale bar indicates 20 μm.

Figure 3. Identification of placental macrophages based on flow cytometry analysis

(A,B) Representative double staining side scatter analysis of isolated cells from placenta of an obese woman (BMI: 31.6) performed with CD14 (red) or CD68 (blue) FITC antibodies and CD133-PE (grey), a marker of trophoblast cells. (C,D) Representative three color analysis of the same cell population performed with CD11b-PE antibody (green) in addition to the three antibodies described in A-B. The turquoise color in panel C indicates that positive cells express both the CD11b and CD68 markers. The yellow color in panel D indicates that positive cells express both the CD11b and CD14 markers. Similar results were obtained from independent analysis of six placenta of obese women. PE: phycoerythrin, FITC: Fluorescein isothiocyanate.

Figure 4. Expression of macrophage markers and inflammary cytokines in CD14+ macrophages from the placenta of obese women

mRNA expression of inflammatory markers was measured in whole placenta villous tissue (white bars) and in isolated placental CD14⁺ macrophages (black bars). Results of duplicate real-time PCR analysis were normalized for actin and expressed as fold change in obese vs lean women. Mean ± SE of duplicate determinations processed from 15 placenta of obese and 15 placentas of lean women. *: p < 0.001 in obese compared to lean women. White bars: lean, grey and block bars: obese.