Molecular phenotype of monocytes at the maternal-fetal interface

Abstract

Objective: The purpose of this study was to gain insight into the pathways that are associated with inflammation at the maternal-fetal interface. This study examined the molecular characteristics of monocytes that were derived from the maternal circulation and the placenta of obese women.

Study design: Mononuclear cells were isolated from placenta, venous maternal, and umbilical cord blood at term delivery; activated monocytes were separated with CD14 immunoselection. The genotype and expression pattern of the monocytes were analyzed by microarray and real-time reverse transcriptase-polymerase chain reaction.

Results: The transcriptome of the maternal blood and placental CD14 monocytes exhibited 73% homology, with 10% (1800 common genes) differentially expressed. Genes for immune sensing and regulation, matrix remodeling, and lipid metabolism were enhanced 2-2006 fold in placenta, compared with maternal monocytes. The CD14 placental monocytes exhibited a maternal genotype (9% DYS14 expression) as opposed to the fetal genotype (90% DYS14 expression) of the trophoblast cells.

Conclusion: CD14 monocytes from the maternal blood and the placenta share strong phenotypic and genotypic similarities with an enhanced inflammatory pattern in the placenta. The functional traits of the CD14 blood and placental monocytes suggest that they both contribute to propagation of inflammation at the maternal-fetal interface.

Figure 1. global pattern of gene expression of monocytes from maternal blood and the placenta

The whole transcriptome of CD14 monocytes from placenta (22794 genes) and maternal blood (23750 genes) was obtained by screening oligonucleotide arrays containing a total of 47,000 transcripts. Filtering of raw data identified 16739 genes common to the 2 data sets. 1800 genes having a false discovery rate <0.05 and a fold change >2 were considered differentially expressed between groups.

Figure 2. Flow cytometry analysis of isolated placental and maternal monocytes

Representative double color fluorescent-activated cell sorter analysis using CD14 - PE and CD68-FITC labeled antibodies. Left panel : dot plot side scattered (SSC) of isolated placental double staining with CD14 and CD68 shows wide distribution and size of CD14 positive cells. Right panel: double staining of maternal mononuclear blood cells. Six cell preparations from independent placentas and blood of women with BMI >25 were analyzed.

Figure 3. principal component analysis (PCA) of the systemic mononuclear cells and placental macrophages

Three dimensional scatter plots PCA analysis of the transcriptome of CD14 immnuno-selected monocytes from maternal blood (MMNC) and the placenta (PMNC) and non immuno-selected monocytes from maternal and umbilical blood. A: Analysis of full data set shows a segregation pattern between the CD14 immunoselected MMNC and PMNC cells and the non immunoselected cells (mat-MNC and fet-MNC) closely grouped together. B: Analysis of the 200 top regulated genes showed the expression trends in MMNC and PMNC are superimposable. Each knot represents one microarray with a pool of independent cell samples obtained from 4–6 subjects.

Figure 4. Hierarchical clustering of the top 200 most induced genes in placental macrophages of obese women

Clustering generated heat map identified four primary functional clusters based on biological annotation of the genes up-regulated in CD14 immunoselected compared to unselected monocytes cells from maternal and cord blood.

Figure 5. Differential expression of genes regulating immune response in monocytes of maternal blood and the placenta

GO annotation of immune function and pathways studio significantly up-regulated identified 150 genes based on p values < 10.E⁻⁵.

Figure 6. gender characterization of tissue resident macrophages

Expression of DYS14 markers for the Y chromosome was quantitated by RT-PCR. PMNC of male fetuses exhibited a low Y copy numbers (p<0.001) compared to cord blood mononuclear cells. The level of Y expression was undetectable in MMNC of maternal blood. The expression of the house keeping gene glyceraldehyde -3-P(GAPDH) is gender independent i.e. same level in maternal and fetal cells. Results are means ± SEM of 6 independent samples from male fetuses with determinations performed in duplicate. All values were normalized for actin.