Hofbauer cells and fetal brain microglia share transcriptional profiles and responses to maternal diet-induced obesity

Abstract

Maternal immune activation is associated with adverse offspring neurodevelopmental outcomes, many mediated by in utero microglial programming. As microglia remain inaccessible throughout development, identification of noninvasive biomarkers reflecting fetal brain microglial programming could permit screening and intervention. We used lineage tracing to demonstrate the shared ontogeny between fetal brain macrophages (microglia) and fetal placental macrophages (Hofbauer cells) in a mouse model of maternal diet-induced obesity, and single-cell RNA-seq to demonstrate shared transcriptional programs. Comparison with human datasets demonstrated conservation of placental resident macrophage signatures between mice and humans. Single-cell RNA-seq identified common alterations in fetal microglial and Hofbauer cell gene expression induced by maternal obesity, as well as sex differences in these alterations. We propose that Hofbauer cells, which are easily accessible at birth, provide novel insights into fetal brain microglial programs, and may facilitate the early identification of offspring vulnerable to neurodevelopmental disorders in the setting of maternal exposures.

Keywords: Hofbauer cells; Single-cell RNA sequencing; microglia; neuroimmune; obesity; sex differences.

Figure 1.. Placental macrophages are yolk sac derived

A. Schematic of fetal yolk sac macrophage labeling. Briefly, male Csf1R-Cre^ER;tdTomato^f/f mice were timedmated to female tdTomato^f/f mice. Pregnant females were injected with 4-hydroxytamoxifen (4-OHT) at gestational day 8.5. Embryos were collected at embryonic day 17.5. B. Percent of macrophages (Iba1+ cells) labeled with tdTomato in embryonic placenta and hippocampus following 4-OHT administration at e8.5. Open circles represent individual female embryos and closed diamonds represent individual male embryos (n=4 litters). Pl = placenta; Br = brain (hippocampus) C–C’. Representative images of Iba1 and tdTomato in control (C) and reporter (C’) placenta from e17.5 embryos. D–D’. Representative images of Iba1 and tdTomato in control (D) and reporter (D’) hippocampus from e17.5 embryos. Arrowheads indicate double-positive (Iba1+ tdTomato+) macrophages/microglia in reporter tissue. Scale 50μm, inset scale 10μm.

Figure 2.. Fetal placental and brain macrophages are heterogeneous populations with shared cluster-specific signatures.

A (left). Uniform Manifold Approximation and Projection (UMAP) visualization of Cd11b+ macrophage enriched fetal brain microglia/monocyte cells reveals 8 distinct clusters. Unless otherwise specified, clusters are named as “cell-type-prefix_top-marker-gene”. Mg: microglia; Mono_FBr: fetal brain monocytes; YSI: yolk sac imprint. A (right). Cluster-average expression of the top 3 marker genes for each cluster (right), dot size indicates the percent of cells expressing the given gene, color intensity represents the scaled average gene expression. B (left). UMAP visualization of Cd11b+ macrophage-enriched placenta macrophage/monocyte populations reveals 4 fetal and 6 maternal clusters. Fetal clusters were determined by significantly higher expression of Y chromosome markers Eif2s3y and Ddx3y relative to expression of X chromosome marker Xist (Supplemental Figure 2D–E). Unless otherwise specified, clusters are named as “cell type prefix_top marker gene”. HBC: Hofbauer cell; PAMM: placenta-associated maternal monocyte/macrophages; Mono_FPl: fetal placental monocytes. Color scheme indicates cell origin (purple=fetal macrophages; red=maternal; gray=fetal monocytes). B (right). Cluster-averaged gene expression of the top 3 marker genes for each cluster. Dot size indicates the percent of cells expressing the given gene. C. Module score for yolk sac-derived macrophages and embryonic liver monocytes^, D.Spearman correlation coefficients of cluster-averaged gene expression between brain and placenta clusters shown in A, B. For each brain cluster, the placenta cluster with the highest correlation is shown with a dot. E. Expression levels of canonical microglia (top, blue) and Hofbauer cell marker genes (bottom, orange) F. UMAP visualization including both brain and placenta clusters shown in A,B shows similarity across brain-placenta compartments. G. Gene Ontology (GO) Biological Process enrichment analysis for select Microglia and HBC cluster marker genes. The terms displayed here were curated from among the top 25 most significant GO terms, selecting the processes most relevant to macrophage function, and reducing redundancy. Gene Count gives the number of genes in the query set that are annotated by the relevant GO category. GO terms with an adjusted p-value < 0.05 were considered significant. Full results shown in SI table 2.

Figure 3.. Maternal obesity alters gene expression in fetal microglia and placental macrophages.

A. Fraction of significantly DEG in obesity-exposed fetal microglia that are also significantly differentially expressed in obesity-exposed placental Hofbauer cells. Differentially expressed genes (DEG) between offspring of obese and control dams are shown. DEG with adjusted p-value <0.05, abs(log2FC)>0.25 are considered significant. B. Network plot of the top 15 Gene Ontology Biological Processes enriched in the DEG in fetal MgYSI_Pf4, shown in both MgYSI_Pf4 and placental HBC_Cd72. Nodes correspond to enriched GO categories, node size is proportional to number of genes and edges thickness is proportional to the number of overlapping genes between the two categories. C. Enriched Gene Ontology biological processes in the DEG of obesity-exposed microglia and Hofbauer cells. Select results shown, full results shown in SI Table 4. Shading corresponds to manual grouping of GO categories. Gene Count gives the number of genes in the query set that are annotated by the relevant GO category. D. Up- and downregulation of DEG implicated in ATP metabolism, response to oxidative stress, and regulation of inflammatory response in microglia and Hofbauer cells. DEG fold changes in obesity-exposed microglia and HBCs for three significantly enriched GO biological processes relevant to microglial function.* DEG with adjusted p-value < 0.05. E. Select IPA Canonical Pathways enriched in obesity-exposed microglial and Hofbauer cell cluster DEG. Positive z-score means pathway is activated in fetal macrophages of obese dams, and negative z-score means pathway is suppressed. F. Up- and downregulation of genes in fetal macrophages of obese dams for two significantly activated or inhibited IPA Canonical Pathways relevant to microglial function. DEG fold changes depicted. * DEG with adjusted p-value < 0.05.

Figure 4.. Sex differences in the response of microglia and placental macrophages to maternal diet-induced obesity.

Differentially-expressed genes (DEG) between microglia and placental macrophages in fetuses of obese and control dams are shown, calculated separately for male and female offspring. A. The number of DEG between obesity-exposed versus control macrophages/monocytes is shown as a bar, colored by cell type, and shaded according to sex. * indicates p<0.05 difference between male and female as given by a 2x2 contingency table Fishers-exact test. B (left). The table considers the total number of DEG in yolk-sac imprint microglia (MgYSI) and HBC cell types, that are DEG in male and/or female cells. Genes are categorized as either having a fold change in the same direction (sex-consistent) or different direction (sex-dimorphic) in males and females. B (right). GO Biological process enrichment results for the set of sex-dimorphic and sex-consistent DEG. C. Comparison of the top 12 IPA Canonical Pathways that are enriched in both Mg and HBC. The sum of the Z-score from the female analysis and male analysis is shown, with positive Z-score indicating activation and negative Z-score indicating inhibition. (Full results shown in Supplemental Table 5) D. Fold changes in fetal macrophages in offspring of obese versus control dams for the IPA Canonical Pathway NeuroInflammatory Response Pathway for Males and Females.* DEG with adjusted p-value < 0.05.

Figure 5.. Comparison of maternal and fetal placental macrophages

A. Cluster-average expression of top marker genes distinguishing PAMM from HBC. Dot size indicates the percent of cells expressing the given gene. B. Gene Ontology (GO) Biological Process enrichment analysis for for HBC and PAMM marker genes. Gene Count gives the number of genes in the query set that are annotated by the relevant GO category. GO terms with an adjusted p-value < 0.05 were considered significant. C. Cluster-average expression of genes that distinguish PAMM and HBC populations in human studies. D. Detailed expression of genes Folr2 and Mrc1 that distinguish PAMM and HBC populations in Thomas 2020. E. Markers with valid antibodies that best separate HBC, placental monocyte and PAMM populations.