RNA Sequencing Reveals Diverse Functions of Amniotic Fluid Neutrophils and Monocytes/Macrophages in Intra-Amniotic Infection

Abstract

Intra-amniotic infection, the invasion of microbes into the amniotic cavity resulting in inflammation, is a clinical condition that can lead to adverse pregnancy outcomes for the mother and fetus as well as severe long-term neonatal morbidities. Despite much research focused on the consequences of intra-amniotic infection, there remains little knowledge about the innate immune cells that respond to invading microbes. We performed RNA-seq of sorted amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection to determine the transcriptomic differences between these innate immune cells. Further, we sought to identify specific transcriptomic pathways that were significantly altered by the maternal or fetal origin of amniotic fluid neutrophils and monocytes/macrophages, the presence of a severe fetal inflammatory response, and pregnancy outcome (i.e., preterm or term delivery). We show that significant transcriptomic differences exist between amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection, indicating the distinct roles these cells play. The transcriptome of amniotic fluid immune cells varies based on their maternal or fetal origin, and the significant transcriptomic differences between fetal and maternal monocytes/macrophages imply that those of fetal origin exhibit impaired functions. Notably, transcriptomic changes in amniotic fluid monocytes/macrophages suggest that these immune cells collaborate with neutrophils in the trafficking of fetal leukocytes throughout the umbilical cord (i.e., funisitis). Finally, amniotic fluid neutrophils and monocytes/macrophages from preterm deliveries display enhanced transcriptional activity compared to those from term deliveries, highlighting the protective role of these cells during this vulnerable period. Collectively, these findings demonstrate the underlying complexity of local innate immune responses in women with intra-amniotic infection and provide new insights into the functions of neutrophils and monocytes/macrophages in the amniotic cavity.

Keywords: Chorioamnionitis; Fetal inflammatory response; Funisitis; Innate immunity; Microbial invasion of the amniotic cavity; Pregnancy; Preterm; Transcriptome.

Fig. 1

Transcriptomic differences between amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection. a Experimental design showing the FACS of amniotic fluid neutrophils (n = 6) and monocytes/macrophages (n = 6) for RNA-seq. b Volcano plot showing the DEGs between amniotic fluid neutrophils and monocytes/macrophages. c Hierarchical dendrogram showing KEGG pathway impact analysis between amniotic fluid neutrophils and monocytes/macrophages. Pink circle indicates the most significantly affected pathway, and the lightest blue circles indicate the least significantly affected pathways. d Predicted activated and inhibited upstream regulators for DEGs in amniotic fluid neutrophils compared to monocytes/macrophages. e, f GO analysis using genes significantly upregulated (e) or genes significantly downregulated (f) in amniotic fluid neutrophils compared to monocytes/macrophages. The top 10 significantly affected biological processes from each set of DEGs are shown. FC, fold change; FACS, fluorescence-activated cell sorting; DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Fig. 2

Transcriptomic differences between amniotic fluid neutrophils and monocytes/macrophages of predominantly fetal or maternal origin. a Experimental design demonstrating the transcriptomic comparison between amniotic fluid neutrophils or monocytes/macrophages of predominantly fetal and maternal origin. b Table and pie charts representing the maternal (neutrophils, n = 4; monocytes/macrophages, n = 3) or fetal (neutrophils, n = 2; monocytes/macrophages n = 3) origins of amniotic fluid neutrophils and monocytes/macrophages determined using DNA fingerprinting. c, d Volcano plots showing the differentially expressed genes among amniotic fluid neutrophils (c) and monocytes/macrophages (d) of fetal origin compared to those of maternal origin. M, predominantly maternal origin; F, predominantly fetal origin; FC, fold change.

Fig. 3

Transcriptomic differences between amniotic fluid monocytes/macrophages of predominantly fetal or maternal origin. a pORA and total pAcc showing KEGG pathway impact analysis between predominantly fetal (n = 3) and maternal (n = 3) amniotic fluid monocytes/macrophages. Significantly impacted pathways are shown in red. b Predicted activated and inhibited upstream regulators of DEGs in amniotic fluid monocytes/macrophages of predominantly fetal origin compared to those of predominantly maternal origin. c GO analysis using genes significantly downregulated in amniotic fluid monocytes/macrophages of predominantly fetal origin compared to those of predominantly maternal origin. The top 10 significantly enriched biological processes are shown. d STRING analysis showing a subset of genes downregulated in amniotic fluid monocytes/macrophages of predominantly fetal origin compared to those of predominantly maternal origin, represented as nodes in the network. The edges between nodes represent high-confidence protein-protein interactions for the corresponding genes. Red, “response to stimulus” network; blue, “developmental process” network; green, “cellular component organization” network; pORA, plot based on gene overrepresentation; pAcc, pathway accumulation; DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Fig. 4

Transcriptomic differences between amniotic fluid neutrophils or monocytes/macrophages from cases of severe and mild fetal inflammatory response. a Experimental design demonstrating the transcriptomic comparison of amniotic fluid neutrophils or monocytes/macrophages between cases of mild (n = 2) and severe (n = 4) fetal inflammatory response. b, c Representative images of umbilical cord hematoxylin and eosin (H&E) staining showing mild (b) and severe (c) fetal inflammatory response indicated by leukocyte infiltration in the Wharton's jelly localized near the umbilical artery (arrows). Magnification = ×10, scale bar = 100 μm. d, e Volcano plots showing the differentially expressed genes among amniotic fluid neutrophils (d) and monocytes/macrophages (e) from cases of severe fetal inflammatory response compared to those from cases of mild fetal inflammatory response. FC, fold change.

Fig. 5

Transcriptomic differences between amniotic fluid monocytes/macrophages from cases of severe and mild fetal inflammatory response. a pORA and total pAcc showing KEGG pathway impact analysis between amniotic fluid monocytes/macrophages from cases of severe fetal inflammatory response (n = 4) compared to those from cases of mild fetal inflammatory response (n = 2). Significantly impacted pathways are shown in red. b Predicted activated and inhibited upstream regulators of DEGs in amniotic fluid monocytes/macrophages from cases of severe fetal inflammatory response compared to those from cases of mild fetal inflammatory response. c GO analysis using genes significantly upregulated in amniotic fluid monocytes/macrophages from cases of severe fetal inflammatory response compared to those from cases of mild fetal inflammatory response. The top 10 significantly enriched biological processes are shown. d STRING analysis showing a subset of genes upregulated in amniotic fluid monocytes/macrophages from cases of severe fetal inflammatory response compared to those from cases of mild fetal inflammatory response, represented as nodes in the network. The edges between nodes represent high-confidence protein-protein interactions for the corresponding genes. Red, “cytokine-mediated signaling pathway” network; blue, “regulation of cell population proliferation” network; and green, “extracellular matrix assembly” network; pORA, plot based on gene overrepresentation; pAcc, pathway accumulation; DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Fig. 6

Transcriptomic differences between amniotic fluid neutrophils and monocytes/macrophages from preterm or term deliveries. a Experimental design demonstrating the transcriptomic comparison of amniotic fluid neutrophils and monocytes/macrophages between preterm (n = 4) and term (n = 2) deliveries. b, c Volcano plots showing the differentially expressed genes in amniotic fluid neutrophils (b) and monocytes/macrophages (c) between preterm and term deliveries. d, e pORA and total pAcc showing KEGG pathway impact analysis of amniotic fluid neutrophils (d) and monocytes/macrophages (e) between preterm and term deliveries. Significantly impacted pathways are shown in red. f GO analysis using genes significantly upregulated in amniotic fluid neutrophils from preterm deliveries compared to those from term deliveries. The top 10 enriched biological processes are shown. g STRING analysis showing a subset of genes upregulated in amniotic fluid neutrophils from preterm deliveries compared to those from term deliveries, represented as nodes in the network. The edges between nodes represent high-confidence protein-protein interactions for the corresponding genes. Red, “Mitochondrial gene expression” network; blue, “organonitrogen compound metabolic process” network; green, “small molecule metabolic process” network; FC, fold change; NS, not significant; pORA, plot based on gene overrepresentation; pAcc, pathway accumulation; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.