Disrupted metabolic signatures in amniotic fluid associated with increased risk of intestinal inflammation in cesarean section offspring

Abstract

Introduction: Children born by cesarean section (CS) are at a greater risk of inflammatory bowel disease (IBD). However, the mechanisms underlying the association are not yet well understood. Herein, we investigated the impact of CS delivery on colonic inflammation and mechanisms underlying these effects in offspring.

Methods: CS mice model and dextran sulfate sodium (DSS)-induced colitis model were constructed and used to analyze the impact of CS on the development of colitis. Colonic tight junction markers and epithelium differentiation markers were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Levels of zonulin in serum were detected by enzyme-linked immunosorbent assay (ELISA). Immune cells in colon were analyzed by flow cytometry. Metabolic profiling between human vaginal delivery (VD) and CS AF were analyzed by using mass spectrometry. Transcriptome changes between VD AF- and CS AF-treated human intestine epithelial cells were analyzed by RNA-sequencing. A multi-omics approach that integrated transcriptomics with metabolomics to identify the pathways underlying colonic inflammation associated with delivery modes. Then, the identified pathways were confirmed by immunoblotting and ELISA.

Results: Mice pups delivered by CS exhibited a defective intestinal homeostasis manifested by decreased expression of tight junction markers of ZO-1 and Occludin in the colons, increased levels of zonulin in serum and dysregulated expression of intestinal epithelium differentiation markers of Lysozyme, Mucin2, and Dipeptidyl peptidase-4. CS pups were more susceptible to DSS-induced colitis compared to VD pups. The proportion of macrophage, dendritic cells (DCs), and natural killer cells (NKs) in the colons were altered in an age-dependent manner compared with pups born naturally. The metabolites in AF differed between CS and VD cases, and the CS AF-induced differentially expressed genes (DEGs) were significantly enriched in pathways underlying IBD. Signal transducer and activator of transcription 3 (STAT3) signaling was downregulated in NCM460 intestinal epithelial cells by CS AF compared to VD AF and in colon of CS pups compared to VD pups. Deficiency in metabolites like vitamin D2 glucosiduronate in CS AF may attribute to the risk of inflammatory intestine through STAT3 signaling.

Conclusion: Our study provides a novel insight into the underlying mechanisms of CS-associated intestinal inflammation and potential prevention strategies.

Keywords: amniotic fluid; cesarean section; intestinal inflammation; multi-omics; vaginal delivery.

Figure 1

Body weight, the length of colons, and expression of intestinal epithelium differentiation genes in a murine model. Litter size (A) and the survivability of the fostered pups (B) between the cesarean section (CS) group (n = 6) and the vaginal delivery (VD) group (n = 6). (C), body weight between pups delivered by CS (n = 6) and VD (n = 6) at day 0, day 10, day 20, and day 30 after birth. (D), Colon length for CS-delivered (n = 6) compared with VD (n = 6) pups at day 10, day 20, and day 30 after birth. Relative mRNA expression of intestinal epithelium differentiation biomarkers, Lysozyme (Lyz1, E), Mucin2 (Muc2, F), Chromogranin-A (ChgA, G), Dipeptidyl peptidase-4 (Dpp4, H), and Villin (I) in colons of CS delivered pups (n = 6) compared with VD pups (n = 6). Significance was calculated with a two-tail student’s t test. Error bars represent SEM. *p < 0.05, **p < 0.01, ns indicates no significance.

Figure 2

Pups delivered by CS exhibited a defective epithelial barrier and an increased susceptibility to DSS-induced colitis. Relative mRNA expression of tight junction protein 1 (ZO-1, (A) and Occludin (B) in colons of CS delivered pups compared with VD pups. (C), Concentrations of zonulin were quantified by ELISA in serum of VD (n = 6) and CS (n = 6) pups at day 10 and day 20. (D), Protein expression of ZO-1 and Occludin in CS amniotic fluid (AF)-treated NCM460 cells. (E) Weight loss compared to day 0 after administration of DSS in VD (n = 5) or CS (n = 5) pups for 7 days. (F) Disease activity index (DAI) scores for mice in VD and CS groups. (G) Representative H&E-stained colon sections of VD and CS mice after administration of DSS. (H) Representative images of colons of VD and CS mice after administration of DSS. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Cesarean section altered the proportions of intestinal immune cells in mice offspring. (A), Representative flow cytometric analysis of macrophage from lymphocyte of colons from CS and VD pups at day 10, day 20, and day 30 after birth (n = 6). (B), Representative flow cytometric analysis of dendritic cells from lymphocyte of colons from CS and VD pups at day 10, day 20, and day 30 after birth (n = 6). (C), Representative flow cytometric analysis of NK cells from lymphocyte of colons from CS and VD pups at day 10, day 20, and day 30 after birth (n = 6). Significance calculated with a two-tail student’s t test. Error bars represent SEM. *p < 0.05, **p < 0.01.

Figure 4

Metabolomics analysis for AF metabolites profiles in CS women compared with VD women. PCA (A) and OPLS-DA (B) score plots for differentiating the metabolites in each group by GC-MS/MS. (C), Volcano plot showing the differentially accumulated metabolites in the CS AF (n = 7) and VD AF (n = 7) by GC-MS/MS. PCA (D) and OPLS-DA (E) score plots for differentiating the metabolites in each group by LC-MS/MS. (F), Volcano plot showing the differentially accumulated metabolites in the CS AF and VD AF by LC-MS/MS. (G), Hierarchical clustering analysis (HCA) of the metabolites that differed between the CS (n = 7) and VD (n = 7) subjects. (H), Enriched KEGG pathways in CS AF group compared with VD AF group. (I), Abundance comparisons of metabolites between CS AF group and VD AF group. Error bars represent SEM. **p < 0.01, ***p < 0.001.

Figure 5

Transcriptome analyses of human intestinal epithelial NCM460 cells after CS AF stimuli. (A), Correlation heatmap analysis between CS AF-treated cells (n = 3) and VD AF-treated cells (n = 3). (B), PCA score plots for differentiating the genes in each group. (C), Volcano plot showing the differentially expressed genes (DEGs) in the CS AF-treated group (n = 3) and VD AF-treated cells (n = 3). Red dots represent the DEGs up-regulated in the CS AF-treated cells, and green dots represent the DEGs down-regulated in the CS AF-treated cells. (D), KEGG classification analysis of DEGs. (E), Validation of DEGs between CS AF-treated group (n = 3) and VD AF-treated group (n = 3) using RT-qPCR. The change in fold-expression was calculated by the 2-∆∆CT method using GAPDH as a reference gene. Error bars represent SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

Correlation analyses of differential transcripts and metabolites. (A), Heat map comparing the expression of DEGs between CS AF- (n = 3) and VD AF-treated (n = 3) human intestinal epithelial NCM460 cells. In the heat map, yellow represents high expression, and blue represents low expression. (B), Gene Set Enrichment Analysis (GSEA) for the potential altered pathways between CS AF- and VD AF-treated NCM460 cells. (C), KEGG pathway enrichment analysis indicated that the lysosome, glycosaminoglycan degradation, and complement and coagulation cascades were significantly enriched by DEGs. (D), Validation of DEGs involved in above pathways between CS AF- (n = 3) and VD AF-treated (n = 3) group by using RT-qPCR. The change in fold-expression was calculated by the 2-∆∆CT method using GAPDH as a reference gene. Error bars represent SEM. **p < 0.01; ***p < 0.001. (E), Correlation-based joint analysis between the representative differentially expressed metabolites (DEMs) and DEGs.

Figure 7

CS abrogated IL-6 signaling through inhibition of both STAT3 protein and STAT3 phosphorylation. (A), Relative mRNA expression of STAT3 in CS AF-treated (n = 3) NCM460 intestinal epithelial cells compared to VD AF-treated group (n = 3); (B), Immunoblotting analyses of the expression of STAT3 and p-STAT3 in colon of CS pups; (C), IL-6 levels in AF samples of CS women (n = 7) compared to the VD women (n = 7); (D), IL-10 and TNF-α levels in serum of CS-born cord blood from CS women (n = 7) compared to VD women (n = 7). Correlation analyses of AF vitamin D2 glucosiduronate and levels of IL-10 (E) and TNF-α (F) in the cord blood. Significance calculated with a two-tail student’s t test. Error bars represent SEM. *p < 0.05, **p < 0.01.