Prenatal Maternal Stress Exacerbates Experimental Colitis of Offspring in Adulthood

Abstract

The prevalence of inflammatory bowel disease (IBD) is increasing worldwide and correlates with dysregulated immune response because of gut microbiota dysbiosis. Some adverse early life events influence the establishment of the gut microbiota and act as risk factors for IBD. Prenatal maternal stress (PNMS) induces gut dysbiosis and perturbs the neuroimmune network of offspring. In this study, we aimed to investigate whether PNMS increases the susceptibility of offspring to colitis in adulthood. The related index was assessed during the weaning period and adulthood. We found that PNMS impaired the intestinal epithelial cell proliferation, goblet cell and Paneth cell differentiation, and mucosal barrier function in 3-week-old offspring. PNMS induced low-grade intestinal inflammation, but no signs of microscopic inflammatory changes were observed. Although there was no pronounced difference between the PNMS and control offspring in terms of their overall measures of alpha diversity for the gut microbiota, distinct microbial community changes characterized by increases in Desulfovibrio, Streptococcus, and Enterococcus and decreases in Bifidobacterium and Blautia were induced in the 3-week-old PNMS offspring. Notably, the overgrowth of Desulfovibrio persisted from the weaning period to adulthood, consistent with the results observed using fluorescence in situ hybridization in the colon mucosa. Mechanistically, the fecal microbiota transplantation experiment showed that the gut microbiota from the PNMS group impaired the intestinal barrier function and induced low-grade inflammation. The fecal bacterial solution from the PNMS group was more potent than that from the control group in inducing inflammation and gut barrier disruption in CaCo-2 cells. After treatment with a TNF-α inhibitor (adalimumab), no statistical difference in the indicators of inflammation and intestinal barrier function was observed between the two groups. Finally, exposure to PNMS remarkably increased the values of the histopathological parameters and the inflammatory cytokine production in a mouse model of experimental colitis in adulthood. These findings suggest that PNMS can inhibit intestinal development, impair the barrier function, and cause gut dysbiosis characterized by the persistent overgrowth of Desulfovibrio in the offspring, resulting in exacerbated experimental colitis in adulthood.

Keywords: colitis; early life; microbiota; mucosal barrier; prenatal maternal stress.

Figure 1

Prenatal maternal stress impaired the intestinal development of 3-week-old offspring. (A) Flow diagram of the experimental procedure. (B) Microscopic observation of the intestinal and colonic development of 3-week-old pups with hematoxylin and eosin (HE) staining. (C) Changes in the body weight of offspring mice from week 0 to week 8. (D) Measurement of the villus lengths in the jejunum and ileum and the crypt depths of the colon. PNMS, prenatal maternal stress; DSS, dextran sulfate sodium. Scale bar, 100 μm. Magnification, ×200. *p < 0.05, **p < 0.01. PNMS (n = 6) vs. control (n = 6).

Figure 2

Prenatal maternal stress inhibited the proliferation and differentiation of the intestinal tract in 3-week-old offspring mice. (A) Cell proliferation in the small intestine was assessed through Ki-67 immunohistochemical staining. (B) Paneth cells in the small intestine were evaluated by lysozyme immunohistochemical staining and the quantification of positively stained cells. (C) Relative mRNA expression levels of Muc2 in the colon and defensin, cryptdin, and Reg3γ in the small intestine. (D) Immunohistochemical staining for MUC2 mucin (brown) in colon tissue section. (E) Periodic acid–Schiff (PAS) staining for goblet cells in colon tissue. Quantification of positive cells per villus as shown by the histogram. At least 100 villi were randomly selected for each sample for calculation. PNMS, prenatal maternal stress. Scale bar, 100 μm. Magnification, ×200. *p < 0.05, **p < 0.01, ^nsp > 0.05. PNMS (n = 6) vs. control (n = 6).

Figure 3

Prenatal maternal stress impaired the intestinal barrier and induced colonic mucosal low-grade inflammation in 3-week-old offspring. (A) Intestinal permeability was measured through fluorescein isothiocyanate–dextran (FITC-D) assay. (B) Quantification of the relative mRNA expression levels of ZO-1, occludin, and claudin 3. (C) Western blot bands and relative quantification of claudin 3 in the colon. (D) Immunofluorescence staining of sIgA (green) and nuclear staining with DAPI (blue) were visualized via a fluorescent microscope. (E) Number of sIgA-positive cells per intestinal villus. (F) mRNA levels of the inflammatory cytokines in the colon. PNMS, prenatal maternal stress; sIgA, secretory immunoglobulin (A) Scale bar, 100 μm. Magnification, ×200. *p < 0.05, **p < 0.01, ^nsp > 0.05. PNMS (n = 6) vs. control (n = 6).

Figure 4

Prenatal maternal stress induced dysbiosis of the gut microbiota in 3-week-old offspring. (A) Venn diagram of the operational taxonomic units (OTUs). (B) Microbial composition at the level of bacterial phylum. (C, D) Cladogram and the linear discriminant analysis (LDA) scores showed significant bacterial differences between the two groups by linear discriminant analysis effect size (LEfSe). (E) Fluorescence in situ hybridization (FISH) detection of Desulfovibrio in colon tissues of the control and PNMS offspring using a fluorescein isothiocyanate (FITC)-conjugated universal bacterial 16S rDNA-directed oligonucleotide probe (EUB338, green) and Cy5-conjugated Desulfovibrio specific probe (red). PNMS, prenatal maternal stress. Scale bar, 50 μm. Magnification, ×400. Control-3w (n = 15) vs. PNMS-3w (n = 15).

Figure 5

Fecal microbiota from PNMS offspring impaired the gut barrier function and induced low-grade inflammation. (A) Experimental workflow. (B) Detection of claudin 3 protein by Western blotting. (C) Quantification of the relative mRNA expression levels of claudin 3, occludin, and Muc2. (D) mRNA levels of the inflammatory cytokines in the colon. (E–I) Treatment of Caco-2 cells with the fecal bacterial solution from the control and 3-week-old PNMS offspring mice alone or in combination with adalimumab. (E, F) Expressions of TNF-α and IL-1β assessed by quantitative PCR (qPCR). (G, H) Expression of claudin 3 assessed by Western blot and qPCR. (I) Fluorescence images for ZO-1. FMT, fecal microbiota transplantation; PNMS, prenatal maternal stress; Ada, adalimumab. Scale bar, 50 μm. Magnification, ×400. *p < 0.05, **p < 0.01, ***p < 0.001. FMT-control (n = 5) vs. FMT-PNMS (n = 5). ns, P < 0.05.

Figure 6

Prenatal maternal stress has a long-term impact on the gut microbiota in the offspring. (A) Venn diagram of the operational taxonomic units (OTUs). (B) Gut microbiota composition at the phylum level. (C, D) Linear discriminant analysis (LDA) scores and the cladogram showed significant bacterial differences between the two groups by linear discriminant analysis effect size (LEfSe). (E, F) The gut microbiota of the 3- and 8-week-old offspring were put together for weighted Unifrac principal coordinate analysis (PCoA) and weighted Unifrac analysis of similarities (ANOSIM). PNMS, prenatal maternal stress. Control-3w (n = 15) vs. PNMS-3w (n = 15) vs. Control-8w (n = 8) vs. PNMS-8w (n = 8).

Figure 7

Prenatal maternal stress aggravated the severity of experimental colitis in adult mice. (A) Disease activity index (DAI) scores during the process. (B) Colon appearance after treatment. (C) Colon lengths. (D) Histopathological changes of the colon section detected by hematoxylin and eosin (HE) staining. (E) Histological scores of colonic inflammation. (F) Ratio of spleen weight to body weight. (G) Fluorescein isothiocyanate–dextran (FITC-D) intestinal permeability assay. (H) Relative mRNA expressions of the inflammation factors in the colon. PNMS, prenatal maternal stress; DSS, dextran sulfate sodium. Scale bar, 100 μm. Magnification, ×200. *p < 0.05, **p < 0.01, ***p < 0.001. Control+Water (n = 8), PNMS+Water (n = 8), Control+DSS (n = 10), PNMS+DSS (n = 10). ns, P < 0.05.

Figure 8

The relative abundance of Desulfovibrio increased in PNMS offspring mice and was associated with intestinal inflammation. (A) Fluorescence in situ hybridization (FISH) detection of Desulfovibrio in colon tissues in 8-week-old colitis model offspring. A fluorescein isothiocyanate (FITC)-conjugated universal bacterial 16S rDNA-directed oligonucleotide probe (EUB338, green) and a Cy5-conjugated Desulfovibrio specific probe (red) were applied. (B, C) Heatmap of Spearman’s correlations between the differential bacteria and markers of intestinal inflammation in 3- and 8-week-old offspring. DSS, dextran sulfate sodium; PNMS, prenatal maternal stress. Scale bar, 50 μm. Magnification, ×400. ⁺p < 0.05, *p < 0.01 (Spearman’s test).

Figure 9

Schematic overview of how prenatal maternal stress increases the susceptibility to colitis in adult offspring. Prenatal maternal stress can impair the intestinal development, disturb the mucosal barrier function, induce low-grade inflammation, alter the composition of the microbiota in offspring, and increase the susceptibility to colitis in adulthood.