Chronic stress promotes colitis by disturbing the gut microbiota and triggering immune system response

Abstract

Chronic stress is known to promote inflammatory bowel disease (IBD), but the underlying mechanism remains largely unresolved. Here, we found chronic stress to sensitize mice to dextran sulfate sodium (DSS)-induced colitis; to increase the infiltration of B cells, neutrophils, and proinflammatory ly6C^hi macrophages in colonic lamina propria; and to present with decreased thymus and mesenteric lymph node (MLN) coefficients. Circulating total white blood cells were significantly increased after stress, and the proportion of MLN-associated immune cells were largely changed. Results showed a marked activation of IL-6/STAT3 signaling by stress. The detrimental action of stress was not terminated in IL-6^-/- mice. Interestingly, the composition of gut microbiota was dramatically changed after stress, with expansion of inflammation-promoting bacteria. Furthermore, results showed stress-induced deficient expression of mucin-2 and lysozyme, which may contribute to the disorder of gut microbiota. Of note is that, in the case of cohousing, the stress-induced immune reaction and decreased body weight were abrogated, and transferred gut microbiota from stressed mice to control mice was sufficient to facilitate DSS-induced colitis. The important role of gut microbiota was further reinforced by broad-spectrum antibiotic treatment. Taken together, our results reveal that chronic stress disturbs gut microbiota, triggering immune system response and facilitating DSS-induced colitis.

Keywords: DSS-induced colitis; chronic stress; gut microbiota; immune reaction; mucin-2.

Fig. 1.

Chronic stress accelerated DSS-induced colitis. (A) Body weight of mice in normal and stress groups before DSS and immobility time of mice after 1 mo stress in TST and FST using an EthoVision XT 11.5 system (n = 10). (B) Representative colon pictures and colon length, colon weight, bleeding score, and body weight (n = 9–10). (C) Representative H&E staining, Ki-67 staining, pathological score, and mean positive Ki-67 staining cells counted in 20 crypts (n = 4–5; *P < 0.05, **P < 0.01, and ***P < 0.001). (Scale bars: H&E stain, 100 μm; Ki-67 stain, 50 μm.)

Fig. 2.

Chronic stress disturbed the immune system. (A) Flow cytometric analysis of B cells (CD45⁺, CD19⁺), NEUs (CD45⁺, Ly6G⁺), and ly6c^hi macrophages in colonic lamina propria (n = 4–5). (B) Representative F4/80 staining images and mean positive cells counted in five high-power fields (HFPs; n = 4–5). (Scale bar: 20 μm.) (C) Thymus coefficient, MLN coefficient, and spleen coefficient (n = 9–10). Thymus/MLN/spleen coefficient is calculated as thymus/MLN/spleen weight/body weight × 100. (D) WBC count in peripheral blood (n = 4–5). (E) Flow cytometric analysis of B (CD45⁺, CD19⁺), CD4⁺ T (CD45⁺, CD4⁺), CD8⁺ T (CD45⁺, CD8⁺), and NEU (CD45⁺, Ly6G⁺) cells in MLN (n = 4–5). (F) RT-PCR and ELISA results of IL-6 in colon (n = 5). (G) Western blot results of p-STAT3 in colon (n = 5; *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 3.

Genetic deletion of IL-6 failed to terminate the effect of chronic stress.(A) Body weight before DSS and body weight at the time of euthanasia (n = 5). (B) Colon length, bleeding score, and MLN coefficient (n = 5). (C) Flow cytometric analysis of CD8⁺ T, NEU, and NK cells in MLN (n = 5). (D) Thymus coefficient (n = 5). (E) Flow cytometric analysis of B and CD4⁺ T cells in MLN (n = 5). (F) WBC counts in the peripheral blood (n = 5). (G) Flow cytometric analysis of B, NEU, CD4⁺ T, and CD8⁺ T cells and ly6c^hi macrophages in colon (n = 5; *P < 0.05 and **P < 0.01).

Fig. 4.

Chronic stress changed composition of gut microbiota and degraded the colonic mucus barrier. (A) Representative images and numbers of bacterial colonies in MLN (n = 4; *P < 0.05). (B) PCoA (n = 4). (C) UPGMA method (n = 4). (D) Composition of microbiota at phylum level (n = 4). (E) Composition of microbiota at genus level (n = 4). (F) Bacterial species from colon content that had the greatest increase in abundance between different groups and their relative abundance. OTU#120 is related to Helicobacter, OTU#76 to Peptostreptococcaceae, OTU#41 to Streptococcus, and OTU#38 to E. faecalis (n = 4). (G) Bacterial species from colon content that had the greatest decrease in abundance between different groups and their relative abundance (*P < 0.05 and ***P < 0.001 vs. normal group; ^###P < 0.001 vs. DSS group). OTU#23 is related to Rikenella, OTU#24 to Rikenella, OTU#66 to Roseburia, and OTU#69 to Lachnospiraceae bacterium 6-1 (n = 4).

Fig. 5.

Chronic stress degraded the colonic mucus barrier. (A) RT-PCR results of MUC2, Cdx2, Klf3, and Tff3 (n = 5). (B) Representative images of Alcian blue-stained inner mucus layer. (Scale bar: 20 μm.) (C) Representative PAS-stained goblet cell pictures. (Scale bar: 20 μm.) (D) Quantification of inner mucus layer thickness and mean PAS⁺ goblet cells in the colon (n = 4). (E) RT-PCR results of lysozyme and CAMP (n = 5; *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 6.

Gut microbiota is responsible for the susceptibility of DSS colitis to chronic stress. (A) PCoA of each mouse (n = 4). (B) Bacterial species from colon content that had the greatest increase in abundance between different groups and their relative abundance. OTU#120 is related to Helicobacter, OTU#76 to Peptostreptococcaceae, OTU#41 to Streptococcus, and OTU#38 to E. faecalis (n = 4). (C) Body weight before DSS. (D) Body weight at the time of euthanasia, colon length, bleeding score, and WBC count in peripheral blood. (E) MLN and thymus coefficient. (F) Flow cytometric analysis of B cells, CD4⁺ T cells, NEUs, and CD8⁺ T cells in MLN (n = 5–6). (G) Bacterial species from colon content in the DSS+stress group in H₂O and antibiotic-treated conditions (n = 4). (H) Body weight before DSS (n = 6). (I) Body weight at the time of euthanasia, bleeding score, and WBC count in the peripheral blood (n = 6; *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 7.

A proposed model illustrating influences of chronic stress on colitis. Chronic stress disturbs gut microbiota and impairs the mucus layer, which then triggers the immune system and facilitates colitis.