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, 10 (6), 391

Intestinal Autophagy Links Psychosocial Stress With Gut Microbiota to Promote Inflammatory Bowel Disease

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Intestinal Autophagy Links Psychosocial Stress With Gut Microbiota to Promote Inflammatory Bowel Disease

Shu-Ling Wang et al. Cell Death Dis.

Abstract

Psychosocial stress is a critical inducing factor of inflammatory bowel diseases (IBD), while autophagy is a novel central issue of IBD development. The present study investigated the potential role of autophagy in stress-related IBD in patients and animal model. The correlation between psychosocial stress and intestinal autophagy was determined in 23 patients with IBD. Corticotropin-releasing hormone (CRH), a well-established inducer of psychosocial stress, was administrated in dextran sulfate sodium (DSS)-induced IBD mice and lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDM). In IBD patients, the autophagy markers beclin-1, LC3-II/I ratio, Atg16L1, and Atg4B were significantly enhanced. The psychosocial stress score was positively associated with the levels of beclin-1 and the LC3II/I ratio in intestinal biopsy specimens. In IBD mouse model, CRH significantly aggravated intestinal inflammation, increased Paneth cell metaplasia, and enhanced intestinal autophagy (beclin-1, Atg16L1, PIK3R4, and Atg4B upregulation; GAA, CTSD, and PPKAA1 downregulation). Additionally, the CRH-induced gut microbial dysbiosis was evidenced by a marked increase in the number of detrimental bacteria. In LPS-stimulated BMDM, CRH substantially increased M1/M2 polarization and thus promoted inflammation. In both IBD mice and LPS-treated BMDM, blockade of autophagy by chloroquine abrogated the unbeneficial effects of CRH, whereas autophagy inducer rapamycin resulted in a pronounced protective effect against IBD lesion. Our data demonstrate that psychosocial stress may link the enhanced intestinal autophagy by modulating gut microbiota and inflammation to aggravate IBD. These data indicate autophagy as a promising therapeutic target for psychosocial stress-related IBD.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Stress contributes to the aggravation of IBD and enhancement of autophagy in IBD patients.
Ten mild/moderate IBD and 13 severe IBD patients were enrolled. The basic characteristics, and endoscopic pictures were obtained during the colonoscopy. The colonic biopsy tissue was fixed, and H&E staining was used for assessment of histological changes. Immunohistochemical staining for LC3 was used for identification of autophagy and immunohistochemical staining for CD68 for detection of M1. Western blotting was applied for the detection of autophagy-related Beclin-1 and LC3-II/I ratio and correlation between autophagy-related proteins (Beclin-1 and LC3-II/I ratio) and IBD severity index (IBD activity and score) was analyzed by correlation analysis. a, b Compared to the mild and moderate IBD group, the severe IBD patient group showed a significant increase in CPSS (P = 0.039) and there was a moderate relationship between the CPSS and Mayo Score (R = 0.625, P = 0.0015). c Compared to the patients with lower CPSS, those with higher CPSS showed aggravated inflammatory infiltration in the colonoscopic view and HE staining, as well as enhancement of the levels of CD68 and LC3 in the colonic tissue (n = 6 per group). *P < 0.05 vs. the patients with low CPSS, **P < 0.01 vs. the patients with low CPSS. d Western blotting analysis for Beclin-1, LC3-II/I ratio in biopsy specimens from the healthy controls, mild/moderate IBD patients, and severe IBD patients. *P < 0.05, **P < 0.01 vs. the control group; #P < 0.05, ##P < 0.01 vs. the mild and moderate IBD group. e Correlation analysis between psychosocial stress and autophagy (evaluated with Beclin-1 and LC3-II/I ratio) and correlation analysis between IBD score and autophagy (evaluated with Beclin-1 and LC3-II/I ratio). *P < 0.05, **P < 0.01 vs. the control group; #P < 0.05, ##P < 0.01 vs. the mild and moderate IBD group. f Western blotting analysis for Atg16L1, Atg4B, and ATF4 in biopsy specimens from health control, mild/moderate IBD patients and severe IBD patients (n = 6 per group). *P < 0.05, **P < 0.01 vs. the control group; #P < 0.05, ##P < 0.01 vs. the mild and moderate IBD group
Fig. 2
Fig. 2. Severity of IBD is aggravated by peripheral administration of CRH.
DSS (3%) was given to C57BL/6 mice for 6 days while water was given to the control group. For certain groups, CRH was intraperitoneally given at the dose of 50 μg/kg body weight from day 1 to day 6 and saline was injected as vehicle. Body weight, the presence of occult or gross blood per rectum, stool consistency, and colon length were determined by two investigators blinded to the treatment groups. ac Compared to the DSS + Vehicle group, the mice in the DSS + CRH group exhibited significant deterioration in body weight loss, bloody stool score, and colon length (n = 8 per group). **P < 0.01 vs. the control group, #P < 0.05 vs. the DSS + Vehicle group, ##P < 0.01 vs. the DSS + Vehicle group. d ELISA and the O-dianisidine method were used for the detection of the levels of TNF-α, IL-18, IL-7, MPO, and MPO activity in the left colon. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed aggravated enhancement of the levels of TNF-α, IL-18, IL-7, MPO, and MPO activity in serum (n = 6 per group). **P < 0.01 vs. the control group, ##P < 0.01 vs. the DSS + Vehicle group. e The left edge of the left colon was isolated and fixed and H&E staining was used for the detection of histological score. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed aggravated inflammatory infiltration in the left colon (n = 8 per group). **P < 0.01 vs. the control group, ##P < 0.01 vs. the DSS + Vehicle group
Fig. 3
Fig. 3. Paneth cell metaplasia is enhanced by peripheral administration of CRH in the epithelium of left the colon from IBD mice.
DSS (3%) was given to C57BL/6 mice for 6 days while water was given to the control group. For certain groups, CRH was intraperitoneally given at the dose of 50 μg/kg body weight from day 1 to day 6 and saline was injected as vehicle. a The left edge of the left colon was separated and fixed and immunohistochemical staining was used for the detection of Paneth cell. IOD values were analyzed. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed enhancement of Paneth cell metaplasia in the epithelium of the left colon (n = 6 per group). **P < 0.01 vs. the control group, ##P < 0.01 vs. the DSS + Vehicle group. b The left edge of the left colon was further detected by immunofluorescence staining and IOD values were analyzed. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed enhancement of Paneth cell metaplasia in the epithelium of the left colon (n = 6 per group). **P < 0.01 vs. the control group, ##P < 0.01 vs. the DSS + Vehicle group
Fig. 4
Fig. 4. Intestinal microflora homeostasis is deteriorated by peripheral administration of CRH in IBD mice.
DSS (3%) was given to C57BL/6 mice for 6 days while water was given to the control group. For certain groups, CRH was intraperitoneally given at the dose of 50 μg/kg body weight from day 1 to day 6 and saline was injected as vehicle. Fecal samples were analyzed by performing high-throughput 16S rRNA gene sequencing (n = 5). a α-Diversity, illustrated by microbiota richness (number of observed operational taxonomic unit (OTU)), Shannon and ace index were reduced in the DSS group, and peripheral administration of CRH aggravated this downward trending (P = 0.035, 0.012, and 0.094, respectively, Wilcoxon rank-sum test analysis), *P < 0.05, **P < 0.01. b Principal coordinate analysis (PCoA) of the overall microbial composition of the DSS and DSS + CRH groups deviated from the Control and CRH groups (ANOSIM R: 0.7887, P = 0.001). c Compared to the control group, microbiome alterations at the genus level in the DSS group were significantly changed, and this trend was further obvious in the DSS + CRH group. d, e Linear discriminant analysis (LDA) effect size (LEfSe) analysis illustrated marked bacterial differences in fecal microbiota among these four groups. f The abundance of Turicibacter, Ruminococcaceae, and Lactobacillus were decreased, while the genera Klebsiella and Parabacteroides were increased in the DSS group. Moreover, peripheral administration of CRH further aggravated these changes in IBD
Fig. 5
Fig. 5. The level of autophagy is increased by peripheral administration of CRH in the left colon from IBD mice.
DSS (3%) was given to C57BL/6 mice for 6 days while water was given to the control group. For certain groups, CRH was intraperitoneally given at the dose of 50 μg/kg body weight from day 1 to day 6 and saline was injected as vehicle. a The left colon was separated and the levels of Beclin-1, LC3-II/I ratio, and p62/SQSTM1 were analyzed by western blotting. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed an increase in Beclin-1 and LC3-II/I ratio while a decrease in p62/SQSTM1 (n = 5 per group). **P < 0.01 vs. the control group, ##P < 0.01 vs. the DSS + Vehicle group. Electronic microscope was used for the detection of autophagosome number (b) and immunofluorescence staining for LC3 was used for the analysis of autophagy level (c). Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed an increase in the number of autophagosome and LC3 dots (n = 6 per group). **P < 0.01 vs. the control group, ##P < 0.01 vs. the DSS + Vehicle group. The left colon was separated and the total RNA (125 ng) was extracted and reverse transcribed to cDNA using miScript II RT kit (Qiagen, Valencia, CA, USA), and the expression profiles of autophagy gene, including a total of 84 genes were analyzed. d Heatmap displaying the relative expression of autophagy-related genes in the control, CRH, DSS + Vehicle and DSS + CRH groups (n = 3 per group). e Relative gene expression profiles between the DSS + Vehicle vs. the DSS + CRH mice (n = 3). The red lines indicate positive or negative fold changes (upregulated twofold or downregulated 0.5-fold) for selected genes in the DSS vs. DSS + CRH mice. f Most significant increased genes were mainly associated with autophagy, the process utilizing autophagic mechanism, and macroautophagy. g Compared to the DSS group, the levels of several proteins vital in the induction and regulation of autophagy were significantly changed (red nodes for genes with higher expression and blue nodes for genes with lower expression). Compared to the DSS + Vehicle group, several proteins positively correlated to autophagy such as PIK3R4, Atg4B, and DRAM1 were upregulated and those negatively correlated to autophagy such as GAA, HSP, CTSD, PPKAA1 were downregulated in the DSS + CRH group
Fig. 6
Fig. 6. Chloroquine ameliorates CRH-induced colonic damage and Paneth cell metaplasia in IBD mice.
DSS (3%) was given to C57BL/6 mice for 6 days while water was given to the control group. For certain groups, CRH (50 μg/kg body weight) and/or chloroquine (60 mg/kg body weight) was intraperitoneally given from day 1 to day 6 and saline was injected as vehicle. ac Body weight, the presence of occult or gross blood per rectum, stool consistency, and colon length were determined by two investigators blinded to the treatment groups. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group exhibited significant deterioration in body weight loss, bloody stool score, and colon length, while the administration of chloroquine largely attenuated the aggravated effect of CRH (n = 8 per group). **P < 0.01 vs. the control group, ##P < 0.01 vs. the DSS + Vehicle group, $$P < 0.01 vs. the DSS + CRH group. dh The left edge of the left colon was isolated and the levels of TNF-α, IL-18, IL-7, MPO, and the MPO activity in the left colon were analyzed by ELISA and O-dianisidine method. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed aggravated colonic inflammation, while the administration of chloroquine largely attenuated the effect of CRH (n = 6 per group). *P < 0.05, **P < 0.01. i The left edge of the left colon was isolated and fixed and H&E staining was used for the detection of histological score. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed aggravated inflammatory infiltration in the left colon, while the administration of chloroquine largely attenuated the effect of CRH (n = 8 per group). *P < 0.05, **P < 0.01. j The left edge of the colon was separated and fixed and immunohistochemical staining was used for the detection of Paneth cell. IOD values were analyzed. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed enhancement of Paneth cell metaplasia in the epithelium of the left colon, while the administration of chloroquine largely attenuated the effect of CRH (n = 6 per group). *P < 0.05, **P < 0.01
Fig. 7
Fig. 7. Rapamycin aggravated CRH-induced Paneth cell metaplasia but not colonic damage in IBD mice.
DSS (3%) was given to C57BL/6 mice for 6 days while water was given to the control group. For certain groups, CRH (50 μg/kg body weight) and/or rapamycin (1.25 mg/kg body weight) was intraperitoneally given from day 1 to day 6 and saline was injected as vehicle. ac Body weight, the presence of occult or gross blood per rectum, stool consistency, and colon length were determined by two investigators blinded to the treatment groups. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group exhibited significant deterioration in body weight loss, bloody stool score, and colon length, which were not significantly affected by rapamycin (n = 8 per group). *P < 0.05 vs. the control group. **P < 0.01 vs. the control group. ##P < 0.01 vs. the DSS + Vehicle group. dh The left edge of the left colon was isolated and the levels of TNF-α, IL-18, IL-7, MPO, and MPO activity in the left colon were analyzed by ELISA and the O-dianisidine method. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed aggravated colonic inflammation, which were not significantly affected by rapamycin (n = 6 per group). **P < 0.01. i The left edge of the colon was isolated and fixed and H&E staining was used for the detection of histological score. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed aggravated inflammatory infiltration in the left colon, which were not significantly affected by rapamycin (n = 8 per group). **P < 0.01. j The left edge of the left colon was separated and fixed and immunohistochemical staining was used for the detection of Paneth cell. IOD values were analyzed. Compared to the DSS + Vehicle group, the mice in the DSS + CRH group showed enhancement of Paneth cell metaplasia in the epithelium of the left colon, while the administration of rapamycin further aggravated the effect of CRH (n = 6 per group). **P < 0.01
Fig. 8
Fig. 8. Chloroquine attenuates CRH-induced enhancement of M1/M2 ratio in the left colon from IBD mice in murine BMDM under the challenge of LPS.
a, b DSS (3%) was given to C57BL/6 mice for 6 days while water was given to the control group. For certain groups, CRH (50 μg/kg body weight) and/or chloroquine (60 mg/kg body weight) was intraperitoneally given from day 1 to day 6 and saline was injected as vehicle. The left edge of colon was separated, and fixed and immunofluorescence staining was used for the detection of CD68 (M1 cells) and CD206 (M2 cells). Compared to the DSS + Vehicle group, the mice in the DSS + CRH group had an increased ratio of CD68+ cells and a decreased ratio of CD206+ cells in the left colon, while the administration of chloroquine largely attenuated the effect of CRH (n = 6 per group). c, d Murine BMDM were obtained and stimulated with LPS (100 ng/ml), CRH (10−8 M), and/or chloroquine (10 μM), and immunofluorescence staining was used for the detection of CD68 (M1 cells), and CD206 (M2 cells). Compared to the LPS + Vehicle group, the LPS + CRH group had an increased ratio of CD68+ cells and a decreased ratio of CD206+ cells, while the administration of chloroquine largely attenuated the effect of CRH (n = 6 per group). e, f Murine BMDM were obtained and stimulated with LPS (100 ng/ml), CRH (10−8 M), and/or chloroquine (10 μM). FACS analysis was conducted for the detection of CD68+ cell and CD206+ cell ratios. Compared to the LPS + Vehicle group, the LPS + CRH group had an increased ratio of CD68+ cells and a decreased ratio of CD206+ cells, while the administration of chloroquine largely attenuated the effect of CRH (n = 6 per group). **P < 0.01.

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