Gut microbe-derived milnacipran enhances tolerance to gut ischemia/reperfusion injury

Abstract

There are significant differences in the susceptibility of populations to intestinal ischemia/reperfusion (I/R), but the underlying mechanisms remain elusive. Here, we show that mice exhibit significant differences in susceptibility to I/R-induced enterogenic sepsis. Notably, the milnacipran (MC) content in the enterogenic-sepsis-tolerant mice is significantly higher. We also reveal that the pre-operative fecal MC content in cardiopulmonary bypass patients, including those with intestinal I/R injury, is associated with susceptibility to post-operative gastrointestinal injury. We reveal that MC attenuates mouse I/R injury in wild-type mice but not in intestinal epithelial aryl hydrocarbon receptor (AHR) gene conditional knockout mice (AHR^flox/flox) or IL-22 gene deletion mice (IL-22^-/-). Collectively, our results suggest that gut microbiota affects susceptibility to I/R-induced enterogenic sepsis and that gut microbiota-derived MC plays a pivotal role in tolerance to intestinal I/R in an AHR/ILC3/IL-22 signaling-dependent manner, revealing the pathological mechanism, potential prevention and treatment drugs, and treatment strategies for intestinal I/R.

Keywords: IL-22; aryl hydrocarbon receptor; enterogenic sepsis; intestinal flora; milnacipran.

Graphical abstract

Figure 1

Mice have significant differences in susceptibility to intestinal I/R-induced enterogenic sepsis (A) Schematic diagram of differences in susceptibility to enterogenic sepsis in mice. (B) Mouse survival curve (n = 20; chi-square test). (C and D) Hematoxylin and eosin (HE) staining (C) and the pathological damage scores (D) of intestinal tissue sections. Scale bar, 100 μm (n = 8). (E–G) The fluorescein isothiocyanate (FITC)-dextran levels (E) and 16S/18S levels in blood cells (F) and endotoxin levels in plasma (G) reflect the permeability of the intestinal barrier and bacterial translocation (n = 8). (H–J) Mouse lung tissue HE staining (H); the pathological damage scores (I) and wet/dry weight ratios (J). Scale bar, 100 μm (n = 8). (K–N) Mouse liver tissue HE staining (K); the pathological damage scores (L) and plasma ALT (M) and AST levels (N). Scale bar, 100 μm (n = 8). (O–Q) Mouse kidney tissue HE staining (O); the pathological damage scores (P) and plasma BUN levels (Q). Scale bar, 100 μm (n = 8). The results are expressed as the median and quartile. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by were determined by the Mann-Whitney test.

Figure 2

Differences in the gut microbiota and metabolites in mice with different susceptibilities to enterogenic sepsis (A) Alpha diversity analysis (Chao1 index, n = 6; Wilcoxon test). (B) Principal coordinate analysis (PCoA) of 16S rRNA gene sequencing data (n = 6; Adonis analysis and Anosim analysis). (C) The composition of the gut bacterial abundance at the phylum level (n = 6). (D) Linear discriminant analysis (LDA) effect size (LEfSe) of different species between groups (n = 6). (E) PCoA of metabolomics (n = 6; Adonis analysis and Anosim analysis). (F) Volcano plot of differential metabolites between groups (n = 6). (G) The differential metabolite Kyoto Encyclopedia of Genes and Genomes (KEGG) bubble plot between groups (n = 6). (H) Chemical structure of MC (n = 6). (I) Liquid chromatography-tandem mass spectrometry (LC–MS/MS)-targeted detection of the MC content in feces of WT mice or germ-free (GF) mice (n = 6). (J) LC-MS/MS-targeted detection of the MC content in the feces. The results are expressed as the median and quartile. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by were determined by the Mann-Whitney test in (A), (I), (J), and adonis analysis and anosim analyses in (B) and (E).

Figure 3

Gut microbiota affects susceptibility to enterogenic sepsis induced by intestinal I/R (A) Feces from the ESTM or the ESSM were transplanted into pseudosterile mice to establish a model of enterogenic sepsis. (B) The relative abundance of Bacteroidetes and Verrucomicrobiota in the feces. (C and D) Hematoxylin-eosin (HE) staining (C) and pathological damage scores (D) of intestinal tissue sections. Scale bar, 100 μm. (E–G) The fluorescein isothiocyanate (FITC)-dextran levels (E) and 16S/18S levels in blood cells (F) and endotoxin levels in plasma (G) reflect the permeability of the intestinal barrier, bacterial translocation, and infection. (H) The pre-operative stools of patients undergoing CPB from the AGI group or the NAGI group were transplanted into pseudosterile mice to establish a model of enterogenic sepsis. (I) The MC content in the feces 7 days after transplantation. (J and K) HE staining (J) and pathological damage scores (K) of intestinal tissue sections. Scale bar, 100 μm. (L–N) The FITC-dextran levels (L) and 16S/18S levels in blood cells (M) and endotoxin levels in plasma (N) reflect the permeability of the intestinal barrier, bacterial translocation. The results are expressed as the median and quartile, n = 8 for each group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 were determined by the Mann-Whitney test.

Figure 4

MC ameliorates intestinal I/R injury in mice by activating the intestinal epithelial AHR (A and B) Hematoxylin-eosin (HE) staining (A) and pathological damage scores (B) of intestinal tissue sections. Scale bar, 100 μm (n = 8). (C–E) The fluorescein isothiocyanate (FITC)-dextran levels (C), and 16S/18S levels in blood cells (D) endotoxin levels in plasma (E) reflect the permeability of the intestinal barrier and bacterial translocation (n = 8). (F and G) Immunofluorescence (F) and relative quantitative analysis (G) of the cell proliferation marker Ki67 in the intestine. Scale bar, 100 μm (n = 5). (H) Relative mRNA level of the intestinal stem cell self-renewal marker Lgr5 in the intestine (n = 8). (I) HE staining of intestinal organoids. Scale bar, 20 μm (n = 6). (J) Intestinal organoid viability (n = 6). (K) LDH levels in the medium (n = 6). (L–M) Immunofluorescence (L) and relative quantitative analysis (M) of Ki67 in intestinal organoids. Scale bar, 20 μm (n = 5). (N) Relative mRNA level of Ki67 in intestinal organoids (n = 6). (O) Relative mRNA level of Lgr5 in intestinal organoids (n = 6). The results are expressed as the median and quartile. ∗, #, and & p < 0.05; ∗∗, ##, and && p < 0.01; ∗∗∗, ###, and &&& p < 0.001 were determined by two-way ANOVA and Tukey’s post hoc test. ∗p < 0.05 compared with WT mouse Sham group; #p < 0.05 compared with WT mouse I/R group; &p < 0.05 compared with WT mouse I/R + MC group. I/R + MC group: WT mice or AHR^flox/flox mice were injected intraperitoneally with 10 mg/kg MC 1 h before intestinal I/R; H/R + MC group: 10 μmol/L MC was added to intestinal organoids 1 h before H/R.

Figure 5

MC promotes increased NCR⁺ILC3/ILC3 ratio and IL-22 release by activating intestinal epithelial AHR (A and B) Flow cytometry to detect the ratio of NCR⁺ILC3s/ILC3s (n = 3). (C and D) Flow cytometry to detect the ratio of IL-22⁺NCR⁺ILC3s/NCR⁺ILC3s (n = 3). (E) The mRNA levels of IL-22 in intestinal tissue (n = 8). (F and G) Immunohistochemical results (F) and relative quantitative analysis (G) of IL-22 in intestinal tissue (n = 5). (H) IL-22 levels in intestinal organoid culture medium (n = 6). (I) The 7-day survival curve of mice (n = 20; chi-square test). The results are expressed as the median and quartile. ∗, #, and & p < 0.05; ∗∗, ##, and && p < 0.01; ∗∗∗, ###, and &&& p < 0.001 were determined by two-way ANOVA and Tukey’s post hoc test. ∗p < 0.05 compared with WT mouse Sham group; #p < 0.05 compared with WT mouse I/R group; &p < 0.05 compared with WT mouse I/R + MC group.

Figure 6

AHR activator Ficz reduces intestinal I/R injury in mice by releasing IL-22 (A and B) Hematoxylin-eosin (HE) staining (A) and the pathological damage score (B) of intestinal tissue sections. Scale bar, 100 μm (n = 8). (C–E) The fluorescein isothiocyanate (FITC)-dextran levels (C) and 16S/18S levels in blood cells (D) and endotoxin levels in plasma (E) reflect the permeability of the intestinal barrier and bacterial translocation (n = 8). (F and G) Immunofluorescence (F) and relative quantitative analysis (G) of the cell proliferation marker Ki67 in the intestine. Scale bar, 100 μm (n = 5). (H) Relative mRNA level of the intestinal stem cell self-renewal marker Lgr5 in the intestine (n = 8). (I) HE staining of intestinal organoids. Scale bar, 20 μm (n = 6). (J) Intestinal organoid viability (n = 6). (K) LDH levels in the medium (n = 6). (L and M) Immunofluorescence (L) and relative quantitative analysis (M) of Ki67 in intestinal organoids. Scale bar, 20 μm (n = 5). (N) Relative mRNA level of Ki67 in intestinal organoids (n = 8). (O) Relative mRNA level of Lgr5 in intestinal organoids (n = 8). The results are expressed as the median and quartile. ∗, #, and & p < 0.05; ∗∗, ##, and && p < 0.01; ∗∗∗, ###, and &&& p < 0.001 by were determined were determined by two-way ANOVA and Tukey’s post hoc test. ∗p < 0.05 compared with WT mouse Sham group; #p < 0.05 compared with WT mouse I/R group; &p < 0.05 compared with WT mouse I/R + Ficz group. I/R + Ficz group: mice were treated with daily intraperitoneal injection of 50 μg/kg Ficz for 7 consecutive days before induction of intestinal I/R; H/R + Ficz group: 200 nM Ficz was added to ILC3s and the intestinal organoid co-culture system 1 h before H/R.

Figure 7

IL-22 promotes the self-renewal of intestinal stem cells by activating the Wnt and Notch pathways (A and B) The relative quantitative analysis (A) and representative immunohistochemical map (B) of β-catenin expression in intestinal tissues. (C) The mRNA level of Wnt3 in intestinal tissues (n = 7–8). (D and E) The relative quantitative analysis (D) and representative immunohistochemical map (E) of Notch1 expression in intestinal tissues. (F) The mRNA level of Notch1 in intestinal tissues (n = 7–8). (G and H) The relative quantitative analysis (G) and representative immunohistochemical map (H) of β-catenin expression in intestinal organoids. (I) The mRNA level of Wnt3 in intestinal organoids (n = 7–8). (J and K) The relative quantitative analysis (J) and representative immunohistochemical map (K) of Notch1 expression in intestinal organoids. (L) The mRNA level of Notch1 in intestinal organoids (n = 7–8). The results are expressed as the median and quartile. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by were determined by one-way ANOVA (Tukey’s test). I/R + rmIL-22 group: mice were injected intraperitoneally with 0.25 mg/kg rmIL-22 1 h before intestinal I/R; H/R + rmIL-22 group: organoids were supplemented with 5 ng/mL rmIL-22 1 h before H/R.