ACE2 shedding exacerbates sepsis-induced gut leak via loss of microbial metabolite 5-methoxytryptophan

Abstract

Background: Sepsis, a critical organ dysfunction resulting from an aberrant host response to infection, remains a leading cause of mortality in ICU patients. Recent evidence suggests that angiotensin-converting enzyme 2 (ACE2) contributes to intestinal barrier function, the mechanism of which is yet to be explored. Additionally, alterations in intestinal microbiota and microbial metabolites could affect gut homeostasis, thus playing a potential role in modulating sepsis progression.

Results: ACE2 shedding weakens the integrity of the intestinal barrier in sepsis. Mice deficient in ACE2 exhibited increased intestinal permeability and higher mortality rates post-operation compared to their wild-type counterparts. Notably, ACE2 deficiency was associated with distinct alterations in gut microbiota composition and reductions in protective metabolites, such as 5-methoxytryptophan (5-MTP). Supplementing septic mice with 5-MTP ameliorated gut leak through enhanced epithelial cell proliferation and repair. The PI3K-AKT-WEE1 signaling pathway was identified as a key mediator of the beneficial effects of 5-MTP administration.

Conclusion: ACE2 plays a protective role in maintaining intestinal barrier function during sepsis, potentially through modulation of the gut microbiota and the production of key metabolite 5-MTP. Our study enriched the mechanisms by which ACE2 regulates gut homeostasis and shed light on further applications. Video Abstract.

Keywords: 5-methoxytryptophan; Angiotensin-converting enzyme 2; Bacterial metabolite; Gut leak; Sepsis.

Fig. 1

Leaky gut exacerbates sepsis and is highly positively correlated with shed ACE2 levels in sepsis. A Serum ACE2 levels of healthy volunteers and septic patients (n (healthy volunteer) = 18, n (septic patient) = 71). B Serum LPS-binding protein (LBP) levels of healthy volunteers and septic patients (n (healthy volunteer) = 16, n (septic patient) = 71). C Serum ACE2 levels in sham and CLP (cecal ligation and puncture) mice (n(sham) = 7, n(CLP) = 17). D, E ACE2 protein levels of the lung, small intestine, colon, liver, gallbladder, and kidney by Western blot (n = 4). F–J ACE2 protein levels of the small intestine, kidney, and lung and ADAM17 protein levels of the small intestine (n = 6). K Feces ACE2 levels of sham and CLP mice (n = 6). L Immunohistochemistry (IHC) staining of ACE2 in the ileum (scale bar = 50 μm). M IL-6, Pro-BNP, lactic acid, LBP, urea, age, WBC, PO₂/FiO₂ ratio, IBIL, and PCT levels were shown between survivors and non-survivors by fold change. N The receiver operating characteristic (ROC) curve of LBP for predicting in-hospital death of patients with sepsis (n = 71). O Correlation analysis between LBP, ACE2, and clinical tests (n = 71). Data are presented as mean ± SD by the Student’s t test. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001. Abbreviations: IL-6, interleukin-6; pro-BNP, pro-brain natriuretic peptide; Lac, lactic acid; LBP, lipopolysaccharide-binding protein; WBC, white blood cell count; IBIL, indirect bilirubin; PCT, procalcitonin

Fig. 2

ACE2 contributes to gut integrity in septic mice. A A schematic diagram of the experimental procedures. B Survival rates of Sham or CLP operation of both WT mice and Ace2^-/y mice (n(Sham) = 5, n(CLP) = 15). C–E Serum TNF, PCT, and glucose concentration of WT mice and Ace2^-/y mice underwent Sham or CLP operation (n = 4–15). F Fluorescein isothiocyanate-dextran (FITC-dextran; FD-4) was gavaged 3 h before sacrifice (20 mg/kg), and serum FD-4 concentration was examined (n = 4–15). G Hematoxylin and eosin (H&E) staining (upper panel), alcian blue periodic acid Schiff (AB-PAS) staining (middle panel), and IF staining of tight junction (TJ) protein OCLN (lower panel) in the ileum (scale bar = 50 μm). H–J Ileal ZO-1, OCLN, and ACE2 levels by Western blot (n = 6). K Quantification of goblet cells (n = 6). Data are presented as mean ± SD. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-tailed Student’s t test, Mann–Whitney test, or one-way ANOVA and Sidak’s post hoc analysis. Intergroup survival differences were analyzed by a log-rank test. Abbreviations: CLP, cecal ligation and puncture; TNF, tumor necrosis factor; PCT, procalcitonin; FD-4, fluorescein isothiocyanate-dextran; OCLN, occludin; ZO-1, zona occludens 1

Fig. 3

ACE2 reduces bacteria translocation and alleviates organ dysfunction in sepsis. A–C In vivo imaging and photon quantifications of bioluminescent E. coli (n = 3). D, E Quantification of bacterial load in blood, peritoneal lavage fluid (PLF), and lung samples from mice who underwent sham or CLP operation (n = 3). F H&E staining and histopathological evaluation of the lungs (scale bar = 50 μm, n = 5). G Wet/dry ratio of the lungs (n = 6). H Periodic acid Schiff (PAS) staining and the histopathological evaluation of the kidneys. Tubular structural disruption was assessed with a semi-quantitative scale (0: no lesion; 1: < 20%; 2: 20–40%; 3: 40–60%; 4: 60–80%; 5: 80–100% (scale bar = 50 μm, n = 4–6). I Serum creatinine levels of the mice (n = 6). Data are presented as the mean ± SD. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by one-way ANOVA and Sidak’s post hoc analysis. Abbreviations: PLF, peritoneal lavage fluid; Scr, serum creatinine

Fig. 4

Microbial composition and metabolites undergo alterations in Ace2^−/y mice. A Shannon index of Ace2^−/y mice and WT littermates (n = 4). B–E Beta-diversity analyzed by 3D principal component analysis (PCA), principal coordinate analysis (PCoA), cluster tree barplot, and Lefse analysis (LDA score > = 4.5) of WT and Ace2^−/y mice. F Regulated metabolites detected by untargeted metabolomics (n = 4). G PCoA plot (PCo1 vs. PCo2) of metabolite composition in Ace2^−/y mice and WT littermates (n = 4). H Volcano plot representation of regulated metabolite (black arrow indicating 5-methoxytryptophan). I Bacterial species highly correlate with 5-methoxytryptophan (5-MTP) abundance (n = 8). Data are presented as the mean ± SD. Alpha-diversity was calculated by two-tailed Student’s t test, **p < 0.01, and beta-diversity was calculated by the binary Jaccard method. Abbreviation: 5-MTP, 5-methoxytryptophan

Fig. 5

The worsening of sepsis-induced gut leak is transferrable by fecal transplantation partially via 5-MTP. A A schematic diagram of fecal microbiota transplantation (FMT) and pretreatments. B Survival rates of WT-FMT mice and Ace2^−/y -FMT mice underwent CLP (n = 10). C, D Serum glucose and TNF concentration of WT-FMT mice and Ace2^−/y -FMT mice underwent CLP (n = 6). E Serum FD-4 concentration of WT-FMT mice and Ace2^−/y -FMT mice underwent CLP (n = 5–9). F–H Ileal TJ protein (ZO-1 and OCLN) levels by Western blot (n = 6). I H&E staining (left panel), AB-PAS staining (middle panel), and IF staining of TJ protein OCLN (right panel) in the ileum. J Quantification of goblet cells (n = 6). K Typical multiple reaction monitoring chromatogram and chemical structural formula of pure 5-MTP. L Extracted ion chromatogram (XIC) overlap diagram targeting fecal 5-MTP of antibiotic (ABX) mice, WT-FMT mice, and Ace2^−/y -FMT mice. M Fecal 5-MTP quantification of ABX-mice and FMT-mice (n = 5). Data are presented as the mean ± SD. **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-tailed Student’s t test or Mann–Whitney test. Intergroup survival differences were analyzed by a log-rank test. Abbreviations: ABX, antibiotic; FMT, fecal microbiota transplantation; PCT, procalcitonin; TNF, tumor necrosis factor; FD-4, fluorescein isothiocyanate-dextran; TJ, tight junction; OCLN, occludin; ZO-1, zona occludens 1; 5-MTP, 5-methoxytryptophan

Fig. 6

5-MTP protects the gut barrier by promoting epithelial cell proliferation. A Survival rates of sham or CLP mice treated with 5-MTP or phosphate-buffered saline (PBS) (n=5–10). B–D Serum TNF, PCT, and glucose concentration of CLP mice treated with 5-MTP or PBS (n=6). E Serum FD-4 concentration in CLP mice treated with 5-MTP or PBS (n=6). F Ileal H&E staining (left panel), AB-PAS staining (middle panel), and IF staining of TJ protein OCLN (right panel). The bar chart shows the quantification of ileal goblet cells (n=6). G H&E staining, histopathological evaluation, and the wet/dry ratio of the lungs (scale bar= 50 μm, n=4–6). H PAS staining, histopathological evaluation of the kidneys, and the serum creatinine levels of the mice (scale bar= 50 μm, n=4–6). I Quantification of ileal 5-ethynyl-20-deoxyuridine (EdU) assay of 5-MTP or PBS-treated CLP mice (scale bar= 50 μm, n=6). J Ileal proliferating cell nuclear antigen (PCNA) protein level by Western blot (n=6). K A schematic diagram illustrating the role of ACE2 in promoting intestinal epithelial cell repair in sepsis via the key microbial metabolite 5-MTP. Data are presented as the mean ± SD. ns=no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-tailed Student’s t test or Mann-Whitney test. Intergroup survival differences were analyzed by the log-rank test. Abbreviations: 5-MTP, 5-methoxytryptophan; TNF, tumor necrosis factor; PCT, procalcitonin; FD-4, fluorescein isothiocyanate-dextran; Scr, serum creatinine

Fig. 7

The PI3K-AKT-WEE1 pathway participated in 5-MTP-induced epithelial cell proliferation and repair. A, B RNA-sequencing reveals enriched pathways and cellular components after 5-MTP administration. C Gene set enrichment analysis (GSEA) of regulated genes. D Heatmap visualizes regulated gene expression after 5-MTP administration (n = 3). E Cell cycle-related genes of CLP mice treated with 5-MTP or PBS were analyzed in ileal (n = 5). F, G Protein levels and mRNA levels of the cell cycle and PI3K-AKT-WEE1 signaling pathway were measured in vitro (n = 6–9). H, I Cell cycle analysis by flow cytometry (n = 3). Data are presented as the mean ± SD. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-tailed Student’s t test or one-way ANOVA and Sidak’s post hoc analysis