ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

Abstract

Malnutrition affects up to one billion people in the world and is a major cause of mortality. In many cases, malnutrition is associated with diarrhoea and intestinal inflammation, further contributing to morbidity and death. The mechanisms by which unbalanced dietary nutrients affect intestinal homeostasis are largely unknown. Here we report that deficiency in murine angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), which encodes a key regulatory enzyme of the renin-angiotensin system (RAS), results in highly increased susceptibility to intestinal inflammation induced by epithelial damage. The RAS is known to be involved in acute lung failure, cardiovascular functions and SARS infections. Mechanistically, ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. Transplantation of the altered microbiota from Ace2 mutant mice into germ-free wild-type hosts was able to transmit the increased propensity to develop severe colitis. ACE2-dependent changes in epithelial immunity and the gut microbiota can be directly regulated by the dietary amino acid tryptophan. Our results identify ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis. These results provide a molecular explanation for how amino acid malnutrition can cause intestinal inflammation and diarrhoea.

Figure 1. Ace2 deficiency and protein malnutrition worsen DSS-induced colitis.

a, Colon histopathology, b, percentage weight loss, and c, diarrhoea scores in control and DSS-treated Ace2^+/y and Ace2^−/y littermates. In a, note crypt damage (arrowheads), ulcerations (arrow), and infiltration of inflammatory cells (asterisks) in DSS-treated Ace2^−/y mice. Haematoxylin and eosin staining on day 7 after DSS challenge. Scale bars, 100 μm. d, Colon histopathology (haematoxylin and eosin staining, day 4 after DSS challenge; scale bars, 100 μm), e, percentage weight loss, and f, diarrhoea scores of DSS-treated Ace2^+/y and Ace2^−/y littermates fed either normal chow (Control) or a protein free diet (PFD; <0.2% protein). All values are mean ± s.e.m. of 5–9 mice per group. *P < 0.05, **P < 0.01 comparing DSS-treated Ace2^+/y with Ace2^−/y littermates, or Ace2^+/y mice on normal diet with those on PFD (paired-t-test). PowerPoint slide

Figure 2. Rescue of severe colitis with nicotinamide or tryptophan di-peptides.

a, Colon histopathology (haematoxylin and eosin, day 10 after DSS challenge; scale bars, 100 μm), b, percentage weight loss, and c, diarrhoea scores of DSS-treated Ace2^+/y and Ace2^−/y littermates that received vehicle or nicotinamide (NAM) in their drinking water. Nicotinamide treatment was started 3 days before DSS challenge. d, Colon histopathology (haematoxylin and eosin, day 7; scale bars, 100 μm), e, percentage weight loss, and f, crypt injury scores of Ace2^+/y and Ace2^−/y mice fed a di-peptidic tryptophan diet (Trp+) or normal chow (Control). Values are mean ± s.e.m. of 3–10 mice per group. *P < 0.05, **P < 0.01 comparing Ace2^−/y mice on a normal diet with those on Trp+ diet, or vehicle- versus nicotinamide-treated Ace2^−/y mice. ##P < 0.01 comparing Ace2^+/y versus Ace2^−/y mice (paired-t-test). PowerPoint slide

Figure 3. Tryptophan controls antimicrobial peptides and mTOR activity.

a, b, mRNA expression levels of antimicrobial peptides in epithelial cells isolated from the small intestine of a, unchallenged Ace2^+/y and Ace2^−/y littermates, and b, Ace2^+/y mice fed a tryptophan-free diet (Trp−) or normal chow (Control). c, mRNA expression levels of antimicrobial peptide Defa1 in Ace2^+/y and Ace2^−/y littermates fed a Trp+ diet or normal chow (Control) for 10 days. d, e, Immunohistochemistry to detect levels of phosphorylated S6 (brown) in the small intestine of d, unchallenged Ace2^+/y and Ace2^−/y littermates or e, Ace2^−/y mice fed a Trp+ or normal chow diet (Control). Scale bars, 200 μm. f, Colon histopathology (haematoxylin and eosin, day 8; scale bars, 100 μm) of DSS treated wild-type mice receiving vehicle or rapamycin (RAPA) i.p., initiated 6 days before DSS challenge. Values are mean ± s.e.m. of 5–6 mice per group. *P < 0.05, **P < 0.01 comparing Ace2^+/y with Ace2^−/y mice; ^#P < 0.05, ^##P < 0.01 comparing Ace2^+/y mice on normal diet with those on Trp− diet (paired-t-test). PowerPoint slide

Figure 4. Altered gut bacteria from Ace2 mutant mice can confer susceptibility to colitis.

a, b, Principal coordinate analysis plots; a, calculated by Bray–Curtis algorithm and b, based on unweighted UniFrac analysis. Plots show the similarity among ileocaecal bacterial communities in Ace2^+/y and Ace2^−/y mice fed a Trp+ diet or normal chow (Control) for 10 days. Only the two axes with high R² values are shown (axis 1, R² = 0.335; axis 2, R² = 0.8116). Each dot represents data from an individual animal. c, Comparison of microbial communities in Ace2^+/y and Ace2^−/y mice fed a Trp+ diet or normal chow (Control). The heat map depicts abundance of the top 25 species level OTUs contributing significantly to the axis shown in the weighted principal coordinate analysis plot (a). d, Diarrhoea scores and e, colon histopathology (haematoxylin and eosin, day 7; scale bars, 100 μm) of DSS challenged germ-free mice that received intestinal microbiota from Ace2^+/y or Ace2^−/y littermates. Values are mean ± s.e.m. of 4–6 mice per group. *P < 0.05 (paired-t-test). PowerPoint slide