Impairments of intestinal arginine and NO metabolisms trigger aging-associated intestinal barrier dysfunction and 'inflammaging'

Abstract

Aging is considered a state of low grade inflammation, occurring in the absence of any overt infection often referred to as 'inflammaging'. Maintaining intestinal homeostasis may be a target to extend a healthier status in older adults. Here, we report that even in healthy older men low grade bacterial endotoxemia is prevalent. In addition, employing multiple mouse models, we also show that while intestinal microbiota composition changes significantly during aging, fecal microbiota transplantation to old mice does not protect against aging-associated intestinal barrier dysfunction in small intestine. Rather, intestinal NO homeostasis and arginine metabolism mediated through arginase and NO synthesis is altered in small intestine of aging mice. Treatment with the arginase inhibitor norNOHA prevented aging-associated intestinal barrier dysfunction, low grade endotoxemia and delayed the onset of senescence in peripheral tissue e.g., liver. Intestinal arginine and NO metabolisms could be a target in the prevention of aging-associated intestinal barrier dysfunction and subsequently decline and 'inflammaging'.

Keywords: Aging; Endotoxin; Intestinal permeability; Microbiota; Nitric oxide.

Graphical abstract

Fig. 1

Effect of aging on markers of intestinal permeability in young and old male healthy humans. (A) Study design, (B) endotoxin concentration and (C) levels of toll-like receptor 2 (TLR2) and (D) TLR4 ligands, (E) lipopolysaccharide binding protein (LBP) concentration and (F) cluster of differentiation 14 (CD14) concentration in plasma of young and old humans (n = 15–16). Values are means ± sem. *p < 0.05 due to unpaired two-tailed students t-test. mo: months.

Fig. 2

Effect of aging on markers of intestinal permeability, NO metabolism and arginase activity in intestine of young and old male healthy mice. (A) Study design, (B) endotoxin concentration in plasma of portal vein and (C) xylose permeation of everted sacs of 3 months and 24 months old mice. Densitometric analysis and representative pictures (small intestine: 400x, colon: 100x) of (D) occludin as well as (E) zonula occludens-1 (ZO-1) in intestine of young and old mice. (F) PCoA plot showing the microbial communities of each sample and (G) significant microbial communities at genus and family level. (H) Evaluation of 3-nitrotyrosine (3-NT) positive cells; (I) inducible nitric oxide synthase (inos) mRNA expression and (J) NO₂⁻ concentration in proximal small intestine. (K) Arginase activity and (L) densitometric analysis of arginase-2 Western blot as well as (M) representative Western blots and (N) representative pictures of arginase-1 (200x) and arginase-2 (630x) protein staining of proximal small intestine of 3 months and 24 months old mice (n = 5–9). Values are means ± sem. *p < 0.05 due to unpaired two-tailed students t-test. mo: months.

Fig. 3

Effect of FMT on markers of intestinal barrier function in mice. (A) Endotoxin concentration in vena fascialis of mice from SPF facility at different age (n = 5–9; *p < 0.05 due to one-way ANOVA) as well as (B) representative blot of p16 protein in plasma. (C) Study design, (D) hepatic p16 mRNA expression, (E) xylose permeation of everted gut sac, (F) occludin and (G) zonula occludens-1 (Zo1) mRNA expression of proximal small intestine, (H) endotoxin concentration, (I) levels of toll-like receptor 4 (TLR4) ligands (J) and densitometric analysis of cluster of differentiation 14 (CD14) Western blot in plasma from portal vein, (K) Tlr4 and (L) Myeloid differentiation primary response 88 (Myd88) mRNA expression of hepatic tissue of old (o) mice receiving young and old FMT (+yFMT, +oFMT), (n = 7–10). Values are means ± sem. *p < 0.05 due to unpaired two-tailed students t-test. AB: antibiotics, mo: months, FMT: fecal microbiota transfer.

Fig. 4

Effect of arginase inhibition on markers of intestinal barrier in proximal small intestine of mice. (A) Study design, (B) hepatic p16 mRNA expression, (C) xylose permeation in everted gut sacs and (D) occludin and (E) zonula occludens-1 (Zo1) mRNA expression in proximal small intestine, (F) endotoxin concentration, (G) levels of toll-like receptor 4 (TLR4) ligands in plasma of portal vein, (H) cluster of differentiation 14 (CD14) concentration in plasma determined by Western blot, (I) Cd14, (J) Tlr4 and (K) Myeloid differentiation primary response 88 (Myd88) hepatic mRNA expression and (L) NO₂⁻ concentration of scraped mucosa of intestinal tissue in old (o) mice treated with NaCl or N(ω)-hydroxy-nor-l-arginine (norNOHA) (n = 5–7). Values are means ± sem. *p < 0.05 due to unpaired two-tailed students t-test. mo: months.

Fig. 5

Effect of inhibition of arginase and ROCK signaling cascade in old everted gut sacs. (A) arginase signaling cascade, (B) study design, (C) pROCK2/ROCK2 Western blot in proximal small intestine of 3 months and 24 months old mice (n = 5–6, p = 0.06 due to unpaired two-tailed students t-test). (D) Schematic explanation of everted gut sacs, (E) Xylose permeation of everted gut sacs, (F) arginase activity and (G) NO₂⁻ concentration in tissue of everted gut sacs from 21 to 24 months old mice incubated with KRH buffer (C), 1 μM N(ω)-hydroxy-nor-l-arginine (norNOHA) or 20 μM Y-27632 (n = 4, *p < 0.05 due to one-way ANOVA). (H) Xylose permeation of everted gut sacs, (I) NO₂⁻ concentration in tissue of everted gut sacs from 21 to 24 months old mice incubated with KRH buffer (C), 1 μM N(ω)-hydroxy-nor-l-arginine (norNOHA) or 1 mM difluoromethylornithine (DFMO) (n = 3–5, *p < 0.05 due to one-way ANOVA). Values are means ± sem. mo: months.