Short-chain fatty acids ameliorate necrotizing enterocolitis-like intestinal injury through enhancing Notch1-mediated single immunoglobulin interleukin-1-related receptor, toll-interacting protein, and A20 induction

Abstract

Single immunoglobulin interleukin-1-related receptor (SIGIRR), toll-interacting protein (TOLLIP), and A20 are major inhibitors of toll-like receptor (TLR) signaling induced postnatally in the neonatal intestine. Short-chain fatty acids (SCFAs), fermentation products of indigestible carbohydrates produced by symbiotic bacteria, inhibit intestinal inflammation. Herein, we investigated the mechanisms by which SCFAs regulate SIGIRR, A20, and TOLLIP expression and mitigate experimental necrotizing enterocolitis (NEC). Butyrate induced NOTCH activation by repressing sirtuin 1 (SIRT1)-mediated deacetylation of the Notch intracellular domain (NICD) in human intestinal epithelial cells (HIECs). Overexpression of NICD induced SIGIRR, A20, and TOLLIP expression. Chromatin immunoprecipitation revealed that butyrate-induced NICD binds to the SIGIRR, A20, and TOLLIP gene promoters. Notch1-shRNA suppressed butyrate-induced SIGIRR/A20 upregulation in mouse enteroids and HIEC. Flagellin (TLR5 agonist)-induced inflammation in HIEC was inhibited by butyrate in a SIGIRR-dependent manner. Neonatal mice fed butyrate had increased NICD, A20, SIGIRR, and TOLLIP expression in the ileal epithelium. Butyrate inhibited experimental NEC-induced intestinal apoptosis, cytokine expression, and histological injury. Our data suggest that SCFAs can regulate the expression of the major negative regulators of TLR signaling in the neonatal intestine through Notch1 and ameliorate experimental NEC. Enteral SCFAs supplementation in preterm infants provides a promising bacteria-free, therapeutic option for NEC.NEW & NOTEWORTHY Short-chain fatty acids (SCFAs), such as propionate and butyrate, metabolites produced by symbiotic gut bacteria are known to be anti-inflammatory, but the mechanisms by which they protect against NEC are not fully understood. In this study, we reveal that SCFAs regulate intestinal inflammation by inducing the key TLR and IL1R inhibitors, SIGIRR and A20, through activation of the pluripotent transcriptional factor NOTCH1. Butyrate-mediated SIGIRR and A20 induction represses experimental NEC in the neonatal intestine.

Keywords: Notch1; butyrate; intestinal epithelium; necrotizing enterocolitis; short-chain fatty acids.

Graphical abstract

Figure 1.

SCFAs enhance A20 and SIGIRR expression in association with activated Notch intracellular domain (NICD) in HIECs. A: expression of A20, SIGRR, TOLLIP, and HEY2, a Notch downstream target in HIECs treated with sodium butyrate for 48 h at different concentration as indicated, was quantified by real-time PCR. n = 4. B: gene expression was quantified by real-time PCR in HIECs treated with 5 mM butyrate for different times. n = 3. C: HIECs were treated with 5 mM butyrate or 10 mM propionate for 16 h, and cell lysates were analyzed by Western blotting using the indicated antibodies. n = 3. Densitometry quantification is shown graphically. D: gene expression was quantified by real-time PCR in colon epithelial cells treated for 16 h at indicated concentrations. n = 4. Data shown as means ± SD, *P < 0.05, **P < 0.01, ***P < 0.005. ****P < 0.001. HIEC, human intestinal epithelial cell; NICD, Notch intracellular domain; SIGIRR, single immunoglobulin interleukin-1-related receptor; TOLLIP, toll-interacting protein.

Figure 2.

NICD transcriptionally regulates expression of TLR-negative regulator. A: ChIP-qPCR assay was performed using anti-NICD antibody to analyze the recruitment of NICD on promoters of SIGIRR, A20, and HEY2 in HIECs with 5 mM butyrate treatment, and values were quantified against IgG controls. n = 3. B: whole cell lysate of HIECs with 5 mM butyrate or 10 mM propionate was immunoblotted for SIRT1 and β-actin. Densitometry quantification is shown graphically. n = 4. C: NICD acetylation in HIECs with butyrate treatment. NICD was immunoprecipitated by NICD antibody from HIECs with 6 h of treatment of butyrate, then analyzed by Western blotting with NICD antibody and acetylated lysine antibody. Densitometry quantification is shown graphically. n = 3. D: gene expression was quantified by real-time PCR in HIECs transfected with pCDNA3 empty vector (control), pCDNA3-NICD, or pCDNA3 NICD 14KR. n = 5. E: overexpression of SIRT1 in HIEC suppress butyrate-induced expression of SIGIRR, A20, and TOLLIP. HIECs were transfected with pcDNA3-empty vector (EV) or FLAG-SIRT1(Addgene) for 48 h and then treated with butyrate at 5 mM for 8 h. Gene expression were quantified by real-time PCR. n = 4. Data shown as means ± SD, *P < 0.05, **P < 0.01, ***P < 0.005. ****P < 0.001. ChIP, chromatin immunoprecipitation; HIEC, human intestinal epithelial cell; NICD, Notch intracellular domain; SIGIRR, single immunoglobulin interleukin-1-related receptor; TOLLIP, toll-interacting protein; TLR, toll-like receptor.

Figure 3.

Notch1 is required for butyrate-induced TLR-negative regulator expression in mouse enteroids and HIECs. A: HIECs stably expressing lentiviral Notch1 shRNA, Notch2 shRNA, or scramble control shRNA were treated with 5 mM butyrate for 16 h. TLR inhibitors and Notch1 expression were quantified by real-time PCR. n = 3. B: Immunofluorescent confocal images of mouse enteroids stained with antibodies against indicated proteins. n = 3. Scale bar = 50 µm. C: SIGIRR and A20 expression in mouse enteroids treated with 5 mM butyrate for 48 h were quantified by real-time PCR. *P < 0.05. n = 4. D: ADAM17 expression in enteroids with 48 h butyrate treatment was quantified by real-time PCR. *P < 0.05. n = 3. E: whole cell lysate of HIECs with 5 mM butyrate was immunoblotted for AMAM17 and β-actin. n = 3. F: enteroids transduced by or scramble shRNA or shRNA specificaly targeting Notch1 mRNA were treated with 5 mM butyrate for 48 h, then lysed for mRNA exrepssion by real-time PCR. *P < 0.05. n = 3. Data shown as means ± SD, *P < 0.05, **P < 0.01. EV, empty vector, HIEC, human intestinal epithelial cell; SIGIRR, single immunoglobulin interleukin-1-related receptor; TOLLIP, toll interacting protein.

Figure 4.

SCFAs suppress TLR- and IL1R-mediated inflammation in cell culture. A: expression of A20, SIGRR, TOLLIP, and HEY2 in 293/TLR4 treated with sodium butyrate for 48 h at different concentration as indicated was quantified by real-time PCR. n = 3. B: 293/TLR4 were treated with 5 mM butyrate or 10 mM propionate for 48 h, and cell lysates were analyzed by Western blotting using the indicated antibodies. n = 3. C: SCFAs suppressed TLR4-mediated inflammation in HEK293. 293/TLR4 were stimulated with 100 ng/mL LPS, a TLR4 agonist for 16 h after 48-h treatment with 5 mM butyrate or 10 mM propionate. SIGIRR, IL8, NFκB1, and iNOS expression were analyzed by real-time PCR. n = 4. D: butyrate inhibits flagellin-induced inflammatory gene expression. HIECs were treated with 5 mM butyrate for 16 h and then were stimulated with flagellin, a TLR5 agonist. Proinflammatory cytokines and inflammatory maker expression were quantified by real-time PCR. n = 3. E: butyrate reduced IL1R-mediated inflammation. HIECs were treated with 5 mM butyrate for 16 h and then were stimulated with IL1β, an IL1R agonist. Proinflammatory cytokines and inflammatory maker expression were quantified by real-time PCR. n = 3. F: SIGIRR is involved in butyrate-mediated inflammation suppression. HIECs stably expressing lentiviral SIGIRR shRNA or scramble shRNA (SCR) were treated with 5 mM butyrate for 16 h, followed by flagellin at 100 ng/mL for 8 h. Proinflammatory cytokines expression were quantified by real-time PCR. *P < 0.05. n = 3. Data shown as means ± SD, *P < 0.05, **P < 0.01. HIEC, human intestinal epithelial cell; SCFA, short-chain fatty acid; SIGIRR, single immunoglobulin interleukin-1-related receptor; TOLLIP, toll-interacting protein; TLR, toll-like receptor.

Figure 5.

SCFAs promote A20 and SIGIRR expression in neonatal intestine. A: immunofluorescent confocal images of DOL5, 10, and 14 mouse terminal ilea stained with Notch1 antibody. n = 3 mice per time point. Scale bar = 50 µm. B: A20 and SIGIRR expression in IECs isolated from terminal ileum of DOL8 mice with 3 days butyrate oral gavage were quantified by real-time PCR. n = 6 or 7 mice/group. C: mouse IEC cell lysates were analyzed by immunoblotting with indicated antibodies. n = 3 mice/group. Data shown as means ± SD, *P < 0.05, ****P < 0.005. DOL, day of life; IEC, intestinal epithelial cell; NICD, Notch intracellular domain; SCFA, short-chain fatty acid; SIGIRR, single immunoglobulin interleukin-1-related receptor.

Figure 6.

Butyrate alleviated NEC-like intestinal injury in neonatal mice. A: hematoxylin and eosin staining of terminal ileum from littermate control (control), experimental NEC (NEC) and NEC with butyrate administration (NEC + butyrate). Quantification of intestinal injury by a validated scoring tool shows decrease in injury score following butyrate administration. n = 5–7 mice/group. B: TUNEL staining of terminal ileum of indicated group, with quantification shown graphically. n = 5 mice/group. Scale bar = 50 µm. C: real-time PCR analysis of proinflammatory cytokines expression in terminal ileum from indicated mouse group. n = 4 or 5 mice/group. D: mouse terminal ileum lysates from control group, NEC group, and NEC plus enteral butyrate administration group were analyzed by immunoblotting with indicated antibodies. n = 3 mice/group. Data shown as means ± SD, **P < 0.01, ***P < 0.005. ****P < 0.001. NEC, necrotizing enterocolitis.