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. 2018 May;11(3):752-762.
doi: 10.1038/mi.2017.118. Epub 2018 Feb 7.

GPR43 mediates microbiota metabolite SCFA regulation of antimicrobial peptide expression in intestinal epithelial cells via activation of mTOR and STAT3

Ye Zhao  1   2 , Feidi Chen  3 , Wei Wu  2   4 , Mingming Sun  2   4 , Anthony J Bilotta  2 , Suxia Yao  2 , Yi Xiao  2   5 , Xiangsheng Huang  2 , Tonyia D Eaves-Pyles  2 , George Golovko  6 , Yuriy Fofanov  6 , Warren D'Souza  7 , Qihong Zhao  8 , Zhanju Liu  4 , Yingzi Cong  2   3
Affiliations

GPR43 mediates microbiota metabolite SCFA regulation of antimicrobial peptide expression in intestinal epithelial cells via activation of mTOR and STAT3

Ye Zhao et al. Mucosal Immunol. 2018 May.

Abstract

The antimicrobial peptides (AMP) produced by intestinal epithelial cells (IEC) play crucial roles in the regulation of intestinal homeostasis by controlling microbiota. Gut microbiota has been shown to promote IEC expression of RegIIIγ and certain defensins. However, the mechanisms involved are still not completely understood. In this report, we found that IEC expression levels of RegIIIγ and β-defensins 1, 3, and 4 were lower in G protein-coupled receptor (GPR)43-/- mice compared to that of wild-type (WT) mice. Oral feeding with short-chain fatty acids (SCFA) promoted IEC production of RegIIIγ and defensins in mice. Furthermore, SCFA induced RegIIIγ and β-defensins in intestinal epithelial enteroids generated from WT but not GPR43-/- mice. Mechanistically, SCFA activated mTOR and STAT3 in IEC, and knockdown of mTOR and STAT3 impaired SCFA induction of AMP production. Our studies thus demonstrated that microbiota metabolites SCFA promoted IEC RegIIIγ and β-defensins in a GPR43-dependent manner. The data thereby provide a novel pathway by which microbiota regulates IEC expression of AMP and intestinal homeostasis.

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Conflict of interest statement

Conflict of Interest: No authors have conflicting financial interests.

Figures

Figure 1
Figure 1. Decreased production of RegIIIγ and β-defensins in IEC of GPR43−/−mice
IEC from small intestine were isolated from WT and GPR43−/−mice respectively. (A) IEC expression of RegIIIγ and β-defensins 1, 3, 4 in GPR43−/−and WT mice determined by qRT-PCR and normalized against gapdh. Pooled data from 2 independent experiments. n=5 mice/group. (B) Protein levels of RegIIIγ and β-defensins were determined by Western blot. Data are reflective of 2 independent experiments. n=4 mice/group. (C) Protein/β-actin relative expression of RegIIIγ and β-defensins were compared between GPR43−/−and WT mice. Data were combined from 2 independent experiments. *p<0.05; **p<0.01.
Figure 2
Figure 2. Butyrate feeding promotes IEC expression of RegIIIγ and β-defensins in vivo
WT and GPR43−/−C57BL/6 mice were treated with antibiotics in drinking water for 10 days, and then fed with or without 300 mM butyrate in drinking water for 21 days. (A) IEC expression of RegIIIγ and β-defensins 1, 3, 4, and CRAMP in WT mice were determined by qRT-PCR and normalized against gapdh. Data are reflective of 2 independent experiments. n=4 mice/group. (B) Protein levels of RegIIIγ and β-defensins determined by Western blot in WT mice. Data are reflective of 2 independent experiments. (C) Protein/β-actin relative expression of RegIIIγ and β-defensins were compared between control and butyrate-treated WT mice. Data were combined from 2 independent experiments. *p<0.05. (D) IEC expression of RegIIIγ and β-defensins 1, 3, 4 in GPR43−/−mice were determined by qRT-PCR and normalized against gapdh. Data are reflective of 2 independent experiments. n=4 mice/group.
Figure 3
Figure 3. Butyrate feeding changes gut microbiota
WT and GPR43−/−C57BL/6 mice were fed with 300 mM butyrate for 21 days. Gut microbiota prior and after feeding butyrate was determined by 16s rRNA sequencing. Bar charts for fecal bacterial composition of phylum level (A), family level (B), and genus level (C). N=4–5 mice/group.
Figure 4
Figure 4. Butyrate and GPR43 agonist induce expression of RegIIIγ and β-defensins in mouse IEC
MSIE cells were treated with 0.5 mM butyrate for 48 h. (A) The expression of RegIIIγ and β-defensins mRNA determined by qRT-PCR and normalized against gapdh. (B) Protein levels of RegIIIγ and β-defensin 1 determined by Western blot. (C) MSIE cells were treated with 5 μM GPR43 agonist. The expression of RegIIIγ and β-defensins were determined by qRT-PCR at 48 h. *p<0.05; **p<0.01. Data are reflective of 3 independent experiments.
Figure 5
Figure 5. Butyrate and GPR43 agonist induce expression of RegIIIγ and β-defensins in human IEC
HT-29 cells were treated with 0.5 mM butyrate for 48 h. (A) The expression of α-defensins, β-defensins, RegIIIα, and LL37 were determined by qRT-PCR and normalized against gapdh. (B) HT-29 cells were treated with 5 μM GPR43 agonist 48 h, and the expression of α-defensin 6, β-defensin 1, RegIIIα, and LL37 was determined by qRT-PCR. *p<0.05; **p<0.01. Data are reflective of 3 independent experiments.
Figure 6
Figure 6. Butyrate induces expression of RegIIIγ and β-defensins in WT but not GPR43−/−intestinal epithelial enteroids
Intestinal epithelial enteroids were generated from either WT or GPR43−/−mice, and treated with 0.5 mM butyrate. The expression of RegIIIγ and β-defensins were determined by qRT-PCR at 48 h and normalized against gapdh. *p<0.05. Data are reflective of 2 independent experiments.
Figure 7
Figure 7. mTOR regulates butyrate induction of RegIIIγ and β-defensins in IEC
MSIE cells were treated with 0.5 mM butyrate for 1 h. (A) Phosphorylation of mTOR and 4E-BP1 were determined by Western blot, with β-actin as a loading control. (B) The expression of phosphorylated S6K1 was determined by flow cytometry at 48 h post butyrate treatment. Combined median fluorescence intensity (MFI) was presented. (C and D) MSIE cells were transfected with mTOR siRNA or control non-targeting siRNA, followed by 0.5 mM butyrate for 48 h. (C) siRNA knockdown efficiency was confirmed by RT-PCR at 40 h post-transfection. (D) The expression of RegIIIγ and β-defensins were determined by qRT-PCR and normalized against gapdh 48 h post-treatment. *p<0.05; **p<0.01. Data are reflective of 3 independent experiments.
Figure 8
Figure 8. STAT3 regulates butyrate induction of RegIIIγ and β-defensin in IEC
MSIE cells were treated with 0.5 mM butyrate for 1 h. (A) Phosphorylation of STAT3 was determined by Western blot, with total STAT3 and β-actin as loading controls. (B) MSIE cells were treated with 0.5 mM butyrate in the presence or absence of 5 μM STAT3 inhibitor HJC0152. The expression of RegIIIγ and β-defensins was determined by qRT-PCR at 48 h and normalized against gapdh. (C and D) MSIE cells were transfected with STAT3 siRNA or control non-targeting siRNA and then treated with 0.5 mM butyrate. (C) siRNA knockdown efficiency was confirmed by RT-PCR at 40 h post-transfection. (D) The expression of RegIIIγ and β-defensins were determined by qRT-PCR 48 h post-transfection and normalized against gapdh. (E) Protein levels of RegIIIγ and β-defensin were determined by Western blot and combined protein/β-actin relative expression was presented. *p<0.05; **p<0.01. Data are reflective of 3 independent experiments.
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