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. 2017 Oct 15;199(8):2976-2984.
doi: 10.4049/jimmunol.1700105. Epub 2017 Sep 11.

Microbial-Derived Butyrate Promotes Epithelial Barrier Function through IL-10 Receptor-Dependent Repression of Claudin-2

Leon Zheng  1   2 , Caleb J Kelly  1   2 , Kayla D Battista  1   2 , Rachel Schaefer  1   2 , Jordi M Lanis  1   2 , Erica E Alexeev  1   2 , Ruth X Wang  1   2 , Joseph C Onyiah  1   2 , Douglas J Kominsky  3 , Sean P Colgan  4   2   5
Affiliations

Microbial-Derived Butyrate Promotes Epithelial Barrier Function through IL-10 Receptor-Dependent Repression of Claudin-2

Leon Zheng et al. J Immunol. .

Abstract

Commensal interactions between the enteric microbiota and distal intestine play important roles in regulating human health. Short-chain fatty acids (SCFAs), such as butyrate, produced through anaerobic microbial metabolism represent a major energy source for the host colonic epithelium and enhance epithelial barrier function through unclear mechanisms. Separate studies revealed that the epithelial anti-inflammatory IL-10 receptor α subunit (IL-10RA) is also important for barrier formation. Based on these findings, we examined if SCFAs promote epithelial barrier through IL-10RA-dependent mechanisms. Using human intestinal epithelial cells (IECs), we discovered that SCFAs, particularly butyrate, enhanced IEC barrier formation, induced IL-10RA mRNA, IL-10RA protein, and transactivation through activated Stat3 and HDAC inhibition. Loss and gain of IL-10RA expression directly correlates with IEC barrier formation and butyrate represses permeability-promoting claudin-2 tight-junction protein expression through an IL-10RA-dependent mechanism. Our findings provide a novel mechanism by which microbial-derived butyrate promotes barrier through IL-10RA-dependent repression of claudin-2.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Butyrate enhances intestinal epithelial cell barrier formation
T84 cells were treated with HHBS (Hanks’ Buffer with 10mM HEPES) at time 0h ± (A) butyrate (5mM), (B) propionate (20mM), or (C) acetate (20mM). Transepithelial electrical resistance (TEER) was measured and analyzed over 72h. Data are representative of at least two independent experiments. Error bars represent mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001; n.s., not significant (unpaired Student’s t test and ANOVA).
Figure 2
Figure 2. Butyrate upregulates interleukin-10 receptor α (IL-10RA)
(A) Caco-2 and (B) T84 cells were treated with HHBS ± acetate (20mM), propionate (20mM), or butyrate (5mM) for up to 24h. RNA was isolated, cDNA was synthesized and IL-10RA mRNA was quantitated by qPCR (data normalized to β-actin). (C, D) In a separate experiment, cells were treated with HHBS ± butyrate (0–10mM) for 18h and IL10RA mRNA was analyzed by qPCR (data normalized to β-actin). (E) Caco-2 and (F) T84 cells were treated for 24h with HHBS ± butyrate (0–20mM). Whole-cell lysates were collected and probed for IL-10RA by immunoblot (β-actin was used as loading control). (G) Representative photographs of immunofluorescence staining of IL-10RA (red) in T84 cells following HHBS ± butyrate (10mM) for 24h. Nuclei stained with DAPI (blue). (H) Caco-2 cells were transfected with empty vector control (EV) and an IL10RA promoter reporter (IL10RA-luc). After 24h, cells were treated with HHBS ± butyrate (0–10mM) for another 24h and luciferase activity was quantified. (I) T84 cells were treated with HHBS ± butyrate (10mM) for 24h and stimulated with PBS ± IL-10 (20ng/ml) for 2h. SOCS3 mRNA measured by qPCR (data normalized to β-actin). Data are representative of two or three independent experiments. Error bars represent mean ± SEM.
Figure 3
Figure 3. Butyrate regulates IL-10RA through Stat3 activation and histone deacetylase inhibition
(A) T84 cells were pretreated with DMSO ± Stattic (20mM) for 30min followed by HHBS ± butyrate (10mM) for 24h. Cells were stimulated with PBS ± IL-10 (20ng/ml) for 1h prior to harvesting the whole-cell lysates and probing for pStat3 by immunoblotting (β-actin was used as loading control). (B) T84 cells were pretreated with DMSO ± Stattic (20mM) for 30min followed by HHBS ± butyrate (5mM) for 6h. IL10RA mRNA was measured by qPCR (data normalized to β-actin). (C) T84 cells were stimulated with DMSO control or TSA (100, 250, or 500ng/ml) for 18h. RNA was isolated, cDNA was synthesized, and IL10RA mRNA was quantitated by qPCR (data normalized to β-actin). (D) T84 cells were treated with HHBS ± butyrate (2.5, 5mM) for 24h, then whole-cell lysates were collected and acH3K9 was probed by immunoblot (β-actin was used as loading control). Data are representative of at least three independent experiments. Error bars represent mean ± SEM.
Figure 4
Figure 4. IL-10RA plays a pivotal role in formation and integrity of the intestinal epithelial cell barrier
(A) Relative IL10RA mRNA in T84 cells transduced with lentivirus shRNA targeting IL10RA (shIL10RA) v. shControl by qPCR. (B) IL10RA gene expression, by qPCR, in T84 cells transduced with IL10RA ORF lentivirus (LentiORF; oeIL10-RA) v. shControl cells (data normalized to β-actin). (C) T84 shControl (shCtrl) and shIL10RA were treated with HHBS ± butyrate (5mM) at 0h. TEER was measured and analyzed over 75h. (D) T84 shCtrl and oeIL10RA were treated with HHBS ± butyrate (5mM) at 0h. TEER was recorded over 75h. Data are representative of three independent experiments. (A, B) Error bars represent mean ± SEM. (C, D) Error bars represent mean ± SD.
Figure 5
Figure 5. Butyrate represses Claudin-2 through IL-10RA and pStat3
(A) Caco-2 and (B) T84 cells were stimulated with HHBS ± butyrate (5mM) for 18h. cDNA generated from RNA isolation was analyzed by qPCR and data was normalized to β-actin. (C) Claudin-2 (Cldn2) was probed by immunoblot in T84 cells treated with HHBS ± butyrate (2.5 or 5mM) for 24h and also T84 shCtrl, shIL10RA, and oeIL10RA cell lines (β-actin was loading control). (D) T84 cells were pretreated with DMSO ± Stattic (20mM) for 30min followed by HHBS ± butyrate (5mM) for 6h. Cldn2 mRNA was measured by qPCR (data normalized to actin). Data are representative of at least three independent experiments. Error bars represent mean ± SEM.
Figure 6
Figure 6. Microbial-derived butyrate enhances Il10ra in an in vivo system
C57BL/6 mice were gavaged with broad-spectrum antibiotics for 3d and euthanized. (A) Colon and (B) ileal tissue collected from the control and antibiotic treated mice were lysed, RNA was isolated, cDNA was synthesized, and Il10ra mRNA was analyzed by qPCR. (C and D) Data collected from Geo Profiles of both Rank % and transformed counts of germ free mice colonic Il10ra during conventionalization were graphed. Data are representative of three independent experiments (A, B). Error bars represent mean ± SEM.
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