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. 2021;11(5):1327-1345.
doi: 10.1016/j.jcmgh.2021.01.004. Epub 2021 Jan 19.

Exposure to p40 in Early Life Prevents Intestinal Inflammation in Adulthood Through Inducing a Long-Lasting Epigenetic Imprint on TGFβ

Yilin Deng  1 , Oliver G McDonald  2 , Anna L Means  3 , Richard M Peek Jr  4 , M Kay Washington  5 , Sari A Acra  1 , D Brent Polk  6 , Fang Yan  7
Affiliations

Exposure to p40 in Early Life Prevents Intestinal Inflammation in Adulthood Through Inducing a Long-Lasting Epigenetic Imprint on TGFβ

Yilin Deng et al. Cell Mol Gastroenterol Hepatol. 2021.

Abstract

Background & aims: Colonization by gut microbiota in early life confers beneficial effects on immunity throughout the host's lifespan. We sought to elucidate the mechanisms whereby neonatal supplementation with p40, a probiotic functional factor, reprograms intestinal epithelial cells for protection against adult-onset intestinal inflammation.

Methods: p40 was used to treat young adult mouse colonic (YAMC) epithelial cells with and without deletion of a methyltransferase, su(var)3-9, enhancer-of-zeste and trithorax domain-containing 1β (Setd1β), and mice in early life or in adulthood. Anti-transforming growth factor β (TGFβ)-neutralizing antibodies were administered to adult mice with and without colitis induced by 2,4,6-trinitrobenzenesulfonic acid or dextran sulfate sodium. We examined Setd1b and Tgfb gene expression, TGFβ production, monomethylation and trimethylation of histone H3 on the lysine 4 residue (H3K4me1/3), H3K4me3 enrichment in Tgfb promoter, differentiation of regulatory T cells (Tregs), and the inflammatory status.

Results: p40 up-regulated expression of Setd1b in YAMC cells. Accordingly, p40 enhanced H3K4me1/3 in YAMC cells in a Setd1β-dependent manner. p40-regulated Setd1β mediated programming the TGFβ locus into a transcriptionally permissive chromatin state and promoting TGFβ production in YAMC. Furthermore, transient exposure to p40 during the neonatal period and in adulthood resulted in the immediate increase in Tgfb gene expression. However, only neonatal p40 supplementation induced the sustained H3K4me1/3 and Tgfb gene expression that persisted into adulthood. Interfering with TGFβ function by neutralizing antibodies diminished the long-lasting effects of neonatal p40 supplementation on differentiation of Tregs and protection against colitis in adult mice.

Conclusions: Exposure to p40 in early life enables an epigenetic imprint on TGFβ, leading to long-lasting production of TGFβ by intestinal epithelial cells to expand Tregs and protect the gut against inflammation.

Keywords: Histone Methyltransferase; Intestinal Epithelial Cell; Probiotic Function Factor; Regulatory T Cell.

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Figures

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Graphical abstract
Figure 1
Figure 1
p40 stimulates TGFβ production in IECs. (A and B) Enteroids and colonoids from WT mice were cultured in IntestiCult organoid growth medium in the presence and absence of p40 (100 ng/mL). (A) Representative images of colonoids at the indicated days after culture are shown. Images were taken using a light microscope at a magnification of 10×. (C and D) YAMC cells were treated with p40 at 10 ng/mL for the indicated times. RNA was isolated from (B) enteroids and colonoids cultured for 9 days, and (C) YAMC cells for RT-PCR analysis of the expression levels of Tgfb mRNA. The Tgfb mRNA expression levels in the control groups were set as 1. The mRNA expression levels in treated groups were compared with the control group. (D) Supernatants from YAMC cell culture with and without p40 treatment were collected for assessing the amount of TGFβ release using enzyme-linked immunosorbent assay. The TGFβ concentration is presented as picograms per 106 cells. (B–D) Data are quantified from 3 independent experiments. (B) For each experiment, data represent the average of at least 5 enteroids or colonoids in each group. ∗P < .05 compared with the control group. #P < .05 compared with the 2-hour p40 treatment group.
Figure 2
Figure 2
p40-stimulated TGFβ production in IECs contributes to protective epithelial cellular responses. (A) YAMC cells were treated with p40 (10 ng/mL) for the indicated times. Total cellular proteins were prepared for Western blot analysis. β-actin blot was used as the protein loading control. (B) Caco2 cells were treated with H2O2 (20 μmol/L) for 4 hours with or without 1-hour pretreatment of p40 (10 ng/mL) and TGFβ-neutralizing antibody (1 μg/mL). (C) Total cellular proteins from IMCS4fl/fl and IMCS4null cells were prepared for Western blot analysis. (D) IMCS4fl/fl and IMCS4null cells were treated with H2O2 (20 μmol/L) for 4 hours with or without 1-hour pretreatment of p40 (10 ng/mL). The cells were fixed and immunostained to localize ZO-1 using an anti–ZO-1 antibody and a Cy3-conjugated secondary antibody (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Membrane (white arrows) and intracellular (yellow arrows) ZO-1 distribution are shown. Images were taken using a fluorescent microscope at a magnification of 40×. (E) Cells were treated with lipopolysaccharide (LPS) (1 μg/mL) with and without p40 (10 ng/mL) for 24 hours. RNA was isolated for RT-PCR analysis of the expression levels of indicated genes. The mRNA expression levels in the control groups were set as 1. The mRNA expression levels in treated groups were compared with the control group of the same cell line. (A and C) The fold changes of band density are shown under the blot. (E) ∗P < .05 compared with the control group of the same cell line. Data in this figure are representative of at least 3 independent experiments. Ab, antibody; CCL, CC chemokine ligands; KC, keratinocytes-derived chemokine; P-SMAD3, phosphorylated-SMAD3; T-SMAD3, total-SMAD3.
Figure 3
Figure 3
p40 supplementation in early stage has a long-term effect on the increase in Tgfb gene expression in mice. (A) For testing the short-term effects of p40 in neonate and adult mice, WT mice were supplemented with p40 from postnatal day 2 to day 14 or from week 6 to week 8. Mice were killed at the end of treatment. (B) For testing the long-term effects of p40 supplementation in neonatal stage and in adulthood, WT mice were supplemented with p40 from postnatal day 2 to day 21 (neo-p40) and from week 6 to week 8 (adult-p40). Mice supplemented with hydrogels without p40 were used as controls. Mice were killed 4 weeks after the end of p40 supplementation. Each symbol represents one mouse in A and B. (C) For testing the sustained effects of p40 on Tgfb gene expression in mouse enteroids, enteroids from WT mice were treated with p40 (100 ng/mL) and passaged as indicated. RNA was isolated from colonic tissues and enteroids for RT-PCR analysis of Tgfb1 gene expression. The Tgfb1 mRNA expression levels in the control groups were set as 1. The mRNA expression levels in treated groups were compared with the control group. (C) Data are quantified from 3 independent cultures.
Figure 4
Figure 4
p40 up-regulates Setd1β expression in IECs. (A, B, D, and E) YAMC cells were treated with p40 at 10 ng/mL for the indicated times. (C) Enteroids and colonoids were generated and cultured as described in Figure 1, in the presence or absence of p40 (100 ng/mL). (A and C) RNA was isolated from (A) YAMC cells and (C) enteroids and colonoids for RT-PCR analysis of the levels of Setd1b mRNA. Setd1b mRNA expression levels in the control groups were set as 1. The mRNA expression levels in treated groups were compared with the control group. Data are quantified from 3 independent experiments. (C) For each experiment, data represent the average of at least 5 enteroids or colonoids in each group. (B, D, and E) Total cellular proteins were prepared from YAMC cells for Western blot analysis. β-actin blot was used as the protein loading control. The fold changes of band density are shown under the blot. Data are representative of at least 3 independent experiments. ∗P < .05 compared with the control group.
Figure 5
Figure 5
p40 supplementation in early life stimulates short-term up-regulation of Setd1b gene expression, but sustained increase in epigenetic marks in mice. The treatment plans for detecting the short-term (Figure 3A) and long-term effects (Figure 3B) are described. (A and B) RNA was isolated from colonic tissues for RT-PCR analysis of Setd1b gene expression. Each symbol represents one mouse in A and B. (C) Paraffin-embedded tissue sections were prepared for immunohistochemistry using anti-H3K4me1 antibody and fluorescein isothiocyanate–conjugated secondary antibody (green), an epithelial cell marker using anti–E-cadherin antibody and Cy3-conjugated secondary antibody (red), and nuclei using 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). Images were taken using a fluorescent microscope at 40×. (D) The numbers of H3K4me1 and E-cadherin double-positive cells per crypt are shown. N = 5 in each group.
Figure 6
Figure 6
Increase of Setd1b expression in IECs mediates p40-promoted TGFβ production. (A and B) YAMC cells were transduced with lentiviral Setd1b shRNAs to generate a stable cell line with reduced expression of Setd1β. Nontargeting shRNA was used as a control. Knockdown efficiency of Setd1β was determined by assessing (A) mRNA expression and (B) protein levels using RT-PCR and Western blot analysis, respectively. The Setd1b mRNA expression level in the nontarget shRNA transduced cells was set as 1. (C) Total cellular lysates were prepared to detect p40-regulated histone methylation in the indicated cell lines. (D and E) H3K4me3 region within the TGFβ locus in mouse intestinal cells is shown. The enrichment of H3K4me3 in the Tgfb1 promoter was analyzed by ChIP quantitative PCR. The percentage of relative enrichment (H3K4me3/Input) in the control group is set as 1. (F and G) Cells were treated with p40 (10 ng/mL) for the indicated times. (F) RNA was isolated for RT-PCR analysis of the Tgfb1 mRNA level. The Tgfb1 mRNA expression level in the control group with nontarget transduction was set as 1. (G) Supernatants from cell culture were collected for analysis of the amount of TGFβ release using enzyme-linked immunosorbent assay, as described in Figure 1. ∗P < .05 compared with the control group in each cell line. #P < .05 compared with the p40 treatment group in nontargeting shRNA-transduced cell line. (B and C) The fold changes of band density are shown under the blot. Images shown are representative of 3 independent experiments. (A and E–G) Data are quantified from 3 independent experiments.
Figure 7
Figure 7
Neonatal p40 supplementation-promoted Treg expansion in the lamina propria of the colon in adult mice requires sustained increase in TGFβ production. (A) The treatment plan is shown. Foxp3-GFP mice were supplemented with p40 from postnatal day 2 to day 21 and received anti–TGFβ-neutralizing antibodies or isotype-negative control antibodies (IgG) at 50 μg/d at the indicated time points. Lymphocytes were isolated from the lamina propria of the colon. CD4- and Foxp3-expressing cells were assessed using flow cytometry analysis. (B) Representative contour plots of Foxp3 and CD4 are shown. Numbers in quarter 2 of contour plots represent the percentages of CD4+Foxp3+ in total lamina propria cells. (C) The percentages of CD4+Foxp3+ cells in total lamina propria cells are shown. N = 3 samples. Each sample contains cells from 2 to 3 mice. ∗P < .05 compared with the counterpart in the no-p40 group. #P < .05 compared with the Neo-p40 group with TGFβ antibody co-treatment. Ab, anti-TGFβ antibody.
Figure 8
Figure 8
Sustained TGFβ production by neonatal p40 supplementation mediates prevention of colitis in adult mice. (A) The treatment plan is shown. Mice were supplemented with p40 from postnatal day 2 to day 21 and received TGFβ-neutralizing antibodies or isotype control antibodies (IgG) at 50 μg/d, at the indicated time points. Colitis was induced by TNBS in ethanol intrarectally. Mice receiving ethanol were used as controls for TNBS treatment. Mice were killed 4 days after TNBS treatment. (B) Colon sections were stained with H&E for assessment of inflammation. (C) The inflammation scores are shown. (D) RNA was isolated from the colonic tissues for RT-PCR analysis of the indicated cytokine mRNA expression levels. The average cytokine mRNA expression level in the control mice of the no-p40 group was set as 1, and the mRNA expression level of each mouse was compared with this average. ∗P < .05 compared with the control mice in the no-p40 group. #P < .05 compared with the p40 group with TNBS or TNBS and IgG co-treatment. (E) Paraffin-embedded colon tissues were used to determine ZO-1 distribution by immunohistochemistry using an anti–ZO-1 antibody and a Cy3-labeled secondary antibody (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Slides with H&E staining and immunostaining were scanned and images were exported at 10X magnification. Membrane (white arrowheads) and intracellular (yellow arrowheads) ZO-1 distributions are shown. Ab, antibody; Sac, sacrifice.
Figure 9
Figure 9
Sustained TGFβ production by neonatal p40 supplementation mediates. prevention of colitis in adult mice. (A) The treatment plan is shown. Mice were supplemented with p40 from postnatal day 2 to day 21 and received TGFβ-neutralizing antibodies or isotype control antibodies (IgG) at 50 g/d, at the indicated time points. Colitis was induced by 3% DSS in drinking water for 4 days. Mice receiving water were used as controls for DSS treatment. Mice were killed at the end of DSS treatment. (B) Colon sections were stained with H&E for assessment of inflammation. Slides were scanned and images were exported at 10X magnification. (C) The inflammation/injury scores are shown. (D) RNA was isolated from the colonic tissues for RT-PCR analysis of the indicated cytokine mRNA expression levels. The average cytokine mRNA expression level in the control mice of the no-p40 group was set as 1, and the mRNA expression level of each mouse was compared with this average. ∗P < .05 compared with the control mice in the no-p40 group. #P < .05 compared with the p40 group with DSS or DSS and IgG co-treatment. Ab, antibody; Sac, sacrifice.
Figure 10
Figure 10
p40 stimulates Tgfb and Setd1b expression and histone modification in small intestinal epithelial cells in vitro and in vivo. (A–C) MSIE cells were treated with p40 at 10 ng/mL for the indicated times. RNA was isolated for RT-PCR analysis of the mRNA expression levels of (A) Setd1b and (C) Tgfb. The mRNA expression level in the control group was set as 1. The mRNA expression level in the treated group was compared with the control group. (B) Total cellular proteins were prepared from MSIE cells for Western blot analysis. β-actin blot was used as the protein loading control. The band density fold changes are shown under the bands. (A and C) Data are quantified from 3 independent experiments. (B) Images represent results in at least 3 independent experiments. (D) The treatment plan. WT mice were supplemented with p40 for testing the short-term and the long-term effects, as shown in Figure 3. Mice supplemented with hydrogels without p40 were used as controls. (E and F) RNA was isolated from small intestinal tissues for RT-PCR analysis of Tgfb1 and Sedt1b gene expression. The mRNA expression level in the control groups was set as 1. The mRNA expression level in treated groups was compared with the control group. Sac, sacrifice.
Figure 11
Figure 11
p40-stimulated EGFR transactivation and Setd1b gene expression in IECs are 2 independent functions. YAMC, Egfr-/- mouse colonic epithelial (MCE), and YAMC transduced with lentiviral Setd1b shRNAs or nontargeting shRNA as used in Figure 6 were treated with p40 at 10 ng/mL for the indicated times. (A) RNA was isolated for RT-PCR analysis of the levels of Setd1b mRNA. The Setd1b mRNA expression level in the control group was set as 1. The mRNA expression levels in treated groups were compared with the control group. (B and C) Western blot analysis of cellular lysates was performed to detect levels of H3K4me3, total H3, phospho-Tyr1068-EGFR (P-EGFR), and total EGFR. β-actin blot was used as a loading control. (A) Data are quantified from 3 independent experiments. (B and C) Images are representative of at least 3 independent experiments.
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