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. 2017 Dec 4;214(12):3507-3518.
doi: 10.1084/jem.20170418. Epub 2017 Oct 24.

R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease

Eiko Hayase  1 , Daigo Hashimoto  2 , Kiminori Nakamura  3 , Clara Noizat  1 , Reiki Ogasawara  1 , Shuichiro Takahashi  1 , Hiroyuki Ohigashi  1 , Yuki Yokoi  4 , Rina Sugimoto  4 , Satomi Matsuoka  1 , Takahide Ara  1 , Emi Yokoyama  1 , Tomohiro Yamakawa  1 , Ko Ebata  1 , Takeshi Kondo  1 , Rina Hiramine  5   6 , Tomoyasu Aizawa  5   6 , Yoshitoshi Ogura  7 , Tetsuya Hayashi  7 , Hiroshi Mori  8 , Ken Kurokawa  8   9 , Kazuma Tomizuka  10 , Tokiyoshi Ayabe  3 , Takanori Teshima  11
Affiliations

R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease

Eiko Hayase et al. J Exp Med. .

Abstract

The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits.

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Figures

Figure 1.
Figure 1.
R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H&E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt (n = 6 per group). (C) Crypt depth (n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H&E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5-EGFP-creER×R26Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5+ ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5+ ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum (n = 3 per group). (H) Numbers of goblet cells per crypt (n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum (n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA (n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method (n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2.
Figure 2.
R-Spo1 protects Paneth cells from GVHD and prevents intestinal dysbiosis. Lethally irradiated B6D2F1 mice were transplanted with BM cells plus splenocytes from B6 (Allo) or B6D2F1 (Syn) donors on day 0. Mice were treated with R-Spo1 or PBS on day −3 to −1 and day 1 to 3. (A) Confocal images of the small intestine on days 0 and 7. Crp1 with DAPI (blue) counterstaining. (B) H&E staining of the small intestine on day 7. Areas in the white squares are magnified and shown to the right of the original images. (C) Numbers of Paneth cells per crypt (n = 4–9 per group). (D) Fecal levels of Crp4 (n = 4–8 per group). (E) A group of mice received a combination of four antibiotics (4ABX) in drinking water from day −7. Ratio of bacterial load in fecal pellets on day 7 determined by quantitative PCR of 16S rRNA gene copies compared with that of naive mice (n = 4 per group). (F–M) Intestinal microbial compositions were determined by 16S rRNA sequencing (Naive, n = 4; Syn, n = 5; Allo, n = 14; R-Spo1, n = 21). (F) Each bar represents bacterial composition of an individual mouse at the genus level. (G) Principal component analysis of genus compositions of intestinal microbiota from each mouse. (H) The p-values of PERMANOVA of the genus compositions. Bonferroni-corrected statistically significant combinations are indicated in yellow. (I–K) Correlation analysis between body weight on day 7 after allogeneic SCT and abundance of the genus Escherichia, Bacteroides, and Lactobacillus determined by a linear regression using the Spearman analysis for nonparametric data. (L) Diversities of intestinal microbiome determined by a Simpson index at the genus level. (M) Abundances of specific bacteria at the genus levels. Data from two (C–E) or six (F–K) independent experiments were combined and are shown as means ± SE. (C–E, L, and M) Mann–Whitney U tests or one-way ANOVA followed by Tukey’s posttest were used to compare the data. Bars, 50 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3.
Figure 3.
Oral administration of Crp4 partially prevents dysbiosis and GVHD. SCT was performed as in Fig. 2. Recipient mice were orally administered 125 µg Crp4 or control twice a day from day 3 to 7 after allogeneic SCT. (A) Fecal levels of Crp4 were measured with ELISA (means ± SE, n = 6 per group). (B, C, and E–H) Bacterial compositions of intestinal microbiota at the genus level determined by 16S rRNA sequencing on days 7 and 18 (B), Simpson diversity index of intestinal microbiota on days 7 and 18 (C), principal component analysis of genus compositions of intestinal microbiota on day 7 (E), the q-values (false discovery rates) of PERMANOVA of genus compositions between groups (F), and abundances of specific bacteria at the genus levels (G) and order levels (H) are shown. Yellow highlighting in F indicates statistically significant values. (D) Ratio of bacterial load in fecal pellets on day 7 by quantitative PCR of 16S rRNA gene copies compared with that of naive mice (means ± SE, n = 4 per group). (I–K) Body weight (I) and clinical GVHD scores (J) on days 21 and 35 and survival curves (K) after SCT (n = 12 per group) are shown. (C–J) Data from one of two independent experiments with similar results are shown. (K) Data from two independent experiments were combined and are shown as means ± SE. (C, D, and G–J) Student’s t tests or Mann–Whitney U tests were used to compare the data. (K) Log-rank test was used to compare survival curves. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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