R-spondin1 Promotes Paneth Cell Growth, Maintains Intestinal Microbial Ecology, and Ameliorates GvHD
Authors: Hayase Eiko , Hashimoto Daigo , Nakamura Kiminori , Ogura Yoshitoshi , Hayashi Tetsuya , Kurokawa Ken , Ayabe Tokiyoshi , Teshima Takanori 
Published: October 2019

Abstract
Introduction: Graft-versus-host disease (GVHD) and infections are the major obstacles to perform allogeneic hematopoietic stem cell transplantation (HSCT). We previously demonstrated that intestinal stem cells (ISCs) and their niche Paneth cells were targeted in GVHD. The intestinal microbial ecology is regulated by Paneth cells through secretion of antimicrobial molecules. Loss of Paneth cells by GVHD frequently leads to intestinal dysbiosis (Eriguchi et al, Blood, 2012). R-spondin1 (R-Spo1) is a growth factor of ISCs and protects ISCs from the damages by conditioning and GVHD (Takashima et al, J Exp Med. 2011). However, the effects of R-Spo1 on Paneth cell biology and intestinal microbial ecology have never been demonstrated.
Method: Naïve B6D2F1 (H-2b/d) mice were intravenously injected with R-Spo1 (200 μg/day) or diluent for 6 days. In HSCT models, lethally irradiated B6D2F1 mice were transplanted with 5 × 106 splenocytes plus 5 × 106 bone marrow cells from allogeneic B6 (H-2b) or syngeneic B6D2F1 donors on day 0. Mice were injected with R-Spo1 or diluent from day -3 to -1 and day +1 to +3 after HSCT. On days +4 and +7, small intestine was harvested to evaluate histology and fecal samples were collected to measure fecal levels of alpha-defensin and to determine composition of microbiota by 16S rRNA gene sequencing.
Result: In naïve mice, R-Spo1 administration significantly increased Paneth cells in the small intestine (Table). Fate mapping analysis using LGR5-creERT2 × Rosa26Tomato mice to visualize LGR5+ ISCs and their progenies revealed that R-Spo1 did not expand pre-existing Paneth cells but stimulated de novo differentiation of Paneth cells from ISCs. R-Spo1-induced Paneth cells produced antimicrobial peptide alpha-defensin and its activator matrix metalloproteinase-7, leading to a significant increase in fecal levels of alpha-defensin (Table). Next, we evaluated the effects of R-Spo1 on Paneth cells and intestinal microbial ecology in a HSCT model. The numbers of Paneth cells in the small intestine and fecal levels of alpha-defensin were significantly reduced in allogeneic controls compared to those in syngeneic controls on day +7 (Table), resulting in dysbiosis with a significant outgrowth of the family Enterobacteriaceae, including the genus Escherichia, and the family Bacteroidaceae in allogeneic animals (Figure). R-Spo1 protected Paneth cells from GVHD, resulting in significantly more Paneth cells persisting on days +4 and +7 compared to allogeneic controls (Table). Importantly, R-Spo1 restored the fecal levels of alpha-defensin in allogeneic recipients (Table), resulted in preservation of intestinal commensals (Figure). R-Spo1 reduced GVHD mortality as previously reported, but this survival benefit mediated by R-Spo1 was abrogated when mice were treated with broad-spectrum antibiotics including ampicillin, streptomycin, vancomycin and metronidazole, suggesting that R-Spo1-mediated GVHD suppression depended on intestinal commensals.
Conclusion: We demonstrated for the first time that R-Spo1 is a growth factor of Paneth cells. R-Spo1 administration in allogeneic recipients restored fecal levels of alpha-defensin, prevented intestinal dysbiosis, and improved the outcome of HSCT in the presence of intestinal commensals. Brief administration of R-Spo1 represents a novel pharmacological approach to modify intestinal microbial ecology. Such strategies to target the intestinal microbiota may have benefits in the treatments of inflammatory bowel diseases other than GVHD.
Table. No. of Paneth cells / crypt Fecal levels of alpha-defensin4(ng / g feces) Naïve B6D2F1 Control R-Spo1 3.6 ± 0.2 10.2 ± 0.7* 46.3 ± 5.3 105.3 ± 28.8* Transplant (day4) Allo Allo+R-Spo1 2.2 ± 0.1 3.9 ± 0.2* 25.9 ± 4.0 108.2 ± 12.6* Transplant (day7) Syn Allo Allo+R-Spo1 3.1 ± 0.1 0.7 ± 0.1 1.3 ± 0.2* 18.9 ± 3.8 7.1 ± 2.9 25.6 ± 4.2*
Data are shown as mean ± SE. NA; not assessed, *P<0.05 (Control vs R-Spo1, Allo vs Allo+R-Spo1)
Figure 1. Figure 1. R-Spo1 prevents dysbiosis in allogeneic recipients: Lethallyirradiated B6D2F1 mice were transplanted from syngeneic B6D2F1 or allogeneic B6 mice. A group of allogeneic recipients were treated with R-Spo1 from day -3 to -1 and day +1 to +3 after HSCT. 16S rRNA sequence analysis was performed to assess the intestinal microbiota on day +7 after HSCT.

Disclosures
No relevant conflicts of interest to declare.