Supplementary Materials Supplemental Textiles (PDF) JEM_20170418_sm. -Defensins will be the many bactericidal AMPs released from Paneth cells (Ayabe et al., 2000; Salzman et al., 2010). Rising proof demonstrates Sstr2 that Paneth cell features are impaired in a variety of inflammatory and metabolic disorders, leading to unfavorably changed intestinal microbiota (dysbiosis; Bevins and Salzman, 2013). Dysbiosis, nevertheless, exacerbates the root diseases, developing a vicious circuit between your web host and microbiota thus. Graft-versus-host disease (GVHD) can be an alloreactive, donor T cellCmediated inflammatory disease occurring after allogeneic hematopoietic stem cell transplantation (SCT), relating to the epidermis, liver, and gastrointestinal tract (Ferrara et al., 2009). We and others have shown that GVHD leads to a loss of Paneth cells and mediates intestinal dysbiosis (Eriguchi et al., 2012; Jenq et al., 2012). The dysbiosis that occurs in MHC-mismatched mouse models of GVHD is definitely Curculigoside remarkable and thus represents a feasible tool to test novel strategies to modulate dysbiosis (Eriguchi et al., 2012). Current strategies to restore the gut ecosystem are bacteriotherapy, using diet, prebiotics/probiotics, and fecal microbiota transplantation; however, no physiological approach has been developed so far. Here, we Curculigoside demonstrate a novel approach to restore intestinal microbial ecology and prevent dysbiosis by Wnt agonist R-Spondin1 (R-Spo1; Kim et al., 2005; Takashima et al., 2011) or recombinant -defensin (Tomisawa et al., 2015) in mice. The Wnt agonist R-Spo1, which binds to leucine-rich repeatCcontaining G proteinCcoupled receptor (Lgr) 5, is one of the essential factors to create intestinal villus-crypt models from a single Lgr5+ intestinal stem cell (ISC; Sato et al., 2009; de Lau et al., 2011; Farin et al., 2016). We found that R-Spo1 stimulates ISCs to differentiate to Paneth cells and enhanced luminal secretion of -defensins. In addition, administration of R-Spo1 or the recombinant mouse -defensin cryptdin-4 (Crp4) helps prevent GVHD-mediated dysbiosis after SCT. Such methods symbolize a physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and hostCmicrobiota cross talk toward restorative benefits. Because dysbiosis has a role in the pathogenesis of many diseases, such methods have broad potential in individuals at risk or with numerous diseases. Results and conversation R-Spo1 stimulates ISC differentiation to Paneth cells and enhances Paneth cell production of -defensins R-Spo1 enhances the proliferation of Curculigoside cycling ISCs via the Wnt/-catenin signaling pathway and generates crypt-villus organoids from ISCs in vitro (Sato et al., 2009). We previously showed that administration of R-Spo1 stimulated proliferation of ISCs and induced crypt cell hyperplasia in vivo (Kim et al., 2005; Takashima et al., 2011). However, the effects of R-Spo1 on Paneth cell proliferation and function remain to be identified. Here, we 1st resolved whether R-Spo1 could increase the number of Paneth cells in vivo. R-Spo1 was i.v. injected to B6D2F1 mice at a dose of 200 g for 6 d. The number of Paneth cells morphologically identified as cells comprising eosinophilic granules in H&E staining was significantly increased in all sites of the small intestine, including duodenum, jejunum, and ileum of R-Spo1Ctreated mice (Fig. 1, A and B). R-Spo1 significantly elongated crypt depth (Fig. 1 C). Although Kim et al. (2005) showed that daily injection of R-Spo1 at a dose of 100 g for 3 d did not increase Paneth cell figures, variations in dose and period of the R-Spo1 used may clarify the discrepancy in the results between studies. Immunofluorescence studies shown that Paneth cells generated by R-Spo1 coexpress lysozyme, Crp1, a subtype of -defensins, and matrix metalloproteinase-7 (MMP-7), which converts proC-defensins into active form (Fig. 1, D and E). These results indicate that they are functionally mature Paneth cells (Wilson et al., 1999). Very similar outcomes were attained in BALB/c mice, ruling out the strain-specific ramifications of R-Spo1 on Paneth cell extension (Fig. S1, ACC). There have been some MMP-7+ Crp1? cells in R-Spo1Ctreated mice (Fig. 1 Fig and E. S1 C). Although features of the cells remain to become elucidated, Wnt activation can lead to precocious differentiation of progenitors into Paneth cells (Tian et al., 2015). Open up in another window Amount 1. R-Spo1 treatment promotes development of Paneth cells from increases and ISCs luminal concentrations of -defensins. (ACE and HCP) B6D2F1 mice had been i.v. injected with Curculigoside R-Spo1 (200 g/d) or PBS for 6 d. 1 Curculigoside d afterwards, the tiny intestine was gathered..