Microbiota-Derived Lactate Accelerates Intestinal Stem-Cell-Mediated Epithelial Development

Article

Microbiota-Derived Lactate Accelerates Intestinal Stem-Cell-Mediated Epithelial Development Graphical Abstract

Authors Yong-Soo Lee, Tae-Young Kim, Yeji Kim, ..., Nobuhiko Kamada, Nan Gao, Mi-Na Kweon

Correspondence [email protected]

In Brief Lee et al. reveal how lactic-acidproducing bacteria, including Bifidobacterium and Lactobacillus spp., support intestinal epithelial cell regeneration. Symbiont-derived lactate is sensed by G-protein-coupled receptor 81 on Paneth and stromal cells to promote regeneration in a Wnt3/ b-catenindependent manner. Lactate preadministration protects mice exposed to radiation- and chemotherapy-induced intestinal damage.

Highlights d

Symbiont-generated lactate is critical for Lgr5+ ISC-mediated epithelial development

d

Lactate signals through the G-protein-coupled receptor Gpr81 to elicit ISC proliferation

d

Lactobacillus plantarum lacking lactate dehydrogenase fails to induce ISC regeneration

d

Pre-feeding of lactate protects mice from chemotherapy- and radiation-induced gut damage

Lee et al., 2018, Cell Host & Microbe 24, 833–846 December 12, 2018 ª 2018 Elsevier Inc. https://doi.org/10.1016/j.chom.2018.11.002

Cell Host & Microbe

Article Microbiota-Derived Lactate Accelerates Intestinal Stem-Cell-Mediated Epithelial Development Yong-Soo Lee,1 Tae-Young Kim,1 Yeji Kim,1 Su-Hyun Lee,1 Seungil Kim,1 Sung Wan Kang,1 Jin-Young Yang,1 In-Jeoung Baek,2 Young Hoon Sung,2 Yun-Yong Park,2 Sung Wook Hwang,3 Eunju O,4 Kwang Soon Kim,4 Siqing Liu,5 Nobuhiko Kamada,6 Nan Gao,7 and Mi-Na Kweon1,8,* 1Mucosal Immunology Laboratory, Department of Convergence Medicine, University of Ulsan College of Medicine/Asan Medical Center, Seoul, Republic of Korea 2Asan Institute for Life Science, Asan Medical Center, Seoul, Republic of Korea 3Department of Gastroenterology, Asan Medical Center, Seoul, Republic of Korea 4Academy of Immunology and Microbiology, Institute for Basic Science, Pohang, Republic of Korea 5National Center for Agricultural Utilization Research, USDA ARS, Peoria, IL, USA 6Department of Internal Medicine, Division of Gastroenterology, University of Michigan Medical School, Ann Arbor, MI, USA 7Department of Biological Sciences, Rutgers University, Newark, NJ, USA 8Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2018.11.002

SUMMARY

Symbionts play an indispensable role in gut homeostasis, but underlying mechanisms remain elusive. To clarify the role of lactic-acid-producing bacteria (LAB) on intestinal stem-cell (ISC)-mediated epithelial development, we fed mice with LAB-type symbionts such as Bifidobacterium and Lactobacillus spp. Here we show that administration of LAB-type symbionts significantly increased expansion of ISCs, Paneth cells, and goblet cells. Lactate stimulated ISC proliferation through Wnt/b-catenin signals of Paneth cells and intestinal stromal cells. Moreover, Lactobacillus plantarum strains lacking lactate dehydrogenase activity, which are deficient in lactate production, elicited less ISC proliferation. Pre-treatment with LAB-type symbionts or lactate protected mice in response to gut injury provoked by combined treatments with radiation and a chemotherapy drug. Impaired ISC-mediated epithelial development was found in mice deficient of the lactate G-proteincoupled receptor, Gpr81. Our results demonstrate that LAB-type symbiont-derived lactate plays a pivotal role in promoting ISC-mediated epithelial development in a Gpr81-dependent manner.

INTRODUCTION Symbionts are live microorganisms that seem to promote host defense systems and regulate intestinal homeostasis, preventing gut infectious and inflammatory diseases (Fuller, 1989; Sartor, 2004). Numerous studies have shown the beneficial effects of different types of symbionts in gut-related diseases such as ulcerative colitis, Crohn’s disease, pouchitis, and irritable bowel

syndrome in human and animal models (Bibiloni et al., 2005; Gionchetti et al., 2000; O’Mahony et al., 2005). The intestinal epithelium contains stem cells in the crypt and their transitamplifying daughter cells as well as terminally differentiated functional cells in the villus that help to prevent intestinal pathology (Clevers, 2013). Through a self-renewal process, the intestinal epithelium is continuously renewed every 3 to 5 days (Stevens and Leblond, 1947). Intestinal stem cells (ISCs) differentiate into functional intestinal cells, such as Paneth, goblet, enteroendocrine, enterocytes, and tuff cells, and enterocytes (Cheng and Leblond, 1974; Gerbe et al., 2011; Li and Clevers, 2010). ISCs express leucine-rich repeat-containing G-proteincoupled receptor 5 (Lgr5) on their surfaces, which is located between Paneth cells that secrete anti-microbial peptides such as lysozyme and defensins (Barker et al., 2007; Sato et al., 2009). A previous study demonstrated that Paneth cells regulate the maintenance and differentiation of Lgr5+ ISCs (Sato et al., 2011). Among the earliest studies, Wnt signaling was found to play an indispensable role for stem cell niches and for supporting stem cell self-renewal within the wide spectrum of Wnt effects on various target cells (Farin et al., 2012; Kabiri et al., 2014; Pinto et al., 2003). Another study showed mutations in TCF4, the ultimate outcome of the Wnt pathway, resulting in loss of ISCs and subsequent breakdown of the intestine (Korinek et al., 1998). Conversely, stimulation of Wnt signaling by the addition of b-catenin, another outcome of the Wnt pathway, could lead to expansion of stem cells in both hematopoietic and hair follicle systems (Gat et al., 1998). Treatment with isolated Wnt3a protein on the hematopoietic stem cells enhances the self-renewal function (Willert et al., 2003). A more recent study revealed that Wnt/b-catenin signaling supports gut homeostasis by maintaining self-renewal of Lgr5+ stem cells in the crypt (Clevers and Nusse, 2012). Secreted Wnt proteins bind to LRP5/6 and Frizzled co-receptors present on epithelial crypt cells, leading to an increase in b-catenin protein (de Lau et al., 2011).

Cell Host & Microbe 24, 833–846, December 12, 2018 ª 2018 Elsevier Inc. 833

Gut microbiota affects nutrient acquisition and energy regulation of the host. The microbiota produces short-chain fatty acid (SCFA; i.e., acetate, butyrate, and propionate) and lactate, which are important energy sources for the host (Muegge et al., 2011; Turnbaugh et al., 2006). The G-protein-coupled receptor (Gpr) is indispensable for activating signaling molecules for many aspects of immunologic and metabolic functions. Several studies have sought a cross-relationship between commensal bacteria-derived metabolites and the Gprs. Of these receptors, Gpr41 and -43 are specific for SCFA (Brown et al., 2003). SCFA-Gpr43 interactions profoundly influence the inflammatory responses, which showed exacerbated inflammation in various disease models, such as colitis, arthritis, and asthma, in Gpr43
/
mice (Maslowski et al., 2009). Activation of SCFAGpr43 interactions suppresses insulin signaling in adipocytes, which inhibits fat accumulation in adipose tissue and promotes the metabolism of glucose in other tissues (Kimura et al., 2013). In addition, Gpr120, a receptor for the u-3 fatty acids, exerts potent anti-inflammatory effects on macrophages in vitro and on obese mice in vivo (Oh et al., 2014). Previous studies found that Gpr81 is a specific receptor for lactate (Cai et al., 2008; Ge et al., 2008; Liu et al., 2009); however, the relationship between the lactate derived from symbionts and Gpr81 signaling associated with cellular functions in the gut is not known. Among the symbiotic bacteria contained in the probiotics, lactic-acid-producing bacteria (LAB) have been suggested to confer broad spectrums of health benefits, such as activation of mucosal and systemic immunity and resistance to infectious illnesses (Schiffrin et al., 1995; Wells and Mercenier, 2008). Daily feeding of LAB containing Lactobacillus rhamnosus, Lactobacillus acidophilus, and Bifidobacterium lactis resulted in significant enhancement of innate and acquired immunity (Gill et al., 2000). Lactate produced by the probiotic Lactobacillus casei was critical in modulating inflammation in a model of indomethacin-induced gut damage with decreased neutrophil infiltration and cytokine expression (Watanabe et al., 2009). Although there is strong evidence for the beneficial roles of symbionts, neither the exact role of the differentiation of Lgr5+ ISCs nor the relevance of regulation of the intestinal environments is clear. RESULTS Lactate Produced by Symbionts Is Critical for ISCMediated Epithelial Regeneration To identify the effectiveness of symbionts on gut epithelial regeneration, we orally administered human-use probiotics (VSL#3) containing LAB, such as Bifidobacterium and Lactobacillus spp., to Lgr5-GFP mice for 5 days. Mice fed probiotics had increased crypt height and more Lgr5+ ISCs, Paneth cells (Lyz1+), and goblet cells (Muc2+) in the small intestine (SI) than mice fed PBS (Figures S1A and S1B). We also found that probiotics significantly induced Ki-67-positive cells (Figure S1C), Olfm4-positive cells (Figure S1D), and anti-bacterial peptide expressions in the SI (Figure S1E). Among several probiotic metabolites, significantly higher levels of lactate were determined in the contents of the SI and large intestine (LI) of probiotic-fed mice than in PBS-fed mice (Figure 1A). The SI crypts treated with probiotic culture supernatant or lactate in medium containing epidermal growth factor, noggin, and R-spondin 1 (ENR) for 834 Cell Host & Microbe 24, 833–846, December 12, 2018

3 days showed significantly increased organoid size (Figure 1B). Treatment with probiotic culture supernatant or lactate yielded increased numbers of Lgr5+ ISCs and organoid growth for 3 and 5 days of cultivation when compared with groups treated with ENR alone (Figures 1C and S1F). By contrast, culture in the presence of lactate antagonist (3-hydroxy-butyrate [3-OBA]) inhibited the growth of SI organoids generated in lactate-sufficient conditions (Figure 1D). Similar results were obtained when SI organoids were co-cultured with gut contents from newborn mice (NBGC) (Figures 1C and S1F) that contained high levels of lactate (Figure S1G). We found that treatment of the SI organoids with probiotic culture supernatant or lactate significantly enhanced expression of Wnt/b-catenin pathway-related genes (i.e., Wnt3, Ctnnb1, Axin2, and GSK3b) and that lactate antagonist (3-OBA) completely blocked activation of those genes (Figure 1E). In addition, treatment with probiotic culture supernatant, lactate, or NBGC resulted in enhanced numbers of Lgr5+ cells and CD24+ Paneth cells and Wnt3 expression in SI organoids (Figure S2A). We further confirmed in vivo that predominant expression of Axin2 and nuclear localization of b-catenin occurred in the SI crypts of mice fed probiotic or lactate, unlike control mice fed PBS (Figures S2B and S2C). In vitro treatment of lactate enhanced protein levels of b-catenin and p-GSK3ab in the SI organoids in a time-dependent manner (Figure S2D). Overall, lactate produced by probiotic treatment accelerated differentiation of ISCs in a Wnt3/b-catenin pathway-dependent manner. Oral Feeding of Lactate Can Reach the Bottom of Crypts To determine how LAB-producing lactate reaches the intestinal crypts, we fed mice drinking water containing lactate for 5 days before isolating crypts from their SIs and LIs. We measured the oxygen consumption rate (OCR; an indicator of oxidative phosphorylation [OXPHOS]) and the extracellular acidification rate (ECAR; an indicator of glycolysis) in the crypts. Despite plating the same numbers of crypts, the basal levels of OCR in the small and large intestinal crypts were higher in lactate-fed mice than in PBS-fed mice (Figure S3). No significant differences in the ECAR levels were found (data not shown). Overall, these results demonstrate that mitochondrial functionrelated respiration was increased in the intestinal crypts of mice after lactate feeding, and thus, we suggest that lactate can indeed reach the bottom of crypts. Interaction of Lactate with Gpr81 in Intestinal Stromal Cells Is Important for Differentiation of Lgr5+ ISCs We next addressed predominant induction of Lgr5-GFP and Wnt3 expressions on Lyz1+ Paneth cells on SI organoids by lactate or Gpr81 agonist (3-chloro-5-dihydroxybenzoic acid [3,5-DHBA]) (Figures S4A and S4B). There were more Lgr5+ cells and proliferating Ki67+ cells in the SI crypts of mice fed with 3,5-DHBA than in those given only water (Figure S4C). We next assessed Gpr81 expression in the Paneth cells, which are well known to support ISC proliferation (Sato et al., 2011). CD24+ Paneth cells, but not Lgr5+ ISCs isolated from the SIs of the naive mice, expressed Gpr81 (Figure S5A). None of the CD24+Gpr81+ cells were CD45+ hematopoietic but all expressed high levels of Wnt3 (Figure S5A). Although some CD24
cells expressed high levels of Gpr81, Wnt3 expression was not detectable.

A

B

C

D

E

Figure 1. Probiotic-Derived Lactate Enhances ISC-Mediated Epithelial Differentiation (A) Measurement of lactate in SI and LI of mice fed PBS or human-use probiotics (Pro) for 5 days. Each 100 mg of gut content was diluted in 1 mL of DMEM. (B) Size of SI organoids cultured in the presence of ENR medium with culture supernatant from Pro (1:100) or lactate (Lac; 5 mM) (n = 40 organoids/group). Scale bar, 200 mm. (C) Bright field images (upper) and confocal images for GFP (bottom) of SI organoids from Lgr5-GFP mice in the presence of ENR culture medium with Pro, Lac, or gut contents of newborn mice (NBGC; 1:100). NBGC was from the stomachs of 3-day-old mice. Scale bar, 100 mm. (D) Size of SI organoids co-cultured with lactate inhibitor 3-OBA (3 mM) (n = 20 organoids/group). (E) qPCR of relative mRNA expression of Wnt3, Ctnnb1, Axin2, and GSK3b genes of the Wnt/b-catenin axis in SI organoids. Expression shown is relative to b-actin gene. Data are represented as mean ± SEM; comparisons were made by two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3–5. Data were combined from R3 independent experiments.

Expression of Gpr81 in the Paneth cells and stromal cells in the submucosal layer was confirmed by in situ hybridization (Figure S5B) and by immunofluorescence (Figure S5C) staining in the naive mice. Co-expression of Gpr81 on Lyz1+ Paneth cells of purified crypts and aSMA+ intestinal stromal cells was confirmed by immunohistochemistry staining (Figure S5D). Another study demonstrated that intestinal stromal cells express diverse cytokines related to Wnt signaling and thus can support the growth of intestinal epithelium in culture (Gregorieff et al., 2005). A recent study reported that subepithelial mesenchymal cells express high levels of Wnt2b, which is essential for the renewal of the intestinal epithelium (Valenta et al., 2016). We

therefore hypothesized that, in addition to Paneth cells, stromal cells might play an important role in proliferation of ISCs with lactate-Gpr81 interaction. To test our hypothesis, stromal cells isolated from the SIs of naive mice were added to SI organoids in the presence of lactate or 3-OBA. Lactate was significantly induced, as shown by the size of SI organoids, the numbers formed, and the amount of budding; however, 3-OBA inhibited induction completely (Figure 2A). Interestingly, while organoids with lactate grow as mini-guts with visible crypt structures and budding, the addition of intestinal stromal cells and/or lactate resulted in spherical organoids (Figure 2A). Another recent study revealed that similar phenotypes of organoids in Cell Host & Microbe 24, 833–846, December 12, 2018 835

A

B

C

D

Figure 2. Lactate-Gpr81 Interaction in Stromal Cells Supports ISC-Mediated Epithelial Differentiation (A) Size of SI organoids cultured with lactate (Lac), Gpr81 antagonist (3-OBA), stromal cells, and their combinations (n = 20 organoids/group). Scale bar, 200 mm. (B) qPCR of relative mRNA expression of Wnt3 and Wnt2b of intestinal stromal cells co-cultured with or without 5 mM lactate for 3 days. mRNA expression shown is relative to b-actin gene. (C) Immunofluorescence analysis for internalization of Gpr81 (red) and b-arrestin (green) in intestinal stromal cells lactate. Scale bar, 50 mm. (D) Confocal image of aSMA (green) and Wnt3 (red) or PORCN (red) in intestinal stromal cells in the presence of lactate, 3-OBA, Gpr81 agonist (3,5-DHBA), and combinations. Quantitation for Wnt3 and PORCN was measured by mean fluorescence intensity (MFI) (n = 80 cells/group). Scale bar, 100 mm. Data are represented as mean ± SEM; comparisons were made by two-tailed t test, n = 3–5. **p < 0.01, ***p < 0.001. Data were combined from R3 independent experiments.

Wnt3a-conditioned ENR media grow spherical organoids that lack differentiated cell types and proliferate homogeneously (Rodriguez-Colman et al., 2017). Expression levels of the Wnt3 836 Cell Host & Microbe 24, 833–846, December 12, 2018

and Wnt2b genes in intestinal stromal cells (Figure 2B) were significantly increased after addition of lactate. When lactate interacted with intestinal stromal cells, expression of b-arrestin

A

B

E

C

D

F

Figure 3. Lactobacillus plantarum Lacking Lactate Dehydrogenase Activity Failed to Elicit ISC Proliferation (A) Measurement of lactate in supernatant of L. plantarum strains after cultivation for 18 hr. (B and C) Bright field images (B) and confocal image (C) for Lgr5-positive (green) and Ki67-positive (red) cells of SI organoids obtained from Lgr5-GFP mice in presence of ENR medium with culture supernatant of L. plantarum wild-type or mutant strains (1:100) at 5 days. Scale bar, 200 mm (B) and 50 mm (C). (D) Lgr5+ cells were counted in the SI crypt of Lgr5-GFP mice post oral administration with L. plantarum wild-type or mutant strains (3 3 109 colony-forming units [CFU] per mouse) for 5 days (n = 40 cells/group). Scale bar, 20 mm. (E and F) PAS staining and quantification of Paneth cells (E), and Ki67 staining (F) in mice after oral administration with L. plantarum wild-type or mutant strains (3 3 109 CFU per mouse) every day for 5 days (n = 40 cells/group). Scale bar, 50 mm. Data are represented as mean ± SEM; comparisons were made by two-tailed t test, n = 3–5. ***p < 0.001. Data were combined from R2 independent experiments.

and Gpr81 increased in the cytosol region (Figure 2C). To determine whether intestinal stromal cells directly express Wnt3 and porcupine (PORCN), which is required for secretion of Wnt protein, we isolated stromal cells from the SIs of naive mice and stimulated them with lactate, Gpr81 agonist, or Gpr81 antagonist (3-OBA). In addition, expression levels of Wnt3 and PORCN in the stromal cells were highly activated after addition of lactate or Gpr81 agonist. Because Gpr81 antagonist (3-OBA) completely inhibited activation (Figure 2D), Wnt3 and PORCN activation in the stromal cells are highly specific responses that are induced by lactate. To directly address the role of PORCN, we next adopted the PORCN inhibitor (i.e., Wnt-C59). Treatment with Wnt-C59 completely inhibited SI organoid growth induced by lactate alone or by lactate and intestinal stromal cells (Figure S6A). By use of in situ hybridization staining, we screened expression patterns of a variety of Wnt signals. Wnt3 and Wnt2b, but not Wnt6 and Wnt9, were activated by feeding with probiotics or lactate (Figure S7). We also found that higher levels of Wnt2b were spontaneously expressed on intestinal stromal cells, and, similarly to Wnt3, lactate also accelerated expression of Wnt2b on intestinal stromal cells (Figure S6B). These results

indicate that both Paneth cells and intestinal stromal cells are indispensable for proliferation of Lgr5+ ISCs, which are activated via the lactate-Gpr81 pathway. Lactobacillus plantarum Lacking Lactate Dehydrogenase Activity Failed to Elicit ISC Proliferation To further confirm a role of microbiota-derived lactate on the ISC niche, we used L. plantarum wild-type and L. plantarum lacking lactate dehydrogenase activity (L. plantarum DldhD-DldhL) strains (Liu et al., 2006). As expected, defective lactate secretion was confirmed in the culture supernatant of L. plantarum DldhDDldhL (Figure 3A). Significantly smaller organoids and fewer Lgr5+ and Ki67+ cells in organoids were detected in the co-cultures with culture supernatant of L. plantarum DldhD-DldhL strain when compared with those of L. plantarum parent strain (Figures 3B and 3C). In addition, mice fed the L. plantarum DldhD-DldhL strain daily for 5 days had significantly fewer Lgr5+ cells (Figure 3D), Paneth cells (Figure 3E), and Ki67+ cells (Figure 3F) in the SI crypt than mice fed the wildtype L. plantarum strain. We further compared and addressed the broad phenotypic characterization of known epithelial cell Cell Host & Microbe 24, 833–846, December 12, 2018 837

markers in mice after administration of probiotic, lactate, L. plantarum wild-type, or L. plantarum DldhD-DldhL strains (Figure S8). Expression levels of Lyz1, Muc2, and Olfm4 were drastically enhanced in the SIs of mice fed probiotic, lactate, or L. plantarum wild-type, but not L. plantarum DldhD-DldhL strain. These results suggest that lactate plays a crucial role in ISC proliferation. ISCs in Newborn Mice Are Hyper-responsive to Lactate Because the gut microbiota in newborn mice is dominated by LAB, we speculated that lactate would be one of the most abundant gut metabolites and likely important in promoting epithelial proliferation in mature gut organs. To evaluate the effect of lactate-Gpr81 interaction on the proliferation of ISCs in newborn mice, we obtained SIs from 3-day-old Lgr5-GFP mice and made organoids. Similar to findings in adult mice, treatment with lactate, Gpr81 agonist, NBGC, or intestinal stromal cells with or without lactate promoted proliferation of ISCs in SI organoids (Figures 4A and 4B). Stimulation of the intestinal stromal cells with lactate produced the most significant effects with spherical organoids. In addition, stimulation with intestinal stromal cells plus lactate highly activated genes associated with the Wnt/ b-catenin pathways (Figure 4C) in newborn SI organoids. The effect of lactate on the proliferation of ISCs was much higher in the newborn mouse SI organoids than in those from adult mice (Figure 4D). Pre-exposure to Gut Microbiota Results in Positive Feedback on ISC Proliferation To address the role of pre-exposure of gut microbiota to lactate on the proliferation of ISCs, we adopted germ-free (GF) and antibiotic-treated mice. We obtained SI organoids from 3-day-old newborn specific pathogen-free (SPF) and GF mice. ISC proliferation was defective in the newborn GF mice when compared with newborn SPF mice, but lactate treatment compensated for the defect in the newborn GF mice (Figure 5A). In addition, expression levels of CD24 and Wnt3 were lower in SI organoids from 3-day-old GF mice than in those from SPF mice (Figure 5B). As expected, Wnt3 expression recovered after addition of lactate in SI organoids from 3-day-old GF mice (Figure 5B). We also fed antibiotics to adult Lgr5-GFP mice before and during lactate treatment and analyzed gut pathology. We found distorted crypts and fewer granules in the Paneth cells of mice treated with antibiotics than in the PBS-fed controls; however, Paneth cells were recovered in mice fed with lactate during antibiotic treatment (Figure 5C). Lactate feeding also led to recovery of goblet cells numbers, which had been reduced by antibiotic treatment (Figure 5D). To further address a role of other gut bacteria on the proliferation of ISCs, we fed 6-week-old GF mice with L. plantarum or L. plantarum DldhD-DldhL strains. At 1 and 3 weeks after oral administration, numbers of Paneth cells in the SI crypts were significantly increased in GF mice fed L. plantarum when compared with those fed L. plantarum DldhD-DldhL (Figure 5E). These results suggest that lactate is sufficient to stimulate ISC proliferation in vivo in GF condition. Overall, pre-exposure of symbiont-derived lactate resulted in positive feedback on ISC proliferation through Wnt3 activation in the absence and presence of gut commensal bacteria. 838 Cell Host & Microbe 24, 833–846, December 12, 2018

Pre-feeding with Probiotics or Lactate Protects Mice from Gut Damage Provoked by Radiation and Chemotherapy To further clarify the protective role of lactate derived from probiotics, we treated mice with radiation (10 Gy) and methotrexate (MTX) to destroy the SI epithelium as described previously (Bismar and Sinicrope, 2002; Fox et al., 1988). Groups of mice were fed probiotics before or after treatment (Figure 6A). Feeding of probiotics before treatment with radiation and MTX (Pro-R + M and Pro-R + M-Pro) significantly protected mice against destruction of villi and crypts when compared with non-treated mice (R + M) (Figure 6B). Likewise, mice pre-fed probiotics had no abnormalities in crypt height. Pre-feeding of probiotics protected mice from loss of Lgr5+ ISCs due to treatment with radiation and MTX (Figure 6C). We next obtained SI crypts from each group of mice and manipulated organoids (500 crypts/well) and counted the number of budding-formed organoids. Most importantly, pre-feeding of probiotics before treatment with radiation and MTX (Pro-R + M and Pro-R + M-Pro) resulted in more budding-formed organoids than were seen in the treatmentalone group (R + M) (Figure 6D). To further address clinical indices, we increased radiation doses (i.e., 12 Gy). As expected, mice fed probiotics before treatment with radiation and MTX (Pro-R + M and Pro-R + M-Pro) had less body weight loss (data not shown) and higher survival rates (Figure S9A) and improved gut barrier function/gut permeability (Figure S9B) than mice in the treatment-alone group (R + M). Of note, lactate feeding resulted in identical protective roles for gut damage and maintenance of Lgr5+ ISC numbers after exposure with radiation and MTX (Figures 6E and 6F). Regeneration of SI organoids continued when mice were pretreated with probiotics or lactate before treatment with radiation and MTX (Figure 6G). We also confirmed the protective role of probiotics in a bacterial infection model that provoked SI injury (Chang et al., 2013). As expected, oral administration with probiotics protected gut injury against infection with enterohemorrhagic Escherichia coli (EHEC) (Figures S9C and S9D). When the results are considered together, it is evident that probiotic or lactate feeding promoted proliferation of Lgr5+ ISCs and regeneration of intestinal epithelium under steady-state and damaged conditions such as irradiation and anti-cancer drug treatment. Depletion of Gpr81 Impairs Regeneration of ISCMediated Epithelial Cells through a Defect of Wnt3 Activation To determine the exact role of lactate receptor Gpr81 on the development of ISC-mediated epithelial regeneration, we constructed Gpr81
/
mice using CRISPR/Cas9 technology (Figure S10). Of note, there were significantly fewer Lgr5+ cells, Olfm4+ cells, Lyz1+ cells, Muc2+ cells (Figures S10A–S10C), and goblet and Paneth cells (Figure 7A) in the SIs of Gpr81
/
than in Gpr81+/
mice. The Gpr81
/
mice also had significantly smaller and fewer SI organoids (Figure 7B). Immuno-histochemical studies using SI organoids revealed reduced expression levels of Lgr5 and Wnt3 in Gpr81
/
mice when compared with Gpr81+/
mice (Figure 7C). In addition, Gpr81
/
mice had significantly lower levels of Wnt3a protein in SI tissue homogenates than were found in Gpr81+/
mice (Figure 7D). Because there were significant defects in SI organoid formation in Gpr81
/


A

B

C

D

Figure 4. Lactate-Gpr81 Interaction in Stromal Cells Supports ISC-Mediated Epithelial Differentiation in Newborn Mice (A and B) Size (n = 30 organoids/group) (A) and GFP expression (B) in SI organoids derived from newborn Lgr5-GFP mice after co-culture with lactate, Gpr81 agonist (3,5-DHBA), NBGC, intestinal stromal cells, and their combinations for 3 days. Scale bars, 200 mm (A) and 100 mm (B). (C) qPCR of relative mRNA expression of Wnt3, Ctnnb1, Axin2, and GSK3b genes of the Wnt/b-catenin axis in SI organoids derived from newborn Lgr5-GFP mice. Expression shown is relative to b-actin gene. (D) Comparison of regeneration of epithelium cells in SI organoids derived from adult and newborn mice in the presence of lactate, stromal cells, and their combinations for 3 days (n = 20 organoids/group). Scale bar, 200 mm. Data are represented as mean ± SEM; comparisons were made by two-tailed t test, n = 3–5. *p < 0.05, **p < 0.01, ***p < 0.001. Data were combined from R3 independent experiments.

Cell Host & Microbe 24, 833–846, December 12, 2018 839

A

B

C

D

E

Figure 5. Pre-exposure to Gut Microbiota Supports Lactate Signals for ISC Proliferation (A and B) Photographs and surface area (A) and confocal images for CD24 and Wnt3 (B) in SI organoids derived from newborn SPF and GF mice after co-culture with lactate (n = 30 organoids/group). Scale bar, 100 mm. (C and D) Adult Lgr5-GFP mice were fed PBS or antibiotics (Abx) before and during lactate treatment, and gut pathology was analyzed. Histological staining by PAS was done to show Paneth cells per crypt (C) and goblet cells per villus (D). Scale bar, 50 mm. (legend continued on next page)

840 Cell Host & Microbe 24, 833–846, December 12, 2018

mice under steady-state conditions (ENR alone), we attempted to stimulate the SI organoids with lactate, Gpr81 agonist, and probiotic culture supernatant. All stimulators (lactate, Gpr81 agonist [3,5-DHBA], and probiotic culture supernatant) failed to activate Wnt3a secretion in SI organoids from Gpr81
/
mice, while predominant levels of Wnt3a were found in SI organoids of Gpr81+/
mice (Figure 7E). Of importance, Wnt3a treatment, but not lactate, led to recovery of SI organoid formation in Gpr81
/
mice (Figure 7F). Similar to the findings at steady-state conditions, there were fewer Lyz1+ and Ki67+ cells in the SI crypts in the Gpr81
/
mice than in Gpr81+/
mice after PBS feeding (Figure 7G). Most importantly, while probiotic feeding accelerated Ki67+ stem cells and stem cell niche activation in Gpr81+/
mice, it failed in Gpr81
/
mice (Figure 7G). We confirmed that probiotic feeding led to more Paneth cells as measured by periodic acid-Schiff (PAS) staining in Gpr81+/
mice, but not in Gpr81
/
mice (Figure 7H). Furthermore, the protective role of LAB-type symbionts against infection with EHEC was not found in Gpr81
/
mice (Figure S10D). Thus, depletion of Gpr81 impairs regeneration of ISC-mediated epithelial cells through a defect of Wnt3 activation. DISCUSSION In this study, we demonstrate that oral feeding of human-used symbionts containing LAB, such as Bifidobacterium and Lactobacillus spp., promotes significant acceleration of Lgr5+ ISC proliferation and epithelial development in vivo and ex vivo. Among several metabolites from LAB-type symbionts, lactate is associated with ISC-mediated epithelial development. Paneth cells and intestinal stromal cells highly express Gpr81, a known lactate receptor, and lactate treatment promotes ISC-mediated epithelial regeneration in a Gpr81-dependent manner. Furthermore, feeding of LAB-type symbionts or lactate protects mice from severe gut injury induced by radiation exposure and chemotherapy drug treatment. Thus, we propose that lactate and the receptor Gpr81 promote ISC-mediated epithelial regeneration by stimulation of the Wnt/b-catenin signal pathway in Paneth and intestinal stromal cells. An earlier study demonstrated that supplemental Wnt agonist R-spondin-1 can maintain ISCs in a self-renewing state that are able to fully differentiate epithelial cells in SI organoids (Sato et al., 2009). A subsequent study revealed that Paneth cells, which constitute the crypt niche, support a growth of Lgr5 stem cells by providing Wnt3 (Sato et al., 2011). Those researchers found that Paneth cell-null mice have concomitant loss of Lgr5 stem cells. In contrast, others reported that depletion of Paneth cells or knockout of the Wnt3 gene in the intestinal epithelium did not lead to obvious phenotype changes in vivo, suggesting the possibility of another functional cell niche (Durand et al., 2012; Farin et al., 2012). If so, intestinal myofibroblast-enriched stromal cells may play a functional role in ISC maintenance and differentiation (Kabiri et al., 2014). Intestinal stromal cells express diverse cytokines related to Wnt signaling

(Gregorieff et al., 2005) and thus can support the growth of intestinal epithelium in culture (Lahar et al., 2011). We found that expression levels of Wnt/b-catenin pathway-related genes were significantly enhanced in SI organoids in the presence of culture from symbionts or lactate (Figure 1E), and those expression levels were more activated when organoids were cocultured with intestinal stromal cells (Figure 2B). In parallel, Wnt3 protein was highly expressed in intestinal stromal cells by stimulation with lactate (Figure 2D). As reported by others (Sato et al., 2011), we also confirmed that Paneth cells express high levels of Wnt3 protein (Figure S2). Those results were confirmed in the SI crypts by in situ hybridization staining (Figure S7). We therefore conclude that lactate derived from LAB-type symbionts stimulate both Paneth cells and intestinal stromal cells to secrete Wnt3 and support ISC maintenance and differentiation. Although lactate has been regarded as an intermediate of carbon metabolism contributing to anti-microbial effects of fermented food, recent accumulated evidence suggests that it has unique bioactive properties, such as regulation of immune responses and tissue regeneration in the gut mucosa (Garrote et al., 2015). Others demonstrated that lactate produced by microbial fermentation reduced inflammatory responses in intestinal epithelial cells and myeloid cells by abrogation of TLR and IL-1b-dependent activation (Iraporda et al., 2015). Furthermore, transient starvation enhanced colon epithelial cell turnover on refeeding in the presence of Lactobacillus murinus and its metabolite, lactate (Okada et al., 2013). Orally administered L. rhamnosus GG probiotic and probiotic-derived products protected the murine SI from radiation injury (Ciorba et al., 2012). In addition, treatment with probiotics containing L. acidophilus and Bifidobacterium bifidum resulted in mild diarrhea in cancer patients receiving radiation therapy (Chitapanarux et al., 2010). Taken together with our results, these data indicate that administration of LAB-type symbionts or lactate may be a useful prophylactic strategy to limit gut injury to humans during radiation therapy and chemotherapy. Several studies have sought a cross-relationship between commensal bacteria-derived metabolites and the Gprs (Blad et al., 2012). Gpr81 is expressed primarily in adipocytes, and hence, lactate-Gpr81 interaction inhibits lipolysis (Cai et al., 2008; Liu et al., 2009). Lactate reduced inflammation by Gpr81-mediated negative regulation of innate immunity and ameliorated liver and pancreatic injury (Hoque et al., 2014). In our study, we found that lactate receptor Gpr81 is broadly expressed in the intestinal stromal cells adjacent to ISCs (Figure S5). The addition of lactate could accelerate the differentiation and proliferation of ISCs through Wnt3 and PORCN signaling in a Gpr81-dependent manner (Figure 2E). Importantly, defective ISCs mediated epithelial differentiation and Wnt3 expression in Gpr81
/
mice under steady-state conditions (Figure 7). Overall, lactate derived from a LAB-type symbiont plays an indispensable role in ISC-mediated epithelial differentiation in Gpr81-dependent activation.

(E) Numbers of Paneth cells were determined in the SI crypts from GF mice at 1 and 3 weeks after oral administration with L. plantarum or L. plantarum DldhD-DldhL strains. Scale bar, 100 mm. Data are represented as mean ± SEM; comparisons were made by two-tailed t test, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001. Data were combined from R2 independent experiments.

Cell Host & Microbe 24, 833–846, December 12, 2018 841

A

B

C

D

E

F

G

Figure 6. Pre-treatment with Probiotics or Lactate Prevents Gut Injury (A) Experimental schedule for induction of gut injury by irradiation and MTX. (B) Pathology and pathology score of SI by H&E staining. Scale bar, 100 mm. (C and D) GFP-expressing Lgr5+ cells in the SI (n = 30 crypts/group) (C) and number of SI organoids from probiotic-fed Lgr5-GFP mice (n = 4 wells/group) (D). Scale bar, 50 mm. (E and F) GFP-expressing Lgr5+ cells in the SI (n = 30 crypts/group) (E) and number of SI organoids from lactate-fed Lgr5-GFP mice (n = 4 wells/group) (F). Scale bar, 50 mm. (G) Regeneration of SI organoids isolated from probiotic- or lactate-fed mice before treatment with irradiation and MTX. Bright field images and numbers of SI organoids cultured for 3 days (n = 3 wells/group). Scale bar, 100 mm. Data are represented as mean ± SEM; comparisons were made by two-tailed t test, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001. Data were combined from R2 independent experiments.

842 Cell Host & Microbe 24, 833–846, December 12, 2018

A

C

B

D

E

F

G

H

Figure 7. Gpr81–/– Mice Had Impaired ISC-Mediated Epithelial Regeneration and Wnt3a Secretion (A) PAS staining of SI and quantification of goblet cells per villus and Paneth cells per crypt in Gpr81+/
and Gpr81
/
mice at age 6 weeks (n = 40 crypts/group). Scale bar, 100 mm. (legend continued on next page)

Cell Host & Microbe 24, 833–846, December 12, 2018 843

We discovered that stimulation with lactate resulted in internalization of Gpr81 to the cytoplasmic membrane of intestinal stromal cells and induced expression of Wnt3 cytokine by activation of the PORCN signaling pathway (Figure 2D). We also showed PORCN inhibitor completely inhibited SI organoid growth in the presence of lactate and intestinal stromal cells (Figure S6A). The PORCN protein is a membrane-bound O-acyltransferase that resides in the endoplasmic reticulum and is known to be involved with acylation and secretion of all 19 Wnt ligands (Kadowaki et al., 1996; Najdi et al., 2012). Wnt secretion from Paneth cells or other epithelial cells is required to form SI organoids from purified crypts (Sato et al., 2011), but crypts isolated from PORCN-depleted SI epithelium do not form organoids in vitro (Kabiri et al., 2014). Therefore, development of intestinal tissue by symbiont-derived lactate seems to control the differentiation and proliferation of ISCs by controlling expression of PORCN-dependent Wnt3 in intestinal stromal cells. It has been proposed that Lgr5+ ISCs are irradiation resistant and thus important for gut regeneration after radiation injury (Metcalfe et al., 2014). Due to the intense experimental conditions in this study, most adult Lgr5+ ISCs had disappeared by 5 days after combined treatment; however, pre-feeding of LAB-type symbionts or lactate enhanced survival of Lgr5+ ISCs in vivo and ex vivo (Figure 6). Lactate treatment reduced apoptotic cell death while lactate inhibitor OBA enhanced cell death in the ex vivo SI organoid system (data not shown). Consistent with this, others reported that administration of LAB-type probiotics before irradiation decreased epithelial apoptosis and thus increased crypt survival in TLR-2-, My88-, and COX-2-dependent activation (Ciorba et al., 2012). Although details of the mechanism remain to be clarified, lactate secreted from LAB-type symbionts supports Lgr5+ ISC survival and subsequently results in efficient protection in response to gut injury. One previous study linked Gpr81 and lactate to the regulation of monocarboxylase transporters and lactate metabolizing enzymes (Roland et al., 2014). Therefore, the involvement of Gpr81 may boost lactate uptake and conversion of lactate to pyruvate, as proposed by others (Rodriguez-Colman et al., 2017). We thus tested the role of pyruvate on SI organoids. In contrast to probiotic supernatant or lactate, pyruvate did not have a role in the stem cell niche (data not shown). Therefore, we speculate that the conversion of lactate to pyruvate might not be involved in our study as designed. A study by Kaiko et al. (2016) showed that hydroxy-butyrate, as an antagonist of Gpr81 (i.e., 3-OBA), produced by gut microbiota is indeed deleterious to ISCs because this metabolite

cannot reach the stem cell compartments during homeostasis. That study elegantly showed that differentiated colonocytes metabolized butyrate, likely preventing it from reaching stem cells in the crypt. It seems likely that mammalian crypt architecture protects ISC proliferation in part through a metabolic barrier formed by differentiated colonocytes. Therefore, it is reasonable to question whether the lactate produced by the LAB-type symbiont reaches the crypt. We found that mitochondrial respiration was significantly higher in the crypts of SIs and LIs after lactate treatment (Figure S3). In contrast to metabolizing butyrate, we assume that LAB-produced lactate can reach the bottom of intestinal crypts. Others demonstrated that Paneth cells maintain stem cell function and crypt homeostasis by providing metabolic lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ ISCs (Rodriguez-Colman et al., 2017), which substantially agrees with our findings in this study. Furthermore, deletion of lactate dehydrogenase in Lgr5+ cells prevented the activation of hair follicle stem cells (Flores et al., 2017). We suggest that lactate directly stimulates CD24+ Paneth cells and aSMA+ intestinal stromal cells through Gpr81 and activates the Wnt3-b-catenin pathway, which plays an important role in maintaining stemness of Lgr5+ ISCs. By taking advantage of the essential roles of LAB-derived lactate and Gpr81 interactions in Wnt3 secretion, we propose prophylactic clinical use of LAB-type symbionts or lactate to protect gut injury in response to therapy such as radiation and chemotherapy. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mice B Organoid Culture B Conditions for the HEK293t and Wnt3a-expressing Cell Lines METHOD DETAILS -/B Generation of Gpr81 Mice Using CRISPR/Cas9 B In Vivo Experiments B In Vitro Experiments B In Vitro Measurements QUANTIFICATION AND STATISTICAL ANALYSIS B Statistics

(B) Formation of SI organoids from Gpr81+/
and Gpr81
/
mice (n = 30 organoids/group). Scale bar, 100 mm. (C) Immuno-histochemical analysis for CD24 and Wnt3 expression in SI organoids. Scale bar, 50 mm. (D) Measurement of Wnt3a in SI tissue homogenates from Gpr81+/
and Gpr81
/
mice. (E) Measurement of Wnt3a in intestinal stromal cells after stimulation with lactate (Lac), Gpr81 agonist (3,5-DHBA), or supernatant from probiotics (Pro). (F) Formation of SI organoids from Gpr81+/
and Gpr81
/
mice in the presence of lactate or Wnt3a (n = 20 organoids/group). Scale bar, 100 mm. (G) Confocal image of Paneth cells (Lyz1+) and proliferating Ki67+ cells in SI crypts of Gpr81+/
and Gpr81
/
mice after oral administration of PBS or probiotics daily for 5 days. Yellow asterisks indicate Ki67+ proliferating stem cells. Scale bar, 50 mm. (H) PAS staining of SI and quantification of Paneth cells per crypt in Gpr81+/
and Gpr81
/
mice after oral administration of PBS or probiotics. Scale bar, 50 mm. Data are represented as mean ± SEM; comparisons were made by two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001, n = 5. Data were combined from R2 independent experiments.

844 Cell Host & Microbe 24, 833–846, December 12, 2018

Supplemental Information includes 10 figures and 1 table and can be found with this article online at https://doi.org/10.1016/j.chom.2018.11.002.

Chang, S.Y., Lee, S.N., Yang, J.Y., Kim, D.W., Yoon, J.H., Ko, H.J., Ogawa, M., Sasakawa, C., and Kweon, M.N. (2013). Autophagy controls an intrinsic host defense to bacteria by promoting epithelial cell survival: a murine model. PLoS One 8, e81095.

ACKNOWLEDGMENTS

Clevers, H. (2013). The intestinal crypt, a prototype stem cell compartment. Cell 154, 274–284.

SUPPLEMENTAL INFORMATION

This work was supported by the National Research Foundation of Korea (NRF-2017R1A2B3002132), the Asan Institute for Life Sciences, Asan Medical Center (2018-678), and the Korean Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs, Republic of Korea (HI13C0016). AUTHOR CONTRIBUTIONS Y.-S.L. and M.-N.K. conceived the project, designed and performed experiments, and wrote the manuscript. Y.K., S.-H.L., T.-Y.K., S.W.K., S.K., J.-Y.Y., and Y.-Y.P. performed experiments. I.-J.B. and Y.H.S. manipulated knockout mice. S.W.H. performed human-related experiments. E.O. and K.S.K. provided GF mice. S.L. provided L. plantarum strains. N.K. and N.G. advised on manuscript preparation. DECLARATION OF INTERESTS The authors have no financial interests to declare. Received: August 1, 2018 Revised: September 14, 2018 Accepted: September 27, 2018 Published: December 12, 2018 REFERENCES Andersson-Rolf, A., Fink, J., Mustata, R.C., and Koo, B.K. (2014). A video protocol of retroviral infection in primary intestinal organoid culture. J. Vis. Exp. e51765, https://doi.org/10.3791/51765. Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., et al. (2007). Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007. Bibiloni, R., Fedorak, R.N., Tannock, G.W., Madsen, K.L., Gionchetti, P., Campieri, M., De Simone, C., and Sartor, R.B. (2005). VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis. Am. J. Gastroenterol. 100, 1539–1546. Bismar, M.M., and Sinicrope, F.A. (2002). Radiation enteritis. Curr. Gastroenterol. Rep. 4, 361–365. Blad, C.C., Tang, C., and Offermanns, S. (2012). G protein-coupled receptors for energy metabolites as new therapeutic targets. Nat. Rev. Drug Discov. 11, 603–619. Brown, A.J., Goldsworthy, S.M., Barnes, A.A., Eilert, M.M., Tcheang, L., Daniels, D., Muir, A.I., Wigglesworth, M.J., Kinghorn, I., Fraser, N.J., et al. (2003). The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. J. Biol. Chem. 278, 11312–11319. Cai, T.Q., Ren, N., Jin, L., Cheng, K., Kash, S., Chen, R., Wright, S.D., Taggart, A.K., and Waters, M.G. (2008). Role of GPR81 in lactate-mediated reduction of adipose lipolysis. Biochem. Biophys. Res. Commun. 377, 987–991.

Clevers, H., and Nusse, R. (2012). Wnt/b-catenin signaling and disease. Cell 149, 1192–1205. Das, S., Yu, S., Sakamori, R., Vedula, P., Feng, Q., Flores, J., Hoffman, A., Fu, J., Stypulkowski, E., Rodriguez, A., et al. (2015). Rab8b vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche. Development 142, 2147–2162. de Lau, W., Barker, N., Low, T.Y., Koo, B.K., Li, V.S., Teunissen, H., Kujala, P., Haegebarth, A., Peters, P.J., van de Wetering, M., et al. (2011). Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297. De Vries, W.N., Evsikov, A.V., Haac, B.E., Fancher, K.S., Holbrook, A.E., Kemler, R., Solter, D., and Knowles, B.B. (2004). Maternal b-catenin and E-cadherin in mouse development. Development 131, 4435–4445. Dieleman, L.A., Palmen, M.J., Akol, H., Bloemena, E., Pena, A.S., Meuwissen, S.G., and Van Rees, E.P. (1998). Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin. Exp. Immunol. 114, 385–391. Durand, A., Donahue, B., Peignon, G., Letourneur, F., Cagnard, N., Slomianny, C., Perret, C., Shroyer, N.F., and Romagnolo, B. (2012). Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1). Proc. Natl. Acad. Sci. U S A 109, 8965–8970. Everard, A., Belzer, C., Geurts, L., Ouwerkerk, J.P., Druart, C., Bindels, L.B., Guiot, Y., Derrien, M., Muccioli, G.G., Delzenne, N.M., et al. (2013). Crosstalk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc. Natl. Acad. Sci. U S A 110, 9066–9071. Farin, H.F., Van Es, J.H., and Clevers, H. (2012). Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells. Gastroenterology 143, 1518–1529.e17. Flores, A., Schell, J., Krall, A.S., Jelinek, D., Miranda, M., Grigorian, M., Braas, D., White, A.C., Zhou, J.L., Graham, N.A., et al. (2017). Lactate dehydrogenase activity drives hair follicle stem cell activation. Nat. Cell Biol. 19, 1017–1026. Fox, A.D., Kripke, S.A., De Paula, J., Berman, J.M., Settle, R.G., and Rombeau, J.L. (1988). Effect of a glutamine-supplemented enteral diet on methotrexateinduced enterocolitis. JPEN J. Parenter. Enteral. Nutr. 12, 325–331. Fuller, R. (1989). Probiotics in man and animals. J. Appl. Bacteriol. 66, 365–378. Garrote, G.L., Abraham, A.G., and Rumbo, M. (2015). Is lactate an undervalued functional component of fermented food products? Front. Microbiol. 6, 629. Gat, U., DasGupta, R., Degenstein, L., and Fuchs, E. (1998). De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell 95, 605–614. Ge, H., Weiszmann, J., Reagan, J.D., Gupte, J., Baribault, H., Gyuris, T., Chen, J.L., Tian, H., and Li, Y. (2008). Elucidation of signaling and functional activities of an orphan GPCR, GPR81. J. Lipid Res. 49, 797–803.

Cheng, H., and Leblond, C.P. (1974). Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am. J. Anat. 141, 537–561.

Gerbe, F., van Es, J.H., Makrini, L., Brulin, B., Mellitzer, G., Robine, S., Romagnolo, B., Shroyer, N.F., Bourgaux, J.F., Pignodel, C., et al. (2011). Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium. J. Cell Biol. 192, 767–780.

Chitapanarux, I., Chitapanarux, T., Traisathit, P., Kudumpee, S., Tharavichitkul, E., and Lorvidhaya, V. (2010). Randomized controlled trial of live Lactobacillus acidophilus plus Bifidobacterium bifidum in prophylaxis of diarrhea during radiotherapy in cervical cancer patients. Radiat. Oncol. 5, 31.

Gill, H.S., Rutherfurd, K.J., Prasad, J., and Gopal, P.K. (2000). Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019). Br. J. Nutr. 83, 167–176.

Ciorba, M.A., Riehl, T.E., Rao, M.S., Moon, C., Ee, X., Nava, G.M., Walker, M.R., Marinshaw, J.M., Stappenbeck, T.S., and Stenson, W.F. (2012). Lactobacillus probiotic protects intestinal epithelium from radiation injury in a TLR-2/cyclo-oxygenase-2-dependent manner. Gut 61, 829–838.

Gionchetti, P., Rizzello, F., Venturi, A., Brigidi, P., Matteuzzi, D., Bazzocchi, G., Poggioli, G., Miglioli, M., and Campieri, M. (2000). Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 119, 305–309.

Cell Host & Microbe 24, 833–846, December 12, 2018 845

Gregorieff, A., Pinto, D., Begthel, H., Destree, O., Kielman, M., and Clevers, H. (2005). Expression pattern of Wnt signaling components in the adult intestine. Gastroenterology 129, 626–638. Hoque, R., Farooq, A., Ghani, A., Gorelick, F., and Mehal, W.Z. (2014). Lactate reduces liver and pancreatic injury in Toll-like receptor- and inflammasomemediated inflammation via GPR81-mediated suppression of innate immunity. Gastroenterology 146, 1763–1774.

Najdi, R., Proffitt, K., Sprowl, S., Kaur, S., Yu, J., Covey, T.M., Virshup, D.M., and Waterman, M.L. (2012). A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities. Differentiation 84, 203–213. O’Mahony, L., McCarthy, J., Kelly, P., Hurley, G., Luo, F., Chen, K., O’Sullivan, G.C., Kiely, B., Collins, J.K., Shanahan, F., et al. (2005). Lactobacillus and Bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 128, 541–551.

Iraporda, C., Errea, A., Romanin, D.E., Cayet, D., Pereyra, E., Pignataro, O., Sirard, J.C., Garrote, G.L., Abraham, A.G., and Rumbo, M. (2015). Lactate and short chain fatty acids produced by microbial fermentation downregulate proinflammatory responses in intestinal epithelial cells and myeloid cells. Immunobiology 220, 1161–1169.

Oh, D.Y., Walenta, E., Akiyama, T.E., Lagakos, W.S., Lackey, D., Pessentheiner, A.R., Sasik, R., Hah, N., Chi, T.J., Cox, J.M., et al. (2014). A Gpr120-selective agonist improves insulin resistance and chronic inflammation in obese mice. Nat. Med. 20, 942–947.

Ito, T., Hirose, K., Saku, A., Kono, K., Takatori, H., Tamachi, T., Goto, Y., Renauld, J.C., Kiyono, H., and Nakajima, H. (2017). IL-22 induces Reg3g and inhibits allergic inflammation in house dust mite-induced asthma models. J. Exp. Med. 214, 3037–3050.

Okada, T., Fukuda, S., Hase, K., Nishiumi, S., Izumi, Y., Yoshida, M., Hagiwara, T., Kawashima, R., Yamazaki, M., Oshio, T., et al. (2013). Microbiota-derived lactate accelerates colon epithelial cell turnover in starvation-refed mice. Nat. Commun. 4, 1654.

Kabiri, Z., Greicius, G., Madan, B., Biechele, S., Zhong, Z., Zaribafzadeh, H., Edison, Aliyev, J., Wu, Y., Bunte, R., et al. (2014). Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts. Development 141, 2206–2215.

Pinto, D., Gregorieff, A., Begthel, H., and Clevers, H. (2003). Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 17, 1709–1713.

Kadowaki, T., Wilder, E., Klingensmith, J., Zachary, K., and Perrimon, N. (1996). The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing. Genes Dev. 10, 3116–3128. Kaiko, G.E., Ryu, S.H., Koues, O.I., Collins, P.L., Solnica-Krezel, L., Pearce, E.J., Pearce, E.L., Oltz, E.M., and Stappenbeck, T.S. (2016). The colonic crypt protects stem cells from microbiota-derived metabolites. Cell 165, 1708–1720. Kim, M.Y., Park, B.Y., Lee, H.S., Park, E.K., Hahm, J.C., Lee, J., Hong, Y., Choi, S., Park, D., Lee, H., et al. (2010). The anti-angiogenic herbal composition Ob-X inhibits adipose tissue growth in obese mice. Int. J. Obes. 34, 820–830. Kimura, I., Ozawa, K., Inoue, D., Imamura, T., Kimura, K., Maeda, T., Terasawa, K., Kashihara, D., Hirano, K., Tani, T., et al. (2013). The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43. Nat. Commun. 4, 1829. Korinek, V., Barker, N., Moerer, P., van Donselaar, E., Huls, G., Peters, P.J., and Clevers, H. (1998). Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat. Genet. 19, 379–383. Lahar, N., Lei, N.Y., Wang, J., Jabaji, Z., Tung, S.C., Joshi, V., Lewis, M., Stelzner, M., Martin, M.G., and Dunn, J.C. (2011). Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium. PLoS One 6, e26898. Li, L., and Clevers, H. (2010). Coexistence of quiescent and active adult stem cells in mammals. Science 327, 542–545. Liu, C., Wu, J., Zhu, J., Kuei, C., Yu, J., Shelton, J., Sutton, S.W., Li, X., Yun, S.J., Mirzadegan, T., et al. (2009). Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81. J. Biol. Chem. 284, 2811–2822. Liu, S., Nichols, N.N., Dien, B.S., and Cotta, M.A. (2006). Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production. J. Ind. Microbiol. Biotechnol. 33, 1–7. Maslowski, K.M., Vieira, A.T., Ng, A., Kranich, J., Sierro, F., Yu, D., Schilter, H.C., Rolph, M.S., Mackay, F., Artis, D., et al. (2009). Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 461, 1282–1286. Metcalfe, C., Kljavin, N.M., Ybarra, R., and de Sauvage, F.J. (2014). Lgr5+ stem cells are indispensable for radiation-induced intestinal regeneration. Cell Stem Cell 14, 149–159. Muegge, B.D., Kuczynski, J., Knights, D., Clemente, J.C., Gonzalez, A., Fontana, L., Henrissat, B., Knight, R., and Gordon, J.I. (2011). Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans. Science 332, 970–974.

846 Cell Host & Microbe 24, 833–846, December 12, 2018

Rodriguez-Colman, M.J., Schewe, M., Meerlo, M., Stigter, E., Gerrits, J., PrasRaves, M., Sacchetti, A., Hornsveld, M., Oost, K.C., Snippert, H.J., et al. (2017). Interplay between metabolic identities in the intestinal crypt supports stem cell function. Nature 543, 424–427. Roland, C.L., Arumugam, T., Deng, D., Liu, S.H., Philip, B., Gomez, S., Burns, W.R., Ramachandran, V., Wang, H., Cruz-Monserrate, Z., et al. (2014). Cell surface lactate receptor GPR81 is crucial for cancer cell survival. Cancer Res. 74, 5301–5310. Sartor, R.B. (2004). Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: antibiotics, probiotics, and prebiotics. Gastroenterology 126, 1620–1633. Sato, T., van Es, J.H., Snippert, H.J., Stange, D.E., Vries, R.G., van den Born, M., Barker, N., Shroyer, N.F., van de Wetering, M., and Clevers, H. (2011). Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418. Sato, T., Vries, R.G., Snippert, H.J., van de Wetering, M., Barker, N., Stange, D.E., van Es, J.H., Abo, A., Kujala, P., Peters, P.J., et al. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265. Schiffrin, E.J., Rochat, F., Link-Amster, H., Aeschlimann, J.M., and DonnetHughes, A. (1995). Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J. Dairy Sci. 78, 491–497. Stevens, C.E., and Leblond, C.P. (1947). Rate of renewal of the cells of the intestinal epithelium in the rat. Anat. Rec. 97, 373. Sung, Y.H., Kim, J.M., Kim, H.T., Lee, J., Jeon, J., Jin, Y., Choi, J.H., Ban, Y.H., Ha, S.J., Kim, C.H., et al. (2014). Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 24, 125–131. Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., and Gordon, J.I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031. Valenta, T., Degirmenci, B., Moor, A.E., Herr, P., Zimmerli, D., Moor, M.B., Hausmann, G., Cantu, C., Aguet, M., and Basler, K. (2016). Wnt ligands secreted by subepithelial mesenchymal cells are essential for the survival of intestinal stem cells and gut homeostasis. Cell Rep. 15, 911–918. Watanabe, T., Nishio, H., Tanigawa, T., Yamagami, H., Okazaki, H., Watanabe, K., Tominaga, K., Fujiwara, Y., Oshitani, N., Asahara, T., et al. (2009). Probiotic Lactobacillus casei strain Shirota prevents indomethacin-induced small intestinal injury: involvement of lactic acid. Am. J. Physiol. Gastrointest. Liver Physiol. 297, G506–G513. Wells, J.M., and Mercenier, A. (2008). Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nat. Rev. Microbiol. 6, 349–362. Willert, K., Brown, J.D., Danenberg, E., Duncan, A.W., Weissman, I.L., Reya, T., Yates, J.R., 3rd, and Nusse, R. (2003). Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452.

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Mouse CD24 (clone # 910019)

R&D systems

Cat# FAB8547P; RRID: AB_2753323

Mouse Lgr5/GPR49 (clone # 803420)

R&D systems

Cat# FAB8240P; RRID: AB_2753324

Anti-Lysozyme (clone BGN/06/961)

Abcam

Cat# ab36362; RRID: AB_776115

Anti-Wnt3

Abcam

Cat# ab50341; RRID: AB_883541

Anti-Wnt3a

Abcam

Cat# ab28472; RRID: AB_2215308

Anti-OLFM4

Abcam

Cat# ab85046; RRID: AB_10670544

Anti-LGR5

Abcam

Cat# ab75732; RRID: AB_1310281

Anti-aSMA (clone E184)

Abcam

Cat# ab196919; RRID: AB_2753325

Anti-PORCN

Abcam

Cat# ab105543; RRID: AB_10860951

Anti-bArrestin 2

Abcam

Cat# ab54790; RRID: AB_2060273

Goat anti-Mouse IgG

Abcam

Cat# ab150113; RRID: AB_2576208

Rabbit anti-Goat IgG

Abcam

Cat# ab150147

FKSG80/GPR81

LifeSpan Bio Sciences

Cat# LS-C243939; RRID: AB_2753327

LYZ/Lysozyme

LifeSpan Bio Sciences

Cat# LS-C420109; RRID: AB_2753328

Anti-mouse/human Ki-67 (clone 11F6)

BioLegend

Cat# 151202; RRID: AB_2566621

Goat anti-rat IgG (clone poly4054)

BioLegend

Cat# 405422; RRID: AB_2563301

Antibodies

Anti-Mouse CD45 (clone 30-F11)

BD Biosciences

Cat# 557659; RRID: AB_396774

Anti-b-Catenin (clone 14/b-Catenin)

BD Biosciences

Cat# 610154; RRID: AB_397555

Mucin 2 (clone H-300)

Santa Cruz Biotechnology

Cat# SC-15334; RRID: AB_2146667

CD24 (clone M1/69)

eBioscience

Cat# 17-0242; RRID: AB_10852841

Donkey anti-Rabbit IgG

Thermo Fisher Scientific

Cat# A10040; RRID: AB_2534016

Donkey anti-Rat IgG

Thermo Fisher Scientific

Cat# A21208; RRID: AB_141709

Donkey anti-Goat IgG

Thermo Fisher Scientific

Cat# A11056; RRID: AB_2534103

Goat anti-Rabbit IgG

Thermo Fisher Scientific

Cat# A11034; RRID: AB_2576217

Goat anti-Mouse IgG

Thermo Fisher Scientific

Cat# A865; RRID: AB_2536211

Rabbit anti-Goat IgG

Thermo Fisher Scientific

Cat# A11078; RRID: AB_2534122

Phospho-GSK-3a/b

Cell Signaling Technology

Cat# 9327S; RRID: AB_2753330

Anti-Tubulin (clone TU-20)

EMD Millipore

Cat# MAB1637; RRID: AB_2210524

Bacterial and Virus Strains Lactobacillus plantarum

Liu et al., 2006

N/A

Lactobacillus plantarum DldhD-DldhL

Liu et al., 2006

N/A

VSL#3

Sigma-TauPharmaceuticals

N/A

Enterohemorrhagic E. coli (EHEC, O157:H7)

The Korea Center for Disease Control and Prevention

N/A

Chemicals, Peptides, and Recombinant Proteins Sodium DL-lactate solution

Sigma-Aldrich

Cat# 71723

Lactobacilli MRS Broth

BD Difco

Cat# 288130

3-hydroxybutyrate (3-OBA)

Sigma-Aldrich

Cat# 54965-10G-F

3-Chloro-5-hydroxybenzoic acid (3, 5-DHBA)

Sigma-Aldrich

Cat# SML0447

Advanced DMEM-F/12 media

Gibco

Cat# 12634-010

Matrigel

Corning

Cat# 356231

Vancomycin

Sigma-Aldrich

Cat# V1130-1G

Neomycin sulfate

USB

Cat# 19435

Metronidazole

Nacalai Tesque

Cat# 23254-22 (Continued on next page)

Cell Host & Microbe 24, 833–846.e1–e6, December 12, 2018 e1

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Ampicillin

Amresco

Cat# 0339

N-Acetyl-L-cysteine

Sigma-Aldrich

Cat# A9165

B27 supplement

Thermo Fisher Scientific

Cat# 12587-010

N2 supplement

Thermo Fisher Scientific

Cat# 17502-048

EGF

Thermo Fisher Scientific

Cat# PMG8043

Noggin

R&D Systems

Cat# 1967-NG-025

TrypLE Express

Thermo Fisher Scientific

Cat# 12604-013

Antibiotic-Antimycotic

Thermo Fisher Scientific

Cat# 15240-062

Collagenase/Dispase

Sigma-Aldrich

Cat# 11097113001

SuperScript II Reverse Transcriptase

Thermo Fisher Scientific

Cat# 18064-014

Oligo(dT)12–18 Primer

Thermo Fisher Scientific

Cat# 18418-012

HotStart-IT SYBR Green qPCR Mix (2X)

Affymetrix

Cat# 75762

Oligomycin

Sigma-Aldrich

Cat# O4876

FCCP

Sigma-Aldrich

Cat# C2920

Rotenone

Sigma-Aldrich

Cat# R8875

Antimycin

Sigma-Aldrich

Cat# A8674

2-Deoxy-D-glucose (2-DG)

Sigma-Aldrich

Cat# D6134

Methotrexate

Sigma-Aldrich

Cat# M9929

Fluorescein isothiocyanate–dextran

Sigma-Aldrich

Cat# FD4

Wnt3a assay kit

Cusabio

Cat# CSB-EL026136MO

RNeasy mini kit

QIAGEN

Cat# 74104

RNAscope 2.5 HD Detection Reagents

ACDbio

Cat# 322300

RNAscope Probe-Mm-Axin2

ACDbio

Cat# 400331

RNAscope Probe-Mm-Olfm4

ACDbio

Cat# 311831

RNAscope Probe-Mm-Gpr81

ACDbio

Cat# 317421

RNAscope Probe-Mm-Wnt3

ACDbio

Cat# 312241

RNAscope Probe-Mm-Wnt2b

ACDbio

Cat# 405031

RNAscope Probe-Mm-Wnt6

ACDbio

Cat# 401111

RNAscope Probe-Mm-Wnt9b

ACDbio

Cat# 405091

Seahorse XF24 FluxPak

Agilent

Cat# 100850-001

Critical Commercial Assays

BCA Protein Assay Kit

Thermo Fisher Scientific

Cat# 23225

Lactate assay kit

Biovision

Cat# K607-100

MEGAshortscript T7 kit

Ambion

Cat# AM1345

mMESSAGE mMACHINE T7 Ultra Kit

Ambion

Cat# AM1354

HEK293t cell line

ATCC

Cat# CRL-1573

Wnt3a-expressed cell line

ATCC

Cat# CRL-2647

The Jackson Laboratory

Stock No:008875

Experimental Models: Cell Lines

Experimental Models: Organisms/Strains Mouse: Lgr5-EGFP-IRES-creERT2 Mouse: GF mice

POSTECH

N/A

Mouse: Gpr81-/- mice

This paper

N/A

Mouse: C57BL/6N, ICR mice

Orient Bio.

N/A

Primers for Wnt3, see Table S1

Das et al., 2015

N/A

Primers for Wnt2b, see Table S1

Das et al., 2015

N/A

Primers for Axin2, see Table S1

PrimerBank

N/A

Primers for Ctnnb1, see Table S1

PrimerBank

N/A

Oligonucleotides

(Continued on next page)

e2 Cell Host & Microbe 24, 833–846.e1–e6, December 12, 2018

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Primers for GSK3b, see Table S1

De Vries et al., 2004

N/A

Primers for Lyz1, see Table S1

Everard et al., 2013

N/A

Primers for Reg3b, see Table S1

Ito et al., 2017

N/A

Primers for Reg3g, see Table S1

Ito et al., 2017

N/A

Primers for b-actin, see Table S1

Kim et al., 2010

N/A

Primers for Gpr81, see Table S1

This paper

N/A

pUC57-sgRNA vector

Addgene

Cat# 51132

pRGEN-Cas9-CMV plasmid

ToolGen

Cat# TGEN_dRS1

ImageJ

NIH

https://imagej.nih.gov/ij/

Zen image program

Carl Zeiss

https://www.zeiss.com/microscopy/us/downloads/ zen.htm

FlowJo software

Tree Star

https://www.flowjo.com/solutions/flowjo/downloads

GraphPad PRISM

GraphPad Software

https://www.graphpad.com/

Recombinant DNA

Software and Algorithms

Other 0.22-mm syringe filter

Pall Corporation

Cat# PN4612

70-mm cell strainer

BD Falcon

Cat# 352350

Aria II Flow Cytometry

BD Biosciences

N/A

ImageQuant LAS 4000

GE Healthcare Life Sciences

N/A

Fluorescence spectrophotometer

PerkinElmer

VICTOR X3

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, M.-N.K. ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Mice Animal experiments were carried out with adult 6-8-week-old mice and used equal numbers of male and female mice. All mice analyzed were provided sterile pellet food and water ad libitum. C57BL/6N mice were purchased from Orient Bio (South Korea) and Lgr5-EGFP-IRES-CreERT2 (Lgr5-GFP) mice were purchased from Jackson Laboratory (Bar Harbor, ME). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Asan Biomedical Research Center (Approval No: PN 201413-069) and conducted in compliance with regulatory guidelines. Mice were maintained under SPF conditions in the animal facility at the Asan Biomedical Research Center (Seoul, South Korea). GF mice were maintained in the animal facility at POSTECH (Pohang, South Korea). All animal experiments were performed under anesthesia with a mixture of ketamine (100 mg / kg BW) and xylazine (20 mg / kg BW). Organoid Culture Organoids from SIs of age- and sex-matched mice were established and maintained as described previously (Sato et al., 2009). For construction of organoids, 200–500 crypts per well were suspended in Matrigel (Corning). We used the same numbers of crypts per group for comparison study. Complete ENR medium containing advanced DMEM/F12 (Gibco), 3100 penicillin/streptomycin (Gibco), 1 mM N-acetyl cysteine (Sigma-Aldrich), B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), EGF (Thermo Fisher Scientific), Noggin (R&D Systems), and R-spondin-1-conditioned medium. ENR medium was replaced every 2 to 3 days. Between days 5 and 7 of culture, organoids were passaged by mechanical disruption with a pipette and cold medium was added to the Matrigel. After the Matrigel was washed away by spinning at 1,200 rpm, organoid fragments were replaced in new Matrigel. To isolate single cells from organoids, the mechanically dissociated organoid pellet was further incubated in TrypLE Express (Thermo Fisher Scientific) for 5 min at 37 C.

Cell Host & Microbe 24, 833–846.e1–e6, December 12, 2018 e3

Conditions for the HEK293t and Wnt3a-expressing Cell Lines HEK293t cells were cultured in 100mm dish with advanced DMEM/F12 medium containing 10% FBS and 1% Antibiotic-Antimycotic. R-spondin-1 (Sino Biological Inc.) were purchased and transfected into HEK293t cell lines using lipofectamin 3000 (Thermo Fisher Scientific). Conditioned media were obtained from R-spondin-1-expressing HEK293t cell lines and used 1:10 dilution for organoid culture. Wnt3a-expressing cell lines (ATCC CRL2647) were performed according to the manufacturer’s instructions. METHOD DETAILS Generation of Gpr81-/- Mice Using CRISPR/Cas9 Single guide RNAs (sgRNAs) specific for the genomic DNA sequences adjacent to the translation start site of Gpr81 gene (sgRNA1, 50 -ACTGCTATGGACAACGGGTC-30 ; sgRNA2, 50 -CGGGTCGTGCTGTCTCATCG-30 ) were selected using web-based tools (Broad Institute Genetic Perturbation Platform, http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design; CRISPR Design, http://crispr.mit.edu/). Oligomers for the generation of sgRNA template plasmids (50 -TAGGCGGGTCG TGCTGTCTCATCG-30 and 50 -AAACCGATGAGACAGCACGACCCG-30 for sgRNA1; 50 -TAGGGACCCGTTGTCCATAGCAGT-30 and 50 -AAACACTGCTATGGACAACGGGTC-30 for sgRNA2) were annealed and cloned into the BsaI sites of pUC57-sgRNA vector (Addgene 51132). Templates for the in vitro transcription were PCR-amplified by M13F (50 -GTAAAACGACGGCCAGT-30 ) and pCAG-RGEN-R (50 -GCACCGACTCGGTGCCACT-30 ), and sgRNAs were synthesized using the MEGAshortscript T7 kit (Ambion) according to the manufacturer’s instructions. Cas9 mRNA was transcribed in vitro from linearized pRGEN-Cas9-CMV plasmid (ToolGen, Seoul, South Korea) using mMESSAGE mMACHINE T7 Ultra kit (Ambion) according to the manufacturer’s instructions. C57BL/6N and ICR mice (Orient Bio.) were used as embryo donors and foster mothers, respectively, and CRISPR/ Cas9-mediated gene targeting in mice was performed as described previously (Sung et al., 2014). Founder mice with null mutations were identified by sequencing the target region of Gpr81 gene using PCR products amplified from genomic DNA samples isolated from the tail biopsy samples of mouse pups with the following primer pair: 50 -AGCCCTAGTGTTCGGATAGGCA-30 and 50 -AAGCAGAAGCCGCACAGGG-30 . Selected founders with null mutations were crossed to wild-type, and the null mutant alleles were confirmed in F1 progenies by sequencing. These Gpr81 heterozygous mutant mice were bred and then were intercrossed to produce Gpr81 null mice. In Vivo Experiments Treatment with Probiotics, L. plantarum, and Lactate Age- and sex-matched mice were orally inoculated with probiotics VSL#3 (2 3 109 CFU; Sigma-Tau Pharmaceuticals) or L. plantarum wild-type (3 3 109 CFU) or L. plantarum double lactate dehydrogenase mutant (L. plantarum DldhD-DldhL) strain (3 3 109 CFU) per 100 ml of PBS daily for 5 days. L. plantarum strains were grown at 37 C in MRS broth (BD Difco) as described previously (Liu et al., 2006). Mice were fed drinking water containing DL-lactate (5 mM; Sigma-Aldrich) for 5 days. Gut Contents of Adult and Newborn Mice The gut contents were taken from the SI and LI of sex-matched 6-week-old adult mice or from the stomach of breast-fed 3-day-old newborn mice (NBGC). Each 100 mg of gut contents was diluted in 1 ml of serum-free DMEM/F12 (Gibco) medium and spun by vortex for 1 hr. The contents were centrifuged at 4,000 rpm for 10 min and supernatants were passed through a 0.22-mm syringe filter (Pall Corporation) before cultivation. Treatment with Irradiation and MTX Mice were treated intraperitoneally with MTX (150 mg/kg, Sigma-Aldrich) followed by administration of 10 Gy or 12 Gy of total-body irradiation (cesium source irradiator; Precision X-Ray, North Branford, CT). Pathologic Scoring Pathologic scoring was done in a blinded fashion using a scoring system as described previously (Dieleman et al., 1998). In brief, three parameters were measured: severity of inflammation (0, none; 1, slight; 2, moderate; 3, severe), extent of injury (0, none; 1, mucosa; 2, mucosa and submucosa; 3, transmural and epithelium lost), and crypt damage (0, none; 1, basal one-third damaged; 2, basal two-thirds damaged; 3, only surface epithelium intact; 4, entire crypt and epithelium lost). The sum of the three parameter values was multiplied by a factor that reflected the percentage of tissue involvement (1, 0%-25%; 2, 26%-50%, 3, 51%-75%; 4, 76%-100%). Intestinal Permeability Assay Intestinal permeability was examined by oral administration of FITC-dextran (10 mg / 100 ml in PBS). Mouse blood samples were collected retro-orbitally 3 hr after oral inoculation of FITC-dextran. Plasma was separated by centrifuging (12,000 rpm, 5min, 4 C) and analyzed for FTTC-dextran concentration by fluorescence spectrophotometer (PerkinElmer, 2030 multi-label reader, Victor X3). Bacterial Strains Enterohemorrhagic E. coli (EHEC, O157:H7) strain was provided by the Korea Center for Disease Control and Prevention (Chungwon, Chungcheongbuk-do, Korea). Bacteria were grown overnight in LB broth and reinoculated with 1% precultured bacteria in fresh medium (up to an optical density [OD] of 0.8 to 0.9). For oral infection, each mouse was administered 5 3 109 CFU of bacteria.

e4 Cell Host & Microbe 24, 833–846.e1–e6, December 12, 2018

In Vitro Experiments Crypt Isolation SIs were opened longitudinally and washed with PBS. To dissociate the crypts, tissues were incubated at 4 C in 1 mM EDTA in PBS for 30 min, washed in PBS, and transferred into 5 mM EDTA in PBS for an additional 1 hr of incubation at 4 C as described by others (Andersson-Rolf et al., 2014). Samples were then suspended in PBS and filtered out by a 70-mm cell strainer (BD Falcon). Supernatants from Probiotics Supernatants were obtained from cultured medium of VSL#3 (1 3 108 CFU) in DMEM/F12 medium containing 10% FBS. The cultures were grown overnight at 37 C. L. plantarum strains were cultured in MRS broth at 37 C (OD = 0.8  0.9) before supernatants were harvested. All supernatants were centrifuged at 4,000 rpm for 10 min and passed through a 0.22-mm syringe filter. Agonist and Antagonist of Gpr81 We used Gpr81 agonist 3-chloro-5-hydroxy benzoic acid (3, 5-DHBA, 10 mM; Sigma-Aldrich) or Gpr81 antagonist 3-hydroxy-butyrate (3-OBA, 3 mM; Sigma-Aldrich). The latter is also known as a chemical chaperone that inhibits ER stress. Antibiotic Treatment Mice were given antibiotics including vancomycin (10 mg/kg; Sigma-Aldrich), neomycin sulfate (30 mg/kg; USB), metronidazole (50 mg/kg; Nacalai Tesque), and ampicillin (50 mg/kg; Amresco) in drinking water for 5 days. Isolation of Intestinal Stromal Cells As described previously (Kabiri et al., 2014), all tissues remaining after crypt isolation were subjected to an additional pipetting with PBS to remove the epithelial cells and washed with serum-free DMEM containing 1% antibiotic-antimycotic (Thermo Fisher Scientific). Tissues were then digested for 1 hr in DMEM containing 10% FBS and 2 mg/ml of collagenase/dispase (Sigma-Aldrich) on the stirrer. Tissues were further dissociated by vigorous pipetting, passed through a 70-mm cell strainer, centrifuged at 1,200 rpm for 5 min, and washed twice in PBS. To obtain high purity stromal cells, cells were sub-cultured five times with DMEM-F12 containing 10% FBS and 1% antibiotic-antimycotic. To test the ability of intestinal stromal cells to support organoid formation, 200–500 crypts per well were suspended in Matrigel. After the Matrigel polymerized, complete ENR medium containing 1 3 103 stromal cells per well was seeded. Immuno-Histochemistry Staining Tissues were fixed with 4% paraformaldehyde (PFA) and dehydrated with 15% and 30% sucrose in PBS. Dehydrated tissues were then embedded in OCT compound (Sakura Finetek) as a Swiss roll, frozen, and sliced into 5-mm sections. For organoid staining, organoids were removed and fixed in 2% PFA in PBS for 30 min at room temperature (RT). Organoids were collected in PBS and subsequently permeabilized in PBS containing 0.1% Tween 20 for 30 min at RT and blocking solution containing 0.2% BSA in PBS at 4 C for 1 hr. Then, tissue sections or organoids were stained with anti-Lysozyme-1, anti-Ki67, and anti-Muc2 from Santa Cruz Biotechnology; anti-Wnt3, anti-CD44, anti-Olfm4, and anti-b-catenin (Abcam); anti-CD24 (R&D Systems); anti-Lgr5/Gpr49 (R&D Systems); anti-Gpr81 (LifeSpan BioSciences) antibodies and 4’,6-dismifino-2-phemylindole (DAPI; Thermo Fisher Scientific). To determine immunocytofluorescence of intestinal stromal cells, plated cells were fixed with 2% PFA in PBS containing 0.1% Tween 20 for 30 min at RT and subsequently permeabilized in PBS containing 0.2% BSA in PBS at 4 C for 1 hr. After a wash, cells were stained with antiaSMA, anti-Wnt3a, anti-PORCN, and anti-b-arrestin from Abcam; anti-Gpr81 antibodies (LifeSpan BioSciences); and DAPI. Alexa488, -546 and -633–conjugated anti-rabbit IgG, anti-rat-IgG, anti-goat-IgG, and anti-mouse IgG antibodies from Thermo Fisher Scientific were used as secondary antibodies. All samples were viewed under a confocal laser scanning microscope (Carl Zeiss) using 3 20 objective and crop settings. Histology Intestines prepared by the Swiss roll technique were fixed in 4% para-formaldehyde, embedded in paraffin, and stained with hematoxylin-eosin (H&E) or periodic acid-Schiff (PAS). For PAS staining, 5-mm sections were deparaffinized and hydrated, and put in periodic acid solution (1%) for 5 min at RT. After a wash, the slide was immersed in Schiff’s reagent for 5 min at 60 C and washed in hot water for 5min. Then, slides were stained with hematoxylin and HCl-alcohol and microscopic images were obtained (BX53; Olympus Optical, Tokyo, Japan). RNA In Situ Hybridization Tissues that were fixed in 4 % para-formaldehyde and embedded in paraffin were used for RNA in situ hybridization, which was performed using RNAscope 2.5 HD Detection kit from Advanced Cell Diagnostics (ACD bio) according to manufacturer instructions. Total 7 RNA probes (Axin2, Olfm4, Gpr81, Wnt3, Wnt2b, Wnt6, and Wnt9b) designed by ACD were used and hybridized to their target mRNA. Signals from probes were detected by DAB (3,3’-diaminobenzidine). In Vitro Measurements Measurement of Organoid Size The surface area of organoids was measured microscopically by taking several random non-overlapping photos of organoids in a well using an inverted microscope (Carl Zeiss). Each photo was analyzed using ImageJ software (NIH) and the Zen image program (Carl Zeiss). Organoid perimeters for area measurements were defined manually and by automated ImageJ software FACS Analysis Purified cells were blocked with solution containing 0.2% BSA in PBS and stained with direct fluorescence conjugated antibodies; anti-CD24 (R&D Systems), anti-CD45 (BD Bioscience), anti-Gpr81 (LifeSpan BioSciences), and anti-Wnt3a (abcam) antibodies. Flow cytometry data were collected with LSR II (BD Biosciences) and files were analyzed using FlowJo software (Tree Star). Cell Host & Microbe 24, 833–846.e1–e6, December 12, 2018 e5

Real-time PCR RNA was collected from isolated SI crypts or from organoids. Total RNA was extracted using the RNeasy mini kit (QIAGEN) and cDNA was synthesized using Superscript II reverse transcriptase and oligo (dT) primer (Thermo Fisher Scientific). cDNA was used as the template for real-time PCR (qPCR) performed using SYBR green chemistry (Affymetrix) on an ABI 7500 Real Time PCR system (Applied Biosystems). Extracted RNA was stored -80 C. Primers used are shown at Key Resources Table (KRT). Seahorse XF24 Cell Mitochondrial Function Test Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XF24 analyzer (Agilent). Intestinal crypts (250350) were embedded in 3 mL of matrigel in XF24 cell culture microplates (Seahorse Bioscience) and incubated with OCR assay medium (minimal DMEM, Sigma-Aldrich) supplemented with 2 mM L-glutamine, 5 mM pyruvate, 20 mM glucose, pH 7.4) or ECAR assay medium (minimal DMEM supplemented with 2 mM L-glutamine, pH 7.4) in the 37 C non-CO2 incubator for 1 hour. For OCR measurements, we used oligomycin (2 mM), FCCP (5 mM), and rotenone & antimycin (1.5 mM) from Sigma-Aldrich. For ECAR, glucose (20 mM, JUNSEI, Tokyo, Japan) and oligomycin (2 mM) and 2-DG (50 mM; both from Sigma-Aldrich) were used. After measurements, cells were lysed with RIPA buffer (Thermo Scientific) and proteins were quantified with the BCA assay kit (Thermo Fisher Scientific) for normalization. Western Blotting Western blot analysis was performed as described previously using antibodies against b-catenin (Sigma-Aldrich) and p-GSK3ab (Cell Signaling Technology) and a-tubulin (EMD Millipore). Antibodies were used at a 1:1000 ratio on membranes blocked with BSA. Membranes were visualized using ImageQuant LAS 4000. Determination of Lactate Levels Lactate concentration was measured by lactate assay kit (Biovision, Milpitas, CA) per the manufacturer’s instructions. Absorbance was measured at 570 nm using a spectrophotometer microplate reader (Bio-Rad). QUANTIFICATION AND STATISTICAL ANALYSIS Statistics Statistical analyses were conducted with Prism software (GraphPad, La Jolla, CA). The surface area of organoid was measured using Image J (NIH) and the line image program (Carl Zeiss). For pairwise and two independent group comparison two-tailed t-test was used. Data are presented as mean ± SEM and p < 0.05, p < 0.01, p < 0.001 was considered statistically significant. The exact value of n, representing the number of mice in the experiments depicted, was indicated in the figure legends. Any additional technical replicates are described within the Method Details as well as the results.

e6 Cell Host & Microbe 24, 833–846.e1–e6, December 12, 2018