《微生物学》课程教学资源（前沿进展）宿主通过粪便中的microRNA塑造肠道菌群（英文原文）The Host Shapes the Gut Microbiota via Fecal MicroRNA.pdf

Cell Host & Microbe Article The Host Shapes the Gut Microbiota via Fecal MicroRNA Shirong Liu,1 Andre Pires da Cunha,1 Rafael M. Rezende,1 Ron Cialic,1 Zhiyun Wei,1 Lynn Bry,2 Laurie E. Comstock,3 Roopali Gandhi,1 and Howard L. Weiner1, * 1Department of Neurology, Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 2Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 3Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA *Correspondence: hweiner@rics.bwh.harvard.edu http://dx.doi.org/10.1016/j.chom.2015.12.005 SUMMARY The host gut microbiota varies across species and individuals but is relatively stable over time within an individual. How the host selectively shapes the microbiota is largely unclear. Here, we show that fecal microRNA (miRNA)-mediated inter-species gene regulation facilitates host control of the gut microbiota. miRNAs are abundant in mouse and human fecal samples and present within extracellular vesicles. Cell-specific loss of the miRNA-processing enzyme, Dicer, identified intestinal epithelial cells (IEC) and Hopx-positive cells as predominant fecal miRNA sources. These miRNAs can enter bacteria, such as F. nucleatum and E. coli, specifically regulate bacterial gene transcripts, and affect bacterial growth. IEC-miRNA-deficient (Dicer1DIEC) mice exhibit uncontrolled gut microbiota and exacerbated colitis, and WT fecal miRNA transplantation restores fecal microbes and ameliorates colitis. These findings identify both a physiologic role by which fecal miRNA shapes the gut microbiota and a potential strategy for manipulating the microbiome. INTRODUCTION The gut hosts a complex microbiota that is initially comprised of microbes from the mother and continues to develop through feeding and other environmental contacts (Ma¨ ndar and Mikelsaar, 1996). At approximately 3 years of age in humans, the intestinal microbiota resembles an adult-like microbiota that is relatively stable over time (Faith et al., 2013; Schloissnig et al., 2013). Many factors contribute to shaping the mammalian microbiota, including host genetics, diet, and disease states (Goodrich et al., 2014; Ley et al., 2005; Turnbaugh et al., 2009). Broad trends exist within a given species, but interspecies microbial composition often differs dramatically. Reciprocal transplantation of gut microbiota into germ-free zebrafish and mouse recipients revealed that the relative abundance of microbial lineages resembles the composition of the recipient host (Rawls et al., 2006), suggesting that there are selective mechanisms in the host for the maintenance of specific components of the microbiota. Since the gut microbiota play an important role in host metabolism and immunity as well as in disease (An et al., 2014; Belkaid and Naik, 2013; Hooper et al., 2012; Iida et al., 2013; Koren et al., 2011; Smith et al., 2013; Tremaroli and Ba¨ ckhed, 2012; Turnbaugh et al., 2009), it is important to understand the mechanisms by which the microbiota is regulated by the host and to identify ways by which to manipulate the microbiome (Goodrich et al., 2014). MicroRNAs (miRNAs) are small non-coding RNAs, 18–23 nucleotides in length, synthesized in nucleus, that are processed and function in the cytoplasm. However, increasing evidence demonstrates that miRNAs exist extracellularly and circulate in body fluids (Weber et al., 2010). Isolated studies have measured RNA in human stool and identified miRNAs as potential markers of intestinal malignancy (Ahmed et al., 2009; Link et al., 2012). Whether functional fecal miRNA exists in the normal gut is unexplored. Here we identify gut miRNAs in intestinal contents and feces and demonstrate their role in modulating the gut microbiota composition. RESULTS Identification of miRNA in Mouse and Human Feces To determine whether miRNAs could be identified in the stool, we isolated RNA from both human and mouse fecal samples using the mirVana miRNA isolation kit and compared it to splenic RNA isolates. We found that both human and mouse fecal samples contained small RNA species in a pattern similar to extracellular exosome RNA (Figure S1A), whose size pattern lacks peaks for the 18S and 28S rRNA subunits (Valadi et al., 2007) as measured by bioanalyzer. We then performed small RNA bioanalyzer on the fecal samples (Figure S1B) and found three major RNA peaks in the small RNA region: 20 nt, 65 nt, and 100 nt. The majority of these small RNAs were of miRNA size. In order to establish that specific miRNAs were present in mouse fecal samples, we carried out a pilot study and found that specific miRNAs could be identified by real-time quantitative PCR (qPCR) (not shown). Based on these data, we profiled mouse fecal miRNA using the NanoString nCounter platform (Geiss et al., 2008). Of the 566 miRNAs tested in mouse feces, 283 miRNAs were detected (Table S1) with miR-1224, miR-2146, miR-2134, miR- 483, miR-710, miR-2141, miR-720, miR-155, and miR-34c being the most abundant miRNAs (Figure 1A). 32 Cell Host & Microbe 19, 32–43, January 13, 2016 ª2016 Elsevier Inc

The stability of miRNAs is robust compared to mRNA (Jung et al., 2010). Extracellular miRNAs can be released in both extracellular vesicle (EV) form (e.g., microvesicle, exosome) and an EV-free form associating with high-density lipoproteins or argonaute protein. These forms may contribute to extracellular miRNA stability (Creemers et al., 2012). It was reported that epithelial cells could release exosome-like vesicles displaying major histocompatibility complexes (Van Niel et al., 2003). To explore whether EV exist in the feces, we examined the specimen with NanoSight and electron microscopy and observed EVs in the fecal samples (Figures S1C and S1D). Furthermore, we found that the most abundant miRNAs in feces, such as miR-1224, miR-2146, miR-2134, miR-2141, and miR-34c, were abundantly present in EVs (Figure S1E). We then performed Nanostring analyses of human fecal samples to determine which miRNAs are expressed and how they compare to the ones in mouse. Of the 800 miRNAs tested in human feces, 181 miRNAs were detected (Table S2) with miR-1246, miR-601, miR-630, miR-2116-5p, miR-320e, miR-1224-5p, miR-155-5p, and miR-194-5p being the most abundant miRNAs (Figure 1B). When we compared the 50 most abundant miRNAs in mouse and human feces, we found that 17 miRNAs were shared between these species (Figure 1C). Figure 1. Identification of miRNA in Feces and Intestinal Luminal Contents (A) Mean values ± SEM for the 50 most abundant miRNAs in mouse fecal samples (n = 6). See Table S1 for full list. (B) Mean values ± SEM for the 50 most abundant miRNAs in human feces (n = 10). See Table S2 for full list. (C) Venn diagram showing 17 miRNAs from the 50 most abundant fecal miRNAs shared between human and the mouse as shown in (A) and (B). (D–F) Upper panels: volcano plots for miRNA level based on Nanostring detection in ileal luminal contents (n = 5) versus colonic luminal contents (n = 8) (D), germ-free (GF) mouse feces (n = 8) versus SPF colonized (Colonized) mouse feces (n = 8) (E), and antibiotic-treated (ABX) mouse feces (n = 4) versus SPF mouse feces (n = 4) (F). Each dot represents one miRNA; x axis: fold change; y axis: p value comparing individual miRNAs between groups (unequal variance t test followed by Benjamini-Hochberg correction); the color of the dot indicates mean expression (exp.) level of the corresponding miRNA in both groups as shown in side color scale bar. Lower panel: PCA analyses of miRNAs based on the same Nanostring datasets. See also Figure S1. In order to investigate whether the miRNAs are present in different sections of the gut lumen, we collected gut luminal contents from the distal ileum and colon of C57BL/6J mice, isolated RNA, and measured miRNA profiles. We observed that the miRNAs were significantly different between different regions of the intestine. More abundant miRNAs were found in the ileal lumen compared to the colon (Figure 1D). This distribution is opposite to that of the gut microbes, which are more abundant in the colon. The gut microbiota shapes many aspects of gut physiology and immune system maturation (Lee and Mazmanian, 2010). To determine whether resident gut microbes affect fecal miRNA, we compared the fecal miRNA profile of germ-free (GF) mice with that of SPF colonized littermates. We found that the abundance of fecal miRNA in GF mice was higher than in SPF colonized mice and that the miRNA profiles in these two populations differed (Figure 1E). We further clarified this correlation by comparing SPF mice with antibiotic-treated mice and found that removal of microbes in the gut by antibiotics resulted in significantly more luminal miRNA (Figure 1F). Intestinal Epithelial Cells and Hopx-Positive Cells are Two Main Sources of Fecal miRNA The source of fecal miRNA has not been reported. Since intestinal epithelial cells (IECs) were reported to secret exosomes (Van Niel et al., 2003) and we observed miRNA containing exosomelike EVs in the feces (Figures S1C–S1E), we investigated whether fecal miRNAs originated from IECs. Villin protein is universally expressed in IECs. Villin-cre transgenic mice (Vil-cre) express Cell Host & Microbe 19, 32–43, January 13, 2016 ª2016 Elsevier Inc. 33

Cre recombinase under the direction of the villin 1 promoter (Madison et al., 2002). Dicer is required for the processing of miRNAs. FloxP sites on either side of the Dicer1 gene (Dicer1fl/fl) result in the Cre-expressing cell-specific deletion of miRNAs (Harfe et al., 2005). We bred Vil-Cre mice with Dicer1fl/fl mice to generate mice defective in IEC-specific miRNA (Vil-CreTg/, Dicer1fl/fl; referred to as Dicer1DIEC hereafter) (McKenna et al., 2010). We compared the fecal miRNA profiles of Dicer1DIEC mice with miRNA profiles of their wild-type littermates (Vil-Cre/, Dicer1fl/fl; referred to as Dicer1fl/fl or WT hereafter) and found that the miRNA abundance was decreased and the profiles were altered (Figure 2A and Table S3), suggesting that intestinal epithelial cells are a major source of fecal miRNA. We then asked whether Paneth cells and goblet cells also contributed to fecal miRNA. The HOP homeobox gene (Hopx) is expressed in intestinal epithelial +4 niche stem cells and in their derived cells including Paneth and goblet cells (Takeda et al., 2011). We thus bred HopxERCre mice (Takeda et al., 2011) with Dicer1fl/fl mice to generate Dicer1DHopx (HopxERCreTg/, Dicer1fl/fl; referred to as Dicer1DHopx hereafter) mice in which Hopx-expressing cells are deficient in miRNA following tamoxifen induction. We found that most of the detectable fecal miRNAs in Dicer1DHopx mice were decreased as compared to WT littermates and the profiles were changed (Figure 2B and Table S4), suggesting that the Hopx-expressing cells are also a source of fecal miRNAs. The decreased fecal miRNAs in Dicer1DHopx mice were different from those affected in Dicer1DIEC mice (Tables S3 and S4), suggesting that epithelial cells and Hopx-expressing cells are responsible for the generation of different miRNAs in the feces. To further investigate the gut epithelium as a source of fecal miRNAs, we studied dextran sulfate sodium (DSS)-induced colitis in which DSS treatment results in epithelial cell destruction and mild goblet cell loss (Solomon et al., 2010). Destruction of intestinal epithelial cells secondary to DSS treatment caused a marked decrease in fecal miRNAs (Figure 2C), supporting a Figure 2. Intestinal Epithelial Cells and Hopx-Expressing Cells are Two Predominant Fecal miRNA Sources Volcano plot of fecal miRNA levels detected by Nanostring in feces from: (A) Dicer1DIEC (n = 6) versus Dicer1fl/fl (n = 5) mice (see also Table S3); (B) Dicer1DHopx (n = 4) versus Dicer1fl/fl (n = 4) mice (see also Table S4); (C) DSS-treated day 4 (n = 4) versus naive mice (n = 4); and (D) Rag1/ (n = 5) versus wild-type C57Bl/6J (B6) (n = 5) mice. x axis: Log2 (fold change) of expression level between the groups as indicated; y axis: Benjamini-Hochberg corrected unequal variance t test p value of the compared groups. Dotted horizontal line: p = 0.05. The color of the dot indicates expression level of the corresponding miRNA in (A) and (B): Dicer1fl/fl (WT) group; (C): naive group; (D): mean of both Rag1/ and B6 groups. key role of gut epithelial cells in the generation of fecal miRNAs. In order to distinguish whether the identified miRNAs are secreted from epithelial cells or derived from sloughed epithelial cells, we compared the miRNA profile of epithelial cells and luminal content. Although, as expected, many miRNAs were found in both luminal content and epithelial cells, we also found many miRNAs, such as miR-1224, miR-155, miR-710, and miR-2138, at a much higher abundance in the gut luminal contents compared to epithelial cells (Figure S1F), suggesting that these miRNAs are specifically secreted into the gut lumen. To investigate the degree to which fecal miRNAs are derived from lymphocytes that may be present in the gut, we analyzed fecal miRNA in Rag1 gene knockout mice (referred to as Rag1/ hereafter), which have no mature B cells or T cells (Mombaerts et al., 1992). We found no major differences in the abundance of fecal miRNAs as compared to WT mice (Figure 2D), suggesting that these cells are not a major source of fecal miRNA. Increased Gut Microbiota Dissimilarity in IEC miRNADeficient Mice To evaluate whether fecal miRNA affects the gut microbiota, we surveyed the fecal bacterial composition of Dicer1DIEC and Dicer1fl/fl littermate mice by sequencing the V4 region of 16S rRNA gene using an Illumina MiSeq platform and analyzing with the QIIME software following an established protocol (Caporaso et al., 2010). We generated over 250,000 sequences for each sample, and unique sequences were classified and grouped into 1,895 operational taxonomic units based on 97% nucleotide sequence identity (97% ID OTUs). Taxonomic classi- fication revealed a shift of the dominant bacterial phyla Firmicutes and Proteobacteria in Dicer1DIEC mice compared to Dicer1fl/fl mice (Figure S2A). At the family level, we found an increase of Bacteroidaceae and Helicobacteraceae and a decrease of Prevotellaceae, Porphyromonadaceae, Lachnospiraceae, and Ruminococcaceae in Dicer1DIEC mice (Figure 3A). Furthermore, UniFrac metric b-diversity-based principal coordinate analysis (PCoA) showed a phylogenetic architecture that was more dissimilar within Dicer1DIEC mice compared to 34 Cell Host & Microbe 19, 32–43, January 13, 2016 ª2016 Elsevier Inc.

WT littermates (Figure 3B), as indicated by both unweighted UniFrac analysis (Figure 3C), which measures the phylogenetic similarity between microbial communities, and weighted UniFrac analysis (Figure 3D), which is an abundance-based metric (Goodrich et al., 2014). We then constrained the distance metric analyses to the three most dominant bacteria families detected in WT mice: Prevotellaceae, Porphyromonadaceae, and Lachnospiraceae (Figure 3A). We found marked dissimilarity between Dicer1DIEC individuals compared to WT individuals using the weighted UniFrac metric within the Prevotellaceae family, Porphyromonadaceae, and Lachnospiraceae family (Figures 3E–3G). Within the Figure 3. Deficiency of Intestinal Epithelial Cell miRNA Increases the Dissimilarity of the Gut Microbiota Bacterial 16S rDNA sequence-based surveys were performed on the feces of 16 mice (n = 7 Dicer1fl/fl, 9 Dicer1DIEC mice). (A) Relative abundance of bacteria was classified at a family-level taxonomy. (B) Principal coordinates analysis (PCoA) based on weighted UniFrac metrics. Dashed circle indicates the clustering of Dicer1fl/fl samples. (C–H) Box and whiskers plots of b-diversity distances between microbial communities comparing individuals within Dicer1fl/fl mice and between Dicer1DIEC individual mice. (C–G) b-diversity at family level: (C) unweighted and (D) weighted UniFrac of the whole microbiota; (E) the bacterial family Prevotellaceae; (F) the bacterial family Porphyromonadaceae; and (G) the bacterial family Lachnospiraceae. (H) b-diversity at the genus level. (C–H) the specific distance metric used in each analysis is indicated on the axes. Values are: box, median; whiskers, min to max; p value, non-parametric t test). See also Figure S2 and Table S5. Related to Figure 6. Porphyromonadaceae and Lachnospiraceae family, we observed greater dissimilarity between Dicer1DIEC individuals compared to WT individuals using unweighted UniFrac. However, no signifi- cant b-diversity differences were observed within Prevotellaceae families using unweighted UniFrac (Figures S2B– S2D). This indicates that the increased b-diversity within the Dicer1DIEC mice was either from changed bacterial species or the changed abundance of particular bacteria. Furthermore, we confirmed the marked dissimilarity within Dicer1DIEC individuals at the genus level by analyzing the overall OTU counts using Bray-Curtis b-diversity analyses (Figure 3H). Due to high b-diversity in the Dicer1DIEC mice, we were not able to identify major individual OTU differences between Dicer1DIEC mice and WT littermates. However, we did find 10 OTUs that had lower abundance and 2 OTUs with higher abundance in Dicer1DIEC mice compared to WT mice (Figure S2E and Table S5). Host miRNA Affects the Growth of Gut Bacteria We next asked whether host miRNA affects individual microbial species in the gut. To determine whether specific miRNAs can affect bacteria directly in vitro, we examined two gut bacteria: the anaerobic species Fusobacterium nucleatum (Fn) and the facultative anaerobic species Escherichia coli (E. coli). Prior to in vitro culturing, we asked whether bacteria had any nucleic acid sites that could be targeted by miRNAs based on sequence Cell Host & Microbe 19, 32–43, January 13, 2016 ª2016 Elsevier Inc. 35