Journal of Crohn's and Colitis, 2018, 835–848 doi:10.1093/ecco-jcc/jjy038 Advance Access publication March 28, 2018 Original Article

Original Article Loss of MicroRNA-21 Influences the Gut Microbiota, Causing Reduced Susceptibility in a

Murine Model of Colitis Downloaded from https://academic.oup.com/ecco-jcc/article/12/7/835/4955734 by guest on 10 September 2020 Daniel G. W. Johnstona,b, Michelle A. Williamsb, Christoph A. Thaissc, Raul Cabrera-Rubiod, Mathilde Raverdeaua, Craig McEnteea, Paul D. Cotterd, Eran Elinavc, Luke A. J. O’Neilla, Sinéad C. Corrb aSchool of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland bSchool of Genetics and Microbiology, Moyne Institute of Preventative Medicine, Trinity College Dublin, Dublin, Ireland cImmunology Department, Weizmann Institute of Science, Rehovot, Israel dTeagasc Food Research Centre, Moorepark, Fermoy, and APC Microbiome Institute, Cork, Ireland

Corresponding author: Sinéad C. Corr, Department of Microbiology, School of Genetics and Microbiology, Moyne Institute of Preventative Medicine, Trinity College Dublin, Dublin, Ireland. Tel: 353-1-896-1195; Fax: 353-1-679-9294; Email: [email protected]

Abstract Background and Aims: microRNAs regulate gene expression and influence the pathogenesis of human diseases. The present study investigated the role of microRNA-21 [miR-21] in the pathogenesis of intestinal inflammation, because miR-21 is highly expressed in inflammatory bowel disease. Inflammatory bowel disease is associated with intestinal barrier dysfunction and an altered gut microbiota. Recent studies have demonstrated that host microRNAs can shape the microbiota. Thus, we determined the influence of miR-21 on the gut microbiota and observed the subsequent impact in a dextran sodium sulphate [DSS]-induced colitis model. Methods: The influence of miR-21 on the gut microbiota and inflammation was assessed in wild- type [WT] and miR-21–/– mice, in co-housed mice, following antibiotic depletion of the microbiota, or by colonization of germ-free [GF] mice with fecal homogenate, prior to DSS administration. We carried out 16S rRNA sequencing on WT and miR-21–/– mice to dissect potential differences in the gut microbiota. Results: miR-21–/– mice have reduced susceptibility to DSS-induced colitis compared with WT mice. Co-housing conferred some protection to WT mice, while GF mice colonized with fecal homogenate from miR-21–/– were protected from DSS colitis compared with those colonized with WT homogenate. Further supporting a role for the microbiota in the observed phenotype, the protection afforded by miR-21 depletion was lost when mice were pre-treated with antibiotics. The 16S rRNA sequencing revealed significant differences in the composition of WT and miR-21–/– intestinal microbiota. Conclusions: These findings suggest that miR-21 influences the pathogenesis of intestinal inflammation by causing propagation of a disrupted gut microbiota.

Key Words: MicroRNA; microbiota; inflammation

Copyright © 2018 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. 835 All rights reserved. For permissions, please email: [email protected] 836 D. G. W. Johnston et al.

1. Introduction 2. Materials and Methods Inflammatory bowel disease [IBD], which includes Crohn’s disease 2.1. Animals [CD] and Ulcerative colitis [UC], is a lifelong chronic inflamma- miR-21–/– mice were developed by Taconic Artemis using a Cre/lox tion of the gastrointestinal tract. Inflammatory bowel disease is a approach. Briefly, miR-21 was modified by the insertion of two loxP complex and multifactorial disease of unknown etiology; however, sites that enable excision of the floxed miR-21 segment through it is generally believed to arise from a complex interaction between Cre-mediated recombination. Chimeric offspring were backcrossed genetic factors, environmental factors and the host immune sys- onto the C57BL/6J background for a total of eight generations. tem. It is thought that abnormal interaction of the gut microbiota Homozygous deletion of miR-21 was confirmed by PCR genotyp- with the underlying mucosal immune system leads to an aberrant ing. Homozygous miR-21–/– mice and WT littermates were used immune activation and chronic inflammation.1 Indeed, IBD has for animal studies. Animals were maintained in ventilated cages been shown to have an association with an altered gut microbiota at 21 ± 1°C, humidity 50 ± 10% and with a 12 h-light/12 h-dark [dysbiosis], and patients with IBD have an impaired intestinal bar- light cycle under specific pathogen-free conditions, in line with Irish rier, which either contributes to the disease or arises as a result and European Union regulations. Food and water were available Downloaded from https://academic.oup.com/ecco-jcc/article/12/7/835/4955734 by guest on 10 September 2020 of inflammation, subsequently exacerbating the disease. Whether ad libitum throughout all of the experiments. All experiments were microbial dysbiosis causes impaired barrier function and develop- subject to ethical approval by the Trinity College Dublin’s Animal ment of inflammation or whether dysbiosis is a consequence of an Research Ethics Committee [AREC] and were carried out in accord- altered barrier remains to be determined. Recently, there has been ance with the Irish Health Products Regulatory Authority. Germ-free increasing evidence that this inflammation is associated with the [GF] animal experiments were carried out at the Weizmann Institute, altered expression of microRNA-21 [miR-21]. miRNAs are short Revolt, Israel, according to The Institutional Animal Care and Use ~22-nucleotide non-coding RNAs that act as post-transcriptional Committee [IACUC]. regulators of mRNA function by targeting specific RNAs for 2–4 destruction or by repressing their translation. miRNAs regulate 2.2. DSS-induced experimental colitis many cellular processes in response to a wide variety of stimuli, Colitis was induced in age- and sex-matched WT and miR-21–/– mice. including processes associated with the progression of IBD, such as Mice were weighed at the start of the trial. To induce acute experimen- barrier function, mucin secretion, apoptosis and immune cell acti- tal colitis, mice were administered 2.5% [w//v] dextran sodium sul- vation and function.5 However, although altered miRNA expres- phate [DSS, 36–50 kDa; MP Biomedicals, UK] in their drinking water sion profiles have been identified in serum and tissue from IBD ad libitum for 5 days [unless otherwise stated in the figure legend]. To patients, the mechanistic basis underlying some of these associa- assess experimental colitis and repair, a recovery model was imple- tions is not yet understood.4 mented; specifically, mice were administered 2.5% [w/v] DSS in their miR-21 is overexpressed in both IBD patients and experimen- drinking water ad libitum for 5 days followed by 5 days normal water. tal models of IBD.6–9 It has several reported functions that may Control mice were given normal drinking water throughout. In all col- impact on the disease, including roles in apoptosis and cytoskel- itis models, mice were checked daily for morbidity and body weight etal rearrangement in the context of the barrier.8,9 It also has func- was recorded. Each mouse was scored daily for pathological features, tions in both innate and adaptive immunity. In particular, miR-21 including stool consistency [diarrhea score], presence of fecal occult has been shown to modulate the responses of macrophages to [blood score], and weight loss. Individual scores were combined to bacterially derived Toll-like receptor [TLR] agonists, including generate the Disease Activity Index [DAI], which was calculated daily lipopolysaccharides [LPSs].10 Recently, the role of miRNAs in for each mouse. The maximum DAI score was 12, based on assigning interactions between the host and its microbiota has begun to a 1–4 scoring system for each parameter: Score 0, no weight loss, nor- emerge, with the miRNA profile being dependent on the presence mal stool and no blood; Score 1, 1–3% weight loss; Score 2, 3–6% of an endogenous microbiota; furthermore, host miRNAs have weight loss, loose stool [a loose stool was defined as the formation of been shown to influence the activity of the gut microbiota.11–13 a stool that readily becomes paste upon handling] and blood visible in Given these observations and the ever-increasing evidence impli- stool; Score 3, 6–9% weight loss; and Score 4, >9% weight loss, diar- cating the intestinal microbiota in the pathogenesis of IBD, we rhea and gross bleeding. At the end of the trial, mice were euthanized questioned whether miR-21 expression may influence the compos- by CO asphyxiation. The colons were removed and measured as an ition of the microbiota, and whether this was contributing to the 2 indication of colonic inflammation. Subsequently, colonic sections pathogenesis of IBD. were taken and stored in 10% formalin for histological analysis, or Here we demonstrated that miR-21 plays a pathological role in snap-frozen in liquid nitrogen and stored at –80°C. the development of intestinal inflammation. We showed that mice deficient in miR-21 [miR-21–/–] are less susceptible to DSS-induced 2.3. Co-housing experiments colitis compared with wild-type [WT] mice. We demonstrated a protective microbiota in the miR-21–/– mice that contributed to the Prior to induction of DSS experimental colitis, sex-matched WT and –/– observed reduced susceptibility in the DSS-induced colitis model. miR-21 mice aged 4 weeks were co-housed for a minimum of 4 Furthermore, using 16s rRNA sequencing analysis we confirmed weeks, to allow natural transfer of the fecal microbiota. alterations in the miR-21–/– microbial composition that were indi- cative of protection, including a reduced abundance of the phy- 2.4. Colonization of germ-free mice with fecal lum Bacteroidetes and an increased abundance of . microbiota Interestingly, analysis also revealed an increased abundance of the A homogenate of frozen feces from WT or miR-21–/– mice was pre- protective classes upon loss of miR-21. Taken together, pared in sterile PBS under anaerobic conditions and subsequently this work identified a novel role for miR-21 in shaping the gut micro- filtered through a sterile 70 μm strainer to remove soil particles. For biota and in the subsequent development of intestinal inflammation colonization of GF mice, each mouse was administered 100 μL fecal such as IBD. suspension containing 20 mg feces by oral gavage. Mice were then MicroRNA-21 Influences the Gut Microbiota and Colitis 837 allowed to rest for 9 days to ensure colonization. The same dose was of sequences with <200 nucleotides] with prinseq-lite, and joined used to mimic fecal microbial transplant in conventional mice. using fastq-join [https://expressionanalysis.github.io/ea-utils/].15 The sequences were clustered with 97% identity level [calculated in 2.5. Colonoscopy operational taxonomic units; OTUs], using the closed-reference use- 16 Colonoscopy was performed on experimental mice using a high‐ arch v7.0 algorithm, and the cluster sequences assigned using the 17 resolution mouse video endoscopic system [Carl Storz, Tuttlingen, RDP’s Classifier. Alpha and Beta-diversity was determined using 18 Germany]. The severity of colitis was scored in a blinded manner QIIME. Data were statistically analyzed by Adonis for beta-diver- using MEICS [Murine Endoscopic Index of Colitis Severity], which sity analysis. Statistical differences between multiple samples were is based on five parameters: granularity of mucosal surface, vascular estimated using the Kruskal–Wallis test, and the False discovery rate pattern, translucency of the colon mucosa, visible fibrin, and stool [FDR, qvalue] control based on the Benjamini–Hochberg procedure consistency. was used to correct for multiple testing with the R statistical package [https://www.r-project.org/]. 2.6. Antibiotic treatment of mice Mice were put on a 4-way antibiotic treatment course ad libitum for 2.11. Fecal sIgA detection Downloaded from https://academic.oup.com/ecco-jcc/article/12/7/835/4955734 by guest on 10 September 2020 2 weeks prior to DSS treatment. The course of treatment consisted Fecal pellets were taken, weighed and homogenized in prote- of vancomycin [0.5 g/L], ampicillin [1 g/L], kanamycin [1 g/L] and ase inhibition buffer. Homogenate was centrifuged at 16 000g for metronidazole [1 g/L] in their drinking water. 5 min at 4°C before IgA was measured using an IgA ELISA kit [BD Pharmingen] in accordance with the manufacturer’s instructions. 2.7. Histology Sections from the distal colon of each mouse were fixed in 10% buff- 2.12. Myeloperoxidase activity assay ered formalin and analysed using haematoxylin and eosin [H&E] Colon sections were snap-frozen in liquid nitrogen before homogen- staining. H&E-stained sections of the distal colon were blind scored to ization, and myeloperoxidase [MPO] activity was assessed using a 19 ascertain the extent of colitis, using a previously established protocol.14 slightly modified version of a previously described assay. The modi- A combined score of inflammatory cell infiltration and tissue dam- fications are as follows: 3,3',5,5'-tetramethylbenzidine [TMB] was age was determined as follows: (i) cell infiltration [Score 0, occasional used a substrate for the assay, and MPO activity was expressed in inflammatory cells in the lamina propria [LP]; Score 1, increased infil- arbitrary units [AUs]. trate in the LP, predominantly at the base of crypts; Score 2, confluence of inflammatory infiltrate extending into the mucosa; Score 3, trans- 2.13. Statistical analysis mural extension of infiltrate]; (ii) tissue damage (Score 0, no mucosal Numerical results are given as arithmetic means ± standard error of damage; Score 1, partial [up to 50%] loss of crypts in large areas; Score the means. Statistical differences were analysed by GraphPad Prism 2, partial to total [50–100%] loss of crypts in large areas, epithelium 5.0 statistical software [GraphPad Software Inc., San Diego, USA] intact; Score 3, total loss of crypts in large areas and epithelium lost). to perform Student’s t-tests or the Chi squared test for the Kaplan– Meier survival curve. A p-value of <0.05 [p ≤ 0.05] was considered 2.9. qRT-PCR statistically significant. Total RNA was isolated from colonic sections using the RNeasy PlusMini kit [QIAGEN] and quantified using a Nanodrop 2000 UV-visible spectrophotometer. cDNA was prepared using 20–100 ng/ 3. Results mL total RNA by RT-PCR using a high capacity cDNA reverse tran- 3.1. miR-21 deletion is protective in DSS-induced scription kit [Applied Biosystems], according to the manufacturer’s colitis instructions. Real-time qPCR for miR-21 and IL-1β expression was We initially compared WT and miR-21–/– mice in an acute model of performed on cDNA using Taqman probes. qPCR was performed DSS-induced colitis. It was evident that miR-21–deficient mice were on a 7900 HT Fast Real-Time PCR System [Applied Biosystems] significantly protected from DSS-induced colitis, as evidenced by sig- using KAPA FAST Master Mix High ROX [Kapa Biosystems]. Fold nificantly reduced weight loss Figure 1A[ ], strikingly reduced levels of changes in expression were calculated by the Delta Delta Ct method, occult blood in the stool [Figure 1B] and a reduction in diarrhea score using mouse Rps18 or RNU6B as an endogenous control for mRNA [Figure 1C]. Indeed, miR-21–/– mice had a significantly reduced DAI expression. score [Figure 1D], indicative of reduced susceptibility to disease. The colons of miR-21–/– mice were also significantly longer Figure 1E[ ]. 2.10. 16S sequencing and analysis miR-21 expression was elevated in DSS-treated WT mice when com- To determine the gut microbiota composition of WT and miR-21–/– pared with control animals [Figure 1F]. In a DSS-recovery model, mice, 16S rRNA sequence analysis was performed on fecal samples in which mice received normal drinking water following 5 days from each cohort. Fecal samples were snap-frozen in liquid nitrogen, treatment with 2.5% DSS, miR-21–/– mice again demonstrated pro- and DNA extraction was performed using a MOBIO PowerLyser tection from the DSS-induced colitis, recovering faster and showing DNA extraction kit according to the manufacturers’ instructions. reduced disease symptoms for each parameter [Figure 2A– D]. While The 16S rRNAgene [V3–V4 region] was PCR amplified, follow- there was no significant difference in colon length between WT and ing the Illumina 16S Sample Preparation Guide, and the result- miR-21–/– mice treated with DSS, there was a significant difference ing amplicons were sequenced on the Illumina MiSeq platform between WT control mice and WT mice treated with DSS, which was using v3 sequencing chemistry with 2 × 250 bp paired-end reads. absent between the miR-21–/– groups [Figure 2E]. Histological ana- Sequences were further filtered on the basis of quality [removal of lysis was performed to assess the inflammation in the colon of WT low-quality nucleotides at the 3' end, and removal of windows of and miR-21–/– mice post-DSS treatment. Control mice displayed no 20 nucleotides with a low average quality] and length [removal inflammation, whereas WT mice treated with DSS had significantly 838 D. G. W. Johnston et al.

A Days B 0 12345 115 5 WT *** WT * 110 4 miR21–/– miR21–/– * *** 105 3 ***

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Figure 1. Loss of miR-21 reduces susceptibility to DSS-induced colitis. WT and miR-21–/– mice aged between 8 and 12 weeks were weighed on Day 0 and given 2.5% DSS [w/v] ad libitum for 5 days. Weight [A], blood score [B] and diarrhea score [C] were determined daily throughout the experiment and combined to generate a disease activity index score [DAI] [D]. On Day 5, the mice were sacrificed and their colons measured [E]. miR-21 expression in WT mice post-DSS treatment was compared with that in water controls [F]. [A–E] n = 5 mice per group, mean values ± SEM are presented; p values were calculated using an unpaired t-test. A single asterisk indicates p < 0.05; two asterisks indicate p < 0.01; three asterisks indicate p < 0.001. [F] miR-21 expression was assessed relative to RNU6B in mice treated with water [n = 2] or 2.5% DSS for 5 days [n = 4]. Mean values ± SD are presented; p values were calculated using an unpaired t-test. A single asterisk indicates p < 0.05; two asterisks indicate p < 0.01. more inflamed colons than miR-21–/– mice, as assessed by a cumula- weeks prior to DSS treatment. Control mice co-housed with mice tive histology score [Figure 2F] generated from crypt damage score of the same genotype displayed a similar disease phenotype to that and inflammation score [Figure 2G and 2H]. Representative images observed in previous experiments [Figure 3A, B]. However, there show this difference in inflammation Figure 2I[ ]. was a slight reduction in disease severity in WT mice that had been co-housed with miR-21–/– [CH-WT], as indicated by a reduced DAI 3.2. Co-housing confers protection to WT mice score compared with WT housed with other WT mice [Figure 3A, C]. against DSS-induced colitis Although there was no reduction in weight loss [Figure 3D], CH-WT To determine any influence of miR-21 expression on the composition had reduced diarrhea and blood scores compared with WT controls and activities of the gut microbiota and subsequent development of [Figure 3E, F]. Furthermore, the colon lengths of the CH-WT mice DSS-induced colitis, WT and miR-21–/– mice were co-housed for 4 were longer than those of the WT controls [Figure 3G]. Although MicroRNA-21 Influences the Gut Microbiota and Colitis 839

A DSS Water B WT WT [H2O] Days miR21–/– miR21–/– [H2O] 4 0 246 810 ** ** 110 105 3 100 95 2 * ** * 90 Blood score * 85 * 1 80 75 0 % Initial weight 0 246810 70 WT WT [H2O] Days 65 miR21–/– miR21–/– [H2O]

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Figure 2. miR-21–deficient mice were found to be protected compared with WT mice in a DSS-induced colitis recovery model. WT and miR-21–/– mice aged between 8 and 12 weeks were weighed on Day 0 and given 2.5% DSS [w/v] ad libitum for 5 days, followed by normal drinking water for a further 5 days. Control mice were given normal drinking water throughout. Weight [A], blood score [B] and diarrhea score [C] were determined daily throughout the experiment and combined to generate a disease activity index [DAI] score [D]. On Day 10, the mice were sacrificed and their colons measured [E]. Colitis severity of H&E- stained colonic tissues was assessed by the combined histological score [F] of tissue disruption [crypt damage score; 0–3, according to the severity of mucosal and crypts damages] [G] and colon cellular infiltration [inflammation score; 0–3, according to the extent of inflammation throughout the intestinal wall] [H]. Representative microscopic pictures of H&E-stained colon sections [×100 magnification] from water and 2.5% DSS–treated WT and miR-21–/– mice are shown [I]. Data represent mean values ± SEM of n = 3 mice per group [n = 2 in the miR-21–/– water control group]. Student’s t-test was used to calculate p values. A single asterisk indicates p < 0.05; two asterisks indicate p < 0.01. subtle, these results suggested that natural transfer of the fecal 3.3. Colonization of GF mice with miR-21 fecal microbiota from miR-21–/– mice offers some protection against the microbiota offers protection against DSS-induced severity of DSS-induced colitis. As such, the miR-21–/– fecal micro- colitis biota appears to display a less colitogenic phenotype compared with Having observed protection being partially transferred to WT mice WT fecal microbiota, and can protect against development of colitis via co-housing, we wished to explore whether or not this effect in recipient WT mice. We next attempted to determine whether the could be seen using a GF setting. GF mice were colonized with fecal –/– miR-21 fecal microbiota could also influence the course of estab- homogenates from WT or miR-21–/– animals for 9 days prior to lished colitis [Supplementary figure S1]. To do this, WT mice were DSS treatment. Upon DSS treatment, mice colonized with WT fecal given DSS for 5 days prior to inoculation with either PBS [control] or homogenate exhibited significantly greater weight loss, significantly –/– a homogenate of miR-21 feces [prepared in the same way as for the higher occult blood scores and higher diarrhea scores [Figure 4A–C]. GF colonization model]. On the day of inoculation, DSS was removed This culminated in significantly higher DAI scores Figure [ 4D]. and mice received normal drinking water ad libitum for a further Overall colitis severity was assessed by colonoscopy, where scores 5 days. Mice were scored daily throughout the model, as before, were combined from multiple disease parameters [Figure 4E]. Mice for disease parameters and DAI generated. No difference in the colonized with WT homogenate had a higher disease score than their –/– rates of recovery between control and miR-21 fecal homogenate– miR-21–/– colonized counterparts [Figure 4E] and this can be seen in treated mice was observed, other than reduced shortening of representative images [Figure 4F]. Interestingly, miR-21 expression is –/– colons in miR-21 fecal homogenate–treated mice [Supplementary induced by DSS in the colons of GF mice [Figure 4G]. figure S1E]. 840 D. G. W. Johnston et al.

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Figure 2. (Continued)

3.4. Protection of miR-21–/– mice against DSS- its phylum name followed by its genus name [e.g. Actinobacterium: induced colitis is lost after antibiotic treatment Bifidobacterium] [Figure 6A]. There were considerable differ- In order to ascertain the extent to which the intestinal microbiota ences between the two groups, with the miR-21–/– group display- was influencing the disease severities between the different groups, ing a higher proportion of (i) Actinobacterium: Bifidobacterium; WT and miR-21–/– mice were put on a course of oral antibiotics (ii) Firmicutes: Coprococcus; (iii) Firmicutes: Lactonifactor; (iv) prior to DSS treatment. WT and miR-21–/– control mice had different Firmicutes; Oscilibacter; (v) Firmicutes: Clostridium sensu stricto; weight loss levels, blood and diarrhea scores, as seen before, with (vi) Firmicutes: Anaerosporobacter; and (vii) Firmicutes: Catonella; miR-21–/– mice again showing reduced susceptibility to DSS-induced and a lower proportion of (i) Firmicutes: Clostridium XIVa; (ii) colitis [Figure 5A–D]. However, interestingly, these differences Firmicutes: Tannerella; (iii) Firmicutes: Dorea; (iv) Bacteroidetes: between WT and miR-21–/– mice were lost following antibiotic pre- Barnesiella; and (v) Bacteroidetes: Prevotella, relative to WT mice treatment, as can be seen in the DAI score [Figure 5A–D]. The dif- [Figure 6A]. Beta diversity is represented by a Principal Coordinates ference in colon length between the WT and miR-21–/– mice was also Analysis [PCoA], performed using all 16S rRNA reads clustered at lost following antibiotic pre-treatment [Figure 5E]. Strikingly, miR- 97% similarity [Figure 6B]. Clear phylogenetic separation between 21–/– mice displayed a significantly greater loss of mobility than their the WT and miR-21 knockout groups was evident [ANOSIM WT counterparts upon pre-treatment with antibiotics, and this led p value = 0.001]. There were also several differences apparent at to a significantly higher morbidity in this group [Figure 5F and G]. various taxonomic levels: at the phylum level, Proteobacteria [q = 0.00079935] were present in higher proportions in the miR- 3.5. Altered composition of the miR-21–/– microbiota 21–/– microbiota. At the family level, the miR-21–/– samples con- We employed 16S rRNA sequencing of fecal samples to investigate tained higher proportions of Bifidobacteriaceae[q = 0.01181564] the possibility of differences in the composition of the microbiota and Peptostreptococcaceae [q = 0.14586726], and reduced propor- between WT and miR-21–/– mice. After quality filtering and length tions of Verrucomicrobiaceae [q = 0.07853655] and Bacteroidaceae trimming, an average of 37 313 [±7123 SD] 16S rRNA high-quality [q = 0.05746273]. At the genus level, the WT samples group con- sequences were generated per sample. The average number of OTUs tained higher proportions of Enterorhabdus [q = 0.00036569], per sample was 355 [±16 SD]. The top 25 most abundant OTUs and reduced proportions of Odoribacter [q = 7.896e-05] and were compared between the two groups, with each OTU being given Bifidobacterium [q = 0.00143734] [Figure 6C–E]. MicroRNA-21 Influences the Gut Microbiota and Colitis 841

A WT B 4 miR-21–/– 4 CH-WT WT 3 CH-miR-21–/– 3 miR-21–/– I 2 I 2 DA DA

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Figure 3. WT mice co-housed with miR-21–deficient mice were found to be protected from DSS-induced colitis. WT and miR-21–/– mice aged 4–6 weeks were co-housed for 4 weeks with either mice of the opposite genotype [e.g. WT mouse co-housed with miR-21–/– = CH-WT] or other mice of the same genotype [e.g. WT mouse co-housed with another WT = WT] before being weighed on Day 0 and given 3.5% DSS [w/v] ad libitum for 7 days. Weight, diarrhea score and blood score was determined daily and combined to generate a disease activity index [DAI] score [A]. Individual plots of selected groups’ DAI, weight loss, diarrhea score and blood score are shown [B–F]. On Day 7, the mice were sacrificed and their colons measured [G]. Data represent mean values ± SEM of n = 4 mice per group [except miR-21–/– control group, for which n = 3]. Student’s t-test was used to calculate p values. A single asterisk indicates p < 0.05; two asterisks indicate p < 0.01.

4. Discussion mechanistically influenced the disease by allowing the intestinal epi- thelial barrier to become more permeable via enhancing epithelial Over the past decade there has been an increasing appreciation of apoptosis and targeting the tight junction protein RhoB.6,8 These the crucial roles played by miRNAs in a wide variety of cellular results indicate to us that miR-21 overexpression in IBD is unlikely processes. Among the biggest discoveries in that time was the role to be simply a bystander effect caused by an inflammatory milieu, as of miRNAs in inflammatory processes, in which they regulate both has been suggested,21 but an active component of disease. pro- and anti-inflammatory pathways.20 miR-21 is an extensively Another field of study that has exploded in recent years is the studied molecule with a known association with human IBD.6,8 With growing understanding that the gut microbiota greatly influences this in mind, we generated a knockout mouse to study the disease. human health and disease. Microbial dysbiosis has been found to Our initial finding that miR-21 was pathological in the DSS mouse play a significant contribution in a number of DSS-induced colitis model of colitis was surprising, as miR-21 is widely regarded as an phenotypes exhibited by transgenic mice with alterations in immune anti-inflammatory mediator. These findings were supported by Shi function. One such study demonstrated that the inflammasome et al., who described the same phenotype and suggested that miR-21 842 D. G. W. Johnston et al.

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0 -/- ater DSS ) W –/– -21

Ex-GF (WT) Ex-GF (miR21 Ex-GF (miR

Figure 4. Colonization of germ-free [GF] mice with the fecal microbiota of miR-21–deficient mice protected them against DSS-induced colitis. Germ-free Swiss– Webster mice were colonized with fecal preparations from WT or miR-21–/– mice by oral gavage. Nine days later, mice were given 3% DSS [w/v] ad libitum for 13 days. Weight [A], blood score [B] and diarrhea score [C] were determined daily and combined to generate a disease activity index [DAI] score [D]. On Day 13, the mice were anesthetized and underwent a colonoscopy using the ColoView system. Colons were blind scored for a number of clinical parameters that were combined to give a colitis severity score [E]. Representative images are shown [F]. Data are representative of two independent experiments, presented as mean values ± SEM of n = 5 mice per group. Student’s t-test was used to calculate the p values for A–E. A single asterisk indicates p < 0.05; two asterisks indicate p < 0.01. miR-21 expression relative to RNU6B was assessed by qPCR in colonic tissue from GF mice given 1.5% DSS ad libitum for 10 days; data represent mean values ± SD of n = 3 mice per group. Student’s t-test was used to calculate the p values. A single asterisk indicates p < 0.05 [G]. component NLRP6 was required for intact host–microbe interaction, reasoned that this might be a crucial aspect of the disease phenotype. and that the exacerbated colitis phenotype exhibited by NLRP6–/– First, we observed that, following co-housing of mice, WT mice co- mice in response to treatment with DSS could be transferred to housed with miR-21–/– mice displayed a reduced colitis phenotype WT mice by co-housing.22,23 As miR-21 is a known regulator of the when compared with the WT control group, which was evident in immunological activity of the bacterial sensor TLR4, we sought to the fecal occult blood and in the diarrhea score. This indicated that ascertain the significance of the gut microbiota in our mice, and we the mice lacking miR-21 had an altered microbiota compared with MicroRNA-21 Influences the Gut Microbiota and Colitis 843

A Days B 01235567 4 WT –/– 110 miR21 3 WT[ABX] miR21–/–[ABX] 100 2

90 WT Blood score 1 miR21–/–

% Initial weight 80 WT[ABX] miR21–/–[ABX] 0 0 1234567 Days

C D WT Downloaded from https://academic.oup.com/ecco-jcc/article/12/7/835/4955734 by guest on 10 September 2020 –/– 4 WT 4 miR21 miR21–/– WT[ABX] miR21–/–[ABX] 3 WT[ABX] ** 3 miR21–/–[ABX] I 2 2 DA

Diarrhea score 1 1

0 0 0 1234567 012 34567 Days Days

E F G Days n/s 0 1 23456 7 4 WT 8 n/s –/– miR21 100 6 * 3 WT[ABX] ** miR21–/–[ABX] 80 2 ** 4 60

Mobility loss WT 2 1 40 –/–

Colon length [cm] miR21 Percent survival 20 WT [ABX] *** 0 0 miR21–/– [ABX] –/– 0 1234567 0 WT -21 [ABX] Days –/– miR WT[ABX]

miR21

Figure 5. Protection of miR-21–deficient mice against DSS-induced colitis is lost after antibiotic treatment. WT and miR-21–/– mice aged between 8 and 12 weeks were given a 4-way antibiotics mix ad libitum for 2 weeks. The mice were then weighed on Day 0 and given 3% DSS [w/v] ad libitum for 7 days. Weight [A], blood score [B] and diarrhea score [C] were determined daily and combined to generate a disease activity index [DAI] score [D]. The mice were sacrificed on Day 8 and their colons measured [E]. In addition, the mice were scored for mobility loss [F] and morbidity [G]. Data represent mean values ± SEM pooled from two experiments of n = ≥5 mice per group. Student’s t-test was used to calculate the p values, and the Chi squared test was used to generate the Kaplan–Meier survival curve. A single asterisk indicates p < 0.05; two asterisks indicate p < 0.01; three asterisks indicate p < 0.001 for comparisons between WT and miR-21–/–, except in panel G where they compare WT [ABX] and miR-21–/– [ABX]. their WT counterparts, which offered protection against the devel- To further investigate the protective microbiota observed in the opment of colitis; it also indicated that this protection was transfer- miR-21–/– mice, we colonized GF mice with the fecal microbiota of able. There is a sparse but growing literature regarding the potential WT or miR-21–/– mice prior to a DSS challenge. In this experiment, of miRNAs to modulate the gut microbiota, but given the variety of mice colonized with a miR-21–/– fecal microbiota exhibited protection roles in the regulation of bacterial sensing, it is likely that miRNAs from DSS-induced colitis relative to the mice colonized with the WT play an indirect role. In one interesting study, conducted by Lui et al., microbiota. A colonoscopy confirmed that the miR-21–/––colonized the authors demonstrated that host miRNAs, including miR-21, are mice displayed fewer signs of inflammation. Colonoscopy is the cur- present in mouse feces and that they can be taken up by commensal rent gold standard in IBD diagnosis, and it is becoming increasingly to alter their gene expression. In addition, this study showed utilized in studies modelling the disease.24 It had been previously a significant microbial dysbiosis was present inDicer knockout mice, been reported that miR-21 expression was downregulated in the which lack all functional miRNAs, and that this dysbiosis could be caecum of GF mice relative to that in conventional controls, imply- rescued by re-introducing synthetic miRNAs to the mice.11 ing that the caecal microbiota impacts on its expression.12 In order 844 D. G. W. Johnston et al.

A Bacteroidetes; Barnesiella 37.9 42.1 Firmicutes; Lachnospiracea_incertae_sedis 8.7 5 Bacteroidetes; Alistipes 6 5.5 Bacteroidetes; Prevotella 4.2 6.5 Bacteroidetes; Bacteroides 3.1 6.3 Firmicutes; Clostridium_XIVa 2.4 5.8 Bacteroidetes; Tannerella 2.6 4.6 Firmicutes; Coprococcus 2.4 1.8 Firmicutes; Lactonifactor 2.2 1.3 Firmicutes; Dorea 1.4 2 % Read Firmicutes; Oscillibacter 2 1.1 Abundance Actinobacteria; Bi€dobacterium 2.4 0.2 10.0 Firmicutes; Clostridium_IV 1.3 1.2 Firmicutes; Marvinbryantia 1.4 0.9 1.0 Firmicutes; Pseudoƒavonifractor 0.9 1.2 0.1

Firmicutes; Clostridium_sensu_stricto 1.9 0.1 Downloaded from https://academic.oup.com/ecco-jcc/article/12/7/835/4955734 by guest on 10 September 2020 Firmicutes; Anaerosporobacter 1.5 0.3 Firmicutes; Clostridium_XIVb 0.8 0.9 Firmicutes; Anaerotruncus 0.9 0.6 Firmicutes; Alkalibaculum 0.9 0.5 Firmicutes; Ruminococcus 0.9 0.5 Firmicutes; Robinsoniella 0.8 0.5 Firmicutes; Catonella 1.3 0 Firmicutes; Blautia 0.5 0.7 Actinobacteria; Olsenella 0.8 0.4 21KOWT

B Phyloseq’s Unweighted Unifrac

0.05

Group 21KO WT 0.00 Sex F Axis 2 [13.2%] M

–0.05

–0.05 0.00 0.05 Axis 1 [17.3%]

Figure 6. 16S rRNA analysis of WT and miR-21–deficient mice fecal microbiota. The 16S rRNA reads were compared with the RDP database, and WT and miR-21–/– microbiota samples were compared for OTU analysis [A], beta diversity [B] and taxa analysis at phylum [C], family [D] and genus [E] levels. Data is representative of n = ≥9. to establish whether miR-21 is induced in the DSS colitis model due consequence of depleting this microbiota by administering antibi- to increased penetration of the epithelium by commensal bacteria otics to mice prior to challenge with DSS. Both WT-depleted and and subsequently enhanced immune activation, miR-21 expression miR-21–/––depleted mice displayed increased detectable fecal occult levels were compared between GF mice given DSS or water. It was blood and higher diarrhea scores earlier in the experiment. However, observed that miR-21 expression remained enhanced in these mice interestingly, the protective phenotype previously observed in the upon DSS treatment. This was interesting and pointed to a mech- miR-21–/– mouse was lost following antibiotic treatment, as demon- anism of induction more related to the epithelial damage caused by strated in each of the disease scores and in the overall DAI score. DSS administration.25 Antibiotic depletion of the microbiota has been shown to increase As a final confirmation that the microbiota of miR-21–/– mice intestinal haemorrhaging and mortality in mice subsequently treated confers protection against DSS-induced colitis, we determined the with DSS.26,27 These effects have been demonstrated to be due to MicroRNA-21 Influences the Gut Microbiota and Colitis 845

CDProteobacteria Bi†dobacteriaceae Verrucomicrobiaceae Bacteroidaceae Peptostreptococcaceae q = 0.00079935 q = 0.01181564 q = 0.07853655 q = 0.05746273 q = 0.14586726

–7 –4 –4 –3 –6 –8 –5 Type Type 21KO 21KO –9 WT –6 WT –6 –8 –4 Downloaded from https://academic.oup.com/ecco-jcc/article/12/7/835/4955734 by guest on 10 September 2020 Log–relative normalised Log–relative normalised

–10

–7 –8 –11 –10

FM FM FM F MFM Type Type

E Odoribacter Enterorhabdus Bi†dobacterium q = 7.896e–05 q = 0.00036569 q = 0.00143734 –3 –3

–2 –4

–5 –4

–4 Type –6 21KO WT

–7 Log–relative normalised –5

–6 –8

–9 –6

FM FM FM Type Type Type

Figure 6. Continued a weakened intestinal barrier and the absence of tonic microbial miR-21–/– mice is, at least in part, dependent on the presence of the signals to the epithelium, respectively. Our results demonstrated an microbiota. increase in haemorrhaging [as measured by fecal occult blood], in Having shown that the microbiota of the miR-21–/– mouse deter- keeping with these studies. Most striking of all the observations in mined the outcome of DSS-induced colitis, 16S rRNA sequencing this experiment was the highly significant increase in sickness behav- was employed to compare the fecal microbial profiles of miR-21–/– iour [measured by loss of mobility] and associated mortality dis- and WT mice. The top 25 most abundant OTUs in WT and miR-21–/– played in the miR-21–/– mice treated with antibiotics. This effect has mice were compared and several significant differences were noted. previously been described in WT mice at slightly later time points, Several of the observed differences pointed towards a potentially but interestingly we saw mortality as early as Day 5 in the miR-21 –/– protective microbial signature in the miR-21–/– mouse, including a mice, indicating that in the absence of microbial signaling, miR-21 generally higher abundance of genera within the Firmicute phylum, appears to play a protective role in this disease model. This find- which correlates with protection against IBD, while there was a lesser ing confirms that the microbiota of miR-21–/– mice is protective in abundance of Bacteroidetes and Prevotella, which correlates with DSS-induced colitis, and that the protection demonstrated by the development of IBD.28,29 The higher abundance in the gram-positive 846 D. G. W. Johnston et al. probiotic genera Actinobacterium and Bifidobacterium can also more favorable niche for colitis-protective bacteria.6,8 miRNAs have be interpreted as evidence of a protective microbiota, as several also been reported to alter the expression of specific mucins in the studies have indicated a reduction in the severity of DSS-induced gut, which may also have a bearing on the niche occupied by these disease in mice treated with members of these genera.30–32 In order bacteria.40,41 It is also possible that miR-21 impacts the commensal to assess whether or not these populations shared similar species, bacteria more directly, by targeting secreted bactericidal mediators: beta diversity was assessed and found to differ between WT and for example, miR-21 has been shown to target CAMP and DEFB4A miR-21–/– fecal bacterial populations. This supported the case for an in humans.42 T-cells play an important role in immune homeostasis altered, protective microbiota being present in the miR-21–/– intes- in the gut, and have a crucial role in shaping the gut microbiota.43 tinal compartment. We next performed a taxa analysis to compare miR-21 has been demonstrated to impact the differentiation and acti- the relative proportions of the different bacterial groupings at vari- vation of different T-cell subsets via modulation of key genes such ous taxonomic levels. Surprisingly, at the phylum level there was an as IL-12p35 and Stat3. For instance, in different inflammatory con- increased presence of Proteobacteria in the miR-21–/– mice, which texts, miR-21 has been shown to promote Th17 cells, to negatively would run counter to the idea of a protective microbiota. This phy- regulate Treg cells and to promote memory T-cell activation.44–46 In

lum of gram-negative bacteria generally increase in proportion in murine models of colitis, miR-21 deficiency has been shown to skew Downloaded from https://academic.oup.com/ecco-jcc/article/12/7/835/4955734 by guest on 10 September 2020 IBD, with a commensurate decrease in Firmicutes.33,34 Further down T-cells towards Th1 responses.47 It would be interesting to explore the taxa, at family level the fecal microbiota of the mice lacking the potential contribution of T-cell responses to shaping the micro- miR-21 display an increased proportion of Bifidobacteriaceae and biota in this system. These are a few of the possible mechanisms by Peptostreptococcaceae relative to the WT microbiota, and at genus which miR-21 may regulate the shape of the intestinal microbiota. It level it displays an increase in Bifidobacterium and Odoribacter, all may be that one or all of them are involved in this system, and cer- of which correlate with a healthy microbiota according to various tainly it is likely that the altered barrier permeability demonstrated studies.35,36 We examined the fecal microbiota, because this is rou- to be present in the miR-21–/– mouse by other groups is a factor. It tinely performed in the field, and also because the fecal microbiota is also possible that miR-21 may directly influence bacterial gene was implicated in the co-housing and GF colonization studies that expression following secretion into the lumen in exosomes, as has we carried out. However, it should be noted that the fecal micro- recently been demonstrated.11 Clearly further studies are required in biota might differ from that at individual mucosal surfaces through- order to elucidate the pathways by which miR-21 may be exerting its out the gastrointestinal tract. This has been shown to be important influence on the microbiota. in studies using microbial signatures as a biomarker for intestinal Another interesting aspect of the findings presented here are the disease; thus, further investigation may be required in order to eluci- implications of microbial alteration in the divergent phenotype dis- date whether there are important differences in this model.37 Overall, played by miR-21–/– mice when subjected to the T-cell–dependent these data confirmed that there was an altered microbiota in the 2,4,6-trinitrobenzene sulfonic acid [TNBS] colitis and T-cell trans- miR-21–/– mouse, with some of these alterations being in accordance fer colitis models. It has been previously reported that miR-21 dele- with previous studies indicating protective associations in relation to tion exacerbates colitis in these models through aberrant skewing of IBD and mouse models of colitis. T-cell responses. As stated previously, T-cells help shape the micro- An interesting aspect of this work worth considering is the biota, and the microbiota helps shape T-cell responses in inflam- potential alteration of other mediators present in the feces of miR- matory disease.43 Accordingly, it would be interesting to explore 21–/– mice, which might impact the disease phenotype. These could the microbial contribution to disease onset and progression in this include cytokines, antibodies, secreted epithelial factors or indeed model, and whether or not microbial manipulation [e.g. antibiotic other miRNAs, all of which have been demonstrated to be present in depletion] might affect these phenotypes.47 mouse feces and to have a transferable impact on disease outcomes.11 There is a huge body of literature on miR-21’s role in many forms Secretory IgA [sIgA] has been shown to be an important mediator of cancer. miR-21 is upregulated in nearly all solid tumors and is a of microbial composition, a factor in microbial transferability, and a bone fide oncomiR, influencing many cancer-related processes such marker for susceptibility in IBD models.38,39 We measured sIgA levels as cell division, apoptosis and metastasis.21,48 In the context of IBD, in the feces of WT and miR-21–/– mice, and while there appeared miR-21 is upregulated in colitis-associated cancer [CAC] and has also to be reduced levels of sIgA in the feces of the miR-21–/– mice, this been shown to have a negative role in the azoxymethane [AOM]/DSS difference was not statistically significant Supplementary[ figure S2]. model of CAC, with miR-21–/– mice showing reduced susceptibility We were unable to detect differences in fecal cytokines [data not to the disease.7 Our results are interesting in this regard, because shown]. We also measured the inflammatory markers MPO activ- disruption to the microbiota has been reported to be involved in car- ity and IL-1β expression in all mice following DSS-induced colitis, cinogenesis through various mechanisms, including immune–micro- but found little consistent evidence that these mechanisms may be biota crosstalk.49 This is particularly relevant in the case of CAC, influencing our observed phenotype Supplementary[ figure S3]. This because the breakdown of microbial tolerance and homeostasis in is somewhat in keeping with the counterintuitive nature of miR- the gut leads to inflammation and subsequent tumorigenesis. It is 21’s established role as an anti-inflammatory molecule in immune therefore possible that miR-21’s impact on the intestinal microbi- signaling.21 ome may contribute to disease pathogenesis alongside its direct role The mechanisms by which miR-21 may be influencing microbial in tumor formation and persistence. In our model, we have seen composition have yet to be determined, but given that miR-21 has that miR-21 deletion gives rise to a microbiota that correlates with been demonstrated to be an important regulator of immune and epi- healthy individuals and protects from disease initiation, but does not thelial barrier function, it is likely that modulation of these import- remedy established disease [Supplemental figure S1]. Fecal microbial ant mediators of gut homeostasis is involved. For instance, the transplantation [FMT] therapy for IBD is in its infancy, with several enhanced barrier integrity displayed in miR-21–/– mice demonstrated studies reporting mixed efficacy for UC patients.50 A recent large in previous studies via increase in the expression of the miR-21 tar- study has shown efficacy using a multi-donor approach to maximize get RhoB may play a role in shaping the microbiota by creating a bacterial diversity, without identifying any particularly beneficial MicroRNA-21 Influences the Gut Microbiota and Colitis 847 microbes.51 Future approaches for FMT and other microbial thera- References peutics may rely on a more detailed knowledge of which microbes 1. Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflam- are responsible for beneficial effects. Our findings are interesting in matory bowel disease. Nature 2011;474:298–306. this context, because the mix of microbes and other factors present 2. Bartel DP. 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