Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: J Immunol. 2011 Oct 31;187(11):5834–5841. doi: 10.4049/jimmunol.1100922

Selective Upregulation of microRNA Expression in Peripheral Blood Leukocytes in IL-10−/− Mice Precedes Expression in the Colon1

Jeremy S Schaefer *, Dina Montufar-Solis *, Nadarajah Vigneswaran *, John R Klein *
PMCID: PMC3221883  NIHMSID: NIHMS329364  PMID: 22043014

Abstract

IL-10−/− mice, an animal model of Th1-mediated inflammatory bowel disease, were screened for the expression of 600 microRNAs (miRNAs) using colonic tissues and peripheral blood leukocytes (PBLs) from animals having either mild inflammation or severe intestinal inflammation. The development of colonic inflammation in IL-10−/− mice was accompanied by upregulation in the expression of ten miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-223, miR-326, miR-142-3p, miR-142-5p, miR-146a, and miR-155). Notably, the expression of all of these miRNAs plus miR-375 was elevated in PBLs of IL-10−/− mice at a time when colonic inflammation was minimal, suggesting that changes in specific miRNAs in circulating leukocytes may be harbingers of ensuing colonic pathology. In vitro exposure of colonic intraepithelial lymphocytes to IL-10 resulted in down-regulation of miR-19a, miR-21, miR-31, miR-101, miR-223, and miR-155. Interestingly, unlike IL-10−/− mice, changes in miRNAs in PBL of dextran sulfate sodium-treated mice were minimal, but were selectively elevated in the colon after pathology was severe. We further show that miR-223 is a negative regulator of the Roquin ubiquitin ligase, that Roquin curtails IL-17A synthesis, and that the 3′ UTR of Roquin is a target for miR-223, thus defining a molecular pathway by which IL-10 modulates IL-17-mediated inflammation. To identify additional miRNAs that may be involved in the regulation of Roquin, transcriptome analysis was done using cDNAs from HeLa cells transfected with 90 miRNA mimics. Twenty-six miRNAs were identified as potential negative regulators of Roquin, thus demonstrating functional complexity in gene expression regulation by miRNAs.

Introduction

Crohn’s disease (CD) and ulcerative colitis (UC) are prominent members of a suite of inflammatory conditions of the small and large intestines grouped under the moniker of inflammatory bowel disease (IBD). Whereas UC is limited to the colon and rectum and typically affects only the mucosa, CD can affect any portion of the gastrointestinal tract and usually involves the entire bowel wall. The precise cause of CD is not fully understood; however, it is known that inappropriate immune responses within the intestine, in particular IL-17A and other pro-inflammatory responses, are a hallmark of disease (1). A number of mouse models are available that mimic various aspects of IBD. The interleukin-10 knockout (IL-10−/−) mouse animal of Th1-mediated intestinal inflammation has been particularly useful in that regard (2, 3).

MicroRNAs (miRNAs) are short non-coding RNA species of approximately 19-24 nucleotides derived from primary mRNA transcripts of intergenic or intronic sources (4). Processing and cleavage of mRNA transcripts by Drosha, DGCR8, and Dicer yields a mature miRNA that incorporates into an active RNA-induced silencing complex (RISC) (5). Once incorporated into RISC, miRNAs regulate gene expression via two distinct mechanisms based on complementarity between the miRNA and its target, the 3′ untranslated region (UTR) of mRNA transcripts. In the first, complete complementarity between the miRNA and the mRNA results in the target mRNA cleavage and degradation by Argonaute (5). In the second, imperfect or mismatch binding of the 3′-UTR of the target mRNA results in post-translational repression and mRNA destabilization and degradation resulting from deadenylation and decapping of the target mRNA (6). It is predicted that more than 50% of the genome may be actively regulated by miRNAs (7). However, aberrant expression of miRNAs has been linked to a growing number of diseases, including cancers (chronic lymphocytic leukemias, gliomas, colorectal cancer, prostate cancer, and uveal melanoma) in which the miRNAs act as tumor suppressors or oncogenes (8-10), and autoimmune-related diseases such as rheumatoid arthritis (11) and systemic lupus erythematosus (12). A role for miRNAs in UC and CD is now also becoming apparent (13, 14).

The rationale for this study was two-fold. First, we were interested in determining if miRNA expression patterns in colonic tissues in IL-10−/− mice differ depending upon the degree of colonic pathology. Second, we wished to determine if changes in miRNAs that occur in colonic tissues during inflammation are reflective of miRNA changes in PBL. Here, we demonstrate that changes in specific miRNA expression patterns in circulating leukocytes occur prior to their expression in the colon, thus providing a potentially important diagnostic approach for predicting the development of colonic inflammation in IBD.

Materials and Methods

Mice, cell/tissue isolation, dextran sulfate sodium (DSS) treatment, and intestinal pathology scoring

Breeding stocks of homozygous IL-10−/− mice [C.129P2(B6)-Il10tm1Cg/J] on a BALB/cJ background were purchased from The Jackson Laboratories (Bar Harbor, ME). Control BALB/c mice were purchased from Harlan Laboratories (Indianapolis, IN). Mice were used in accord with University of Texas Health Science Center institutional animal welfare guidelines.

Male and female IL-10−/− mice 10-45 weeks of age were used. Whole blood was collected from the heart of anesthetized mice using EDTA-treated needles and transferred to tubes containing 50 μl of 0.5M EDTA. Blood was layered onto Histopaque-1077 (Sigma, St. Louis, MO) and centrifuged at 400 x g for 30 minutes. Cells at the blood-Histopaque interface were collected, washed with PBS, and snap-frozen at −80°C until RNA isolation. Colonic tissues were stored in RNAlater (Ambion; Austin, TX).

Adult 7 wk old female C57BL/6 mice were given 3% DSS (MW 36,000 – 50,000; MP Biomedicals, Solon, OH) in drinking water for 0, 1, 2, or 7 days. At the designated time, animals were euthanized, PBL were collected from blood, and colonic tissues were taken for histopathological analysis and RNA extraction for miRNA analyses.

Representative H&E stained tissue sections (3 μM) from mid-portions of the proximal and distal colons of IL-10−/− mice and control BALB/c mice were used for histopathologic evaluation. Histopathologic scoring was performed in a blinded fashion by an experienced board-certified pathologist. For IL-10−/− mice, the degree of inflammation and associated crypts architectural distortion were scored microscopically on cross-sections of the colon using a 5-tier scoring system established based on the published criteria for grading of IBD intestinal pathology (2). Score 0: no signs of inflammation or distortion of crypts architecture; Score 1: very low level of mononuclear leukocytes (MLs) in the lamina propria; Score 2: low level of MLs infiltration in the lamina propria; Score 3: moderate level of MLs infiltrate in the lamina propria with occasional crypt distortion; Score 4: high levels of MLs infiltrate within the lamina propria with crypt distortion, high vascular density and thickening of the colon wall; and Score 5: transmural MLs infiltration, widespread crypt distortion/abscess with loss of goblet cells, high vascular density and thickening of the colon wall.

For DSS-treated mice, histological scoring of mucosal injury and the degree of inflammation in the colon was performed as previously reported (2) with some modifications taking into account the histopathologic differences between colitis in DSS-treated mice and IL-10−/− mice. H&E-stained sections were scored for intraepithelial edema, inflammation, erosions, ulceration and abscesses in the proximal, transverse, and distal colon. Mucosal injury in DSS-induced colitis is more severe and occurs in the early stages than IL-10−/− mice. Hence, the severity of inflammation and mucosal injury were each scored independently from 0 to 3; the total pathologic grade of a section was obtained by summing the two scores. Severity of mucosal injury and the degree of inflammation increased steadily in a time-dependent manner starting at day 1 and reaching peak at 7th day, the last time-point examined in these experiments.

Cell staining, stimulation, transfection, and ELISA

Colonic intraepithelial lymphocytes (cIELs) used for intracellular expression of IL-17 and IFNγ were isolated and stained as previously described (2). For in vitro experiments of cIELs, 5 × 106 cells/ml were cultured for 24 hrs in a 37°C incubator with 5% CO2 in RPMI 1640 containing 10% FBS, penicillin/streptomycin and L-glutamine (2). In some experiments, IL-10−/− cIEL cells were cultured with 50 ng/ml recombinant mouse IL-10 (Biolegend, San Diego, CA) or with PBS for control stimulation. Cells were assayed for IL-17A using an ELISA Ready-SET-Go! Kit (eBioscience, San Diego, CA), were assayed by qRT-PCR for Roquin or IL-17A gene expression, or were used to assess the effects of IL-10 on miRNA expression.

EL4 cells were transfected using siPORT NeoFX (Applied Biosystems; Austin, TX) or Oligofectamine (Invitrogen, Carlsbad, CA) with 240 nM of Roquin-specific siRNA or control siRNA (Santa Cruz Biotechnology; Santa Cruz, CA), or were mock transfected. 48 hours post-transfection, RNA was isolated and assayed for Roquin and IL-17A gene expression. For determination of the effects of miR-223 on Roquin expression, cIELs were transfected with 30 nM of anti-miR-223, miR-223 mimic, or matching Cy-3-labeled negative controls (Applied Biosystems).

miRNA profiling, transcriptome analysis, and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated using the miRNeasy Minikit (Qiagen; Valencia, CA) according to the manufacturer’s instructions. cDNA was synthesized using either the High-Capacity cDNA Reverse Transcription Kit or the Taqman miRNA Reverse Transcription Kit (Applied Biosystems).

miRNA profiling of colonic tissue RNAs from normal BALB/c mice, IL-10−/− mice with pathology score = 1.0, and IL-10−/− mice with pathology score ≥3.0 were done by Exiqon (Woburn, MA) using the miRCURY™ LNA array version 11.0, with dChip software, that contained capture probes targeting all miRNAs for human, mouse, or rat registered in the miRBASE version 14.0 at the Sanger Institute. Data sets have been deposited in at NCBI Gene Expression Omnibus with accession number GSE 31706 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=vjytpikguqckytg&acc=GSE31706).

The SureFIND Immunopathology miRNA Transcriptome PCR Array (Qiagen) was used according to the manufacturer’s instructions. Briefly, a multiplex qPCR assay was set up using the TaqMan Universal Master Mix II, No UNG reagent and TaqMan gene expression assays for human Roquin (VIC-labeled probe) and GAPDH (FAM-labeled probe) (Applied Biosystems) to screen HeLa cell cDNAs transfected with a panel of 90 miRNA mimics. Samples were analyzed using the StepOnePlus real-time thermal cycler and the Excel-based data analysis software provided with the SureFIND Immunopathology miRNA Transcriptome PCR Array. Roquin gene expression was compared after normalization of samples to GAPDH.

To measure Roquin and IL-17A transcript levels, the Power SYBR Green PCR Master Mix (Applied Biosystems) was used according to the manufacturer’s instructions. miRNA expression was quantified using miRNA-specific primers and probes (TaqMan microRNA assays; Applied Biosystems) with the TaqMan Universal Master Mix II, No UNG (Applied Biosystems). Samples were analyzed using the StepOnePlus real-time thermal cycler (Applied Biosystems) and software. Relative gene expression was normalized to either GAPDH or U6 snRNA. Roquin, IL-17A, and GAPDH gene specific primers were designed and purchased from Integrated DNA Technologies (Coralville, IA). Primers used were:

mouse Roquin forward: 5′-GGCTGCTCGATCTTTAGGTG-3′

mouse Roquin reverse 5′-TGTTCTCTCCTCAGAGCTTCG-3′

mouse IL-17a forward: 5′-CTCCAGAAGGCCCTCAGACTAC-3′

mouse IL-17a reverse: 5′-GGGTCTTCATTGCGGTGG-3′

mouse GAPDH forward: 5′-AGAACATCATCCCTGCATCC-3′

mouse GAPDH reverse: 5′-AGCCGTATTCATTGTCATACC-3′

human Roquin forward: 5′-ACCAACCTTGCCTCCTACCT-3′

human Roquin reverse: 5′-TAATCGCTGGTCCCTCATTC-3′

human GAPDH forward: 5′-TGCACCACCAACTGCTTAGC-3′

human GAPDH reverse: 5′-GGCATGGACTGTGGTCATGAG-3′.

Luciferase assay

The 599 base pair section (P6496-7095) of the Rc3h1 3′ untranslated region (UTR) incorporating the predicted miR-223 site at position 6638 was PCR-cloned into the HindIII site of the pMIR-REPORT Luciferase plasmid (Applied Biosystems, Carlsbad, CA) to yield the Rc3h1 P6496 3′UTR pMIR plasmid. 293T cells were plated in 24-well plates and co-transfected with 400 ng of Rc3h1 P6496 3′UTR pMIR, 400 ng of the pMIR-REPORT β-galactosidase vector, and 30 nM of either the miR-223 or Cy3-labeled Negative Control Pre-miR miRNA Precursors (Applied Biosystems) using 5 μl/well of Endofectin Lenti (Genecopoeia, Rockville, MD). Forty-eight hours later, the cells were lysed using a 5X Cell Culture Lysis Reagent (Promega, Madison, WI) and the luciferase activities were measured on a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA) using an Enhanced Luciferase Assay kit (BD Biosciences, San Diego, CA). Luciferase activities were normalized to β-galactosidase activity. Luciferase activity with the Rc3h1 P6496 3′UTR pMIR vector alone was regarded as 1.0.

Statistical analysis

The statistical significance of data was determined using an unpaired two-sided Student’s t-test. Data are presented as mean values ± SEM.

Results

Regulated expression of miRNAs in the colon and PBL of IL-10−/− mice

Previous studies have demonstrated extensive cytokine disruption in the colon of IL-10−/− mice (2, 15). This led us to hypothesize that miRNAs may also be dysregulated in a disease-specific pattern. To address this, we examined global expression patterns of miRNAs in colon samples from IL-10−/− and parental BALB/c mice by miRNA microarray analysis. To compare miRNA expression in IL-10−/− mice with mild intestinal pathology to mice with severe intestinal pathology, PBLs were collected and frozen and colonic tissues were stored in RNAlater until scoring for pathology had been completed for colonic tissues. Analysis of miRNA expression was then done using tissues from normal BALB/c mice (Fig. 1A), IL-10−/− mice that had intestinal pathology scores = 1.0 (Fig. 1B), and IL-10−/− mice that had intestinal pathology scores ≥3.0 (Fig. 1C). Each group consisted of RNA pooled from three mice. Out of over 600 miRNAs analyzed, 43 miRNAs were differentially expressed at least 2-fold between the comparison groups as represented in the heat map (Fig. 1D). This identified several miRNAs that were elevated in IL-10−/− mice having high colonic pathology compared to IL-10−/− mice with minimal pathology and to normal BALB/c mice.

FIGURE 1.

FIGURE 1

Open in a new tab

Colonic tissues from (A) normal BALB/c mice without pathology, (B) grade 1.0 pathology of colon tissue from IL-10−/− mouse, and (C) grade 4 pathology of colon tissue from IL-10−/− mouse. (D) Heat Map and Unsupervised Hierarchical Clustering of miRNAs in IL-10−/− and BALB/c mouse colon samples indicate dysregulation of miRNA expression. The miRCURY LNA microarray miRNA profiling service was used to examine miRNA expression in pooled total RNA samples (3 mice each) from colonic tissue sections from normal BALB/c mice, IL-10−/− mice with intestinal pathology score = 1 (IL-10ko 1.0), and IL-10−/− mice with intestinal pathology scores ≥3 (IL10ko ≥3.0). Red color represents an expression level above mean, blue color represents expression lower than the mean. A Delta LogMedian ratio of +/− 1.0 is equal to a fold change of +/− 2.0.

qRT-PCR was done to validate expression levels of eleven miRNAs that were specifically elevated or lowered in the heat map (miR-19a, miR-21, miR-31, miR-142-3p, miR-142-5p, miR-155, miR-223, and miR-375), or have been shown to be linked to chronic inflammatory conditions (miR-101, miR-146a, miR-326) (16-18). In colonic tissues of IL-10−/− mice with pathology score ≥3.0, miR-19a, mir-21, miR-31, miR-101, miR-223, miR-142-3p, miR-142-5p, miR-146a, and miR-155 were significantly elevated compared to colonic tissues from normal mice. miR-326 expression was significantly elevated in mice with pathology score = 1.0, but not in mice with scores ≥3.0. miR-375 expression was significantly lower in mice with pathology scores ≥3.0 compared to normal mice (Fig. 2A).

FIGURE 2.

FIGURE 2

Open in a new tab

Total RNA from IL-10−/− and BALB/c colon were used for TaqMan qRT-PCR analysis of eleven miRNAs in (A) colonic tissues and (B) PBLs from normal mice (score 0) and IL-10−/− mice with low pathology (score 1) or high pathology (score ≥3.0). Expression values were calculated by normalizing to U6 snRNA levels in a sample, and recorded as values relative to the expression of normal mice, which was arbitrarily designated as 1.0. Note that miRNAs were elevated in PBLs of IL-10−/− mice with mild intestinal pathology, but were elevated in IL-10−/− mice with severe intestinal pathology. Determination of statistical significance was calculated using Student’s t-test relative to BALB/c control values.

To determine if changes in miRNA expression in colonic tissues also were present in PBLs, miRNA expression levels for the eleven miRNAs used in Fig. 2A were measured in PBLs from normal mice, IL-10−/− with pathology score = 1.0, and IL-10−/− mice with pathology score ≥3.0. Importantly, miRNAs expression levels were highest in PBLs of IL-10−/− mice with low intestinal pathology (score = 1.0) compared to mice with severe intestinal pathology (score ≥3.0) (Fig. 2B), suggesting that the elevation of specific miRNAs in circulating leukocytes may be an early indicator of developing intestinal pathology.

To test whether exposure to IL-10 would alter miRNA expression, cIELs from IL-10−/− mice were cultured overnight with 50 ng/ml rIL-10 or PBS. miRNA expression was quantified relative to that of PBS control cells. Expression levels for six of eleven miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-155, and miR-223) were significantly suppressed in cIELs from IL-10−/− mice following exposure to IL-10 compared to cIELs cultured with PBS (Fig. 3A). IL-10 exposure had no significant effect on miR-142-3p, miR-142-5p, miR-146, miR-326 and miR-375 expression. To test for the specificity of IL-10, cIELs were cultured with IL-2, which did not alter miRNA expression levels relative to PBS-supplemented cultures (data not shown).

FIGURE 3.

FIGURE 3

Open in a new tab

(A) In vitro treatment of cIELs from IL-10−/− mice with severe pathology overnight with recombinant 50 ng/ml rIL-10 resulted in the suppression of 6 of 11 miRNAs relative to cells cultured with PBS (* ≤ 0.05). (B) TargetScan (Release 5.1) analysis of the mouse Roquin (Rc3h1) 3′UTR revealed a potential target site for mmu-miR-223. Alignment of mmu-miR-223 to the conserved site is shown. (C) Intracellular expression of IL-17 and IFNγ in IL-10−/− cIELs. In vitro IL-10 treatment of cIELs from IL-10−/− mice resulted in significant reduction in (D) IL-17 mRNA expression and (E) IL-17A secretion as determined by ELISA. (F) cIELs from IL-10−/− mice have lower Roquin gene expression. (G) Culture of cIELs from IL-10−/− mice for 24 hours with 50 ng rIL-10 results in an increase in Roquin gene expression. (H-I) Transient transfection of EL4 cells with Roquin-specific siRNA oligonucleotides resulted in suppression of Roquin gene expression and enhanced IL-17 gene expression. Determination of statistical significance was calculated using Student’s t-test.

Roquin modulates IL-17 expression via IL-10 and is negatively regulated by miR-223

We used web-based algorithms (19) to identify potential targets for miRNAs that were elevated in this study. From that, miR-223 was predicted to target Roquin (Fig. 3B). Dysregulation of Roquin expression has been linked to various autoimmune diseases (20), although an association between Roquin and IL-17 expression has not been established. IL-17 production by cIELs is a common feature of IL-10−/− mice (2) (Fig. 3C). The ameliorating effects of IL-10 on IL-17 synthesis can be seen by ex vivo treatment of cIELs with IL-10 (Fig. 3D and E). Of interest, Roquin expression was suppressed in cIELs of IL-10−/− mice (Fig. 3F), and was restored following exposure to IL-10 (Fig. 3G), thus indicating that IL-10 influences Roquin expression. Further documentation of this was evident in experiments in which EL4 cells transfected with Roquin siRNA had significantly lower levels of Roquin gene expression (Fig. 3H), and significantly higher levels of IL-17A gene expression (Fig. 3I).

Transfection of cIELs with anti-miR-223 resulted in an increase in Roquin gene expression and a decrease in IL-17A gene expression; miR-223 suppressed Roquin expression and enhanced IL-17A expression (Fig. 4A), suggesting a role for miR-223 in Roquin regulation. This was confirmed by luciferase reporter assays, which demonstrated that the 3′ UTR of the Roquin gene was a target for miR-223 as seen by the negative regulatory effects (Fig. 4B).

FIGURE 4.

FIGURE 4

Open in a new tab

(A) Transient transfection of cIELs with anti-miR-223 resulted in enhanced Roquin expression and suppressed IL-17A expression, whereas transfection with miR-223 suppressed Roquin expression and enhanced IL-17A expression. (B) Results of luciferase assay using the 3′ UTR of Roquin cloned into the pMIR-REPORT luciferase plasmid and transfected into 239T cells for 48 hr with either 30 nM of miR-223 or pre-miR control. miR-223 suppressed luciferase activity relative to non-transfected and pre-miR control levels.

To identify additional miRNAs that may be involved in the regulation of Roquin, transcriptome analysis was done using cDNAs from HeLa cells transfected with 90 miRNA mimics (Table S1). Twenty-six miRNAs were identified as potential negative regulators of Roquin (Table I). This confirmed the involvement of miR-223, as described above, and it identified twenty-five other functionally-important miRNAs, two of which (miR-146a and miR-155) are associated with IL-17 regulation (11, 21).

Table I.

Negative regulators of Rc3h1

Symbol Fold change
miR-191 −2.02
miR-302b −2.02
miR-105 −2.03
miR-181a −2.04
miR-29c −2.04
miR-142-3p −2.04
miR-20a −2.05
miR-182 −2.08
miR-223 −2.09
miR-200a −2.12
miR-194 −2.13
miR-205 −2.15
miR-302c −2.24
miR-125b −2.27
miR-99b −2.37
miR-451 −2.38
miR-135b −2.39
miR-149 −2.44
miR-155 −2.46
miR-27a −2.70
miR-183 −2.78
miR-184 −2.78
miR-147 −3.14
miR-31 −3.16
miR-185 −3.82
miR-146a −3.95
Open in a new tab

Colonic inflammation in DSS-treated mice is accompanied by selective changes in miRNA expression in the colon but not in PBL

To determine if the patterns of miRNA expression observed in IL-10−/− mice also occurred in other animals models of colonic inflammation, miRNA expression was examined in PBL and colonic tissues of DSS-treated mice at early (days 1 and 2) and late (day 7) times of exposure to DSS. The miRNAs studied were the same as those used for IL-10−/− mice (see Fig. 2). The severity of mucosal injury and the degree of inflammation increased in a time-dependent manner during days 1 through 7 of DSS treatment. Untreated mice had no inflammation (Fig. 5A). At day 1, the colonic mucosa had surface erosion, intraepithelial edema, and increased infiltration of polymorphonuclear and mononuclear leukocytes within the lamina propria (Fig. 5B). By day 2, there was diffuse mucosal ulceration, crypt and submucosal abscess, and diffuse infiltrate of leukocytes extending into the submucosa (Fig. 5C). Maximal colitis resulting in ulceration with extensive submucosal and transmural inflammation, abscess formation, and necrosis occurred at day 7 (Fig. 5D). Similar to other studies of experimental colitis (22), numerous eosinophils were present among the inflammatory cell infiltrate starting at day 2 of DSS exposure. Pathology scores for all mice per group are shown in Fig. 5E.

FIGURE 5.

FIGURE 5

Open in a new tab

Mice were given DSS in the drinking water as described in the Materials and Methods. Representative colonic tissues from (A) day 0, pathology score 0, (B) day 1, pathology score 1 (C) day 2, pathology score 3, and (D) day 7, pathology score 6. (E) Average pathology scores from 3 mice per group. * Statistically-significant difference (p<0.01) compared to days 0 and 1. ▲Statistically-significant difference (p<0.01) compared to day 2.

Using PBL and colonic tissues from DSS-treated animals on days 0, 1, 2, and 7 of DSS treatment, qRT-PCR analysis was done for the same eleven miRNAs studied for IL-10−/− mice. Although some miRNAs were elevated in PBL early during the treatment period (day 1), these were not statistically-significant differences compared to non-treated mice (Fig. 6A). However, five miRNAs (miR-31, miR-223, miR-142-3p, miR-146a, and miR-155) were significantly elevated in the colon of DSS-treated mice at day 7, a time when inflammation was most severe (Fig. 6B). Of these, it was particularly interesting that miR-223 expression was elevated in the colon of both IL-10−/− mice and DSS-treated mice given the relationship of that miRNA to Roquin and IL-17 expression as described above.

FIGURE 6.

FIGURE 6

Open in a new tab

qRT-PCR analysis of the same eleven miRNAs studied for IL-10−/− mice using (A) PBL and (B) colonic tissues of DSS-treated animals on days 0, 1, 2, and 7. Although some miRNAs were elevated in PBL early during the treatment period (day 1), these were not statistically-significant differences. Five miRNAs (miR-31, miR-223, miR-142-3p, miR-146a, and miR-155) were significantly elevated in the colon when inflammation was severe. Expression values were calculated by normalizing to U6 snRNA levels in a sample, and recorded as values relative to the expression of day 0 (non-DSS-treated) mice, which was arbitrarily designated as 1.0.

Discussion

The findings reported here refine our understanding of the molecular pathway by which IL-17 is controlled, and they identify an approach for predicting the development of chronic colonic inflammation based on the temporal appearance of miRNAs in circulating leukocytes. Our data suggest a system in which IL-10 exerts negative regulatory effects on IL-17 and miR-223 expression, and positive effects on Roquin expression. A key and early component of this pathway appears to be miR-223 as seen by its potential to target and regulate Roquin expression, by binding to the 3′ UTR of the Roquin gene. Thus, in the presence of IL-10, miR-223 would be suppressed and Roquin would be maintained at sufficient levels to hold IL-17 in check. In the absence of adequate levels of IL-10, however, high levels of miR-223 would suppress Roquin and increase IL-17A synthesis. Accordingly, there are multiple situations that could independently or collectively lead to chronic IL-17-driven intestinal pathology, including suppression of IL-10 synthesis, over-expression of miR-223, or suppression of Roquin.

Elevated levels of miR-223 also are significant due to its link to myelopoiesis, erythropoiesis and lymphopoiesis (23-26). miR-223, which has been shown to be upregulated in patients with Crohn’s disease (17), is involved in progenitor cell and granulocyte differentiation and function (27). Ectopic expression of miR-223 was shown to cause a 30-40% increase in T cells (25), and to reduce LMO2, an essential protein of erythropoiesis (28). However, the complexity of the functional involvement of miR-223 is witnessed by its ability to negatively regulate miR-142 through LMO2 and CEBP-β, and to attenuate hematopoietic cell proliferation (26). Thus, elevated levels of miR-223 in IL-10−/− mice with severe colonic pathology would promote and sustain the expansion of colonic leukocytes, leading to a chronic inflammatory condition. miR-101 has been linked to the regulation of ICOS expression (29), ICOS+ being a primary source of IL-17 (2, 30). miR-146a, miR-155 and mir-326 have been linked to Th17 differentiation (16, 18, 21). That multiple genes may be regulated by the same miRNA is not surprising and indeed is predictable given the disproportionately few number of miRNAs available for gene regulation considering that as much as 30-90% of human genes are believed to be regulated by miRNAs (19).

It is noteworthy that several other miRNAs were elevated in IL-10−/− mice having severe intestinal pathology. Elevated expression levels of miR-19a, miR-21, and miR-31 are associated with cancer, including gastric and colon cancers (31-36). This may occur by a failure to activate tumor suppressor and anti-apoptotic responses via programmed cell death protein 4, phosphatase and tensin homologue, and/or tropomyosin 1 (37-40). Elevated levels of miR-19a, miR-21, and miR-31 in IL-10−/− mice may have relevance to the increased incidence of colorectal cancer in patients with IBD (41-43) and in IL-10-deficient mice (44-46).

Although our data reveal a temporal pattern of miRNA regulation in IL-10-deficient mice that was first evident in circulating leukocytes, this was not observed in the DSS model of colonic inflammation. Those differences may be reflective of variations in the pathophysiological basis of inflammation in IL-10−/− vs. DSS-treated mice. Development of inflammation in IL-10−/− mice occurs gradually and thus may require the continual seeding of leukocytes from the circulation to the colonic mucosa during the early phase of disease for pathology to be manifest. Colonic pathology induced by DSS treatment occurs rapidly, reaching peak levels within a few days. IL-10−/− mice also differ from DSS-treated mice in that pathology in the former is principally the consequence of cytokine dysregulation resulting in the synthesis of powerful proinflammatory cytokines, whereas inflammation in DSS-treated mice occurs once the integrity of the intestinal mucosa is disrupted. Therefore, changes in miRNA expression in PBL may have value for predicting the underlying basis of inflammation, and may aid in the design of therapeutic protocols in that regard. Finally, it is significant that miR-223, the miRNA that from our study was most definitively associated with IL-17A regulation via Roquin, was dysregulated in the colon of both IL-10−/− and DSS-treated mice, suggesting that miR-223 may serve as a common marker of local intestinal inflammation.

In summary, these findings point to the potential use of monitoring miRNA expression levels in circulating leukocytes as predictive indicators of the development or recurrence of colonic inflammation IBD.

Supplementary Material

1

Abbreviations used in this article

cIELs

colonic intraepithelial lymphocytes

CD

Crohn’s disease

miRNA

microRNA

MLs

mononuclear leukocytes

qRT-PCR

quantitative real-time PCR

RISC

RNA-induced silencing complex

UC

ulcerative colitis

Footnotes

1

This work was supported by National Institutes of Health grant R01 DK035566 (to J.R.K.) and R21 DE019956 (to N.V.), and a Bar Levy Research Award from the Department of Diagnostic Sciences, and an Institution-funded Center for Clinical and Translational Sciences/K12 career development grant (to J.S.S.)

References

  • 1.Fujino S, Andoh A, Bamba S, Ogawa A, Hata K, Araki Y, Bamba T, Fujiyama Y. Increased expression of interleukin 17 in inflammatory bowel disease. Gut. 2003;52:65–70. doi: 10.1136/gut.52.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Schaefer JS, Montufar-Solis D, Vigneswaran N, Klein JR. ICOS promotes IL-17 synthesis in colonic intraepithelial lymphocytes in IL-10−/− mice. J Leukoc Biol. 2010;87:301–308. doi: 10.1189/jlb.0409238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Seiderer J, Elben I, Diegelmann J, Glas J, Stallhofer J, Tillack C, Pfennig S, Jurgens M, Schmechel S, Konrad A, Goke B, Ochsenkuhn T, Muller-Myhsok B, Lohse P, Brand S. Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): upregulated colonic IL-17F expression in active Crohn’s disease and analysis of the IL17F p.His161Arg polymorphism in IBD. Inflamm Bowel Dis. 2008;14:437–445. doi: 10.1002/ibd.20339. [DOI] [PubMed] [Google Scholar]
  • 4.Guarnieri DJ, DiLeone RJ. MicroRNAs: a new class of gene regulators. Ann Med. 2008;40:197–208. doi: 10.1080/07853890701771823. [DOI] [PubMed] [Google Scholar]
  • 5.Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol. 2009;11:228–234. doi: 10.1038/ncb0309-228. [DOI] [PubMed] [Google Scholar]
  • 6.Lin SL, Miller JD, Ying SY. Intronic microRNA (miRNA) J Biomed Biotechnol. 2006;2006:1–13. doi: 10.1155/JBB/2006/26818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Lodish HF, Zhou B, Liu G, Chen CZ. Micromanagement of the immune system by microRNAs. Nat Rev Immunol. 2008;8:120–130. doi: 10.1038/nri2252. [DOI] [PubMed] [Google Scholar]
  • 8.Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, Poon TC, Ng SS, Sung JJ. Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening. Gut. 2009;58:1375–1381. doi: 10.1136/gut.2008.167817. [DOI] [PubMed] [Google Scholar]
  • 9.Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer. 2006;6:857–866. doi: 10.1038/nrc1997. [DOI] [PubMed] [Google Scholar]
  • 10.Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O’Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A. 2008;105:10513–10518. doi: 10.1073/pnas.0804549105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Pauley KM, Satoh M, Chan AL, Bubb MR, Reeves WH, Chan EK. Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients. Arthritis Res Ther. 2008;10:R101. doi: 10.1186/ar2493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Dai R, Phillips RA, Zhang Y, Khan D, Crasta O, Ahmed SA. Suppression of LPS-induced Interferon-gamma and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism of immune modulation. Blood. 2008;112:4591–4597. doi: 10.1182/blood-2008-04-152488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wu F, Zikusoka M, Trindade A, Dassopoulos T, Harris ML, Bayless TM, Brant SR, Chakravarti S, Kwon JH. MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha. Gastroenterology. 2008;135:1624–1635. doi: 10.1053/j.gastro.2008.07.068. [DOI] [PubMed] [Google Scholar]
  • 14.Wu F, Zhu S, Ding Y, Beck WT, Mo YY. MicroRNA-mediated regulation of Ubc9 expression in cancer cells. Clin Cancer Res. 2009;15:1550–1557. doi: 10.1158/1078-0432.CCR-08-0820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Montufar-Solis D, Schaefer J, Hicks MJ, Klein JR. Massive but selective cytokine dysregulation in the colon of IL-10−/− mice revealed by multiplex analysis. Int Immunol. 2008;20:141–154. doi: 10.1093/intimm/dxm126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Niimoto T, Nakasa T, Ishikawa M, Okuhara A, Izumi B, Deie M, Suzuki O, Adachi N, Ochi M. MicroRNA-146a expresses in interleukin-17 producing T cells in rheumatoid arthritis patients. BMC Musculoskelet Disord. 2010;11:209–219. doi: 10.1186/1471-2474-11-209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Wu F, Zhang S, Dassopoulos T, Harris ML, Bayless TM, Meltzer SJ, Brant SR, Kwon JH. Identification of microRNAs associated with ileal and colonic Crohn’s disease. Inflamm Bowel Dis. 2010;16:1729–1738. doi: 10.1002/ibd.21267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Du C, Liu C, Kang J, Zhao G, Ye Z, Huang S, Li Z, Wu Z, Pei G. MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis. Nat Immunol. 2009;10:1252–1259. doi: 10.1038/ni.1798. [DOI] [PubMed] [Google Scholar]
  • 19.Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120:15–20. doi: 10.1016/j.cell.2004.12.035. [DOI] [PubMed] [Google Scholar]
  • 20.Vinuesa CG, Cook MC, Angelucci C, Athanasopoulos V, Rui L, Hill KM, Yu D, Domaschenz H, Whittle B, Lambe T, Roberts IS, Copley RR, Bell JI, Cornall RJ, Goodnow CC. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature. 2005;435:452–458. doi: 10.1038/nature03555. [DOI] [PubMed] [Google Scholar]
  • 21.O’Connell RM, Kahn D, Gibson WS, Round JL, Scholz RL, Chaudhuri AA, Kahn ME, Rao DS, Baltimore D. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity. 2010;33:607–619. doi: 10.1016/j.immuni.2010.09.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Waddell A, Ahrens R, Steinbrecher K, Donovan B, Rothenberg ME, Munitz A, Hogan SP. Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11. J Immunol. 186:5993–6003. doi: 10.4049/jimmunol.1003844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Fazi F, Racanicchi S, Zardo G, Starnes LM, Mancini M, Travaglini L, Diverio D, Ammatuna E, Cimino G, Lo-Coco F, Grignani F, Nervi C. Epigenetic silencing of the myelopoiesis regulator microRNA-223 by the AML1/ETO oncoprotein. Cancer Cell. 2007;12:457–466. doi: 10.1016/j.ccr.2007.09.020. [DOI] [PubMed] [Google Scholar]
  • 24.Fukao T, Fukuda Y, Kiga K, Sharif J, Hino K, Enomoto Y, Kawamura A, Nakamura K, Takeuchi T, Tanabe M. An evolutionarily conserved mechanism for microRNA-223 expression revealed by microRNA gene profiling. Cell. 2007;129:617–631. doi: 10.1016/j.cell.2007.02.048. [DOI] [PubMed] [Google Scholar]
  • 25.Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineage differentiation. Science. 2004;303:83–86. doi: 10.1126/science.1091903. [DOI] [PubMed] [Google Scholar]
  • 26.Sun W, Shen W, Yang S, Hu F, Li H, Zhu TH. miR-223 and miR-142 attenuate hematopoietic cell proliferation, and miR-223 positively regulates miR-142 through LMO2 isoforms and CEBP-beta. Cell Res. 2010;20:1158–1169. doi: 10.1038/cr.2010.134. [DOI] [PubMed] [Google Scholar]
  • 27.Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O, Brummelkamp TR, Fleming MD, Camargo FD. Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature. 2008;451:1125–1129. doi: 10.1038/nature06607. [DOI] [PubMed] [Google Scholar]
  • 28.Felli N, Pedini F, Romania P, Biffoni M, Morsilli O, Castelli G, Santoro S, Chicarella S, Sorrentino A, Peschle C, Marziali G. MicroRNA 223-dependent expression of LMO2 regulates normal erythropoiesis. Haematologica. 2009;94:479–486. doi: 10.3324/haematol.2008.002345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Yu D, Tan AH, Hu X, Athanasopoulos V, Simpson N, Silva DG, Hutloff A, Giles KM, Leedman PJ, Lam KP, Goodnow CC, Vinuesa CG. Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA. Nature. 2007;450:299–303. doi: 10.1038/nature06253. [DOI] [PubMed] [Google Scholar]
  • 30.Nurieva RI, Treuting P, Duong J, Flavell RA, Dong C. Inducible costimulator is essential for collagen-induced arthritis. J Clin Invest. 2003;111:701–706. doi: 10.1172/JCI17321. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 31.Cottonham CL, Kaneko S, Xu L. miR-21 and miR-31 converge on TIAM1 to regulate migration and invasion of colon carcinoma cells. J Biol Chem. 2010;285:35293–35302. doi: 10.1074/jbc.M110.160069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Guo J, Miao Y, Xiao B, Huan R, Jiang Z, Meng D, Wang Y. Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues. J Gastroenterol Hepatol. 2009;24:652–657. doi: 10.1111/j.1440-1746.2008.05666.x. [DOI] [PubMed] [Google Scholar]
  • 33.Wang YX, Zhang XY, Zhang BF, Yang CQ, Chen XM, Gao HJ. Initial study of microRNA expression profiles of colonic cancer without lymph node metastasis. J Dig Dis. 2010;11:50–54. doi: 10.1111/j.1751-2980.2009.00413.x. [DOI] [PubMed] [Google Scholar]
  • 34.Kulda V, Pesta M, Topolcan O, Liska V, Treska V, Sutnar A, Rupert K, Ludvikova M, Babuska V, Holubec L, Jr., Cerny R. Relevance of miR-21 and miR-143 expression in tissue samples of colorectal carcinoma and its liver metastases. Cancer Genet Cytogenet. 2010;200:154–160. doi: 10.1016/j.cancergencyto.2010.04.015. [DOI] [PubMed] [Google Scholar]
  • 35.Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor growth. Oncogene. 2007;26:2799–2803. doi: 10.1038/sj.onc.1210083. [DOI] [PubMed] [Google Scholar]
  • 36.Schetter AJ, Nguyen GH, Bowman ED, Mathe EA, Yuen ST, Hawkes JE, Croce CM, Leung SY, Harris CC. Association of inflammation-related and microRNA gene expression with cancer-specific mortality of colon adenocarcinoma. Clin Cancer Res. 2009;15:5878–5887. doi: 10.1158/1078-0432.CCR-09-0627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Zhu S, Si ML, Wu H, Mo YY. MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1) J Biol Chem. 2007;282:14328–14336. doi: 10.1074/jbc.M611393200. [DOI] [PubMed] [Google Scholar]
  • 38.Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, Lund AH. Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem. 2008;283:1026–1033. doi: 10.1074/jbc.M707224200. [DOI] [PubMed] [Google Scholar]
  • 39.Seike M, Goto A, Okano T, Bowman ED, Schetter AJ, Horikawa I, Mathe EA, Jen J, Yang P, Sugimura H, Gemma A, Kudoh S, Croce CM, Harris CC. MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers. Proc Natl Acad Sci U S A. 2009;106:12085–12090. doi: 10.1073/pnas.0905234106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology. 2007;133:647–658. doi: 10.1053/j.gastro.2007.05.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Bernstein CN, Blanchard JF, Kliewer E, Wajda A. Cancer risk in patients with inflammatory bowel disease: a population-based study. Cancer. 2001;91:854–862. doi: 10.1002/1097-0142(20010215)91:4<854::aid-cncr1073>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  • 42.Xie J, Itzkowitz SH. Cancer in inflammatory bowel disease. World J Gastroenterol. 2008;14:378–389. doi: 10.3748/wjg.14.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Eaden JA, Abrams KR, Mayberry JF. The risk of colorectal cancer in ulcerative colitis: a meta-analysis. Gut. 2001;48:526–535. doi: 10.1136/gut.48.4.526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Hachimine D, Uchida K, Asada M, Nishio A, Kawamata S, Sekimoto G, Murata M, Yamagata H, Yoshida K, Mori S, Tahashi Y, Matsuzaki K, Okazaki K. Involvement of Smad3 phosphoisoform-mediated signaling in the development of colonic cancer in IL-10-deficient mice. Int J Oncol. 2008;32:1221–1226. doi: 10.3892/ijo_32_6_1221. [DOI] [PubMed] [Google Scholar]
  • 45.Sturlan S, Oberhuber G, Beinhauer BG, Tichy B, Kappel S, Wang J, Rogy MA. Interleukin-10-deficient mice and inflammatory bowel disease associated cancer development. Carcinogenesis. 2001;22:665–671. doi: 10.1093/carcin/22.4.665. [DOI] [PubMed] [Google Scholar]
  • 46.Poutahidis T, Haigis KM, Rao VP, Nambiar PR, Taylor CL, Ge Z, Watanabe K, Davidson A, Horwitz BH, Fox JG, Erdman SE. Rapid reversal of interleukin-6-dependent epithelial invasion in a mouse model of microbially induced colon carcinoma. Carcinogenesis. 2007;28:2614–2623. doi: 10.1093/carcin/bgm180. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS329364-supplement-1.doc (57.5KB, doc)

RESOURCES