Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Applied and Environmental Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Food Microbiology

Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes

Susanne Kreuzer-Redmer, Jennifer C. Bekurtz, Danny Arends, Ralf Bortfeldt, Barbara Kutz-Lohroff, Soroush Sharbati, Ralf Einspanier, Gudrun A. Brockmann
J. Björkroth, Editor
Susanne Kreuzer-Redmer
aFaculty of Life Sciences, Thaer Institute, Breeding Biology and Molecular Genetics, Humboldt Universität zu Berlin, Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jennifer C. Bekurtz
aFaculty of Life Sciences, Thaer Institute, Breeding Biology and Molecular Genetics, Humboldt Universität zu Berlin, Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Danny Arends
aFaculty of Life Sciences, Thaer Institute, Breeding Biology and Molecular Genetics, Humboldt Universität zu Berlin, Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ralf Bortfeldt
aFaculty of Life Sciences, Thaer Institute, Breeding Biology and Molecular Genetics, Humboldt Universität zu Berlin, Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Barbara Kutz-Lohroff
bDepartment of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Soroush Sharbati
bDepartment of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ralf Einspanier
bDepartment of Veterinary Medicine, Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gudrun A. Brockmann
aFaculty of Life Sciences, Thaer Institute, Breeding Biology and Molecular Genetics, Humboldt Universität zu Berlin, Berlin, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
J. Björkroth
University of Helsinki
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AEM.04044-15
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Probiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism at the molecular level could be an influence on microRNAs (miRNAs) and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissues from Enterococcus faecium NCIMB 10415-fed piglets versus untreated controls by using next-generation sequencing. We identified miR-423-5p as being greatly affected by the treatment group (2.32-fold; P = 0.014). Validation by reverse transcription-quantitative PCR (RT-qPCR) confirmed a significant upregulation of miR-423-5p (2.11-fold; P = 0.03) and, additionally, downregulation of the important immune-relevant immunoglobulin lambda light C region (IGLC) (0.61-fold; P = 0.03) and immunoglobulin kappa constant (IGKC) (0.69-fold; P = 0.04) target genes. Expression analysis of miR-423-5p and IGLC at different age points shows a clear anticorrelated relationship. Luciferase reporter assays with a HeLa cell line verified IGLC as a target of miR-423-5p. The results provided evidence for an effect of feeding of E. faecium on the expression of miR-423-5p and on the regulation of the IGLC gene through miR-423-5p. This might be a possible mode of action of E. faecium on immune cell regulation in the small intestine.

INTRODUCTION

Weaning of piglets from sows is a critical time point in livestock farming. Symptoms such as diarrhea, weight loss, and feed inefficacy occur as a major result of weaning. These symptoms are summarized as postweaning diarrhea symptoms (PWDS) (1). Until 2006, antibiotics were extensively used as a feed additive and were widely used in the European Union to reduce PWDSs. As a side effect, resistance of microbiota against antibiotics has increased. Since the European Union banned the use of antibiotics as a feed additive (2), pig breeders need alternatives to decrease the effects of PWDS. Feed additives such as prebiotics, probiotics, and trace elements are promising alternatives to overcome PWDS in piglets and livestock production in general.

Probiotics are defined as living organisms that benefit the host when adequate amounts are applied as dietary feed additives (3). Lactobacilli, bifidobacilli, and enterococci belong to this group (4). Several studies have revealed positive effects of probiotics. Investigations with infants fed Lactobacillus reported a reduced duration of diarrhea (5, 6). Studies in pigs showed that Enterococcus faecium NCIMB 10415 reduced the number of mucosa-adherent Escherichia coli pathotypes (7) and that E. faecium reduces Chlamydia loads (8). Those data suggested a potential beneficial effect of the use of E. faecium as a probiotic to reduce infectious diseases. Studies with sows and piglets, including ours, revealed that E. faecium NCIMB 10415 could modulate the intestinal immune system (9–12).

The molecular mechanisms of how ingestion of E. faecium provides these health benefits remain to be elucidated. One possible mechanism could be the modulation of microRNAs (miRNAs) by the probiotic, thereby changing the immune response. miRNAs are a class of endogenous short noncoding RNA molecules (∼21 nucleotides [nt]) that function in posttranscriptional regulation in eukaryotic and prokaryotic cells (13). During the last 2 decades, miRNA regulation has been studied in physiological processes; development of cancer cells; proliferation, apoptosis, and morphogenesis of cells; and neuronal plasticity (reviewed in reference 14) Nowadays, miRNAs, as important regulators of gene expression, are increasingly the focus of research in regard to some unsolved issues in immunology (15, 16). In humans, several studies showed that miRNAs are involved in immunological processes; for example, human miR-181 affects T-cell development (17). Pathogen-directed differential regulation of miRNAs in experimentally infected bovine mammary epithelial cells was found (18).

So far, little is known about miRNA regulation in pigs, even though pigs have been proven to be a suitable model for immunological studies for humans (19). There is some evidence for miRNAs being posttranscriptional regulators of immune responses in pigs. Piglets infected by Actinobacillus pleuropneumoniae and Salmonella enterica serovar Typhimurium showed dysregulation of miR-155 (20) and miR-29a (21), respectively.

The aim of the present study was to analyze (i) if the probiotic E. faecium could alter miRNA expression and (ii) if such an miRNA would affect immune genes. Therefore, we performed a feeding trial with piglets that were fed a diet with or without E. faecium NCIMB 10415 for 54 days ante- and postweaning.

MATERIALS AND METHODS

Animal experiments.The local state office of occupational health and technical safety, Landesamt für Gesundheit und Soziales Berlin (LaGeSo regulation no. 0347/09), approved the study according to international guidelines (EU directive 86/609/EWG), ensued by German law on animal welfare (§8 TierSchG).

The study design, including animals, housing, and feeding conditions, was described previously (10). Briefly, purebred Landrace sows and their piglets were divided into two groups: one group received a diet with the addition of E. faecium NCIMB 10415 (Cylactin; Cerbios-Pharma SA), and another group received no feed intervention and served as controls. The sows were randomly assigned to either the control group (n = 8) or the probiotic group (n = 8) and housed under similar conditions in separate buildings to avoid transfer of the probiotic strain between the treatment groups. E. faecium NCIMB 10415 was mixed into the sows' diet at 4.2 × 106 to 4.3 × 106 CFU/g. Probiotic-supplemented feed was introduced 28 days antepartum and continued until weaning of piglets. Piglets had access to a nonmedicated prestarter diet from the age of 12 days on. The prestarter diet of piglets from the probiotic group contained 5.1 × 106 CFU of E. faecium/g of diet. Piglets were weaned at 26 ± 2 days. Afterwards, the piglets in the probiotic treatment group received a starter diet with 3.6 × 106 CFU/g.

Sampling and RNA isolation.Piglets from each litter per group (probiotic/control) were euthanized at days 12 ± 1 (n = 6), 34 ± 1 (n = 6), and 54 ± 2 (n = 8) for tissue sampling and narcotized with ketamine-azaperone (4 mg/kg [body weight] ketamine; 25 mg/kg azaperone). Following blood sampling, the piglets were euthanized by intracardial injection of a lethal dose of tetracaine hydrochloride, mebezonium iodide, and embutramide (T61; Intervet, Unterschleißheim, Germany). Incision and dissection of the piglets were described in more detail previously (10). Peyer's patches (PPs) and lymph nodes (LNs) were collected from the ileum (ileal PPs [ILPPs] and ileal LNs [ILLNs]) and jejunum (jejunal PPs [JePPs] and jejunal LNs [JeLNs]), rinsed with Hanks' buffered salt solution (HBSS), and shock-frozen in liquid nitrogen. JeLNs and ILLNs were removed from connective tissue and shock-frozen in liquid nitrogen. Total RNA containing small and large RNAs was isolated from the above-mentioned tissues according to the manufacturer's protocols (NucleoSpin miRNA; Macherey & Nagel). Therefore, 30 mg of tissue samples was homogenized in the provided lysis buffer including β-mercaptoethanol in screw-cap tubes containing metal beads with the Fast-Prep 24 instrument (MP Biomedicals, Eschwege, Germany), using 3 bursts of 20 s each at 4.5 m/s. Quantification of RNA was performed by using a NanoDrop instrument (Thermo Fisher) (A260/A280 ratio of >2.0). Quality was checked on a bioanalyzer. For transcriptome sequencing (RNA-Seq), samples with an RNA integrity number (RIN) of >9 were used. These samples and samples from additional animals, which had to have a RIN of >7.5, were taken for testing of expression by quantitative PCR (qPCR). If a sample did not reach the required RIN, RNA isolation was repeated for this sample.

Analysis of RNA sequencing data.Next-generation sequencing (NGS) using the Illumina HiSeq 2000 platform was carried out for miRNA and, in parallel, mRNA to analyze differential expression in the porcine intestine upon feeding of E. faecium. For RNA-Seq, we used pooled RNA samples. We first isolated RNA from four different tissues (jejunal and ileal lymph nodes and Peyer's patches) from randomly chosen piglets at the age of 34 ± 1 days, and we then pooled RNA from the same tissues of three animals belonging to the same feeding group. To analyze gene expression, reads were preprocessed by trimming of low-quality 3′ tails, removal of matching poly(A) sequences, and extraction of residual PCR-Primer-Plus adapter sequences (match of ≥80%). The remaining reads were aligned against 27,928 porcine mRNAs (downloaded from Ensembl v73) by using Bowtie 2 (22) with the parameter sensitive-local. The resulting alignments were quantified by using the eXpress v1.5.0 software package (http://bio.math.berkeley.edu/eXpress/). The quantified transcript reads were taken as an approximation of gene expression.

Additionally, in separate sequencing of the same sample, the fraction of small RNA, including miRNA, was filtered out, according to a minimum length of 15 bp and non-poly(A) sequences. The maximal observed read count per mapped miRNA sequence within a miRNA identification cluster was taken for the calculation of the fold change in expression. The fold change was defined as the ratio of read counts for the probiotic group to that for the control group upon requiring a minimum support of 100 reads. A cutoff of a 1.5-fold upregulation of a miRNA in the probiotic sample was applied for each tissue separately.

Statistical analysis of tissue-specific gene expression.Computational analysis of differential expression between tissues (ILLNs, ILPPs, JeLNs, and JePPs) was performed based on negative binomial distribution implemented in the DEseq2 (v1.0.19) R package (23). Due to the experimental design, different models using subsets of the data were analyzed. Differences in expression levels between probiotic and control samples taken from the ileum and the jejunum were analyzed by using the model Y = tissue type + diet group + e. Tissue type discriminates between the tissue types, Peyer's patches or lymph nodes, from which lymphocytes were isolated for RNA preparation. Differences in expression levels between probiotic and control samples taken from Peyer's patches and lymph nodes were analyzed by using the model Y = localization + diet group + e. Localization describes the localization of the tissue type, Peyer's patches and lymph nodes, along the small intestine. In addition, a full model was run, which included all available samples and compensated for all covariates available, to analyze the difference between probiotic and control animals: Y = localization + tissue type + diet group + e. Due to the small sample size of NGS samples (eight samples, pooled from three individuals each), this model does not include interactions. Y is the normalized gene expression level through RNA read counts mapped for a known transcript (an Excel file including tables of all differentially expressed genes using the different models is available upon request).

miRNA target prediction.Reads that contained poly(A) sequences and were <15 bp were discarded from the RNA-Seq data (as described above) to enrich the data for small RNA reads. Unique sequences were counted for each sample, and only those that were represented at least two times per sample were kept. miRNA reads were then mapped to known Sus scrofa miRNA sequences from miRBASE. In total, 207 known porcine miRNAs were present in the samples and subjected to a target scan using 5′- and 3′-untranslated region (UTR) sequences of the porcine mRNAs. Sequences of 1,000 bp up- and downstream were appended to mRNA sequences without annotated 5′ and 3′ UTRs. The target scan was performed by using the stand-alone version of PITA (24) and miRanda (20).

RT-qPCR for quantification of miRNA and mRNA.Quantification of miRNA was done by a poly(A) technique according to the manufacturer's protocols (miRNA first-strand cDNA synthesis kit; Agilent Technologies). The poly(A) technique includes elongation of miRNAs carried out by a specific poly(A) polymerase (2 U/μl) isolated from E. coli, which elongates the miRNA at the 3′ tail using ATP as the substrate. Therefore, 1 μg of total RNA was polyadenylated for 30 min at 37°C and terminated at 95°C for 5 min in a total volume of 10 μl. In a second step, 10 μl of the elongated miRNA was reverse transcribed by using AffinityScript reverse transcriptase and a universal reverse primer (3.125 μM) in a 20-μl volume. After incubation for 5 min at 55°C and 15 min at 25°C, reverse transcription (RT) was carried out at 42°C for 30 min. Heat inactivation at 95°C for 5 min led to the inactivation of RT. Afterwards, the reaction mixture was diluted in 260 μl RNase-free water. Following reverse transcription, RT-qPCR (Viia 7; Life Technologies) was performed with three technical replicates per sample and two replicates for reference miRNAs by using miRNA qPCR master mix, including reference dye (1:50) and the universal reverse primer provided by Agilent Technologies. One microliter of the reverse transcript miRNA reaction mix served as the template in a 384-reaction plate in 40 cycles. Specific primers used for amplification are given in Table 1. The amplification program comprised an initial step at 95°C for 10 min, followed by an annealing step and an elongation step for 1 min at 60°C. Porcine miR-17, miR-103, and miR-17 served as reference miRNAs (25).

View this table:
  • View inline
  • View popup
TABLE 1

Oligonucleotides used for quantification of porcine cDNA (miRNA/mRNA) and reporter gene assays

For the quantification of mRNA transcripts, 1 μg of total RNA in 10 μl was used for reverse transcription into cDNA by using the oligo(dT) primer according to the manufacturer's protocols for the AffinityScript qPCR cDNA synthesis kit (Agilent Technologies). For qPCR analysis, 10 ng of initial total RNA served as the template, samples were amplified in triplicates, and reference genes were amplified in duplicates. The reaction was done by using 5 μl of SYBR Select master mix containing AmpliTaq DNA polymerase and 6 μmol specific primers (Table 1) in a volume of 10 μl in a 384-reaction plate using the Viia 7 device (Life Technologies). An initialization step was conducted for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of denaturation for 15 s at 95°C and annealing/elongation for 1 min at 60°C. Ribosomal protein L-19 (RPL-19) and TATA-box-binding protein 1 (TBP-1) were chosen as reference genes, since previous studies showed high stability in porcine tissues (26). RNA samples excluding the RT reaction and water served as mock controls. The relative expression level was calculated by the ΔΔCT method (27) and was described in detail previously (28).

Transfection and luciferase assays.Transfection was performed by using the human cervix carcinoma cell line HeLa (ATCC CCL-2) cultivated in RPMI 1640 (Biochrom AG) supplemented with 10% superior fetal bovine serum (Biochrom AG) and 10 μg/ml l-glutamine. The cell culture was incubated in 75-cm2 flasks (Greiner Bio-One GmbH) at 37°C in 5% CO2 and passaged once per week. For transfection, Nucleofector technology (Lonza AG) was used as described in more detail previously (21). The vector pTKGluc (NEB GmbH) was used for the generation of reporter plasmids. Synthetic oligonucleotides (Invitrogen) containing the target sequence/control (Table 1) and the NotI (5′) and XbaI (3′) restriction sites were hybridized and cloned into pTKGluc by using methods comparable to those described previously by Hoeke et al. (21). As a first step, pTKGluc was linearized by using the restriction enzymes NotI and XbaI (NEB GmbH). Fifty nanograms of the linearized vector and 1 pmol of the hybrid were ligated overnight at 16°C by using T4 ligase (NEB GmbH), followed by heat inactivation at 65°C for 10 min, and transformed into E. coli B6. Selection of positive clones was verified by Sanger sequencing (ABI Prism 310 genetic analyzer; Applied Biosystems). Endotoxin-free plasmids were created for transfection according to the manufacturer's protocols for the NucleoBond Xtra Midi kit (Macherey-Nagel GmbH & Co. KG). Cotransfection of the reporter plasmid and miRNA mimics as well as measurement of luciferase activity were described previously (29).

RESULTS

Transcriptome analysis of miRNA and mRNA by next-generation sequencing reveals significant differences between the control and probiotic groups.About 11 million to 12 million and 8 million to 20 million reads were obtained for the different tissue samples from the control and probiotic groups, respectively. Out of these, roughly 156,000 to 183,000 and 162,000 to 283,000 unique sequences with at least 2 reads and fractions of 5,982 to 6,628 and 5,774 to 8,900 reads in the control and probiotic groups, respectively, were identified, which mapped to a total of 207 known Sus scrofa miRNAs (miRBASE v21). In total, 39 porcine miRNAs were upregulated in the E. faecium treatment group versus the control group in at least one of the tissue samples: S. scrofa miR-149 (ssc-miR-149) and ssc-miR-1285 in jejunal tissues, 13 miRNAs in ileal tissues, 22 miRNAs in Peyer's patch samples, and ssc-miR-423-5p and ssc-miR-1285 in lymph nodes.

The scan for target genes was initially performed for all identified known porcine miRNAs against the 5′ and 3′ UTRs of mRNAs that were downregulated in the E. faecium group versus the control group. Considering the confidence cutoffs for PITA and miRanda, ssc-miR-423-5p was identified as the miRNA with the highest number of putative and jointly predicted target genes (133 genes), 11 of which were related to immune system-relevant functions (Fig. 1A). Selection of immune-relevant candidates was performed according to GO annotation including the keywords B cell, T cell, interleukin, and immune for detailed analysis.

FIG 1
  • Open in new tab
  • Download powerpoint
FIG 1

(A) Target genes recognized by porcine microRNAs expressed in gut-associated lymphatic tissues. Each bar represents targets coherently predicted by two independent target scans (PITA and miRanda). Numbers above bars indicate the total number of putative target transcripts regulated by the respective microRNAs. (B) Heat map of downregulated genes within jejunal Peyer's patches (JEPP), jejunal lymph nodes (JELN), ileal Peyer's patches (ILPP), and ileal lymph nodes (ILLN), represented as fold changes generated from RNA-Seq data.

After quality control for transcripts, ∼19 million to 41 million and 37 million to 66 million mRNA reads were obtained for control and probiotic samples, respectively. These reads could be mapped to 27,928 mRNA transcripts annotated in the Ensembl database (v73). Thirty-one transcripts were downregulated in the probiotic group in at least one of the tissues (Fig. 1B); 19 were putative target genes of miR-423-5p.

Enterococcus faecium leads to upregulation of miRNA-423-5p and downregulation of IGLC and IGKC in ILLNs at the age of 34 days.Our study revealed a high number of regulated potential targets for upregulated miR-423-5p. Therefore, we focused on miR-423-5p. To validate the data from RNA-Seq analysis, we used the poly(A) technique of RT-qPCR to quantitate miRNA expression in additional samples. These data indicated a 2.11-fold (P = 0.03) upregulation of miR-423-5p in cells of ileal lymph nodes at day 34 (1 week after weaning) in the probiotic group compared to the control group. No difference was found for ileal Peyer's patches and jejunal lymph nodes and Peyer's patches (Fig. 2A).

FIG 2
  • Open in new tab
  • Download powerpoint
FIG 2

Validation of NGS data by RT-qPCR of ssc-miR-423-5p and its immune-relevant target transcripts in gut-associated lymphatic tissues. (A) Relative expression levels of miR-423-5p at day 34 ± 1 within ileal lymph nodes, ileal Peyer's patches, jejunal lymph nodes, and jejunal Peyer's patches. (B) Relative expression levels of ssc-miR-423-5p target transcripts IGLC, IGKC, IGLV-4, and PTPRCAP at day 34 ± 1 within ileal lymph nodes (control group, n = 6; probiotic group, n = 6). Error bars denote standard deviations. For statistical testing, the Mann-Whitney test (one-sided) was carried out. Significant P values (P < 0.05) are illustrated in each panel.

As we were able to validate an upregulation of miRNA-423-5p within ileal lymph nodes, we also measured the differential expression of immune-relevant target transcripts of miR-423-5p in ileal lymph nodes. We chose four genes, three different immunoglobulin derivates, IGLC (immunoglobulin lambda light C region), IGKC (immunoglobulin kappa constant), and IGLV-4 (immunoglobulin variant 4), and PTPRCAP (protein tyrosine phosphatase, receptor type, C-associated protein), for validation by RT-qPCR. PTPRCAP is associated with the tyrosine phosphatase PTPRC/CD45, which is a key regulator for the activation of B as well as T lymphocytes (http://www.ncbi.nlm.nih.gov/gene/19265). Quantification of mRNA abundances of IGLC and IGKC revealed 0.61-fold (P = 0.03) and 0.69-fold (P = 0.04) downregulation in the probiotic group, respectively (Fig. 2B). For IGLV-4 and PTPRCAP, we did not detect significant differences between the feeding groups.

Expression analysis at different age points reveals anticorrelation of miR-423-5p and IGLC.In a further step, we investigated the expression patterns of miR-423-5 and its putative target transcripts IGLC, IGKC, IGLV-4, and PTPRCAP over the course of time during the feeding experiment. Therefore, we additionally analyzed piglets at the ages of 12 ± 1 days (before weaning) and 54 ± 2 days (3 weeks after weaning). At these time points, we did not find expression differences between diet groups for either miR-423-5p or the IGLC, IGKC, IGLV-4, and PTPRCAP target genes. In contrast, we detected significantly decreased expression levels of miRNA-423-5p in piglets in the probiotic group aged 12 days compared to 34 days (P = 0.007) and in piglets aged 34 days compared to 54 days (P = 0.008). Consistent with the decreased expression levels of miRNA, there was a minor tendency of increased expression levels of IGLC in the probiotic group at day 12 compared to day 34 (P = 0.08) and a significantly higher expression level in samples from piglets at the age of 54 days than at the age of 34 days (P = 0.01) (Fig. 3). The anticorrelation of transcript amounts between miR-423-5p and IGLC meets our expectation of the most often reported direction of effects of gene regulation via miRNA (reviewed in reference 30). A similar change of the expression pattern was found for IGLV-4 and IGKC, but the changes were weaker and not statistically significant (data not shown).

FIG 3
  • Open in new tab
  • Download powerpoint
FIG 3

Analysis of expression of miR-423-5p and its putative target transcript IGLC within ileal lymph nodes at different time points. Shown is the anticorrelation of the relative expression levels of miR-423-5p (black line) and its target gene immunoglobulin lambda constant cluster (IGLC) (gray line) in ileal lymph nodes of piglets at days 12 ± 1, 34 ± 1, and 54 ± 2 of age (n = 5 per group). Error bars show standard deviations. The P values shown are for differences between days (Mann-Whitney test).

IGLC is likely a target of miR-423-5p.Since we observed an anticorrelation of transcribed miR-423-5 and IGLC, in a cell-based experiment, we tested whether the in silico-predicted interaction also occurs in vitro. For binding of miR-423-5 to IGLC, we identified a seed sequence of 6 bp in the 3′ UTR with a PITA value of −11.13 kJ/mol (Fig. 4A). The predicted target site and, in addition, a mutagenized target site, serving as a control, were cloned into a Gaussian luciferase vector for performing reporter assays. As miR-423-5p is conserved between Homo sapiens and Sus scrofa, easy-growing human HeLa cells were used for the experiment. The reporter constructs harboring either the predicted target site of IGLC or a mutagenized target site as well as miR-423-5p mimics (miRNA sequence) or nonsense miRNAs (mutagenized miRNA sequence) were cotransfected into the cells. Significant reductions of luciferase activity of 0.87-fold (P = 0.03) in IGLC–miR-423-5p-cotransfected cells compared to IGLC reporter plasmids cotransfected with a nonsense miRNA and of 0.86-fold (P = 0.01) for the IGLC-mutagenized target site compared to the identified target size were measured (Fig. 4B). These results suggest that IGLC is likely a target of miR-423-5p.

FIG 4
  • Open in new tab
  • Download powerpoint
FIG 4

Analysis of the IGLC–miR-423-5p interaction using a luciferase (LUC) reporter assay. (A) Target prediction by miRanda (ts, target site) referring to ENSSSCT00000011005 (exon 2; Ensembl v72) and miR-423-5p. (B) Luciferase reporter assay with HeLa cells. Relative luciferase activity (LUCGaussia/LUCCypridina) was ascertained to mimic nonsense miRNA (white column) and the mutagenized seed region (shaded black column) serving as controls. The black column represents mimic miR-423-5p. Each column represents the means of data from three replicates. The P values shown for differences between controls and the reporter assay were determined by a Student t test.

DISCUSSION

One important step for understanding the role of probiotics in the immune response is the deciphering of the porcine transcriptome, including microRNA and mRNA, as a major approach to understand the molecular mechanism underlying immune responses. In our study, we were interested in the effect of the addition of E. faecium NCIMB 10415 to the diet on the immune system during the critical phase of weaning.

According to the most often reported direction of regulation of miRNAs, we focused on miRNAs, which are upregulated, and their specific mRNA targets, which are downregulated. Hence, were applied certain criteria to follow the hypothesis revealing dysregulation of miRNA and immune-relevant mRNA in gut-associated lymphatic tissue within the probiotic-fed group. miR-423-5p and its potential target genes were differentially expressed between the probiotic group and the control group. Therefore, we focused on miR-423-5p. This particular miRNA attained interest in human cancer research and heart diseases (31–34). For example, Liu and colleagues (35) found that trefoil factor 1 (TFF1) is a target of miR-423-5p, which is involved in cell proliferation and invasion of cancer cells in the gastrointestinal tract (35). In another study, miR-423-5p was shown to mimic the silencing of IGSF1 (immunoglobulin superfamily member 1), which regulates the interaction between cells (36).

In pigs, there are only a few studies on miRNAs. One study deciphered a high number of novel porcine miRNAs along the intestine and suggested a crucial role of miRNAs in intestinal functions (37). Another study examined the porcine miRNA transcriptome in different tissues and found that miR-423-5p was widely expressed (38). We confirmed the NGS results showing the upregulation of miR-423-5p in lymphocytes of ileal lymph nodes in piglets of the probiotic group at 1 week postweaning that were fed a diet with E. faecium. We did not find differential expression of miR-423-5p in Peyer's patches and jejunal lymph nodes via RT-qPCR. In addition, we measured the expression levels of four immune-relevant, B-cell-associated target genes of miR-423-5p, which were shown to be differentially expressed within the ileal lymph nodes by NGS. We were able to confirm significant downregulation by means of RT-qPCR for two of them: IGLC and IGKC. The IGLC and IGKC genes encode the constant part of immunoglobulins and, therefore, are important for B-cell regulation. Correspondingly, our previous study of the same animals showed a lower relative IgM-positive B-cell count within ileal lymph nodes in the probiotic group 1 and 3 weeks after weaning (10). Genes such as the IGLV-4 and PTPRCAP genes showed no differential expression in ileal lymph nodes.

As a next step, we analyzed the expression patterns of miR-423-5 and its putative target transcripts IGLC, IGKC, IGLV-4, and PTPRCAP at additional time points ante- and postweaning over the course of time during the feeding experiment. These data suggested that miR-423-5p as well as IGLC, IGKC, IGLV-4, and PTPRCAP are not regulated anteweaning at the age of 12 days and at 3 weeks postweaning at the age of 54 days. We found differences in relative expression levels between age groups of 12 and 34 days as well as 34 and 54 days in the probiotic-fed group for miR-423-5p and IGLC (Fig. 3). Scharek and collogues showed similar findings for immunoglobulins, which follow the same pattern over the course of time after feeding of E. faecium (39). Interestingly, the observed age-dependent differences in miR-423-5p and IGLC expression levels are clearly anticorrelated. The anticorrelation of the miRNA and gene transcript amounts was consistently seen over the course of the experiment. Hence, the in silico-predicted interaction of miR-423-5p and IGLC became more likely. To prove this interaction on the gene level, reporter gene assays were performed, which verified in vitro that IGLC is a target of miR-423-5p (Fig. 4). We assume that one possible mechanism of the mode of action of probiotics is mediated through miRNAs. In this particular study, the feeding of E. faecium likely mediates miR-423-5p regulation of IGLC. There might be a physiological role of E. faecium in reducing immunoglobulin levels. In regard to the experimental design used in the present study, it is possible that different molecular mechanism underlying the effect of E. faecium were not taken into account. According to the results, the question of whether feeding of E. faecium NCIMB 10415 has beneficial effects on the immune system is raised. Further research is needed to verify these findings to be able to carry out targeted feed intervention using the probiotic Enterococcus faecium NCIMB 10415.

ACKNOWLEDGMENTS

This study was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG]) within Collaborative Research Group (Sonderforschungsbereich [SFB]) 852/1, Nutrition and Intestinal Microbiota-Host Interactions in the Pig.

The authors are solely responsible for the data and do not represent any opinion of either the DFG or other public or commercial entities.

FOOTNOTES

    • Received 18 December 2015.
    • Accepted 20 January 2016.
    • Accepted manuscript posted online 29 January 2016.
  • Copyright © 2016, American Society for Microbiology. All Rights Reserved.

REFERENCES

  1. 1.↵
    1. Campbell JM,
    2. Crenshaw JD,
    3. Polo J
    . 2013. The biological stress of early weaned piglets. J Anim Sci Biotechnol 4:19. doi:10.1186/2049-1891-4-19.
    OpenUrlCrossRefPubMed
  2. 2.↵
    European Commission. 2003. Regulation (EC) no 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition (text with EEA relevance). Off J Eur Union L 268:29–43.
    OpenUrl
  3. 3.↵
    FAO/WHO. 2006. Probiotics in food—health and nutritional properties and guidelines for evaluation. FAO food and nutrition paper 85. FAO, Rome, Italy.
  4. 4.↵
    1. Teitelbaum JE,
    2. Walker WA
    . 2002. Nutritional impact of pre- and probiotics as protective gastrointestinal organisms. Annu Rev Nutr 22:107–138. doi:10.1146/annurev.nutr.22.110901.145412.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Szajewska H,
    2. Kotowska M,
    3. Mrukowicz JZ,
    4. Armanska M,
    5. Mikolajczyk W
    . 2001. Efficacy of Lactobacillus GG in prevention of nosocomial diarrhea in infants. J Pediatr 138:361–365. doi:10.1067/mpd.2001.111321.
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    1. Weizman Z,
    2. Asli G,
    3. Alsheikh A
    . 2005. Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents. Pediatrics 115:5–9.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    1. Bednorz C,
    2. Guenther S,
    3. Oelgeschlager K,
    4. Kinnemann B,
    5. Pieper R,
    6. Hartmann S,
    7. Tedin K,
    8. Semmler T,
    9. Neumann K,
    10. Schierack P,
    11. Bethe A,
    12. Wieler LH
    . 2013. Feeding the probiotic Enterococcus faecium strain NCIMB 10415 to piglets specifically reduces the number of Escherichia coli pathotypes that adhere to the gut mucosa. Appl Environ Microbiol 79:7896–7904. doi:10.1128/AEM.03138-13.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Pollmann M,
    2. Nordhoff M,
    3. Pospischil A,
    4. Tedin K,
    5. Wieler LH
    . 2005. Effects of a probiotic strain of Enterococcus faecium on the rate of natural chlamydia infection in swine. Infect Immun 73:4346–4353. doi:10.1128/IAI.73.7.4346-4353.2005.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Scharek L,
    2. Guth J,
    3. Reiter K,
    4. Weyrauch KD,
    5. Taras D,
    6. Schwerk P,
    7. Schierack P,
    8. Schmidt MF,
    9. Wieler LH,
    10. Tedin K
    . 2005. Influence of a probiotic Enterococcus faecium strain on development of the immune system of sows and piglets. Vet Immunol Immunopathol 105:151–161. doi:10.1016/j.vetimm.2004.12.022.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Kreuzer S,
    2. Machnowska P,
    3. Assmus J,
    4. Sieber M,
    5. Pieper R,
    6. Schmidt MF,
    7. Brockmann GA,
    8. Scharek-Tedin L,
    9. Johne R
    . 2012. Feeding of the probiotic bacterium Enterococcus faecium NCIMB 10415 differentially affects shedding of enteric viruses in pigs. Vet Res 43:58. doi:10.1186/1297-9716-43-58.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Wang Z,
    2. Burwinkel M,
    3. Chai W,
    4. Lange E,
    5. Blohm U,
    6. Breithaupt A,
    7. Hoffmann B,
    8. Twardziok S,
    9. Rieger J,
    10. Janczyk P,
    11. Pieper R,
    12. Osterrieder N
    . 2014. Dietary Enterococcus faecium NCIMB 10415 and zinc oxide stimulate immune reactions to trivalent influenza vaccination in pigs but do not affect virological response upon challenge infection. PLoS One 9:e87007. doi:10.1371/journal.pone.0087007.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Siepert B,
    2. Reinhardt N,
    3. Kreuzer S,
    4. Bondzio A,
    5. Twardziok S,
    6. Brockmann G,
    7. Nockler K,
    8. Szabo I,
    9. Janczyk P,
    10. Pieper R,
    11. Tedin K
    . 2014. Enterococcus faecium NCIMB 10415 supplementation affects intestinal immune-associated gene expression in post-weaning piglets. Vet Immunol Immunopathol 157:65–77. doi:10.1016/j.vetimm.2013.10.013.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Chen K,
    2. Rajewsky N
    . 2007. The evolution of gene regulation by transcription factors and microRNAs. Nat Rev Genet 8:93–103. doi:10.1038/nrg1990.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    1. Aravin A,
    2. Tuschl T
    . 2005. Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett 579:5830–5840. doi:10.1016/j.febslet.2005.08.009.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    1. Fragoso R,
    2. Mao T,
    3. Wang S,
    4. Schaffert S,
    5. Gong X,
    6. Yue S,
    7. Luong R,
    8. Min H,
    9. Yashiro-Ohtani Y,
    10. Davis M,
    11. Pear W,
    12. Chen CZ
    . 2012. Modulating the strength and threshold of NOTCH oncogenic signals by mir-181a-1/b-1. PLoS Genet 8:e1002855. doi:10.1371/journal.pgen.1002855.
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Hu R,
    2. Kagele DA,
    3. Huffaker TB,
    4. Runtsch MC,
    5. Alexander M,
    6. Liu J,
    7. Bake E,
    8. Su W,
    9. Williams MA,
    10. Rao DS,
    11. Moller T,
    12. Garden GA,
    13. Round JL,
    14. O'Connell RM
    . 2014. miR-155 promotes T follicular helper cell accumulation during chronic, low-grade inflammation. Immunity 41:605–619. doi:10.1016/j.immuni.2014.09.015.
    OpenUrlCrossRefPubMed
  17. 17.↵
    1. Runtsch MC,
    2. Round JL,
    3. O'Connell RM
    . 2014. MicroRNAs and the regulation of intestinal homeostasis. Front Genet 5:347. doi:10.3389/fgene.2014.00347.
    OpenUrlCrossRefPubMed
  18. 18.↵
    1. Jin W,
    2. Ibeagha-Awemu EM,
    3. Liang G,
    4. Beaudoin F,
    5. Zhao X,
    6. Guan LL
    . 2014. Transcriptome microRNA profiling of bovine mammary epithelial cells challenged with Escherichia coli or Staphylococcus aureus bacteria reveals pathogen directed microRNA expression profiles. BMC Genomics 15:181. doi:10.1186/1471-2164-15-181.
    OpenUrlCrossRefPubMed
  19. 19.↵
    1. Meurens F,
    2. Summerfield A,
    3. Nauwynck H,
    4. Saif L,
    5. Gerdts V
    . 2012. The pig: a model for human infectious diseases. Trends Microbiol 20:50–57. doi:10.1016/j.tim.2011.11.002.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    1. Betel D,
    2. Wilson M,
    3. Gabow A,
    4. Marks DS,
    5. Sander C
    . 2008. The microRNA.org resource: targets and expression. Nucleic Acids Res 36:D149–D153.
    OpenUrlCrossRefPubMedWeb of Science
  21. 21.↵
    1. Hoeke L,
    2. Sharbati J,
    3. Pawar K,
    4. Keller A,
    5. Einspanier R,
    6. Sharbati S
    . 2013. Intestinal Salmonella Typhimurium infection leads to miR-29a induced caveolin 2 regulation. PLoS One 8:e67300. doi:10.1371/journal.pone.0067300.
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Langmead B,
    2. Salzberg SL
    . 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi:10.1038/nmeth.1923.
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    1. Anders S,
    2. Huber W
    . 2010. Differential expression analysis for sequence count data. Genome Biol 11:R106. doi:10.1186/gb-2010-11-10-r106.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Kertesz M,
    2. Iovino N,
    3. Unnerstall U,
    4. Gaul U,
    5. Segal E
    . 2007. The role of site accessibility in microRNA target recognition. Nat Genet 39:1278–1284. doi:10.1038/ng2135.
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    1. Timoneda O,
    2. Balcells I,
    3. Cordoba S,
    4. Castello A,
    5. Sanchez A
    . 2012. Determination of reference microRNAs for relative quantification in porcine tissues. PLoS One 7:e44413. doi:10.1371/journal.pone.0044413.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Meurens F,
    2. Berri M,
    3. Auray G,
    4. Melo S,
    5. Levast B,
    6. Virlogeux-Payant I,
    7. Chevaleyre C,
    8. Gerdts V,
    9. Salmon H
    . 2009. Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops. Vet Res 40:5. doi:10.1051/vetres:2008043.
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Livak KJ,
    2. Schmittgen TD
    . 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. doi:10.1006/meth.2001.1262.
    OpenUrlCrossRefPubMedWeb of Science
  28. 28.↵
    1. Kreuzer S,
    2. Rieger J,
    3. Strucken EM,
    4. Thaben N,
    5. Hunigen H,
    6. Nockler K,
    7. Janczyk P,
    8. Plendl J,
    9. Brockmann GA
    . 2014. Characterization of CD4+ subpopulations and CD25+ cells in ileal lymphatic tissue of weaned piglets infected with Salmonella Typhimurium with or without Enterococcus faecium feeding. Vet Immunol Immunopathol 158:143–155. doi:10.1016/j.vetimm.2014.01.001.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Sharbati J,
    2. Lewin A,
    3. Kutz-Lohroff B,
    4. Kamal E,
    5. Einspanier R,
    6. Sharbati S
    . 2011. Integrated microRNA-mRNA-analysis of human monocyte derived macrophages upon Mycobacterium avium subsp. hominissuis infection. PLoS One 6:e20258. doi:10.1371/journal.pone.0020258.
    OpenUrlCrossRefPubMed
  30. 30.↵
    1. Jia S,
    2. Zhai H,
    3. Zhao M
    . 2014. MicroRNAs regulate immune system via multiple targets. Discov Med 18:237–247.
    OpenUrlPubMed
  31. 31.↵
    1. Zhao H,
    2. Gao A,
    3. Zhang Z,
    4. Tian R,
    5. Luo A,
    6. Li M,
    7. Zhao D,
    8. Fu L,
    9. Fu L,
    10. Dong JT,
    11. Zhu Z
    . 2015. Genetic analysis and preliminary function study of miR-423 in breast cancer. Tumour Biol 36:4763–4771. doi:10.1007/s13277-015-3126-7.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Stiuso P,
    2. Potenza N,
    3. Lombardi A,
    4. Ferrandino I,
    5. Monaco A,
    6. Zappavigna S,
    7. Vanacore D,
    8. Mosca N,
    9. Castiello F,
    10. Porto S,
    11. Addeo R,
    12. Prete SD,
    13. De Vita F,
    14. Russo A,
    15. Caraglia M
    . 2015. MicroRNA-423-5p promotes autophagy in cancer cells and is increased in serum from hepatocarcinoma patients treated with sorafenib. Mol Ther Nucleic Acids 4:e233. doi:10.1038/mtna.2015.8.
    OpenUrlCrossRef
  33. 33.↵
    1. Luo P,
    2. He T,
    3. Jiang R,
    4. Li G
    . 2015. MicroRNA-423-5p targets O-GlcNAc transferase to induce apoptosis in cardiomyocytes. Mol Med Rep 12:1163–1168. doi:10.3892/mmr.2015.3491.
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Fang Z,
    2. Tang J,
    3. Bai Y,
    4. Lin H,
    5. You H,
    6. Jin H,
    7. Lin L,
    8. You P,
    9. Li J,
    10. Dai Z,
    11. Liang X,
    12. Su Y,
    13. Hu Q,
    14. Wang F,
    15. Zhang ZY
    . 2015. Plasma levels of microRNA-24, microRNA-320a, and microRNA-423-5p are potential biomarkers for colorectal carcinoma. J Exp Clin Cancer Res 34:86. doi:10.1186/s13046-015-0198-6.
    OpenUrlCrossRefPubMed
  35. 35.↵
    1. Liu J,
    2. Wang X,
    3. Yang X,
    4. Liu Y,
    5. Shi Y,
    6. Ren J,
    7. Guleng B
    . 2014. miRNA423-5p regulates cell proliferation and invasion by targeting trefoil factor 1 in gastric cancer cells. Cancer Lett 347:98–104. doi:10.1016/j.canlet.2014.01.024.
    OpenUrlCrossRefPubMed
  36. 36.↵
    1. Younger ST,
    2. Corey DR
    . 2011. Transcriptional gene silencing in mammalian cells by miRNA mimics that target gene promoters. Nucleic Acids Res 39:5682–5691. doi:10.1093/nar/gkr155.
    OpenUrlCrossRefPubMedWeb of Science
  37. 37.↵
    1. Sharbati S,
    2. Friedlander MR,
    3. Sharbati J,
    4. Hoeke L,
    5. Chen W,
    6. Keller A,
    7. Stahler PF,
    8. Rajewsky N,
    9. Einspanier R
    . 2010. Deciphering the porcine intestinal microRNA transcriptome. BMC Genomics 11:275. doi:10.1186/1471-2164-11-275.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Xie SS,
    2. Li XY,
    3. Liu T,
    4. Cao JH,
    5. Zhong Q,
    6. Zhao SH
    . 2011. Discovery of porcine microRNAs in multiple tissues by a Solexa deep sequencing approach. PLoS One 6:e16235. doi:10.1371/journal.pone.0016235.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Scharek L,
    2. Guth J,
    3. Filter M,
    4. Schmidt MFG
    . 2007. Impact of the probiotic bacteria Enterococcus faecium NCIMB 10415 (SF68) and Bacillus cereus var. toyoi NCIMB 40112 on the development of serum IgG and faecal IgA of sows and their piglets. Arch Anim Nutr 61:223–234. doi:10.1080/17450390701431540.
    OpenUrlCrossRefPubMedWeb of Science
PreviousNext
Back to top
Download PDF
Citation Tools
Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes
Susanne Kreuzer-Redmer, Jennifer C. Bekurtz, Danny Arends, Ralf Bortfeldt, Barbara Kutz-Lohroff, Soroush Sharbati, Ralf Einspanier, Gudrun A. Brockmann
Applied and Environmental Microbiology Apr 2016, 82 (8) 2263-2269; DOI: 10.1128/AEM.04044-15

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Applied and Environmental Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes
(Your Name) has forwarded a page to you from Applied and Environmental Microbiology
(Your Name) thought you would be interested in this article in Applied and Environmental Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes
Susanne Kreuzer-Redmer, Jennifer C. Bekurtz, Danny Arends, Ralf Bortfeldt, Barbara Kutz-Lohroff, Soroush Sharbati, Ralf Einspanier, Gudrun A. Brockmann
Applied and Environmental Microbiology Apr 2016, 82 (8) 2263-2269; DOI: 10.1128/AEM.04044-15
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

About

  • About AEM
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AppEnvMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

 

Print ISSN: 0099-2240; Online ISSN: 1098-5336