Altered Transcription of Murine Genes Induced in the Small Bowel by Administration of Probiotic Strain Lactobacillus rhamnosus HN001

Updated information and services can be found at: http://aem.asm.org/content/80/9/2851 These include: SUPPLEMENTAL MATERIAL REFERENCES

CONTENT ALERTS

Supplemental material This article cites 54 articles, 26 of which can be accessed free at: http://aem.asm.org/content/80/9/2851#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

Gerald W. Tannock, Corinda Taylor, Blair Lawley, Diane Loach, Maree Gould, Amy C. Dunn, Alexander D. McLellan, Michael A. Black, Les McNoe, James Dekker, Pramod Gopal and Michael A. Collett Appl. Environ. Microbiol. 2014, 80(9):2851. DOI: 10.1128/AEM.00336-14. Published Ahead of Print 28 February 2014.

Altered Transcription of Murine Genes Induced in the Small Bowel by Administration of Probiotic Strain Lactobacillus rhamnosus HN001 Gerald W. Tannock,a,e Corinda Taylor,a Blair Lawley,a Diane Loach,a Maree Gould,b Amy C. Dunn,a Alexander D. McLellan,a Michael A. Black,c Les McNoe,c James Dekker,d Pramod Gopal,d Michael A. Collettd

Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli (Lactobacillus-free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus-free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1, angptl4, and hspa1b, associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus-free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

M

embers of the genus Lactobacillus are commonly detected in the stool of vertebrates, including humans (1). Some of these lactobacilli inhabit the digestive tract of specific hosts, where they maintain populations of consistent size throughout the lifetime of the animal host (autochthonous). Other Lactobacillus species present in stool are transient in the bowel and originate from the diet of the host (allochthonous). Probiotic strains of lactobacilli are allochthonous because they are detected in stool only during the period that the probiotic product is consumed (1). Although tantalizing information of the impact of probiotics on the welfare of the consumer continues to be reported (2), in general, there remains an absence of mechanistic explanations of probiotic efficacy. First exposure to probiotic strains probably occurs most commonly in adulthood by ingestion of probiotic yogurts or similar products but could also occur in the case of neonates through prophylactic interventions that are suggested to reduce susceptibility to allergies (3, 4), duration of diarrhea (5), or necrotizing enterocolitis (6) or through inclusion in formulas directed toward “gut comfort” and “immune health” (7). Attempts to explain the efficacy of probiotics have included measurement of the induction of mucosal gene expression in response to the administration of probiotic bacteria in the bowel, especially in relation to genes associated with immunological pathways (8). Probiotic effects on gene expression might be transient, but temporal studies in which gene expression was measured have not been reported. Therefore, we measured transcription of selected genes in the mucosa of the small bowel of mice previously naive to lactobacilli after exposure to probiotic Lactobacillus rhamnosus strain HN001. Animals were investigated in infancy and adulthood, providing a temporal view of gene expression in response to exposure to probiotic bacteria.

May 2014 Volume 80 Number 9

MATERIALS AND METHODS Bacterial strain. Lactobacillus rhamnosus HN001 (DR20) is a commercial probiotic strain that has been extensively characterized and tested for health-promoting efficacy as described in previous publications (9–13). As determined in preliminary experiments, HN001 does not colonize the gut of mice, even previously Lactobacillus-free mice, or humans (14) and must be administered daily to achieve its continuous intestinal transit. Strain HN001 was maintained using Lactobacillus de Man-RogosaSharpe (MRS) medium incubated anaerobically at 37°C. Suspensions of bacteria for addition to drinking water were prepared as follows. Bacterial cells from an MRS culture (200 ml) of strain HN001 were collected by centrifugation and suspended in 20 ml of sterile water. The suspension was added to 180 ml of sterile water per drinking water bottle and mixed. Fresh culture-water mixtures were provided each 4 days. Since HN001 survives well in water, this procedure resulted in the presence of approximately 1 ⫻ 109 viable cells per ml of water each day (determined by the culture method described below). Mouse experiments. Experimentation was approved by the Animal Ethics Committee, University of Otago (AEC 2/08). BALB/c Lactobacillusfree mice were bred and maintained in Trexler isolators and fed a standard rodent diet that had been sterilized by gamma irradiation. All other ma-

Received 29 January 2014 Accepted 21 February 2014 Published ahead of print 28 February 2014 Editor: G. T. Macfarlane Address correspondence to Gerald W. Tannock, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.00336-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.00336-14

Applied and Environmental Microbiology

p. 2851–2859

aem.asm.org

2851

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealanda; Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealandb; Department of Biochemistry, University of Otago, Dunedin, New Zealandc; Fonterra Research and Development Centre, Palmerston North, New Zealandd; Riddet Institute Centre of Research Excellence, Palmerston North, New Zealande

Tannock et al.

2852

aem.asm.org

erated to analyze product specificity. Standard curves were generated using genomic DNA extracted from Lactobacillus rhamnosus HN001 using a Qiagen DNeasy blood and tissue kit and following the protocol for Grampositive bacteria. The standard DNA was quantified spectrophotometrically using a Nanodrop 1000 spectrophotometer (Thermo Scientific) and diluted in 10-fold steps from 5 ⫻ 106 to 5 ⫻ 101 genomes/reaction, calculated using target gene copies per genome obtained from genome sequence information (NCBI). All reactions were carried out in duplicate and were run twice on separate plates. No-template controls were also included on each plate. Pyrosequencing analysis of small-bowel microbiota of infant mice. It was possible that administration of HN001 might alter the acquisition of commensal bacteria in infant mice. Therefore, the phylogenetic composition of the microbiota of the small bowel of Lactobacillus-free and strain HN001-exposed mice was determined by pyrosequencing of amplified 16S rRNA genes in DNA extracted from bowel samples. A portion comprising regions V1 to V3 of the bacterial 16S rRNA gene was amplified using a two-step protocol similar to that described by Dowd et al. (24). First-round PCR was carried out for 15 cycles using the 8fAll (5=-GRGT TYGATYMTGGCTCAG-3=)/HDA2 (5=-GTATTACCGCGGCTGCTGG CAC-3=) primer set under the following conditions: 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min, with a final extension step of 72°C for 5 min. This product was diluted 1/5 with PCR-grade water, and 1 ␮l was used as the template in a 20-␮l secondary PCR. The secondary PCR was carried out for 30 cycles using the 8fAll primer with 454 sequencing Lib-A adapter sequence A (5=-CGTATCGCCTCCCTCGCGCCATCAGGRGTTYGATY MTGGCTCAG-3=) and the HDA2 primer with 454 sequencing Lib-A adapter sequence B plus a 10-base barcode (shown as N=s) (5=-C TATGCGCCTTGCCAGCCCGCTCAGNNNNNNNNNNGTAT TACCGCGGCTGCTGGCAC-3=) using conditions identical to those of the primary PCR. Products were cleaned using Qiagen PCR cleanup columns (Qiagen, Hilden, Germany) and quantified using a Nanodrop 1000 spectrometer. Equivalent quantities of PCR product from each sample were pooled, and the pooled DNA was recleaned through the use of a Qiagen PCR cleanup column, quantified, and sent to Macrogen (Korea) for unidirectional sequencing from the reverse primer using a Roche 454 genome sequencer with Titanium chemistry. Sequences were processed using QIIME v. 1.4 (25). Sequences were excluded from analysis if they were ⬍250 or ⬎550 bases in length, had an average quality score of ⬍25, contained one or more ambiguous bases, had ⬎1 mismatch with the sequencing primer, or had a homopolymer run of ⬎6. Following splitting into barcoded samples and initial quality filtering, the sequences were passed through the QIIME pipeline using default parameters, including chimera checking. After quality screening, an average of 4,311 (range, 1,015 to 15,338) sequences per barcoded sample were recovered for downstream analysis. Thus, a total of 310,393 sequences were obtained from small-bowel-content samples for phylogenetic analysis. Extraction of RNA from bowel tissue. Depending on the experimental requirements, 3 cm of duodenum, jejunum, or ileum was removed from mice at autopsy and placed immediately in 1 ml of TRIzol reagent (Invitrogen Life Technologies). For 10-day-old mice, the jejunum and ileum were not separated because of the small size of the organs. RNA was extracted from bowel mucosal specimens using the Qiagen RNeasy kit procedure according to the manufacturer’s instructions. A DNase treatment (Ambion) was carried out as described by the manufacturer, and the RNA concentration and purity (A260/A280) were determined by spectrophotometric analysis (Nanodrop spectrophotometer). Microarray screen of murine gene expression. Affymetrix microarrays were used to screen the transcriptional responses to exposure to strain HN001 for 6 days relative to those of Lactobacillus-free mice (duodenal mucosa samples were pooled from 5 mice per treatment, and three biological replicates were used). A total of 300 ng of RNA from each sample was amplified and labeled with biotin before being hybridized to mouse genome 430 2.0 GeneChips using an Affymetrix 3= IVT Express kit (catalog no. 901229). T7-tailed cDNA was produced and used as the template

Applied and Environmental Microbiology

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

terials that were used to maintain the animals were sterilized by autoclave, and standard germfree procedures were used in the general care of the animals. Lactobacillus-free mice do not have lactobacilli as gut inhabitants but have a large-bowel microbiota typical of conventional mice (15, 16). The mice provide animal hosts that are naive with respect to lactobacilli but whose tissues have been conditioned by exposure to a complex microbiota. They have been used extensively in studies of Lactobacillus reuteri (for examples, see references 17, 18, and 19). Mice were anesthetized by carbon dioxide anesthesia, after which they were killed by cervical dislocation. The presence (strain HN001) or absence of lactobacilli was confirmed by culturing cecal digesta, collected at necropsy, on Rogosa SL (Difco) agar plates that were incubated anaerobically at 37°C for 48 h. An initial microarray experiment (see below) was conducted using adult (6-week-old) animals whose duodenal tissue was obtained after the mice had consumed strain HN001 in their drinking water for 6 days. This period of exposure was chosen on the basis of previous work in which altered gene transcriptions were detected in relation to colonization of the Lactobacillus-free mouse gut by Lactobacillus reuteri strain 100-23 (20). Duodenal tissue was examined because of a previous report that administration of Lactobacillus plantarum WCFS1 affected gene expression in the human duodenum (8). To confirm and extend the microarray results using reverse transcription-quantitative PCR (RT-qPCR) (see below), adult mice were exposed daily to strain HN001 in the drinking water and duodenal, jejunal, and ileal tissues were collected after 4, 6, 10, and 21 days of administration (n ⫽ 5 per time point). Additional groups of mice were examined at 6 or 10 days to determine the rates of epithelial cell migration and Angptl4 production, respectively (see below). A temporal study to measure the impact of lifelong exposure to an environment containing strain HN001 was also conducted. Breeding pairs of mice and their progeny were exposed daily to strain HN001 in drinking water, and small-bowel specimens were collected from the progeny at 4, 6, 10, 21, 28, 35, and 42 days after birth (n ⫽ 5 per time point). In all experiments, matched (parental lineage, age, and gender) Lactobacillus-free mice were used as comparative controls. Quantification of Lactobacillus populations by culture. To measure the quantity of viable L. rhamnosus in the digestive tract of mice exposed to HN001, specimens (forestomach, duodenum, jejunum, ileum, and proximal colon) were collected and homogenized in sterile deionized water. Dilutions (10-fold) of each sample were prepared, and aliquots were used to inoculate Rogosa SL (Difco) agar plates that were then incubated anaerobically at 37°C for 48 h. CFU per gram of sample was calculated from colony counts. Extraction of DNA from digestive tract samples. DNA was extracted for use in culture-independent measurement of L. rhamnosus abundance and for phylogenetic analysis of microbiota in extruded digestive tract contents. A previously described method that included mechanical disruption of bacterial cells was used (21). Quantification of Lactobacillus populations by qPCR. To quantify the abundance of lactobacilli (alive and dead) in the digestive tract of mice, real-time quantitative PCR was carried out using an ABI 7500 Fast system in MicroAmp Fast optical 96-well plates with optical adhesive film (Applied Biosystems, Foster City, CA). Primers targeting the 16S rRNA gene from Lactobacillus rhamnosus (F3-rha [5=-GTCGGCAGAGTAACT GTTGTCGG-3=] and R1-univ [5=-GACRACCATGMACCACCTGT-3=]) were those described by Klocke and Mundt (22). Universal bacterial primers (8f-All [5=-GRGTTYGATYMTGGCTCAG-3=] and 340R [5=-ACTGC TGCCTCCCGTAGGAGT-3=]) were those described by Tannock et al. (23). All reactions were carried out in a final volume of 20 ␮l containing 1⫻ Fast SYBR green PCR master mix (Applied Biosystems) and a 300 nM concentration of each primer. Template DNA was diluted to 5 ng/␮l, and 10 ng was added to each reaction. The thermocycling profile consisted of an initial activation of the polymerase at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Fluorescence levels were measured after the 60°C annealing/extension step. A melting curve was gen-

Lactobacillus rhamnosus and Murine Gene Transcription

May 2014 Volume 80 Number 9

conditions and images were employed for all preparations using DP Manager software. Measuring migration of enterocytes. Altered expression of genes associated with the apoptosis pathway might affect epithelial replacement (35). Therefore, the results of migration of enterocytes along villi were compared using 5-bromo-2-deoxyuridine (BrdU) labeling of nuclei. BrdU is a synthetic analogue of thymidine that becomes incorporated into new DNA synthesized during the eukaryotic cell cycle. By exposing cells to BrdU for a short period of time, only those cells actively synthesizing DNA are “labeled” by incorporation of the analogue. Hence, the migration of enterocytes can be tracked from crypts to tips of villi (36). Matched Lactobacillus-free and HN001-exposed (6 days) adult mice were injected intraperitoneally with BrdU solution (30 mg dose/kg of body weight). Eighteen hours after injection, the mice were killed and duodenal tissue was removed and fixed in neutral buffered formalin. The fixed tissue was embedded in paraffin. Paraffin-embedded samples of duodenum (n ⫽ 5 per group) were sectioned (5 ␮m thick) and then dewaxed in xylene, rehydrated through to 70% alcohol, and washed twice with 0.01 M Tris-buffered saline (TBS). The following method was used to detect BrdU labeling of nuclei. All incubations were at room temperature unless specified. The sections were incubated for 30 min in proteinase K (20 mg/ml) and then washed in TBS containing 4 mM CaCl2 for 5 min. After two washes in TBS, sections were incubated in 4 mM HCl for 10 min at 37°C and then washed twice in TBS. Sections were then incubated for 10 min in 0.1 M borate, washed three times in TBS, and then flooded with peroxidase block for 5 min. Sections were incubated overnight at 4°C in anti-BrdU solution (antibromodeoxyuridine, mouse IgG1; BD Pharmingen) diluted 1:100. Negative-control slides incubated only with TBS were also prepared. The following day, sections were washed three times in TBS and then incubated with the labeled polymer-horseradish peroxidase anti-mouse antibody (Dako K4007) for 30 min. Sections were washed twice in TBS before being developed for 5 min in DAB (3,3=-diaminobenzidine tetrahydrochloride). This reaction was stopped in tap water, and then sections were stained with Gill’s no. 2 hematoxylin for 6 s, rinsed in tap water until they turned blue, and finally dehydrated in absolute ethanol and cleared in xylene, and coverslips were mounted using DPX mountant. Stained sections were viewed with an Olympus BX-51 microscope. Images were captured using a Spot imaging system (Diagnostic Instruments, Inc., Sterling Heights, MI). To determine the distance that enterocytes had migrated, a measurement was taken from the basement membrane to the top of the nuclear-stained enterocytes, another measurement was taken to the topmost enterocyte, and the percentage was determined. Exactly 50 villi per animal were measured. The observer was blind to the identity of the samples. Inflammation. Altered epithelial integrity might lead to inflammation. Therefore, tissues from duodenum, jejunum, and ileum of matched Lactobacillus-free and HN001-exposed animals (6 days of exposure; n ⫽ 5 per group) were fixed in 10% neutral buffered formalin. The fixed tissue was embedded in paraffin and sectioned (5 ␮m thick), and the sections were stained with hematoxylin and eosin. Gail Williams (a pathologist at the University of Otago) scored the sections in a blind manner for evidence of inflammation, using a well-validated scale (37). Histological scores were based on the degree of lamina propria and submucosal mononuclear cellular infiltration, crypt hyperplasia, goblet cell depletion, and architectural distortion.

RESULTS

Microarray screen of gene expression. The arrays passed standard Affymetrix quality assessment protocols. Interarray correlation ranged from 0.89 to 0.96 across all array pairs. Relative to the results seen with Lactobacillus-free mice, six genes exhibited upregulation of 2-fold or greater in adult mice exposed to strain HN001 for 6 days (Table 1). Three of these genes, sgk1, angptl4, and hspa1b, appeared in GATHER-derived associations with apoptosis (Bayes factor 6 [P ⬍ 0.0001]; Gene Ontology identifiers

aem.asm.org 2853

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

for amplification using in vitro transcription. The resulting biotin-labeled amplified RNA was then purified, quantified, fragmented, and hybridized to the arrays according to the Affymetrix 3= Express kit protocol. Arrays were hybridized for 16 h in an Affymetrix GeneChip 650 hybridization oven with rotating at 60 rpm. Arrays were then washed and stained using an Affymetrix wash and stain kit (catalog no. 900720) in an Affymetrix 450 fluidics wash station using the FS450 0001 wash protocol, as recommended for mouse genome 430 2.0 GeneChips. Arrays were then scanned on a 3000 7G GeneChip scanner using Affymetrix GeneChip Command Console software (AGCC). Subsequent bioinformatic analysis of the microarray data to identify up- or downregulated genes was carried out in R (26) using the affy (27) (robust multiarray average [RMA] normalization) and limma (28) packages, available as part of the Bioconductor project (29). The GATHER web interface (30) was used to identify KEGG pathways (31) that were significantly enriched among the differentially expressed genes. Based on previous published work (32), a 2-fold (greater or lesser) change in gene expression in HN001-exposed animals compared to Lactobacillus-free animals was used as the cutoff value in conjunction with P values (33). RT-qPCR. To extend transcriptional results obtained from the microarray screen, real-time quantitative PCR amplification was performed from reverse-transcribed cDNA in MicroAmp Fast optical 96well or 384-well plates on a 7500 Fast or Viia7 system with optical adhesive film (Applied Biosystems) using TaqMan primers and probes. Template cDNA was generated by reverse transcription of 300 ng of RNA using an Invitrogen SuperScript III first-strand synthesis SuperMix kit according to the manufacturer’s instructions. Two microliters of 2-fold-diluted RT product was used as the template. Assays were performed following the TaqMan assay reagent protocol (Applied Biosystems) with primers and probes specific for the following genes: gapdh (Mus musculus 03302249_g1 [Mm03302249_g1]), angptl4 (Mm00480431_m1), sgk1 (Mm00441380_m1), and hspa1b (Mm3038954_s1). The following conditions were used: 95°C for 1 min and then 40 cycles at 95°C for 30 s and 60°C for 45 s. The efficiency of each primer/probe set was determined by diluting cDNA to generate standard curves. All reactions were performed in duplicate, and angptl4, sgk1, and hspa1b results were normalized to gapdh values. Relative gene expression levels of HN001exposed and nonexposed (Lactobacillus-free) mice were calculated using the ⌬⌬CT (threshold cycle) method. Statistical evaluation was carried out using the relative expression software tool (REST) (34). This is standalone software used to evaluate up- and downregulation for gene expression studies. The software addresses issues surrounding the measurement of uncertainty in expression ratios by using randomization and bootstrapping techniques. Detection of Angptl4 in mouse mucosa. To confirm that upregulated gene transcription resulted in increased translation to protein, frozen duodenum sections derived from test mice (exposed to HN001 for 10 days) and matched Lactobacillus-free mice (both n ⫽ 4) were prepared in a microtome-cryostat. Angptl4 was chosen for study because fluorescent antibody for its detection was obtainable commercially. The sections were fixed in 25% alcohol–75% acetone at room temperature for 10 min and then dried for 10 min. The sections were rehydrated with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 5 min and then immersed in primary antibody solution (goat anti-mouse ABINO angiopoietin-like 4 internal antibody; ABIN337228) at 1/100 in 1% BSA– PBS for 1 h at room temperature. After three washes with 0.05% Tween 20 –PBS, the sections were immersed in 100 ␮l of secondary reagent (Alexa Fluor 594 rabbit anti-goat IgG) diluted 1/200 with 1% BSA–PBS and 0.25 ␮g/ml DAPI (4=,6-diamidino-2-phenylindole) (Norrie Biotech 422801) for 1 h at room temperature. The sections were washed three times as described above and then mounted in Prolong Gold antifade reagent (Invitrogen catalog no. P36930) and examined by fluorescence microscopy using an Olympus BX51 fluorescence microscope with a mercury short-arc burner and UPLAN FL lenses (FN26.5). Identical exposure

Tannock et al.

TABLE 1 Genes upregulated 2-fold or more in DNA microarray experiments comparing mice administered HN001 to Lactobacillus-free mice

Annotation

sgk1 angptl4 pck1 pdk4 cyp2c55 hspa1b

Serum/glucocorticoid-regulated kinase 1 Angiopoietin-like protein 4 Phosphoenolpyruvate carboxykinase 1, cytosolic Pyruvate dehydrogenase kinase isoenzyme 4 Cytochrome P450, family 2, subfamily c, polypeptide 55 Heat shock protein 1b

2.9 (0.11) 2.0 (0.12) 2.0 (0.12) 2.6 (0.19) 2.2 (0.21) 7.5 (0.55)

GO:0043066, GO:0043069, GO:0006915, and GO:0012501), so the transcription of these genes was selected for further study because they might be linked to alteration in the kinetics of epithelial cell replacement in the duodenum. Confirmation and extension of microarray results. RT-qPCR measurements of the transcription of sgk1, angptl4, and hspa1b in the duodenum of adult mice exposed to HN001 were made. Groups of animals were sampled after 4, 6, 10, and 21 days of exposure to determine the temporal effects of HN001 administration. The results confirmed that increased expression of sgk1, angptl4, and hspa1b occurred after 6 to 10 days of administration of HN001. Longer exposure (21 days) led to lower gene expression (Fig. 1). Therefore, the effect on gene transcription was transient and time dependent. Increased production of Angptl4 in duodenal epithelial cells. Comparison of the intensities of fluorescent antibody staining in sections prepared from test and control animals provided evidence of increased expression of Angptl4 in epithelial cells of duodenal mucosa (Fig. 2). This confirmed in an exemplar gene that increased gene transcription led to increased expression of the gene product after exposure to HN001 for 10 days. Migration of epithelial cells in duodenum. BrdU labeling of epithelial cell nuclei showed that epithelial cell migration was faster in adult mice administered HN001 in drinking water for 6 days (Fig. 3). The average migration of labeled enterocytes was 55.4% in Lactobacillus-free mice compared to 75.4% in HN001exposed animals. This result provided a phenotypic change associated with increased transcription, particularly of sgk1 and hspa1b as described above. sgk1 encodes a serine/threonine protein kinase that plays roles in cell survival pathways (38). hspa1b encodes a heat shock protein which chaperones other proteins, ensuring stabilization and correct folding of proteins (39). These two genes, together with angptl4, are associated with apoptosis in GATHER analysis. Inflammation. Histological sections stained with hematoxylin and eosin prepared from duodenal, jejunal, and ileal tissues of both Lactobacillus-free and HN001-exposed animals lacked signs of inflammation. Bacteriology of the small bowel of adult mice. To ensure that changes in gene transcription were concomitant with the presence of lactobacilli in the small bowel, quantitative bacteriological data were gathered from adult mice exposed to HN001 for 6 days. In some mice, numbers of viable lactobacilli were below the level of detection in forestomach (median log10 per gram, 4.4; range, ⬍3.0 to 5.3), duodenal (⬍3.0; ⬍3.0 to 3.2), jejunal (⬍3.0; ⬍3.0 to 3.3), ileal (4.0; ⬍3.0 to 5.7), and colon (3.8; ⬍3.0 to 5.5) samples (n ⫽ 10). However, qPCR data showed that administration of HN001

2854

aem.asm.org

P value

Adjusted P value (false-discovery rate)

1.94E-07 1.63E-05 1.75E-05 5.14E-05 393E-04 473E-04

0.0087 0.2628 0.2628 0.5792 1.0000 1.0000

in drinking water added substantial numbers of lactobacilli to the gut environment as shown in Fig. 4A. Numbers of HN001 were lowest in the jejunum compared to other gut sites, probably due to the rapid passage of digesta in this region. Abundance (the proportion of HN001 in relation to all 16S rRNA gene targets) was highest in the ileum and lowest in the cecum and colon, where obligately anaerobic bacteria predominate (Fig. 4B). Gene transcription in infant mice. Exposure to allochthonous lactobacilli from early life might alter the biological succession in the gut, which might then have an impact on mucosal gene expression. To test this possibility, parents and offspring were exposed to HN001 through the drinking water. As the bowels of the parents contained HN001 (see above), their progeny would be exposed to the bacteria from early life. We extended observations made in adult animals by measuring gene expression in the jejunum and ileum as well as the duodenum. Transcriptional responses in infant mice were variable depending on the smallbowel regions and the ages of the mice examined. sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice 25 days of age or younger, angptl4 was upregulated at 10 days in the duodenum but downregulated in the jejunum and ileum until mice were 25 days old, and hspa1b was upregulated at 10 days in the duodenum but downregulated in jejunal mucosa until day 25. Transcription of these genes in the ileum tended to increase in older animals (Fig. 5). Thus, exposure to HN001 had different transcriptional outcomes in different parts of the small bowel of mice according to age. Bacteriology of the small bowel of infant mice. The composition of bacterial collections in the small bowel was determined by pyrosequencing DNA to determine whether or not microbiota biodiversity in the first 25 days of life, when fluctuations in gene expression levels seemed common, was affected by an altered bacterial succession. L. rhamnosus was detected, in low abundance, in all regions of the small bowel of test animals at all ages sampled (Table 2). There was much variation between animals, probably due to the presence or absence of food boluses in small-bowel regions. Other bacterial groups (Corynebacteriaceae, Clostridiaceae, Pasteurellaceae, Bacteroidales, and Lachnospiraceae) were commonly present in levels of abundance that were variable between animals in both test and control groups (see Fig. S1 to S3 in the supplemental material). Due to large between-animal variations, the abundances of microbiota groups did not differ between Lactobacillus-free and Lactobacillus-exposed mice (Mann-Whitney nonparametric test, P ⬎ 0.05). Pasteurellaceae tended to be more abundant in 10-day-old mice than in older animals (see Fig. S1 to S3).

Applied and Environmental Microbiology

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

Gene

Fold upregulation relative to Lactobacillus-free mice (moderated SE from limma analysis)

Lactobacillus rhamnosus and Murine Gene Transcription

male mouse duodenum. (B) Test male mouse duodenum exposed to HN001 for 10 days. Images show ABINO angiopoietin-like 4 internal antibody staining (red) and DAPI nuclear staining (blue). Images were taken with an Olympus BX51 fluorescence microscope.

FIG 1 Temporal gene transcription in duodenal mucosa of adult mice administered L. rhamnosus HN001 daily in drinking water compared to matched Lactobacillus-free mice. Dots represent individual mice, dotted horizontal lines represent relative expression of 1.0 (gene expression is the same in the test and the control mice), solid horizontal lines represent means, and vertical bars represent standard errors of the means. Relative expression values correspond to 2⫺e⌬⌬CT. Arrows indicate statistically significant differences (P ⬍ 0.001). Statistical evaluation was carried out using the relative expression software tool (REST).

lactobacilli showed differential expression of hundreds to thousands of genes, depending on the human subject. Cellular pathways and processes that were modulated by transcriptional networks were shown to differ according to the probiotic strain that had been tested (42). However, phenotypic changes that could be associated with the transcriptional alterations were not investigated in these studies. In our study, we noted regional differences in transcription of the three selected genes along the length of the small bowel of young mice. Regional differences in gene transcription (for example, transcription of those encoding cytoprotective heat shock proteins) have been reported for the colon of mice and humans (43). These observations may have been related to the differing biodiversities of the microbiota in the different regions. We found that the presence of commensals in the small-bowel regions of mice did not differ between Lactobacillus-free mice and those exposed to L. rhamnosus strain HN001. Therefore, the differential expression of genes that we observed must have been due to the

DISCUSSION

Comparisons of conventional animals (presence of gut microbiota) to germfree animals (absence of microbiota) have shown differences in the expression of a plethora of murine mucosal genes along the length of the small and large bowels (40). These genes were related to adaptive immunity, innate immunity, metabolism, and other aspects of eukaryotic cell physiology. Differential transcription of genes in small-bowel mucosa of experimental animals and humans has also been detected following exposure to lactobacilli used as probiotics. Relative to germfree mice, a large number of genes were differentially regulated in microarray investigations (41). These genes were associated with immune response, energy homeostasis, and host defense. Human studies of gene expression in duodenal mucosa after administration of probiotic

May 2014 Volume 80 Number 9

FIG 3 Higher rate of epithelial cell migration, measured by BrdU incorporation, along the length of villi in adult mice after 6 days of administration of the bacteria daily in drinking water. LF, Lactobacillus-free mice; HN001, L. rhamnosus HN001-exposed mice (five mice per group). The Mann-Whitney nonparametric test was used for evaluations.

aem.asm.org 2855

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

FIG 2 Detection of Angptl4 in mouse mucosa. (A) Control Lactobacillus-free

Tannock et al.

Populations determined by qPCR in relation to the standard curve. The dotted line shows the lower limit of detection. (B) Abundances determined relative to total 16S rRNA genes. Means and standard errors of the means (SEM) are shown for five mice per group. FS, forestomach; DUO, duodenum; JEJ, jejunum; IL, ileum; CAEC, cecum; PROX COL, proximal colon.

presence of Lactobacillus cells. Interestingly, comparison of CFU and qPCR data revealed that it was probable that not of all of the lactobacilli in the bowel were alive. This suggests that even nonviable Lactobacillus cells may stimulate differential gene expression in the small-bowel mucosa. Differential expression of large numbers of genes was not detected in our microarray screen. This may have been due to our use of mice that were Lactobacillus free and yet were colonized by a large-bowel microbiota equivalent to that of conventional animals. Therefore, as a result of coprophagy, the small-bowel environment was already conditioned by exposure to bacteria from feces. This provided more of a “real-life” situation than the use of germfree mice. Three genes (sgk1, angptl4, and hspa1b) were selected for detailed investigation because they were associated with apoptosis, putatively an important regulator of intestinal epithelial homeostasis (35). This association provided a potential marker of phenotypic change in the mouse in relation to differential gene expression resulting from probiotic administration. The migration of enterocytes from the crypt base to villous tips plays a role in the maintenance of an effective barrier at the interface between the digesta and the submucosal tissues (44). Probiotic bacteria could thus influence the integrity of the barrier, possibly enhancing its effectiveness and promoting host defense (45). Indeed, administration of strain HN001 increased the rate of migration of enterocytes in the duodenum of adult mice at a time when genes associated with apoptosis were upregulated and an exemplar protein (Angptl4) was more abundant in duodenal cells. The increased rate of epithelial replacement was not accompanied by inflammation, which suggests no lessening of barrier integrity. Increased enterocyte migration stimulated by Lactobacillus reuteri strains ATCC PTA-6475 and DSM 17938 has been reported to correlate with reduced rotavirus-induced diarrhea in a neonatal mouse model (46, 47). This suggests that an increase in epithelial cell turnover, induced by probiotic bacteria, may contribute to resolution of rotavirus diarrhea. L. rhamnosus strain GG, L. brevis strain SBC8803, and Lactobacillus plantarum strain Lp have been reported to ameliorate inflammatory conditions of the intestine by influencing intestinal barrier function. L. brevis SNC8803 induced heat shock proteins in vitro and decreased DSSinduced intestinal inflammation of mice (48), whereas L. rhamnosus GG appears to have an antiapoptotic effect mediated by a

2856

aem.asm.org

40-kDa soluble protein (49). L. plantarum Lp reduced epithelial apoptosis but upregulated genes encoding tight-junction proteins (50). Although the individual mechanisms may differ, evidence is thus accumulating that effects on epithelial physiology are features of health-promoting qualities of probiotic bacteria. L. rhamnosus HN001 protects mice against infection by Escherichia coli O157:H7, a pathogen that forms attaching and effacing (A/E) lesions on enterocytes (12). HN001 also blocks translocation across the gut epithelia by Salmonella enterica serovar Typhimurium and in so doing reduces the likelihood of infection (10). Additionally, strain HN001 helps maintain enterocyte integrity in an in vitro model of gut barrier function (13). Our current observation that HN001 affects epithelial replacement thus assists in explaining the previous findings of reduced infectivity of epithelium-adherent pathogens and improved gut barrier integrity resulting from probiotic administration. Although focus on pathways rather than individual genes is desirable in gene expression studies (51), it is worth noting that increased transcription of angptl4 has been reported in the HCT116 colonic carcinoma cell line exposed to L. paracasei F19. Further, germfree mice monoassociated with the F19 strain had increased levels of Angplt4 protein in serum (52). Our observations reveal that in mice with a complex microbiota, HN001 can stimulate angptl4 transcription and protein levels, at least in the gut epithelium. These findings are of interest given the reported link between levels of angptl4 transcription, commensals, and fat storage in mice (53). A feature of our transcriptional observations was that upregulation of genes sgk1, angptl4, and hspa1b was dependent on the time of exposure in both adult and infant mice. We consistently found that gene expression and associated phenotypic changes in the duodenum occurred after 6 to 10 days of exposure to the probiotic bacteria. The transiency of the probiotic impact in adult mice, if this same effect were shown to occur in humans, could indicate the need to determine optimal consumption regimens to improve the efficacy of probiotic products. Probiotics such as HN001 have been shown to reduce the occurrence of childhood eczema (4, 54, 55), but the mechanistic explanation for this effect is not known. While transcription of the three genes that we investigated in duodenal mucosa of infant mice (10 days of age) was increased, transcription was either un-

Applied and Environmental Microbiology

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

FIG 4 L. rhamnosus HN001 populations and abundance in the gut of adult mice after 6 days of administration of the bacteria daily in drinking water. (A)

Lactobacillus rhamnosus and Murine Gene Transcription

altered or decreased at other times. The transcription of sgk1 was increased in the duodenum until weaning (about 21 days), but transcription of angptl4 and hspa1b was unaltered or decreased during this period in young mice. Thus, there may have been interplay between probiotic and dietary effects in the developing animal. The mechanism of probiotic action in children is there-

fore more difficult to explain because of the temporal transcriptional features observed in the young mice. Nevertheless, the results of the study provide a link between strain HN001, altered mucosal gene expression in the bowel, and host phenotype (epithelial replacement) which may be useful in further delineating the mechanisms of probiotic action.

TABLE 2 Abundance of Lactobacillus 16S rRNA gene sequences in the small intestine of mice exposed to L. rhamnosus HN001 from birth Median % (25th–75th percentiles) of total 16S rRNA gene targets for mice of indicated age (days)a Organ

10b

15

20

25

Duodenum Jejunum Ileum

0.47 (0.02–74.32) 0.89 (0.41–51.49) 0.89 (0.41–51.49)

3.13 (0.83–5.13) 0.46 (0.05–5.79) 5.35 (0.08–15.2)

0.42 (0.00–6.33) 0.72 (0.46–16.78) 0.35 (0.05–12.00)

1.03 (0.02–38.94) 1.68 (0.44–75.88) 0.14 (0.09–3.60)

a b

n ⫽ 5 mice per sampling time. Jejunum and ileum were combined for 10-day-old mice.

May 2014 Volume 80 Number 9

aem.asm.org 2857

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

FIG 5 Temporal gene expression in duodenal mucosa of mice exposed to L. rhamnosus HN001 from birth to 42 days of age compared to matched Lactobacillusfree mice. Dots represent individual mice, dashed horizontal lines represent relative expression of 1.0 (gene expression is the same in the test and the control mice), solid horizontal lines represent means, and vertical bars represent standard errors of the means. Relative expression values correspond to 2-e⌬⌬CT. Arrows indicate statistically significant differences (P ⬍ 0.001).

Tannock et al.

ACKNOWLEDGMENTS This research was supported by Foundation for Research, Science and Technology program contract DRIX0702 and by funding from Fonterra Co-operative Group Ltd.

REFERENCES

2858

aem.asm.org

19.

20.

21.

22.

23.

24.

25.

26. 27. 28.

29.

30. 31.

32.

33.

Applied and Environmental Microbiology

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

1. Tannock GW. 2004. A special fondness for lactobacilli. Appl. Environ. Microbiol. 70:3189 –3194. http://dx.doi.org/10.1128/AEM.70.6.3189-319 4.2004. 2. Howarth GS, Butler RN, Salminen S, Gibson GR, Donovan SM. 2012. Probiotics for optimal nutrition: from efficacy to guidelines. Adv. Nutr. 3:720 –722. http://dx.doi.org/10.3945/an.112.002501. 3. Wickens K, Black PN, Stanley TV, Mitchell E, Fitzharris P, Tannock GW, Purdie G, Crane J, Probiotic Study Group. 2008. A differential effect of 2 probiotics in the prevention of eczema and atopy: a doubleblind, randomized, placebo-controlled trial. J. Allergy Clin. Immunol. 122:788 –794. http://dx.doi.org/10.1016/j.jaci.2008.07.011. 4. Isolauri E, Rautava S, Salminen S. 2012. Probiotics in the development and treatment of allergic disease. Gastrenterol. Clin. North Am. 41:747– 762. http://dx.doi.org/10.1016/j.gtc.2012.08.007. 5. Bernaola Aponte G, Bad Mancilla CA, Carreazo NY, Rojas Galarza RA. 2013. Probiotics for treating persistent diarrhoea in children. Cochrane Database Syst. Rev. 8:CD007401. http://dx.doi.org/10.1002/14651858 .CD007401.pub3. 6. Li D, Rosito G, Slagle T. 19 July 2013. Probiotics for the prevention of necrotizing enterocolitis in neonates: an 8-year retrospective cohort study. J. Clin. Pharm. Ther. http://dx.doi.org/10.1111/jcpt.12084. 7. Nauta AJ, Ben Amor K, Knol J, Garssen J, van der Beek EM. 2013. Relevance of pre- and postnatal nutrition to development and interplay between the microbiota and metabolic and immune systems. Am. J. Clin. Nutr. 98:586S–593S. http://dx.doi.org/10.3945/ajcn.112.039644. 8. van Baarlen P, Troost FJ, van Hemert S, van der Meer C, de Vos WM, de Groot PJ, Hooiveld GJEJ, Brummer R-JM, Kleerebezem M. 2009. Differential NF-␬B pathways induction by Lactobacillus plantarum in the duodenum of healthy humans correlating with immune tolerance. Proc. Natl. Acad. Sci. U. S. A. 106:2371–2376. http://dx.doi.org/10.1073/pnas .0809919106. 9. Gill HS, Rutherfurd KJ, Prasad J, Gopal PK. 2000. Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019). Br. J. Nutr. 83:167–176. http://dx.doi.org/10.1017/S0007114500000210. 10. Gill HS, Shu Q, Lin HK, Rutherfurd J, Cross ML. 2001. Protection against translocating Salmonella Typhimurium infection in mice by feeding the immuno-enhancing probiotic Lactobacillus rhamnosus strain HN001. Med. Microbiol. Immunol. 190:97–104. 11. Sheih YH, Chiang BL, Wang LH, Liao CK, Gill HS. 2001. Systemic immunity-enhancing effects in healthy subjects following dietary consumption of the lactic acid bacterium Lactobacillus rhamnosus HN001. J. Am. Coll. Nutr. 20:149 –156. http://dx.doi.org/10.1080/07315724.2001 .10719027. 12. Shu Q, Gill HS. 2002. Immune protection mediated by the probiotic Lactobacillus rhamnosus HN001 (DR20) against Escherichia coli O157:H7 infection in mice. FEMS Immunol. Med. Microbiol. 34:59 – 64. http://dx .doi.org/10.1111/j.1574-695X.2002.tb00603.x. 13. Anderson RC, Cookson AL, McNabb WC, Kelly WJ, Roy NC. 2010. Lactobacillus plantarum DSM 2648 is a potential probiotic that enhances intestinal barrier function. FEMS Microbiol. Lett. 309:184 –192. http://dx .doi.org/10.1111/j.1574-6968.2010.02038.x. 14. Tannock GW, Munro K, Harmsen HJ, Welling GW, Smart J, Gopal PK. 2000. Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Appl. Environ. Microbiol. 66:2578 –2588. http://dx.doi.org/10.1128/AEM.66.6.2578 -2588.2000. 15. Tannock GW, Crichton C, Welling GW, Koopman JP, Midtvedt T. 1988. Reconstitution of the gastrointestinal microflora of lactobacillusfree mice. Appl. Environ. Microbiol. 54:2971–2975. 16. Tannock GW. 2009. Research for the 21st century: can we draw a blueprint of the bowel ecosystem? Biosci. Microflora 28:75– 80. 17. Wilson CM, Aggio RB, O’Toole PW, Villas-Boas S, Tannock GW. 2012. Transcriptional and metabolomics consequences of luxS inactivation reveal a metabolic rather than quorum-sensing role for LuxS in Lactobacillus

18.

reuteri 100-23. J. Bacteriol. 194:1743–1746. http://dx.doi.org/10.1128/JB .06318-11. Tannock GW, Wilson CM, Loach D, Cook GM, Eason J, O’Toole PW, Holtrop G, Lawley B. 2012. Resource partitioning in relation to cohabitation of Lactobacillus species in the mouse forestomach. ISME J. 6:927– 938. http://dx.doi.org/10.1038/ismej.2011.161. Frese SA, Benson AK, Tannock GW, Loach DM, Kim J, Zhang M, Oh PL, Heng NC, Patil PB, Juge N, Mackenzie DA, Pearson BM, Lapidus A, Dalin E, Tice H, Goltsman E, Land M, Hauser L, Ivanova N, Kyrpides NC, Walter J. 2011. The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri. PLoS Genet. 7:e1001314. http://dx.doi.org/10.1371/journal.pgen.1001314. Hoffmann M, Rath E, Hölzlwimmer G, Quintanilla-Martinez L, Loach D, Tannock G, Haller D. 2008. Lactobacillus reuteri 100-23 transiently activates intestinal epithelial cells of mice that have a complex microbiota during the early stages of colonization. J. Nutr. 138:1684 –1691. Walter J, Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K, Alatossava T. 2000. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl. Environ. Microbiol. 66:297– 303. http://dx.doi.org/10.1128/AEM.66.1.297-303.2000. Klocke M, Mundt K. 2004. Development of a 16S rDNA-targeted PCR assay for monitoring of Lactobacillus plantarum and Lact. rhamnosus during co-cultivation for production of inoculants for silages. Lett. Appl. Microbiol. 39:267–273. http://dx.doi.org/10.1111/j.1472-765X.2004.015 75.x. Tannock GW, Lawley B, Munro K, Gowri Pathmanathan S, Zhou SJ, Makrides M, Gibson RA, Sullivan T, Prosser CG, Lowry D, Hodgkinson AJ. 2013. Comparison of the compositions of the stool microbiotas of infants fed goat milk formula, cow milk-based formula, or breast milk. Appl. Environ. Microbiol. 79:3040 –3048. http://dx.doi.org/10.1128/AEM .03910-12. Dowd SE, Callaway TR, Wolcott RD, Sun Y, McKeehan T, Hagevoort RG, Edrington TS. 2008. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). BMC Microbiol. 8:125–132. http://dx.doi.org/10 .1186/1471-2180-8-125. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7:335–336. http://dx.doi.org/10.1038/nmeth.f.303. RCore Team. 2013. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. www.r -project.org. Gautier L, Cope L, Bolstad BM, Irizarry RA. 2004. affy—analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20:307–315. http://dx.doi.org/10.1093/bioinformatics/btg405. Smyth GK. 12 February 2004. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3:Article3. http://dx.doi.org/10.2202/1544-6115 .1027. Gentleman RC1, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J. 2004. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5:R80. http://dx.doi.org/10.1186/gb-2004-5-10-r80. Chang JT, Nevins JR. 2006. GATHER: a systems approach to interpreting genomic signatures. Bioinformatics 22:2926 –2933. http://dx.doi.org/10 .1093/bioinformatics/btl483. Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M. 2006. From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res. 34(database issue):D354 –D357. http://dx.doi.org/10.1093/nar/gkj102. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. 2001. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291:881– 884. http://dx.doi.org/10.1126/science.291 .5505.881. Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a

Lactobacillus rhamnosus and Murine Gene Transcription

May 2014 Volume 80 Number 9

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

parasite expulsion. Science 308:1463–1465. http://dx.doi.org/10.1126 /science.1108661. Preidis GA, Saulnier DM, Blutt SE, Mistretta TA, Riehle KP, Major AM, Venable SF, Finegold MJ, Petrosino JF, Conner ME, Versalovic J. 2012. Probiotics stimulate enterocyte migration and microbial diversity in the neonatal mouse intestine. FASEB J. 26:1960 –1969. http://dx.doi.org/10 .1096/fj.10-177980. Preidis GA, Saulnier DM, Blutt SE, Mistretta T-A, Riehle KP, Major AM, Venable SF, Barrish JP, Finegold MJ, Petrosino JF, Guerrant RL, Conner ME, Versalovic J. 2012. Host response to probiotics determined by nutritional status of rotavirus-infected neonatal mice. J. Pediatr. Gastroenterol. Nutr. 55:299 –307. http://dx.doi.org/10.1097/MPG.0b013e318 24d2548. Arvans DL, Vavricka SR, Ren H, Musch MW, Kang L, Rocha FG, Lucioni A, Turner JR, Alverdy J, Chang EB. 2005. Luminal bacterial flora determines physiological expression of intestinal epithelial cytoprotective heat shock proteins 25 and 72. Am. J. Physiol. Gastrointest. Liver Physiol. 288:G696 –G704. http://dx.doi.org/10.1152/ajpgi.00206.2004. Yan F, Cao H, Cover TL, Washington MK, Shi Y, Liu L, Chaturvedi R, Peek RM, Wilson KT, Polk DB. 2011. Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism. J. Clin. Invest. 121:2242– 2253. http://dx.doi.org/10.1172/JCI44031. Mangell P, Nejdfors P, Wang M, Ahrné S, Weström B, Thorlacius H, Jeppsson B. 2002. Lactobacillus plantarum 299v inhibits Escherichia coliinduced intestinal permeability. Dig. Dis. Sci. 47:511–516. http://dx.doi .org/10.1023/A:1017947531536. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. 2005. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U. S. A. 102:15545–15550. http://dx.doi.org/10.1073/pnas.0506580102. Aronsson L, Huang Y, Parini P, Korach-Andre M, Hakansson J, Gustafsson JA, Pettersson S, Arulamalam V, Rafter J. 2010. Decreased fat storage by Lactobacillus paracasei is associated with increased levels of angiopoietin-like 4 protein (ANGPTL4). PLoS One 5:e13087. http://dx .doi.org/10.1371/journal.pone.0013087. Bäckhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, Semenkovich CF, Gordon JI. 2004. The gut microbiota as an environmental factor that regulates fat storage. Proc. Natl. Acad. Sci. U. S. A. 101:15718 – 15723. http://dx.doi.org/10.1073/pnas.0407076101. Wickens K, Black P, Stanley TV, Mitchell E, Barthow C, Fitzharris PG, Purdie R, Crane J. 2012. A protective effect of Lactobacillus rhamnosus HN001 against eczema in the first 2 years of life persists to age 4 years. Clin. Exp. Allergy 42:1071–1079. http://dx.doi.org/10.1111/j.1365-2222.2012 .03975.x. Wickens K, Stanley TV, Mitchell EA, Barthow C, Fitzharris PG, Purdie R, Siebers P, Black N, Crane J. 2013. Early supplementation with Lactobacillus rhamnosus HN001 reduces eczema prevalence to 6 years: does it also reduce atopic sensitization? Clin. Exp. Allergy 43:1048 –1057. http: //dx.doi.org/10.1111/cea.12154.

aem.asm.org 2859

Downloaded from http://aem.asm.org/ on October 1, 2014 by Bibliotheque de l'Universite Laval

practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57:289 –300. 34. Pfaffl MW, Horgan GW, Dempfle L. 2002. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30:e36. http://dx .doi.org/10.1093/nar/30.9.e36. 35. Grossmann J, Walther K, Artinger M, Rummele P, Woenckhaus M, Scholmerich J. 2002. Induction of apoptosis before shedding of human intestinal epithelial cells. Am. J. Gastroenterol. 97:1421–1428. http://dx .doi.org/10.1111/j.1572-0241.2002.05787.x. 36. Boshuizen JA, Reimerink JHJ, Korteland-van Male AM, van Ham VJJ, Koopmans MPG, Buller HA, Dekker J, Einerhand AWC. 2003. Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice. J. Virol. 77:13005–13016. http://dx.doi .org/10.1128/JVI.77.24.13005-13016.2003. 37. Moran JP, Walter J, Tannock GW, Tonkonogy SL, Sartor RB. 2009. Bifidobacterium animalis causes extensive duodenitis and mild colonic inflammation in monoassociated interleukin-10-deficient mice. Inflamm. Bowel Dis. 15:1022–1031. http://dx.doi.org/10.1002/ibd.20900. 38. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME. 2001. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol. Cell. Biol. 21:952– 965. http://dx.doi.org/10.1128/MCB.21.3.952-965.2001. 39. Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, Zamzami N, Mak T, Jaatela M, Penninger JM, Garrido C, Kroemer G. 2001. Heatshock protein 70 antagonizes apoptosis-inducing factor. Nat. Cell Biol. 3:839 – 843. http://dx.doi.org/10.1038/ncb0901-839. 40. Larsson E, Tremaroli V, Lee YS, Koren O, Nooaew I, Fricker A, Nielsen J, Ley RE, Backhed F. 2012. Analysis of gut microbial regulation of host gene expression along the length of the gut and regulation of gut microbial ecology through MyD88. Gut 61:1124 –1131. http://dx.doi.org/10.1136 /gutjnl-2011-301104. 41 Nerstedt A, Nilsson EC, Ohlson K, Hakansson J, Svensson LT, Lowenadler B, Svensson UK, Mahlapuu M. 2007. Administration of Lactobacillus evokes coordinated changes in the intestinal expression profile of genes regulating energy homeostasis and immune phenotypes in mice. Br. J. Nutr. 97:1117–1127. http://dx.doi.org/10.1017/S0007114507682907. 42. van Baarlen P, Troost F, van der Meer C, Hooiveld G, Boekschoten M, Brummer RJ, Kleerebezem M. 2011. Human mucosal in vivo transcriptome responses to three lactobacilli indicate how probiotics may modulate human cellular pathways. Proc. Natl. Acad. Sci. U. S. A. 108(Suppl 1): 4562– 4569. http://dx.doi.org/10.1073/pnas.1000079107. 43. Hu S, Wang Y, Lichtenstein L, Tao Y, Musch MW, Jabri B, Antonopoulus D, Claud EC, Chang EB. 2010. Regional differences in colonic mucosa-associated microbiota determine the physiological expression of host heat shock proteins. Am. J. Physiol. Gastrointest. Liver Physiol. 299: G1266 –G1275. http://dx.doi.org/10.1152/ajpgi.00357.2010. 44. Günther CG, Neumann H, Neurath MF, Becker C. 2013. Apoptosis, necrosis and necroptosis: cell death regulation in the epithelium. Gut 62: 1062–1071. http://dx.doi.org/10.1136/gutjnl-2011-301364. 45. Cliffe LJ, Humphreys NE, Lane TE, Potten CS, Booth C, Grencis RK. 2005. Accelerated intestinal epithelial cell turnover: a new mechanism of