Bifidobacterium bifidum PRL2010 modulates the host innate ...

AEM Accepts, published online ahead of print on 15 November 2013 Appl. Environ. Microbiol. doi:10.1128/AEM.03313-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.

AEM03313-13-REVISED

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Bifidobacterium bifidum PRL2010 modulates the host innate immune response

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Running title: Host response upon bifidobacterial colonization

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Key words: Bifidobacteria, human host’s response, transcriptomics, genomics

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Francesca Turroni1, Valentina Taverniti2, Patricia Ruas-Madiedo 3, Sabrina Duranti4, Simone

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Guglielmetti2, Gabriele Andrea Lugli4, Laura Gioiosa5, Paola Palanza5, Abelardo Margolles3,

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Douwe van Sinderen1 and Marco Ventura3*

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Alimentary Pharmabiotic Centre and Department of Microbiology, Bioscience Institute, National

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University of Ireland, Western Road, Cork, Ireland1; Dipartimento di Scienze e Tecnologie

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Alimentari e Microbiologiche, Università degli Studi di Milano, Milan, Italy2; Departamento de

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Microbiologia y Bioquimica de Productos Lacteos (IPLA – CSIC) Villaviciosa, Asturias, Spain3;

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Laboratory of Probiogenomics, Department of Life Sciences, University of Parma, Italy4;

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Department of Evolutionary and Functional Biology, University of Parma, Italy5.

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*Corresponding author: Laboratory of Probiogenomics, Department of Life Sciences, University of

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Parma, Parco Area delle Scienze 11a, 43124 Parma, Italy. Phone: ++39-521-905666. Fax: ++39-

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521-906014. E.mail: [email protected]

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Downloaded from http://aem.asm.org/ on December 10, 2017 by guest

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Abstract

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Here, we describe data obtained from transcriptome profiling of human cell lines and intestinal cells

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of a murine model upon exposure and colonization, respectively, with Bifidobacterium bifidum

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PRL2010. Significant changes were detected in the transcription of genes that are known to be

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involved in innate immunity. Furthermore, results from ELISA experiments showed that exposure

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to B. bifidum PRL2010 causes enhanced production of IL-6 and IL-8 cytokines, presumably

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through NF-κB activation. The obtained global transcription profiles strongly suggest that

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Bifidobacterium bifidum PRL2010 modulates the innate immune response of the host.

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INTRODUCTION

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During the last decade, bifidobacteria have attracted a lot of scientific attention due to their

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perceived role as health-promoting or probiotic bacteria. Although there is suggestive proof to

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corroborate some of these functional claims, the molecular mechanisms behind such probiotic

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activities remain largely unknown. The decoding and functional analysis of genome sequences of

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probiotic bacteria, i.e. probiogenomics, offers the possibility of accelerating research into the

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mechanisms of probiotic action (1, 2). Probiogenomic investigations have highlighted a plethora of

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genetic features that may explain how bifidobacteria have so well adapted to the human gut.

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Prominent examples are represented by the genes and their products that allow bifidobacteria to

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interact with the human host, either through different morphological structures, such as pili (3, 4) or

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exopolysaccharides (5), but also through the utilization of host-derived glycans such as mucin or

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human milk oligosaccharides (6, 7). Nevertheless, so far relatively little is known about the changes

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that occur in the host’s transcriptome in response to bifidobacterial colonization/exposure (8, 9).

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Among the various human bifidobacteria, a special mention should be reserved for Bifidobacterium

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bifidum PRL2010, whose genome has been subjected to sequencing and functional analysis, thereby

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revealing a number of key traits that would render this bacterium a model organism for the

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investigation of human-microbe co-evolution (6, 10, 11). These analyses of B. bifidum PRL2010

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have provided insights that not only explain the genomic strategy adopted by this strain to

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metabolize host-derived glycans (6, 10), but also the process of host colonization through sortase-

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dependent pili (12).

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B. bifidum strains are claimed to exert a key role in the evolution and maturation of the immune

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system of the host, which is still rather undeveloped following birth (9). The interaction of B.

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bifidum with the host immune system has recently been explored by analyzing the impact of B.

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bifidum Z9 in combination with a second human gut commensal, Lactobacillus acidophilus, on the

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transcriptome of dendritic cells (8). This study revealed that B. bifidum Z9 inhibits expression of 3


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genes related to the adaptive immune system in murine dendritic cells. These findings were in line

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with other publications that employed in vitro assays involving various bifidobacterial strains

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belonging to different species, showing a clear and distinct cytokine profile induced by

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bifidobacteria (9, 13).

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The purpose of the current study was to investigate the particular host gene expression profile

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following exposure to and/or colonization of B. bifidum PRL2010 employing micro array-based

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transcriptome analyses coupled with in vitro (colonic cell lines) and in vivo (murine colonization)

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approaches.

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MATERIALS AND METHODS

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Growth conditions. B. bifidum PRL2010 was cultivated in an anaerobic atmosphere (2.99 % H2,

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17.01 % CO2 and 80 % N2) in a chamber (Concept 400, Ruskin) at 37°C for 32 hours in The Man-

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Rogosa-Sharp (MRS; Scharlau Chemie, Barcelona, Spain) medium, supplemented with 0.05 %

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(w/v) L-cysteine hydrochloride.

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Stimulation of Caco-2 monolayers and enzyme-linked immunosorbent assay measurement of

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cytokine production. A predetermined number of Caco-2 cells was seeded into 24-well plates and

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grown as described previously (14). B. bifidum PRL2010 cells (final concentration of 108 bacterial

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cells ml−1) were added to monolayers of Caco-2 cells at a multiplicity of infection (MOI) of 100 in

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0.5 ml of fresh antibiotic-free Dulbecco's modified Eagle's medium EMEM and incubated 18 h at

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37°C in 5 % CO2. This timing was optimized as described previously (14). Cytokine interleukin

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(IL)-1 (1 ng/ ml), casein (10 g/ml), and BSA (10 g/ml) were used as controls. Microtiter plates

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were kept at −70°C for 4 h to completely disrupt the Caco-2 cells. The supernatant and disrupted

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Caco-2 cells were then collected, and phenylmethylsulfonyl fluoride was added (20 mM final

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concentration). After centrifugation at a relative centrifugal force of 16,000 g for 1 min, levels of

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IL-2, IL-4, IL-6, IL-8, IL-10, IFN- , and TNF- in the supernatants were determined by ELISA

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using Bio-Plex human cytokine multiplex panel (Bio-Rad Laboratories, Segrate, Italy) as instructed

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by the manufacturer. Each sample was processed in duplicate in two independent experiments.

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Evaluation of NF- B activation. A stable recombinant Caco-2 cell line was obtained by

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transfecting cells with the plasmid pNiFty2-Luc (Invivogen, Labogen, Rho, Italy) as described

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previously (15). This plasmid combines five NF- B-dependent transcriptional activation sites

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upstream of the insect luciferase reporter gene luc. The presence of active NF- B molecules in the

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cell activates the promoter, resulting in expression of the luciferase gene. Following growth in the

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presence of 50 g/ml zeocin, cell monolayers (approximately 3 × 105 cells/well) were carefully 5


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washed with 0.1 M Tris-HCl buffer (pH 8.0). Subsequently, 50

l of a bacterial suspension

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containing 2.5 × 108 cells was added to 0.45 ml of fresh EMEM containing 100 mM HEPES (pH

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7.4). The resulting 500 l was finally pipetted into the microtiter plate well containing the Caco-2

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cell layer, resulting in a MOI of 100. Stimulation was conducted both in presence and absence of 2

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ng/ml of (IL)-1 . After incubation at 37°C for 4 h, the samples were treated and the

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bioluminescence was measured as described previously (16). All conditions were analyzed in

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triplicate in at least two independent experiments.

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Tissue culture experiments. About 2 x 105 HT29 cells in 1.5 ml of DMEM (high glucose, HEPES)

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medium supplemented with 10 % heat-inactivated fetal bovine serum (LifeTechnologies, Italy),

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penicillin (100 U/ml), streptomycin (0.1 mg/ml) and amphotericin B (0.25 µg/ml) and 4 mM L-

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glutamine were seeded into the upper compartments of a six-well transwell plate. The lower

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compartments contained 3.0 ml of the same medium. HT29 cells were incubated at 37 °C in a 5 %

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CO2 atmosphere until they reached three days post-confluence. Cells were then washed with Hank’s

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solution and stepped-down in DMEM medium supplemented with L-glutamine (4 mM), sodium

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selenite (0.2 µg/ml) and transferrin (5 µg/ml) for 24 h. These transwell inserts were transferred to an

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anaerobic culture box within an MACS-MG-1000 anaerobic workstation at 37 °C and each insert

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filled with anaerobic DMEM cell medium. A culture of B. bifidum PRL2010 grown to exponential

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phase was harvested by centrifugation at 3500 x g for 5 min and washed with 10 ml of anaerobic

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DMEM medium. The obtained pellet was resuspended in 0.8 ml of the same medium. 100 µl of

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bacterial suspension (108 CFU/ml) was added to wells, with control wells receiving the same

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amount of medium without bacterial cells. An additional control included bacterial cells incubated

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without HT29 cells.

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Tissue culture cells were harvested for analyses after 1h, 2h and 4h of incubation (with bacterial

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cultures), and 1h (un-exposed cell control placed in contact with PBS for 1h) for time course-

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dependent gene expression studies. This study design is similar to those employed in two previous 6


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studies, one involving B. bifidum PRL2010 and a Caco-2 human cell monolayer (6), the second

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involving human gut commensal bacteria and human epithelial cells (17). Non-adherent bacteria

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were carefully aspirated from the wells and pooled, while the adherent fraction was collected after

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washing of the inserts with anaerobic media and also pooled. Each fraction was collected into 1.5

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ml tubes, centrifuged at 3500 x g for 5 min, and the resulting pellet resuspended in 400 µl of

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RNAlater® and submitted to RNA extraction following the protocol described below. Caco-2 cells

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or HT29 cells were harvested from the wells, pooled, and stored in RNA later at 4°C.

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Mouse colonization. All procedures were approved by the University of Parma, as represented by

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the Institutional Animal Care and Use Committee (Dipartimento per la Sanità Pubblica Veterinaria,

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la Nutrizione e la Sicurezza degli Alimenti Direzione Generale della Sanità Animale e del Farmaco

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Veterinario). Two groups of mice were orally inoculated with either 109 CFU of PRL2010 cells

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(test strain) or water (control) according to previously described protocols (12, 18). Each group

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contained five animals of 3 month-old female BALB/c mice. Bacterial colonization was established

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by five consecutive daily administrations whereby each animal received 20 µl of 109 cells using a

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micropipette tip placed immediately behind the incisors (19). Bifidobacterial inocula were prepared

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as described previously (18).

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In order to estimate the number of B. bifidum PRL2010 cells per gram of feces, individual fecal

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samples were serially diluted and cultured on selective agar (MRS) using mupirocin as described

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previously (20). Following enumeration of B. bifidum PRL2010 in fecal samples, 100 random

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colonies were further tested to verify their identity using PCR primers targeting the pil2 and pil3

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loci (3).

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Animals were sacrificed by cervical dislocation and individual gastrointestinal tracts were removed

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and used for RNA extraction.

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Eukaryotic RNA isolation. Human cell lines as well as cells from murine gastrointestinal tracts in

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RNAlater were diluted 1:1 in an equal volume of sterile PBS, followed by centrifugation at 5000 x 7


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g for 10 min at 4ºC. Total RNA from the pellet was isolated using the RNeasy mini kit (Qiagen,

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Italy) according to the manufacturer's instructions, including an RNase-free DNase I (Qiagen, Italy)

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digestion step. Eukaryotic RNA integrity was determined using the Experion (BioRad, Italy).

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Acceptable RNA purity values (OD260/OD280) had to be ≥1.8 with corresponding RNA integrity

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numbers (RIN) ranging from 7 to 9.

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Microarray, description, labelling and hybridizations. Microarray analysis was performed with

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an oligonucleotide array based on the human genome as well as the murine genome from

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CombiMatrix. Oligos were present in triplicate on a 90k CombiMatrix array (CombiMatrix,

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Mulkiteo, USA). Replicates were distributed on the chip at random, non-adjacent positions. A set of

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74 negative control probes designed on phage and plant sequences were also included on the chip.

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Reverse transcription and amplification of 500 ng of total RNA was performed with ImProm-IITM

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Reverse Transcriptase (Promega, Madison, USA) according to the manufacturer’s instructions. 5 µg

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of cDNA was then labelled with ULS Labelling kit for Combimatrix arrays with Cy5 (Kreatech,

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The Netherlands). Hybridizations were performed according to a previously described protocol

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(21).

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Microarray data acquisition and treatment. Fluorescence scanning was performed on an

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InnoScan 710 microarray scanner (Innopsys, France). Signal intensities for each spot were

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determined using GenePix Pro 7 software (Molecular Devices, USA). Signal background was

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calculated as the mean of negative controls plus 2 times the standard deviation (22). A global

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quantile normalization was performed (23) and log2 ratios between the reference sample and the

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test samples were calculated. The distribution of the log2-transformed ratios was separately

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calculated for each hybridization reaction. The fold-change cut-off value for transcriptomic data

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analyses was of ≥ 2 for up-regulated genes and ≤ 0.5 for down-regulated genes. Only those genes

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whose transcription levels were shown to be significantly different (24) were further taken into

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consideration in our analyses. 8


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Statistical analysis. Statistical significance between means was analyzed using the unpaired

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Student's t test with a threshold P value of 24,311 cDNAs.

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Interestingly, a change in the transcription profile occurred when different exposure times to

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PRL2010 cells were used, 1,526 genes exhibited a significant change in their transcription for all

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time points (691 genes were up-regulated and 835 genes were down-regulated) (Figs. 2a and Fig.

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2c). The number of genes whose transcription exhibited significant changes when exposed to

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PRL2010 cells for different lengths of time are also described in Figures 2a and 2c. Functional

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classification of the up- and down-regulated PRL2010-affected human genes according to the Gene

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Ontology database using GO terms (AmiGo) is shown in Figures 2b and 2d. Notably, the majority

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of the host genes whose transcription was influenced by the presence of PRL2010 cells were shown

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to be involved in Immune Response and Positive Regulation of Response to Stimulus, which

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confirmed previously published data (6). Interestingly, we noticed that after 4 hours of treatment of

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HT29 with PRL2010 (time point T4) the number of genes for each GO assignment is consistently

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higher compared to the other time points assayed, thus suggesting that PRL2010 cells is most

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effective in modulating the transcriptome of HT29 human cell monolayer upon 4 hours of contact.

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These results also reinforce also what previously was noticed for similar experiments performed on

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a different type of human cell monolayer (6). In general, analysis of the HT29 monolayer

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transcriptome data suggests that PRL2010 elicits a host innate immune response. This idea is

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supported by the significant down-regulation of several genes coding for cytokines and cytokine

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receptors. Among these, we observed a decrease in transcription of genes specifying cytokines and

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a related receptor belonging to the IL-10 family (33), namely IL-20, IL-24 and the receptor for IL-

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22 (Fig. 3b). For instance, IL-20 has been shown to induce expression of pro-inflammatory

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cytokines, such as tumor necrosis factor (TNF)- in HaCat keratinocytes (34), and to act as a pro12


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inflammatory mediator in rheumatoid and experimental arthritis (35). Furthermore, the IL-22

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receptor, which is found on cells of non-hematopoietic origin in the skin, kidney, liver, lung, and

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gut, allows regulation of local epithelial responses after infection or exposure to inflammatory

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stimuli mediated by IL-22 (36). IL-22 is a pleiotropic cytokine that has been shown to either

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enhance maintenance of the epithelial barrier (37) or exert pro-inflammatory/pathologic properties

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(38) following infection. In addition, PRL2010 was shown to down-regulate transcription of the

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gene specifying IL-25, a member of the IL-17 family known to be expressed in epithelial cells, and

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involved in triggering Th2 type cytokines thus having an implicated role in allergy (39).

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The effect of PRL2010 cells on the HT29 transcriptome also positively affected the transcription of

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certain interleukin-encoding genes. In particular, PRL2010 exposure induced a marked

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transcriptional increase of 4-fold of the IL-11-encoding gene and a modest increase of 2.1-fold in

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transcription of the gene specifying its corresponding receptor IL11RA (Fig. 3a). Interestingly,

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preclinical in vivo and in vitro studies have demonstrated that IL-11 inhibits the secretion of a

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number of proinflammatory cytokines such as TNF, IL-12, IL-1 , and nitric oxide from activated

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macrophages as well as interferon (IFN)- and IL-12 from activated T cells (40-42).

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Intestinal epithelial cells are important players in gut homeostasis due to their role in regulating

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inflammation also through the expression of chemokines and related receptors and the

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consequential interaction with circulating leukocytes (43). Chemokines are a superfamily of

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chemotactic cytokines which are involved in attraction and recruitment of leukocytes at the site of

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inflammation, while they also play a role in angiogenesis and carcinogenesis (44). Our data shows

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that the stimulation of HT29 cells with B. bifidum PRL2010 induced significant transcriptional

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downregulation of genes that encode several chemokines and chemokine receptors (Fig. 3b). For

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instance, PRL2010 was shown to downregulate the genes coding for chemokines of the CXC-

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family, such as CXCL2 and CXCL3. These two chemokines (also known as growth-related

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oncogenes GRO2 and GRO3) have been found to be overexpressed in colorectal carcinoma 13


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compared to normal tissue (45). CXCL2 and CXCL3 are in fact considered biomarkers for the

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angiogenesis of tumors and their expression in HT29 was demonstrated to be triggered by pro-

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inflammatory stimulation (44). Nevertheless, in vivo studies in humans involving PRL2010 should

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be performed in order to further investigate the relevance and importance of these findings.

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Furthermore, PRL2010 was shown to markedly downregulate the transcription of the CXCL16-

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encoding gene. Interestingly, CXCL16 is considered a marker for inflammation due to its perceived

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role in the development of IBD (46). In addition, expression of CXCL16 has been found to be

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upregulated in several types of primary and metastatic cancer tissue and cancer cell lines, and has

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been proposed as a prognostic marker for colorectal cancer (CRC) (47).

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The chemokine whose corresponding gene was most strongly repressed by PRL2010 exposure was

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CCL22 (Fig. 3b). Also known as macrophage-derived chemokine (MDC), CCL22 has been found

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to be expressed in the small intestine (48) and was demonstrated to be a potent chemoattractant of

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immune cells, in particular immature dendritic cells (DCs). High levels of CCL22 have been found

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following stimulation with pro-inflammatory stimuli like TNF , IL-1 , IFN- and LPS (49, 50),

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and in specimens of inflamed mucosa (51).

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Finally, PRL2010 exposure induced a strong transcriptional increase (5-fold) of the gene encoding

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CCL19, which is a chemokine that attracts both dendritic cells (DC) and T lymphocytes. CCL19

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expression has been found to be enhanced during inflammation by cells properly associated to the

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immune system (e.g. dendritic cells) (52). Nonetheless, it is noteworthy that CCL19 has been

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shown to serve as a potential immune stimulator that can reduce tumor burden in a model of

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advanced lung cancer (53). Taken together, these results indicate an opposing effect of PRL2010

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exposure on the production of certain chemokines, such as CCL22 and CCL19, with apparent

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similar function, which supports the idea that PRL2010 has a balanced effect on chemokine-

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mediated immune functions.

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It has been well established that the intestinal mucosa plays a pivotal role in rejecting pathogens,

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dampening inflammatory responses and discriminating between “friends and foes”, thus

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orchestrating tolerance to or mounting immune responses against commensals or pathogens,

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respectively. Consequently, the overall ability of B. bifidum PRL2010 to modulate

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cytokine/chemokine gene expression in HT29 cells would suggest that this bacterium can actively

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exert tolerogenic and potentially immunomodulatory effects.

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The ability of PRL2010 to modulate inflammatory responses is corroborated by the observed

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transcriptional down-regulation of the genes specifying various heat shock proteins (HSPs) of the

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host (Fig. 3b). Although best described as protein chaperones during cellular stress (54), recent

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evidence suggests that HSPs are also important for activation of the innate immune response (55).

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Release of inducible HSPs from necrotic cells is interpreted as a danger signal by antigen-

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presenting cells, and as a result causes cellular activation and cytokine production (56, 57).

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Furthermore, peptides binding to HSPs act as source of antigen and affect the maturation of

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dendritic cells (56, 58). Upregulation of HSP expression is the general response to all types of

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general stresses including infection (59). Consequently, down-regulation of HSPs by B. bifidum

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PRL2010 can be interpreted as a means to affect the host inflammatory response to pathogens. This

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scenario is completely different than that caused by many enteropathogens, which have been shown

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to increase the expression level of HSPs (60). Furthermore, according to a recent model regarding

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tumor development, HSPs have been implicated in tumorigenesis resulting from both infection as

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well as chronic inflammation (60).

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Other genes involved in bacterial-host interaction are genes encoding for integral transmembrane

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proteins identified in tight junctions called cadherin and claudin (61). Cadherins and claudins play

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an important role in the maintenance of cell-cell adhesion and recognition, and we found an up-

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regulation especially for Cadherin type 1 (3.5-fold; p