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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A Commensal <italic>Helicobacter</italic> sp. of the Rodent Intestinal Flora Activates TLR2 and NOD1 Responses in Epithelial Cells</title><author><name sortKey="Chaouche Drider, Nadia" sort="Chaouche Drider, Nadia" uniqKey="Chaouche Drider N" first="Nadia" last="Chaouche-Drider">Nadia Chaouche-Drider</name><affiliation><nlm:aff id="aff1"><addr-line>Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kaparakis, Maria" sort="Kaparakis, Maria" uniqKey="Kaparakis M" first="Maria" last="Kaparakis">Maria Kaparakis</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Karrar, Abdulgader" sort="Karrar, Abdulgader" uniqKey="Karrar A" first="Abdulgader" last="Karrar">Abdulgader Karrar</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fernandez, Maria Isabel" sort="Fernandez, Maria Isabel" uniqKey="Fernandez M" first="Maria-Isabel" last="Fernandez">Maria-Isabel Fernandez</name><affiliation><nlm:aff id="aff3"><addr-line>Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><addr-line>INSERM U389, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Carneiro, Letitia A M" sort="Carneiro, Letitia A M" uniqKey="Carneiro L" first="Letitia A. M." last="Carneiro">Letitia A. M. Carneiro</name><affiliation><nlm:aff id="aff5"><addr-line>Institut Pasteur, Groupe d'Immunité Innée et Signalisation, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Department of Immunology, University of Toronto, Toronto, Ontario, Canada</addr-line></nlm:aff></affiliation></author><author><name sortKey="Viala, Jerome" sort="Viala, Jerome" uniqKey="Viala J" first="Jérôme" last="Viala">Jérôme Viala</name><affiliation><nlm:aff id="aff5"><addr-line>Institut Pasteur, Groupe d'Immunité Innée et Signalisation, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Boneca, Ivo Gomperts" sort="Boneca, Ivo Gomperts" uniqKey="Boneca I" first="Ivo Gomperts" last="Boneca">Ivo Gomperts Boneca</name><affiliation><nlm:aff id="aff1"><addr-line>Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff7"><addr-line>Institut Pasteur, Groupe de Biologie et Génétique de la Paroi Bactérienne, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff8"><addr-line>INSERM, Groupe Avenir, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Moran, Anthony P" sort="Moran, Anthony P" uniqKey="Moran A" first="Anthony P." last="Moran">Anthony P. Moran</name><affiliation><nlm:aff id="aff9"><addr-line>Department of Microbiology, National University of Ireland, Galway, Ireland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Philpott, Dana J" sort="Philpott, Dana J" uniqKey="Philpott D" first="Dana J." last="Philpott">Dana J. Philpott</name><affiliation><nlm:aff id="aff5"><addr-line>Institut Pasteur, Groupe d'Immunité Innée et Signalisation, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Department of Immunology, University of Toronto, Toronto, Ontario, Canada</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ferrero, Richard L" sort="Ferrero, Richard L" uniqKey="Ferrero R" first="Richard L." last="Ferrero">Richard L. Ferrero</name><affiliation><nlm:aff id="aff1"><addr-line>Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19401779</idno><idno type="pmc">2671595</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671595</idno><idno type="RBID">PMC:2671595</idno><idno type="doi">10.1371/journal.pone.0005396</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">001525</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001525</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A Commensal <italic>Helicobacter</italic> sp. of the Rodent Intestinal Flora Activates TLR2 and NOD1 Responses in Epithelial Cells</title><author><name sortKey="Chaouche Drider, Nadia" sort="Chaouche Drider, Nadia" uniqKey="Chaouche Drider N" first="Nadia" last="Chaouche-Drider">Nadia Chaouche-Drider</name><affiliation><nlm:aff id="aff1"><addr-line>Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kaparakis, Maria" sort="Kaparakis, Maria" uniqKey="Kaparakis M" first="Maria" last="Kaparakis">Maria Kaparakis</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Karrar, Abdulgader" sort="Karrar, Abdulgader" uniqKey="Karrar A" first="Abdulgader" last="Karrar">Abdulgader Karrar</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fernandez, Maria Isabel" sort="Fernandez, Maria Isabel" uniqKey="Fernandez M" first="Maria-Isabel" last="Fernandez">Maria-Isabel Fernandez</name><affiliation><nlm:aff id="aff3"><addr-line>Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><addr-line>INSERM U389, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Carneiro, Letitia A M" sort="Carneiro, Letitia A M" uniqKey="Carneiro L" first="Letitia A. M." last="Carneiro">Letitia A. M. Carneiro</name><affiliation><nlm:aff id="aff5"><addr-line>Institut Pasteur, Groupe d'Immunité Innée et Signalisation, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Department of Immunology, University of Toronto, Toronto, Ontario, Canada</addr-line></nlm:aff></affiliation></author><author><name sortKey="Viala, Jerome" sort="Viala, Jerome" uniqKey="Viala J" first="Jérôme" last="Viala">Jérôme Viala</name><affiliation><nlm:aff id="aff5"><addr-line>Institut Pasteur, Groupe d'Immunité Innée et Signalisation, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Boneca, Ivo Gomperts" sort="Boneca, Ivo Gomperts" uniqKey="Boneca I" first="Ivo Gomperts" last="Boneca">Ivo Gomperts Boneca</name><affiliation><nlm:aff id="aff1"><addr-line>Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff7"><addr-line>Institut Pasteur, Groupe de Biologie et Génétique de la Paroi Bactérienne, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff8"><addr-line>INSERM, Groupe Avenir, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Moran, Anthony P" sort="Moran, Anthony P" uniqKey="Moran A" first="Anthony P." last="Moran">Anthony P. Moran</name><affiliation><nlm:aff id="aff9"><addr-line>Department of Microbiology, National University of Ireland, Galway, Ireland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Philpott, Dana J" sort="Philpott, Dana J" uniqKey="Philpott D" first="Dana J." last="Philpott">Dana J. Philpott</name><affiliation><nlm:aff id="aff5"><addr-line>Institut Pasteur, Groupe d'Immunité Innée et Signalisation, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Department of Immunology, University of Toronto, Toronto, Ontario, Canada</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ferrero, Richard L" sort="Ferrero, Richard L" uniqKey="Ferrero R" first="Richard L." last="Ferrero">Richard L. Ferrero</name><affiliation><nlm:aff id="aff1"><addr-line>Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Paris, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Helicobacter</italic> spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal <italic>Helicobacter</italic> spp. are known to induce colitis in mouse models, yet the mechanisms by which these bacteria induce intestinal inflammation are poorly understood. To address this question, we performed <italic>in vitro</italic> co-culture experiments with mouse and human epithelial cell lines stimulated with a selection of <italic>Helicobacter</italic> spp., including known pathogenic species as well as ones for which the pathogenic potential is less clear. Strikingly, a member of the normal microflora of rodents, <italic>Helicobacter muridarum</italic>, was found to be a particularly strong inducer of CXC chemokine (Cxcl1/KC, Cxcl2/MIP-2) responses in a murine intestinal epithelial cell line. Time-course studies revealed a biphasic pattern of chemokine responses in these cells, with <italic>H. muridarum</italic> lipopolysaccharide (LPS) mediating early (24–48 h) responses and live bacteria seeming to provoke later (48–72 h) responses. <italic>H. muridarum</italic> LPS per se was shown to induce CXC chemokine production in HEK293 cells stably expressing Toll-like receptor 2 (TLR2), but not in those expressing TLR4. In contrast, live <italic>H. muridarum</italic> bacteria were able to induce NF-κB reporter activity and CXC chemokine responses in TLR2–deficient HEK293 and in AGS epithelial cells. These responses were attenuated by transient transfection with a dominant negative construct to NOD1, and by stable expression of NOD1 siRNA, respectively. Thus, the data suggest that both TLR2 and NOD1 may be involved in innate immune sensing of <italic>H. muridarum</italic> by epithelial cells. This work identifies <italic>H. muridarum</italic> as a commensal bacterium with pathogenic potential and underscores the potential roles of ill-defined members of the normal flora in the initiation of inflammation in animal hosts. We suggest that <italic>H. muridarum</italic> may act as a confounding factor in colitis model studies in rodents.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article" xml:lang="EN"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title>PLoS ONE</journal-title><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19401779</article-id><article-id pub-id-type="pmc">2671595</article-id><article-id pub-id-type="publisher-id">09-PONE-RA-08434R1</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0005396</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline"><subject>Immunology/Innate Immunity</subject><subject>Microbiology/Cellular Microbiology and Pathogenesis</subject><subject>Gastroenterology and Hepatology/Gastrointestinal Infections</subject><subject>Gastroenterology and Hepatology/Inflammatory Bowel Disease</subject></subj-group></article-categories><title-group><article-title>A Commensal <italic>Helicobacter</italic> sp. of the Rodent Intestinal Flora Activates TLR2 and NOD1 Responses in Epithelial Cells</article-title><alt-title alt-title-type="running-head">TLR/NLR Responses to Bacteria</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Chaouche-Drider</surname><given-names>Nadia</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kaparakis</surname><given-names>Maria</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Karrar</surname><given-names>Abdulgader</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Fernandez</surname><given-names>Maria-Isabel</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Carneiro</surname><given-names>Letitia A. M.</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref><xref ref-type="aff" rid="aff6"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Viala</surname><given-names>Jérôme</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Boneca</surname><given-names>Ivo Gomperts</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff7"><sup>7</sup></xref><xref ref-type="aff" rid="aff8"><sup>8</sup></xref></contrib><contrib contrib-type="author"><name><surname>Moran</surname><given-names>Anthony P.</given-names></name><xref ref-type="aff" rid="aff9"><sup>9</sup></xref></contrib><contrib contrib-type="author"><name><surname>Philpott</surname><given-names>Dana J.</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref><xref ref-type="aff" rid="aff6"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Ferrero</surname><given-names>Richard L.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Paris, France</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></aff><aff id="aff3"><label>3</label><addr-line>Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France</addr-line></aff><aff id="aff4"><label>4</label><addr-line>INSERM U389, Paris, France</addr-line></aff><aff id="aff5"><label>5</label><addr-line>Institut Pasteur, Groupe d'Immunité Innée et Signalisation, Paris, France</addr-line></aff><aff id="aff6"><label>6</label><addr-line>Department of Immunology, University of Toronto, Toronto, Ontario, Canada</addr-line></aff><aff id="aff7"><label>7</label><addr-line>Institut Pasteur, Groupe de Biologie et Génétique de la Paroi Bactérienne, Paris, France</addr-line></aff><aff id="aff8"><label>8</label><addr-line>INSERM, Groupe Avenir, Paris, France</addr-line></aff><aff id="aff9"><label>9</label><addr-line>Department of Microbiology, National University of Ireland, Galway, Ireland</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Ratner</surname><given-names>Adam J.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1">Columbia University, United States of America</aff><author-notes><corresp id="cor1">* E-mail: <email>Richard.Ferrero@med.monash.edu.au</email></corresp><fn fn-type="con"><p>Conceived and designed the experiments: NCD MK RLF. Performed the experiments: NCD MK AK RLF. Analyzed the data: NCD AK RLF. Contributed reagents/materials/analysis tools: MIF LC JV IGB AM DJP. Wrote the paper: RLF.</p></fn></author-notes><pub-date pub-type="collection"><year>2009</year></pub-date><pub-date pub-type="epub"><day>29</day><month>4</month><year>2009</year></pub-date><volume>4</volume><issue>4</issue><elocation-id>e5396</elocation-id><history><date date-type="received"><day>1</day><month>2</month><year>2009</year></date><date date-type="accepted"><day>2</day><month>4</month><year>2009</year></date></history><copyright-statement>Chaouche-Drider et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</copyright-statement><copyright-year>2009</copyright-year><abstract><p><italic>Helicobacter</italic> spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal <italic>Helicobacter</italic> spp. are known to induce colitis in mouse models, yet the mechanisms by which these bacteria induce intestinal inflammation are poorly understood. To address this question, we performed <italic>in vitro</italic> co-culture experiments with mouse and human epithelial cell lines stimulated with a selection of <italic>Helicobacter</italic> spp., including known pathogenic species as well as ones for which the pathogenic potential is less clear. Strikingly, a member of the normal microflora of rodents, <italic>Helicobacter muridarum</italic>, was found to be a particularly strong inducer of CXC chemokine (Cxcl1/KC, Cxcl2/MIP-2) responses in a murine intestinal epithelial cell line. Time-course studies revealed a biphasic pattern of chemokine responses in these cells, with <italic>H. muridarum</italic> lipopolysaccharide (LPS) mediating early (24–48 h) responses and live bacteria seeming to provoke later (48–72 h) responses. <italic>H. muridarum</italic> LPS per se was shown to induce CXC chemokine production in HEK293 cells stably expressing Toll-like receptor 2 (TLR2), but not in those expressing TLR4. In contrast, live <italic>H. muridarum</italic> bacteria were able to induce NF-κB reporter activity and CXC chemokine responses in TLR2–deficient HEK293 and in AGS epithelial cells. These responses were attenuated by transient transfection with a dominant negative construct to NOD1, and by stable expression of NOD1 siRNA, respectively. Thus, the data suggest that both TLR2 and NOD1 may be involved in innate immune sensing of <italic>H. muridarum</italic> by epithelial cells. This work identifies <italic>H. muridarum</italic> as a commensal bacterium with pathogenic potential and underscores the potential roles of ill-defined members of the normal flora in the initiation of inflammation in animal hosts. We suggest that <italic>H. muridarum</italic> may act as a confounding factor in colitis model studies in rodents.</p></abstract><counts><page-count count="10"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p>Mouse models of “spontaneous colitis” have been reported to mimic the lesions observed in human inflammatory bowel disease (IBD) <xref ref-type="bibr" rid="pone.0005396-Blumberg1">[1]</xref>. Though the underlying immunological defects conferring host susceptibility to colitis in these models vary, they all depend on the presence of an intestinal microflora for the initiation of disease <xref ref-type="bibr" rid="pone.0005396-Blumberg1">[1]</xref>. The genus <italic>Helicobacter</italic> contains several intestinal species that have been linked with the development of colitis in mammalian hosts <xref ref-type="bibr" rid="pone.0005396-Ferrero1">[2]</xref>. Two such species, <italic>Helicobacter bilis</italic> <xref ref-type="bibr" rid="pone.0005396-Shomer1">[3]</xref> and <italic>Helicobacter hepaticus</italic> <xref ref-type="bibr" rid="pone.0005396-Cahill1">[4]</xref>, <xref ref-type="bibr" rid="pone.0005396-Kullberg1">[5]</xref>, have been shown to induce particularly severe forms of colitis in mice with defects in cytokine or T-cell functions, whereas <italic>Helicobacter cinaedi</italic> and <italic>Helicobacter fennelliae</italic> have been associated with colitis in humans <xref ref-type="bibr" rid="pone.0005396-Ferrero1">[2]</xref> Many intestinal <italic>Helicobacter</italic> spp., however, do not have a clearly defined role in pathogenesis. One example is <italic>Helicobacter muridarum</italic>, which was initially described as a member of the normal flora of conventional rodents <xref ref-type="bibr" rid="pone.0005396-Phillips1">[6]</xref>. Subsequent studies, however, showed that <italic>H. muridarum</italic> could induce colitis and gastritis in mice, suggesting a potentially pathogenic role for the bacterium <xref ref-type="bibr" rid="pone.0005396-Jiang1">[7]</xref>, <xref ref-type="bibr" rid="pone.0005396-Lee1">[8]</xref>, <xref ref-type="bibr" rid="pone.0005396-Queiroz1">[9]</xref>.</p><p>Intestinal epithelial cells have long been considered to act as a physical barrier that protects the integrity of epidermal or mucosal surfaces. A growing body of evidence now suggests an active role for these cells in host defense. Indeed, intestinal epithelial cells express several types of transmembrane pathogen recognition molecule (PRM) <xref ref-type="bibr" rid="pone.0005396-Cario1">[10]</xref>, <xref ref-type="bibr" rid="pone.0005396-Hornef1">[11]</xref>. Among the best characterized of these molecules are Toll-like receptor 2 (TLR2), which recognizes lipoprotein/lipoteichoic acid as well as atypical forms of lipopolysaccharide (LPS) <xref ref-type="bibr" rid="pone.0005396-Smith1">[12]</xref>, <xref ref-type="bibr" rid="pone.0005396-Triantafilou1">[13]</xref>, and TLR4, which responds to the classical forms of Gram-negative LPS, such as that of <italic>Escherichia coli</italic>. Although PRMs are thought to play a crucial role in host immune responses to microbial pathogens, the mechanisms by which such molecules discriminate between pathogenic organisms and the host commensal microflora remain unclear. Unrestrained activation of intestinal innate immune molecules by micro-organisms plays a fundamental role in the pathophysiology of IBD <xref ref-type="bibr" rid="pone.0005396-MacDonald1">[14]</xref>.</p><p>In addition to the TLRs, a family of intracytoplasmic PRMs with homology to plant resistance proteins, known as the NOD-like receptors (NLRs) <xref ref-type="bibr" rid="pone.0005396-Fritz1">[15]</xref>, has been described. Two members of this family, NOD1 (or CARD4) and NOD2 (or CARD15), were reported to respond to peptidoglycan, a component of bacterial cell walls <xref ref-type="bibr" rid="pone.0005396-Girardin1">[16]</xref>, <xref ref-type="bibr" rid="pone.0005396-Inohara1">[17]</xref>. NOD1 displayed a high specificity for Gram-negative peptidoglycan <xref ref-type="bibr" rid="pone.0005396-Girardin2">[18]</xref>. Moreover, this molecule was implicated in epithelial cell responses to a variety of gastrointestinal bacteria, including the gastric pathogen <italic>Helicobacter pylori</italic> <xref ref-type="bibr" rid="pone.0005396-Fritz1">[15]</xref>.</p><p>Host cell recognition of micro-organisms or their products via PRMs is known to initiate pro-inflammatory signaling events that converge on the transcription factor, nuclear factor-κB (NF-κB), culminating in up-regulated cytokine/chemokine gene expression in cells. Epithelial cell-derived CXC chemokines, such as CXCL8 (or interleukin-8), are critical mediators in the development of local inflammation in human IBD <xref ref-type="bibr" rid="pone.0005396-Mazzucchelli1">[19]</xref>. Mice do not possess a homolog of CXCL8, and produce instead Cxcl1 (KC) and Cxcl2 (MIP-2); these two chemokines are the major promoters of leukocyte recruitment in murine tissues <xref ref-type="bibr" rid="pone.0005396-Ohtsuka1">[20]</xref>, <xref ref-type="bibr" rid="pone.0005396-Remick1">[21]</xref>.</p><p>While the pathophysiology of <italic>Helicobacter</italic>-induced colitis in mouse models is well characterized, the inflammatory mediators and PRMs involved in the development of inflammation in these models is still poorly understood. We sought to address this question by studying NF-κB and/or CXC chemokine responses in epithelial cell lines stimulated with a selection of intestinal <italic>Helicobacter</italic> spp. This work has allowed us to identify, for the first time, the contribution of two PRMs, TLR2 and NOD1, in innate immune recognition of a commensal <italic>Helicobacter</italic> sp. of the normal rodent flora.</p></sec><sec id="s2"><title>Results</title><sec id="s2a"><title>Enterohepatic <italic>Helicobacter</italic> spp. induce CXC chemokine synthesis in murine intestinal epithelial cells</title><p>The effect of murine <italic>Helicobacter</italic> spp. on pro-inflammatory cytokine production in epithelial cells was evaluated in the mouse small intestinal cell line, m-IC<sub>cl2</sub> <xref ref-type="bibr" rid="pone.0005396-Bens1">[22]</xref>. For the purposes of this study, we chose two pathogenic species, <italic>H. bilis</italic> and <italic>H. hepaticus</italic> <xref ref-type="bibr" rid="pone.0005396-Shomer1">[3]</xref>, <xref ref-type="bibr" rid="pone.0005396-Cahill1">[4]</xref>, <xref ref-type="bibr" rid="pone.0005396-Kullberg1">[5]</xref>, and one species of ill-defined pathogenicity, <italic>H. muridarum</italic>.</p><p>All three <italic>Helicobacter</italic> spp. induced increases in gene expression of the CXC chemokines, Cxcl1 and Cxcl2 (<xref ref-type="fig" rid="pone-0005396-g001">Figure 1A and 1B</xref>). <italic>Cxcl1 and Cxcl2</italic> mRNA levels were maximal at 18 h post-stimulation and were greatest in cells stimulated with either <italic>H. bilis</italic> ATCC51630 or <italic>H. muridarum</italic> ATCC49282. The levels of Cxcl1 and Cxcl2 steadily increased over time in <italic>Helicobacter</italic>-stimulated m-IC<sub>cl2</sub> cells, with maximal levels present at between 48 and 72 h post-stimulation (<xref ref-type="fig" rid="pone-0005396-g001">Figure 1C and 1D</xref>). In agreement with the mRNA data, <italic>H. muridarum</italic> ATCC49282 and <italic>H. bilis</italic> ATCC51630 consistently induced 5- to 10-fold greater quantities of Cxcl1 and Cxcl2 production when compared to <italic>H. hepaticus</italic> ATCC51448. Interestingly, Sterzenbach and colleagues <xref ref-type="bibr" rid="pone.0005396-Sterzenbach1">[23]</xref> also reported poor responses of m-IC<sub>cl2</sub> cells to stimulation with live <italic>H. hepaticus</italic>. As previously described for LPS-stimulation <xref ref-type="bibr" rid="pone.0005396-Hornef1">[11]</xref>, stimulation with <italic>Helicobacter</italic> bacteria did not appear to induce interleukin-6 nor tumor necrosis factor production in m-IC<sub>cl2</sub> cells (data not shown).</p><fig id="pone-0005396-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0005396.g001</object-id><label>Figure 1</label><caption><title>Intestinal <italic>Helicobacter</italic> spp. induce CXC chemokine responses in m-IC<sub>cl2</sub> epithelial cells.</title><p>(A) <italic>Cxcl1</italic> and (B) <italic>Cxcl2</italic> mRNA expression was detected by RT-PCR in (1) unstimulated cells and in cells stimulated for 18 h with (2) <italic>H. bilis</italic> ATCC51630, (3) <italic>H. hepaticus</italic> ATCC51448, or (4) <italic>H. muridarum</italic> ATCC49282. The quantities of total RNA were standardized by performing PCR with oligonucleotides specific for β-actin (<italic>ACTB</italic>). These data are representative of two independent experiments. The levels of secreted (C) Cxcl1 and (D) Cxcl2 were measured at the indicated times in the culture supernatants of control unstimulated m-IC<sub>cl2</sub> cells, and cells stimulated with either <italic>H. bilis</italic> ATCC51630, <italic>H. hepaticus</italic> ATCC51448 or <italic>H. muridarum</italic> ATCC49282. Data correspond to the mean±SD (triplicate determinations) and are representative of three independent experiments. Statistical differences were observed between Cxcl1 or Cxcl2 levels following bacterial stimulation compared to control cells (*, <italic>P</italic><0.03; **, <italic>P</italic><0.001).</p></caption><graphic xlink:href="pone.0005396.g001"></graphic></fig></sec><sec id="s2b"><title>Cell contact-dependent chemokine responses in murine epithelial cells to live <italic>H. muridarum</italic> bacteria</title><p>Given that <italic>H. muridarum</italic> induced consistently high levels of chemokine production in m-IC<sub>cl2</sub> cells, and that its potential pathogenicity is poorly understood, we chose to further investigate the interactions of this <italic>Helicobacter</italic> sp. with epithelial cells. The first aim was to determine the role of cell-cell contact in <italic>H. muridarum</italic> induction of CXC chemokine synthesis in m-IC<sub>cl2</sub> cells. The separation of <italic>H. muridarum</italic> ATCC49282 bacteria from m-IC<sub>cl2</sub> cells by Transwell filter membranes was found to completely abrogate the Cxcl1 and Cxcl2 responses observed at 48 h post-incubation (<xref ref-type="fig" rid="pone-0005396-g002">Figure 2</xref>). Moreover, increasing the numbers of Transwell-separated <italic>H. muridarum</italic> bacteria to ten times those required in direct contact assays did not augment CXC chemokine production in the cells (<xref ref-type="fig" rid="pone-0005396-g002">Figure 2</xref>). These data demonstrated that direct bacterial-cell contact was required for epithelial cell responses at 48 h post-incubation with <italic>H. muridarum</italic> ATCC49282. Furthermore, the data suggest that it is unlikely that the responses observed at ≥48 h could be mediated by bacterial products released during bacterial lysis, as these products would not be retained by Transwell filters. In which case, the filters should not have had any effect on chemokine production by the cells.</p><fig id="pone-0005396-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0005396.g002</object-id><label>Figure 2</label><caption><title><italic>H. muridarum</italic> induces CXC chemokine production in m-IC<sub>cl2</sub> cells by a contact-dependent mechanism.</title><p>m-IC<sub>cl2</sub> cells were either left unstimulated (control) or stimulated with different amounts (expressed as the MOI) of live <italic>H. muridarum</italic> ATCC49282 bacteria. The bacteria were incubated in direct contact (dc) or separated from the cells by a transwell (tw) filter membrane. The levels of secreted (A) Cxcl1 and (B) Cxcl2 were measured in culture supernatants at 48 h post-stimulation. Data correspond to the mean±SD (triplicate determinations). Statistical differences were observed between Cxcl1 or Cxcl2 levels following bacterial stimulation compared to control cells (**, <italic>P</italic>≤0.0002).</p></caption><graphic xlink:href="pone.0005396.g002"></graphic></fig></sec><sec id="s2c"><title><italic>H. muridarum</italic> LPS induces pro-inflammatory responses in epithelial cells</title><p>It was previously reported that m-IC<sub>cl2</sub> cells produce Cxcl2 in response to stimulation with <italic>E. coli</italic> LPS <xref ref-type="bibr" rid="pone.0005396-Hornef1">[11]</xref>. We therefore wished to determine whether <italic>H. muridarum</italic> LPS could induce similar responses in this cell line. Indeed, 0.1 µg/ml of highly purified <italic>H. muridarum</italic> ATCC49282 LPS was sufficient to induce a significant increase in Cxcl1 and Cxcl2 production in m-IC<sub>cl2</sub> cells (<italic>P</italic><0.0001; <xref ref-type="fig" rid="pone-0005396-g003">Figure 3A</xref>), with maximal responses detected at 24 h post-stimulation (<xref ref-type="fig" rid="pone-0005396-g003">Figure 3B and 3C</xref>). Interestingly, in contrast to the findings for purified LPS, live <italic>H. muridarum</italic> ATCC49282 bacteria induced significantly higher levels of Cxcl1 and Cxcl2 at the later time-points of 48 and 72 h (<italic>P</italic> = 0.0002 and <italic>P</italic><0.0001; and <italic>P</italic><0.0001 and <italic>P</italic><0.0001, respectively; <xref ref-type="fig" rid="pone-0005396-g003">Figure 3B and 3C</xref>). Thus, the data suggested a biphasic pattern of responses in m-IC<sub>cl2</sub> cells to <italic>H. muridarum</italic>.</p><fig id="pone-0005396-g003" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0005396.g003</object-id><label>Figure 3</label><caption><title><italic>H. muridarum</italic> induces CXC chemokine production in m-IC<sub>cl2</sub> cells by LPS-dependent and -independent mechanisms.</title><p>m-IC<sub>cl2</sub> cells were either left unstimulated (control) or stimulated with purified <italic>H. muridarum</italic> LPS or live <italic>H. muridarum</italic> ATCC49282 bacteria. The levels of secreted (A) Cxcl1 and Cxcl2 in the culture supernatants of cells stimulated for 48 h with different concentrations of <italic>H. muridarum</italic> LPS. Comparison of (B) Cxcl1 and (C) Cxcl2 levels in culture supernatants of cells stimulated for the indicated times with either 0.1 µg/ml <italic>H. muridarum</italic> ATCC49282 LPS or live <italic>H. muridarum</italic> ATCC49282 bacteria. Data correspond to the mean±SD (triplicate determinations) and are representative of three independent experiments. Statistical differences were observed between Cxcl1 or Cxcl2 levels following bacterial stimulation compared to control cells (*, <italic>P</italic> = 0.0002; **, <italic>P</italic><0.0001).</p></caption><graphic xlink:href="pone.0005396.g003"></graphic></fig><p>Given that LPS appeared to be the key agonist involved in the early chemokine responses of m-IC<sub>cl2</sub> cells to <italic>H. muridarum</italic> stimulation, we wished to determine the PRM involved in sensing of <italic>H. muridarum</italic> LPS. Using human embryonic kidney (HEK293) cell lines stably expressing either TLR2 or TLR4, we were able to show dose-dependent CXCL8 responses to <italic>H. muridarum</italic> ATCC49282 and <italic>H. pylori</italic> NCTC11637 LPS only in the TLR2-expressing cells (<xref ref-type="fig" rid="pone-0005396-g004">Figure 4</xref>). <italic>E. coli</italic> 0111:B4 LPS was used as a control in these experiments. As expected, HEK293 cells expressing exogenous TLR4 responded to this classical form of Gram-negative LPS, whereas no significant responses were detected in HEK293 cells, which are naturally deficient in either TLR2 or TLR4 <xref ref-type="bibr" rid="pone.0005396-Girardin2">[18]</xref>, <xref ref-type="bibr" rid="pone.0005396-KurtJones1">[24]</xref>, nor in those expressing exogenous TLR2. As m-IC<sub>cl2</sub> cells were shown to be responsive to the TLR2 agonist, Pam-3-Cys <xref ref-type="bibr" rid="pone.0005396-Hornef1">[11]</xref>, it is likely that TLR2 was responsible for the early responses of these cells to <italic>H. muridarum</italic> LPS.</p><fig id="pone-0005396-g004" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0005396.g004</object-id><label>Figure 4</label><caption><title><italic>H. muridarum</italic> LPS is specifically recognized by TLR2.</title><p>Normal HEK293 cells (A), as well as HEK293 cells stably expressing either TLR2 (B) or TLR4 (C), were either left untreated (control) or stimulated with highly purified LPS from <italic>E. coli</italic> 0111:B4, <italic>H. muridarum</italic> ATCC49282 or <italic>H. pylori</italic> NCTC11637. The levels of secreted CXCL8 were measured in culture supernatants at 24 h post-stimulation. Data correspond to the mean±SD (triplicate determinations) and are representative of two independent experiments. Statistical differences were observed between the CXCL8 responses of LPS-stimulated and control cells (*, <italic>P</italic><0.0001).</p></caption><graphic xlink:href="pone.0005396.g004"></graphic></fig></sec><sec id="s2d"><title>Live <italic>H. muridurum</italic> bacteria induce NOD1-dependent responses in HEK293 and AGS cells</title><p>The studies in m-IC<sub>cl2</sub> cell line suggested that an LPS-independent mechanism was likely to mediate the late chemokine responses to live <italic>H. muridarum</italic> bacteria (<xref ref-type="fig" rid="pone-0005396-g003">Figure 3B and 3C</xref>). To address this question, we used HEK293 cells which are naturally deficient in TLR2 and TLR4 expression <xref ref-type="bibr" rid="pone.0005396-Girardin2">[18]</xref>. As the HEK293 cell line is of human origin, we included in these experiments the human intestinal <italic>Helicobacter</italic> isolates, <italic>H. cinaedi</italic> and <italic>H. fennelliae</italic>.</p><p><italic>H. muridarum</italic> ATCC49282 induced a significant increase in NF-κB reporter activity in HEK293 cells, when compared to unstimulated cells (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5A</xref>; <italic>P</italic><0.05). Similar to the findings for the m-IC<sub>cl2</sub> cell line, HEK293 responses to live <italic>H. muridarum</italic> were cell contact-dependent (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5A</xref>). These responses could not, however, be attributed to the invasive properties of <italic>H. muridarum</italic> as this bacterium was found to be no more invasive than either <italic>H. bilis</italic> or <italic>H. hepaticus</italic> (<xref ref-type="supplementary-material" rid="pone.0005396.s001">Figure S1</xref>). Strikingly, the various other intestinal <italic>Helicobacter</italic> spp. tested here, including the human isolates <italic>H. cinaedi</italic> ATCC35683 and <italic>H. fennelliae</italic> ATCC35684, were very weak inducers of NF-κB reporter activity in HEK293 cells (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5A</xref>).</p><fig id="pone-0005396-g005" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0005396.g005</object-id><label>Figure 5</label><caption><title><italic>H. muridarum</italic> induces NOD1 responses in HEK293 and AGS cells.</title><p>(A) NF-κB-dependent luciferase reporter activity was measured in HEK293 cells that had been incubated for 24 h in direct contact, or separated by a transwell filter membrane, from the following bacteria: <italic>H. bilis</italic> ATCC51630, <italic>H. cinaedi</italic> ATCC35683 (<italic>H. cin</italic>), <italic>H. fennelliae</italic> ATCC35684 (<italic>H. fenn</italic>), <italic>H. hepaticus</italic> ATCC51448 (<italic>H. hep</italic>), or <italic>H. muridarum</italic> ATCC49282 (<italic>H. murid</italic>). Control cells were transfected with the pCDNA3 vector. (B) NF-κB reporter activity in HE293 cells that had been incubated in direct contact for 4 h or 26 h with <italic>H. pylori</italic> (strains 26695 or SS1) or <italic>H. muridarum</italic> ATCC49282 bacteria. Control cells were transfected with the pCDNA3 vector. (C) CXCL8 secretion in culture supernatants of HEK293 cells at 24 h post-stimulation with <italic>H. muridarum</italic> ATCC49282 or <italic>H. pylori</italic> 26695. Control cells were left unstimulated. (D) HEK293 cells were co-transfected with the Igk-<italic>luc</italic> reporter plasmid and increasing amounts (in ng) of a NOD1 dominant-negative construct (ΔCARD NOD1), then either left untreated (control) or stimulated for 24 h with <italic>H. muridarum</italic> ATCC49282 or <italic>H. pylori</italic> 26695 bacteria. The total quantity of DNA was standardized throughout using pCDNA3 alone. (E) AGS cell lines stably expressing siRNA to an irrelevant gene (control siRNA) or to <italic>NOD1</italic> (<italic>NOD1</italic> siRNA) were either left untreated (control), or co-cultured for 1 h with <italic>H. pylori</italic> 251, 251 cagPAI (an isogenic mutant that lacks a functional T4SS) or <italic>H. muridarum</italic> ATCC49282. The medium was changed and the CXCL8 levels measured in supernatants at 24 h post-stimulation. NF-κB and CXCL8 data (triplicate determinations) are representative of 2–3 independent experiments. Statistical differences were observed for NF-κB reporter activity and CXCL8 responses in stimulated versus control cells (*, <italic>P</italic><0.05; **, <italic>P</italic><0.0001; ***, <italic>P</italic> = 0.002). (F) The amino acid sequence of a segment of <italic>H. muridarum</italic> ATCC49282 FlaA (Accession no. FM992115) was deduced by DNA sequencing and compared by CLUSTALW analysis to the corresponding regions of flagellin homologs from <italic>S. typhimurium</italic> <xref ref-type="bibr" rid="pone.0005396-AndersenNissen1">[29]</xref>, <italic>Helicobacter acinonychis</italic> (Hac1403; Accession no. CAK00134), <italic>H. pylori</italic> (HP0601), <italic>Helicobacter felis</italic> (<italic>H. felis</italic>; Accession no. Q9XB38), <italic>Helicobacter mustelae</italic> (<italic>H. mustelae</italic>; Accession no. P50612) and <italic>H. hepaticus</italic> (HH1364). The <italic>H. pylori</italic> and <italic>H. hepaticus</italic> FlaA sequences can be accessed at <ext-link ext-link-type="uri" xlink:href="http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi">http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi</ext-link>. The amino acid residues, corresponding to residues 89–96 of <italic>S. typhimurium</italic> FliC, which have been identified as being critical for TLR5 activation <xref ref-type="bibr" rid="pone.0005396-AndersenNissen1">[29]</xref>, are highlighted by a box. This region shares three amino acid residues with another series (shown by asterisks) that had previously been shown to be important for TLR5 signaling <xref ref-type="bibr" rid="pone.0005396-Smith2">[31]</xref>.</p></caption><graphic xlink:href="pone.0005396.g005"></graphic></fig><p>HEK293 cells are able to respond to bacterial pathogens via the cytosolic PRM, NOD1 <xref ref-type="bibr" rid="pone.0005396-Fritz1">[15]</xref>. As <italic>H. pylori</italic> strains carrying a functional type IV secretion apparatus (T4SS) were shown to trigger NOD1 signaling in these cells <xref ref-type="bibr" rid="pone.0005396-Philpott1">[25]</xref>, <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>, we co-cultured HEK293 cells with either <italic>H. muridarum</italic> ATCC49282, or <italic>H. pylori</italic> bacteria possessing or not a functional T4SS (strains 26695 and SS1, respectively) <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>. <italic>H. muridarum</italic> ATCC49282 was found to induce similar levels of NF-κB-dependent reporter activity, albeit at a slower rate, than <italic>H. pylori</italic> bacteria with a functional T4SS (strain 26695; <xref ref-type="fig" rid="pone-0005396-g005">Figure 5B</xref>). Consistent with the NF-κB reporter studies, <italic>H. muridarum</italic> also up-regulated CXCL8 synthesis in HEK293 cells (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5C</xref>). Transfection of these cells with increasing concentrations of a dominant-negative NOD1 construct, in which the caspase activation recruitment domain (CARD) was deleted <xref ref-type="bibr" rid="pone.0005396-Bertin1">[27]</xref>, significantly abrogated <italic>H. muridarum</italic> effects on NF-κB reporter activity (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5D</xref>; <italic>P</italic><0.05). Furthermore, we demonstrated that AGS cells stably expressing siRNA to NOD1 produced significantly less CXCL8 in response to stimulation with <italic>H. muridarum</italic> (or <italic>H. pylori</italic>), when compared to cells expressing siRNA to an irrelevant control gene (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5E</xref>; <italic>P</italic> = 0.002). The abrogation of NOD1 signaling by ΔCARD NOD1 or NOD1 siRNA resulted in comparable decreases in cell responses to either <italic>H. muridarum</italic> ATCC49282 or T4SS-positive <italic>H. pylori</italic>. Thus, the data suggested that NOD1 was important for epithelial cell responses to <italic>H. muridarum</italic>. As m-IC<sub>cl2</sub> cells express NOD1 mRNA (<xref ref-type="bibr" rid="pone.0005396-Sterzenbach1">[23]</xref>; <xref ref-type="supplementary-material" rid="pone.0005396.s002">Figure S2</xref>), we propose that NOD1 contributed to the LPS-independent responses to live <italic>H. muridarum</italic> bacteria in this cell line (<xref ref-type="fig" rid="pone-0005396-g003">Figure 3B and 3C</xref>). To exclude a role for NOD1 in the observed responses of m-IC<sub>cl2</sub> cells to purified <italic>H. muridarum</italic> LPS (<xref ref-type="fig" rid="pone-0005396-g003">Figure 3</xref>), we delivered the purified material into the cytosol of HEK293 cells, together with NOD1-expressing construct, using a previously described liposome-based technique <xref ref-type="bibr" rid="pone.0005396-Girardin2">[18]</xref>, <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>. Transfection of cells with <italic>H. muridarum</italic> ATCC49282 LPS was shown to have no effect on NF-κB activity, whereas <italic>H. pylori</italic> 26695 peptidoglycan transfected in this way induced a significant response (<xref ref-type="supplementary-material" rid="pone.0005396.s003">Figure S3</xref>). This suggests that the <italic>H. muridarum</italic> LPS preparation was devoid of peptidoglycan muropeptides, which might otherwise induce NOD1 signaling in m-IC<sub>cl2</sub> cells. It also suggests that the observed activity of <italic>H. muridarum</italic> LPS activity on TLR2 signaling (<xref ref-type="fig" rid="pone-0005396-g004">Figure 4</xref>) could not be ascribed to lipoprotein contamination of this LPS preparation.</p><p>Although the data strongly suggested that NOD1 is a key PRM involved in epithelial cell responses to <italic>H. muridarum</italic>, HEK293 and AGS cells both also express the PRM for bacterial flagellin, TLR5 <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>, <xref ref-type="bibr" rid="pone.0005396-Lee2">[28]</xref>. All <italic>Helicobacter</italic> spp. are flagellated and it was therefore possible that this PRM may be involved in the cell responses to <italic>H. muridarum.</italic> To examine this suggestion, we sequenced a region of the gene encoding the major flagellin subunit (FlaA) of <italic>H. muridarum</italic> ATCC49282 (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5F</xref>). We demonstrated that <italic>H. muridarum</italic> FlaA does not possess any of the eight highly conserved amino acid residues, corresponding to residues 89–96 of <italic>Salmonella typhimurium</italic> FliC, which have been found to be required for activation of TLR5 by bacterial flagellins <xref ref-type="bibr" rid="pone.0005396-AndersenNissen1">[29]</xref>, <xref ref-type="bibr" rid="pone.0005396-Gewirtz1">[30]</xref>, <xref ref-type="bibr" rid="pone.0005396-Smith2">[31]</xref>. Instead, the corresponding region of <italic>H. muridarum</italic> FlaA displayed a high degree of conservation with the FlaA homologs of gastric and intestinal <italic>Helicobacter</italic> spp. which are known to evade TLR5 recognition <xref ref-type="bibr" rid="pone.0005396-Sterzenbach1">[23]</xref>, <xref ref-type="bibr" rid="pone.0005396-AndersenNissen1">[29]</xref>, <xref ref-type="bibr" rid="pone.0005396-Gewirtz1">[30]</xref>. These findings, together with the lack of a systematic responsiveness of epithelial cells to other flagellated <italic>Helicobacter</italic> spp. (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5A</xref>; <xref ref-type="bibr" rid="pone.0005396-Sterzenbach1">[23]</xref>, <xref ref-type="bibr" rid="pone.0005396-AndersenNissen1">[29]</xref>), also allow us to formally exclude TLR5 as playing a significant role in <italic>H. muridarum</italic> recognition.</p></sec></sec><sec id="s3"><title>Discussion</title><p>Enterohepatic <italic>Helicobacter</italic> spp. have been reported to induce chronic intestinal inflammation in animal hosts <xref ref-type="bibr" rid="pone.0005396-Ferrero1">[2]</xref>, <xref ref-type="bibr" rid="pone.0005396-Shomer1">[3]</xref>, <xref ref-type="bibr" rid="pone.0005396-Jiang1">[7]</xref>. The role of these bacterial species in the development of intestinal inflammation is, however, poorly understood. By studying epithelial cell responses to various enterohepatic <italic>Helicobacter</italic> spp. of murine and human origin, we showed that <italic>H. muridarum</italic>, an isolate from the normal intestinal flora of conventional rodents <xref ref-type="bibr" rid="pone.0005396-Phillips1">[6]</xref>, <xref ref-type="bibr" rid="pone.0005396-Queiroz1">[9]</xref>, was capable of inducing CXC chemokine production and NF-κB activation in epithelial cells <italic>in vitro</italic>. Collectively, this bacterium was found to be a more potent inducer of pro-inflammatory responses in mouse and human cell lines than either of the two mouse pathogens, <italic>H. bilis</italic> or <italic>H. hepaticus</italic>. Epithelial cell responses to <italic>H. muridarum</italic> were mediated by both LPS-dependent and -independent mechanisms. We propose that the intracellular PRM, NOD1, participates in the LPS-independent responses to <italic>H. muridarum</italic>. While there have been several reports concerning NOD1 signaling to gastrointestinal pathogens <xref ref-type="bibr" rid="pone.0005396-Fritz1">[15]</xref>, this is the first describing the involvement of this molecule in epithelial cell responses to a member of the normal microbial flora.</p><p><italic>H. muridarum</italic> was originally cultivated from the ileal and cecal tissues of conventional rodents <xref ref-type="bibr" rid="pone.0005396-Phillips1">[6]</xref>. <italic>H. muridarum</italic> was also reported to colonize the gastric mucosa of aged conventional mice, and to induce gastritis in these animals <xref ref-type="bibr" rid="pone.0005396-Lee1">[8]</xref>, <xref ref-type="bibr" rid="pone.0005396-Queiroz1">[9]</xref>. Moreover, mice that were monoassociated with <italic>H. muridarum</italic> displayed an accelerated development of IBD-like lesions in a CD45RB<sup>high</sup> CD4<sup>+</sup> T cell transfer model of experimental colitis <xref ref-type="bibr" rid="pone.0005396-Jiang1">[7]</xref>. Hence, it was suggested that the bacterium was a “provocateur” of IBD <xref ref-type="bibr" rid="pone.0005396-Jiang1">[7]</xref>. Consistent with this suggestion, <italic>H. muridarum</italic> was shown here to induce robust pro-inflammatory responses in epithelial cells of human and mouse origins. Co-culture of m-IC<sub>cl2</sub> mouse intestinal cells with <italic>H. muridarum</italic> resulted in increased transcription and synthesis of two CXC chemokines, Cxcl1 and Cxcl2. These chemokines are key promoters of polymorphonuclear leukocyte recruitment in mice <xref ref-type="bibr" rid="pone.0005396-Ohtsuka1">[20]</xref>, <xref ref-type="bibr" rid="pone.0005396-Remick1">[21]</xref> and may thus represent important mediators of the inflammatory responses observed in <italic>Helicobacter</italic> colitis models.</p><p>The m-IC<sub>cl2</sub> cell line synthesizes functional TLR2 and TLR4 molecules <xref ref-type="bibr" rid="pone.0005396-Hornef1">[11]</xref>, <xref ref-type="bibr" rid="pone.0005396-Sterzenbach1">[23]</xref>. Stimulation of these cells with either TLR2 or TLR4 ligands (Pam-3-Cys and LPS, respectively) resulted in increased NF-κB-dependent pro-inflammatory cytokine production <xref ref-type="bibr" rid="pone.0005396-Hornef1">[11]</xref>, <xref ref-type="bibr" rid="pone.0005396-Fernandez1">[32]</xref>. In the current study, we demonstrated a dose-dependent effect of <italic>H. muridarum</italic> LPS on Cxcl1 and Cxcl2 synthesis in m-IC<sub>cl2</sub> cells (<xref ref-type="fig" rid="pone-0005396-g003">Figure 3A</xref>). These responses appear to depend on TLR2 recognition of this LPS (<xref ref-type="fig" rid="pone-0005396-g004">Figure 4</xref>). Interestingly, as found here (<xref ref-type="fig" rid="pone-0005396-g004">Figure 4B</xref>) and elsewhere <xref ref-type="bibr" rid="pone.0005396-Smith1">[12]</xref>, <xref ref-type="bibr" rid="pone.0005396-Triantafilou1">[13]</xref>, <italic>H. pylori</italic> LPS also seems to signal via TLR2, however the situation is not clear as other researchers have claimed TLR4 to be the cognate PRM for this LPS molecule <xref ref-type="bibr" rid="pone.0005396-Mandell1">[33]</xref>. These differences may be attributed to the experimental conditions (<italic>e. g</italic>. LPS concentrations) used in the various studies.</p><p>Although m-IC<sub>cl2</sub> cells were highly responsive to <italic>H. muridarum</italic> LPS, the kinetics and magnitude of these responses were different to those induced by live bacteria (<xref ref-type="fig" rid="pone-0005396-g003">Figure 3B and 3C</xref>). This finding suggested the existence of an LPS-independent mechanism in epithelial cell signaling to <italic>H. muridarum.</italic> Further experiments were undertaken in the HEK293 and AGS cell lines, neither of which express functional TLR2 nor TLR4 <xref ref-type="bibr" rid="pone.0005396-Girardin2">[18]</xref>. Of the five murine and human <italic>Helicobacter</italic> spp. tested, only <italic>H. muridarum</italic> had a dramatic effect on NF-κB reporter activity in HEK293 cells (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5A</xref>). Preliminary studies with three other <italic>H. muridarum</italic> strains suggest that it may be a strain-independent phenomenon (data not shown). Although <italic>H. bilis</italic>, and to a lesser extent, <italic>H. hepaticus</italic>, induced CXC chemokine production in m-IC<sub>cl2</sub> cells, these species were poor agonists of NF-κB responses in HEK293 cells. Taken together, the findings highlight the specific nature of HEK293 cell responses to <italic>H. muridarum</italic>. Moreover, responses to <italic>H. muridarum</italic> were demonstrated to be NOD1-dependent and TLR5-independent (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5</xref>). The latter finding is consistent with the conclusions of several studies reporting that TLR5 is not important for epithelial cell recognition of <italic>Helicobacter</italic> spp. <xref ref-type="bibr" rid="pone.0005396-Sterzenbach1">[23]</xref>, <xref ref-type="bibr" rid="pone.0005396-AndersenNissen1">[29]</xref>, <xref ref-type="bibr" rid="pone.0005396-Gewirtz1">[30]</xref>.</p><p>The present work raises two important questions relating to the bacterial pathogenesis of enterohepatic <italic>Helicobacter</italic> spp. The first of these concerns the mechanism by which <italic>H. muridarum</italic> may trigger NOD1 signaling in epithelial cells. NOD1 responds specifically to Gram-negative peptidoglycan presented within the cytoplasm of epithelial cells by pathogenic bacteria. Until now, this has been reported to occur via either cell invasion or by the actions of a bacterial T4SS, encoded by the <italic>H. pylori cag</italic> pathogenicity island (PAI) <xref ref-type="bibr" rid="pone.0005396-Fritz1">[15]</xref>. <italic>H. muridarum</italic>, however, does not appear to be any more invasive than other <italic>Helicobacter</italic> spp (<xref ref-type="supplementary-material" rid="pone.0005396.s001">Figure S1</xref>) <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>, and does not harbor the genes needed to encode a functional T4SS (RLF, unpublished data). Thus, <italic>H. muridarum</italic> is likely to utilize a different mechanism of peptidoglycan delivery than those described previously. This suggestion would be consistent with the relatively longer incubation periods (>24 h) required for <italic>H. muridarum</italic>-induced NF-κB reporter activity in HEK293 cells, when compared to those used for the T4SS-positive <italic>H. pylori</italic> 26695 strain (≤4 h; <xref ref-type="fig" rid="pone-0005396-g005">Figure 5B</xref>). In AGS cells, however, a 1 h contact time with cells was sufficient for the induction of responses by <italic>H. muridarum</italic>, whereas this was insufficient for <italic>H. pylori</italic> 251<italic>cagPAI</italic>, a T4SS-deficient mutant (<xref ref-type="fig" rid="pone-0005396-g005">Figure 5E</xref>). Nevertheless, when compared to T4SS-positive <italic>H. pylori</italic>, 10-fold more <italic>H. muridarum</italic> bacteria (MOI 1∶100) were required to induce comparable responses in these cells, suggesting that <italic>H. muridarum</italic> is a less efficient initiator of NOD1 signaling. These findings point towards the existence in <italic>H. muridarum</italic> of an alternative mechanism for activation of the NOD1 pathway.</p><p>A second question arising from the work relates to the low pro-inflammatory activity of <italic>H. hepaticus</italic> on epithelial cell lines. Although this bacterium has been shown to induce colitis in certain strains of mice <xref ref-type="bibr" rid="pone.0005396-Cahill1">[4]</xref>, <xref ref-type="bibr" rid="pone.0005396-Kullberg1">[5]</xref>, one study reported that germ-free IL-10<sup>−/−</sup> mice monoassociated with <italic>H. hepaticus</italic> (the same strain as the one used here) did not develop colitis <xref ref-type="bibr" rid="pone.0005396-Dieleman1">[34]</xref>. It is thus possible that the presence of a complex intestinal flora may promote the virulence potential of <italic>H. hepaticus</italic>. It has also been suggested that the level of pathogenicity exhibited by different <italic>H. hepaticus</italic> isolates may be strongly influenced by strain-specific differences in gene content, including the presence or otherwise of a complete PAI <xref ref-type="bibr" rid="pone.0005396-Boutin1">[35]</xref>, <xref ref-type="bibr" rid="pone.0005396-Ge1">[36]</xref>. Conversely, there have been reports that under certain circumstances <italic>H. hepaticus</italic> is able to dampen inflammation <xref ref-type="bibr" rid="pone.0005396-MaggioPrice1">[37]</xref>. Consistent with this suggestion, Sterzenbach <italic>et al</italic>. showed that m-IC<sub>cl2</sub> cells that had been pre-incubated with <italic>H. hepaticus</italic> cell lysates, responded poorly to TLR4 and TLR5 agonists <xref ref-type="bibr" rid="pone.0005396-Sterzenbach1">[23]</xref>. Nevertheless, both that work as well as our own were performed in relatively simple epithelial cell models that mimic only a small aspect of the complex interactions occurring between host cells and intestinal pathogens or commensals. Thus, even if <italic>H. hepaticus</italic> may be able to down-regulate pro-inflammatory responses in epithelial cells, it is plausible that its interactions with other mucosal cell populations and/or other components of the host microflora may contribute to the inflammation normally associated with infection by this bacterium.</p><p>Mazmanian and colleagues <xref ref-type="bibr" rid="pone.0005396-Mazmanian1">[38]</xref> hypothesized that intestinal inflammation may be caused by imbalances between commensal organisms that have a pathogenic potential (so-called “pathobionts”) and those with beneficial potential (“symbionts”). From the evidence presented here, it appears that <italic>H. muridarum</italic> has a pathogenic potential and should therefore be considered a pathobiont. This observation has practical consequences for those who work with rodent models of inflammation and immunity. Indeed, <italic>H. muridarum</italic> may be “missed” by the assays commonly employed to monitor the specific pathogen-free (SPF) status of animals. Furthermore, although some laboratory animal suppliers use <italic>Helicobacter</italic> genus-specific PCR assays to verify the SPF status of their animals, this is not always the case. <italic>H. muridarum</italic> (or indeed other poorly studied <italic>Helicobacter</italic> spp.) may therefore have a confounding effect in mouse colitis models, particularly as <italic>H. muridarum</italic> was shown to engage TLR2 and NOD1, both of which are expressed in intestinal epithelial cells <xref ref-type="bibr" rid="pone.0005396-Hornef1">[11]</xref>, <xref ref-type="bibr" rid="pone.0005396-Sterzenbach1">[23]</xref>, <xref ref-type="bibr" rid="pone.0005396-Hisamatsu1">[39]</xref>, <xref ref-type="bibr" rid="pone.0005396-OrtegaCava1">[40]</xref>. Given that defects in TLR and/or NLR signaling are thought to affect the ability of host cells to respond normally to the intestinal microflora, it is plausible that these together with imbalances within the flora may contribute to the development of inflammatory conditions in the gut. Further investigations should thus be undertaken to elucidate the role of PRMs in epithelial cell sensing of members of the normal microflora.</p></sec><sec sec-type="materials|methods" id="s4"><title>Materials and Methods</title><sec id="s4a"><title>Bacteria</title><p><italic>H. bilis</italic> (CIP204753T, ATCC51630), <italic>H. cinaedi</italic> (CIP103752T, ATCC35683); <italic>H. hepaticus</italic> (ATCC51448), <italic>H. fennelliae</italic> (ATCC35684); and <italic>H. muridarum</italic> (St1, ATCC49282) were kindly provided by the Culture Collection of the Institut Pasteur (Paris, France), Prof. J. G. Fox (Massachusetts Institute of Technology, Boston, MA) and Dr J. O'Rourke (The University of NSW, Sydney, Australia), respectively. <italic>H. pylori</italic> 26695, SS1, and 251 strains were as described previously <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>. The <italic>H. pylori</italic> 251 <italic>cag</italic>PAI deletion mutant was constructed by natural transformation using the kanamycin resistance cassette developed by Odenbreit <italic>et al.</italic> <xref ref-type="bibr" rid="pone.0005396-Odenbreit1">[41]</xref>.</p><p>Bacteria were routinely subcultured on Blood Agar Base No. 2 (Oxoid, Hampshire, UK), supplemented with 8–10% horse blood and an antibiotic cocktail <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>. Broth cultures were prepared in 10 ml Brain Heart Infusion (Oxoid) containing 10% heat-inactivated fetal calf serum (FCS; Invitrogen, Cergy-Pontoise, France and Auckland, New Zealand) <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>. Bacterial suspensions were washed three times in phosphate-buffered saline (pH 7.4), then resuspended in cell culture medium. Viable counts of the bacterial suspensions were determined by serial plating <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>. Highly purified <italic>H. muridarum</italic> ATCC49282 and <italic>H. pylori</italic> NCTC11637 LPS were prepared by hot phenol-water extraction and subsequent enzymatic treatments with DNase, RNase and proteinase K (Sigma Chemical Co., St Louis, MO) and by ultracentrifugation <xref ref-type="bibr" rid="pone.0005396-Moran1">[42]</xref>. <italic>E. coli</italic> 0111:B4 Ultrapure LPS was obtained from InvivoGen (Toulouse, France).</p></sec><sec id="s4b"><title>Cell culture conditions</title><p>The murine m-IC<sub>cl2</sub> epithelial cell line represents a clone of immortalized cells that were derived from the bases of small intestinal villi of transgenic mice <xref ref-type="bibr" rid="pone.0005396-Bens1">[22]</xref>. These cells were routinely grown in a supplemented DMEM-F12 medium (Invitrogen) <xref ref-type="bibr" rid="pone.0005396-Bens1">[22]</xref>. Diluted cell suspensions were seeded onto collagen-coated (2 mg/ml rat tail collagen type 1, Sigma) culture plates, and incubated at 37°C in 5% CO<sub>2</sub>. For co-culture experiments, m-IC<sub>cl2</sub> cells were grown for 1 day then serum-starved overnight. The cells were washed three times in DMEM-F12 medium, and re-incubated in antibiotic-free, serum-free medium, prior to addition of the bacteria or purified <italic>H. muridarum</italic> LPS. Culture supernatants were collected at the indicated times and frozen at −80°C until assayed.</p><p>HEK293 cells, as well as the TLR2- and TLR4-expressing HEK293 cell lines <xref ref-type="bibr" rid="pone.0005396-Latz1">[43]</xref>, were grown routinely in DMEM (Invitrogen) containing 10% FCS, and incubated at 37°C in 5% CO<sub>2</sub> <xref ref-type="bibr" rid="pone.0005396-Philpott1">[25]</xref>. Bacteria were separated from epithelial cells using 0.2 µm Transwell filters (Corning Incorporated, Corning, NY). <italic>Helicobacter</italic> and <italic>E. coli</italic> LPS (0.01–1.0 µg) were added directly to HEK293 cells and the culture supernatants collected 8 h later.</p><p>AGS cells were routinely cultured in serum-supplemented RPMI 1640 cell culture media <xref ref-type="bibr" rid="pone.0005396-Philpott1">[25]</xref>. The AGS control siRNA and <italic>NOD1</italic> siRNA cells were generated by integration of an expression vector containing a small interference RNA (siRNA) directed to either the gene encoding enhanced green fluorescent protein (EGFP) or the CARD of the <italic>NOD1</italic> gene, respectively. A detailed description and characterization of these cell lines is given elsewhere (Grubman <italic>et al</italic>.; manuscript submitted). For co-culture assays, bacteria were added for 1 h to serum-starved cells (MOI 1∶10 for <italic>H. pylori</italic> and 1∶100 for <italic>H. muridarum</italic>), washed off, and the media replaced. Culture supernatants were collected 17–23 h later.</p></sec><sec id="s4c"><title>Cell transfection assays</title><p>HEK293 cells were plated in 24-well plates at a density of 1×10<sup>5</sup> cells and transfected the following day with Igκ-luciferase reporter DNA, using FuGene6 reagent medium (Roche Diagnostics, Meylan, France) <xref ref-type="bibr" rid="pone.0005396-Girardin3">[44]</xref>. For dominant-negative studies, cells were co-transfected with ΔCARD NOD1 DNA <xref ref-type="bibr" rid="pone.0005396-Bertin1">[27]</xref>. The transfected cells were incubated overnight and co-cultured with live bacteria (MOI = 1∶10 to 1∶100) for 2–24 h, prior to lysis of the cells <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>. Lysed cells were assayed for luciferase activities using a 96-well luminometer.</p></sec><sec id="s4d"><title>Reverse Transcriptase–PCR (RT–PCR)</title><p>Total RNA was isolated from epithelial cells using TRIzol reagent (Invitrogen). RNA (1 mg) from each sample was reverse-transcribed using 25 U of superscript II reverse transcriptase (Invitrogen). PCR was conducted using 2 ml of cDNA and 0.25 U Taq DNA polymerase (Amersham Biosciences, Orsay, France). Fifteen pmol each of 5′ and 3′ primers for cytokine genes were used with 3 pmol each of β-actin primers in a multiplex reaction. One PCR cycle consisted of the following: 94°C for 1 min, 64°C for 1 min 15 s, and 72°C for 1 min 15 s. The total cycle numbers were 35. A final elongation step of 7 min at 72°C was then used. Primer sequences were as follows: murine <italic>Cxcl1</italic>, <xref ref-type="bibr" rid="pone.0005396-Savard1">[45]</xref>, <named-content content-type="gene">5′-TTGAAGGTGATGCCGCCAG-3′</named-content> and <named-content content-type="gene">5′-CCCAGACTCTCATCTCTCC-3′</named-content>; murine <italic>Cxcl2</italic>, <xref ref-type="bibr" rid="pone.0005396-Garhart1">[46]</xref>, <named-content content-type="gene">5′-CATCGAATTCGGCAGACTCCAGCCACACTTCAGCCT-3′</named-content> and <named-content content-type="gene">5′-GATCGGATCCGGCAGTTAGCCTTGCCTTTGTTCAGT-3′</named-content>; murine β-actin (<italic>ActB</italic>), <named-content content-type="gene">5′-CCAGAGCAAGAGAGGTATCC-3′</named-content> and <named-content content-type="gene">5′-CTGTGGTGGTGAAGCTGTAG-3′</named-content>. PCR products (with sizes of 205, 358 and 436 bp, respectively) were separated on 1.5% agarose gels with 0.4 mg/ml ethidium bromide. Stained bands were visualized under UV light, and photographed with an Image Master VDS machine (Amersham).</p></sec><sec id="s4e"><title>PCR amplification of <italic>H. muridarum flaA</italic></title><p>PCR was performed on genomic DNA (1–10 ng) that had been purified from <italic>H. muridarum</italic> using the MasterPure™ DNA Purification Kit (EPICENTRE Biotechnologies, Madison, WI). DNA samples from <italic>H. pylori</italic> 26695 and <italic>H. hepaticus</italic> ATCC51448 were used as positive controls. The PCR samples contained 20 µM of each primer, 200 µM of total dNTPs (Promega, Alexandria, NSW, Australia), 0.5 U Taq Polymerase Taq and buffer (both from Roche Applied Science, Castle Hill, NSW, Australia). One PCR cycle consisted of the following: 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min 15 s. The total cycle numbers were 30. A final elongation step of 7 min at 72°C was used. Primer sequences were as follows: oligo A, <named-content content-type="gene">5′-ATGGCTTTTCAGGTCAATAC-3′</named-content>; oligo C, <named-content content-type="gene">5′-CCTACTTGGAATTCTTTG-3′</named-content>. PCR products (449 bp) were separated on 1.5% agarose gels, as above, and the DNA purified using the Perfectprep Gel Cleanup kit (Eppendorf North Ryde, NSW, Australia). DNA sequencing of the <italic>H. muridarum flaA</italic> amplicon was performed by Micromon Services (Monash University, Clayton, VIC, Australia) using BigDye Terminator Cycle Sequencing (Applied Biosystems, Scoresby, VIC, Australia). The deduced <italic>H. muridarum</italic> FlaA sequence was aligned with those of <italic>S. typhimurium FliC</italic> and the FlaA homologs of other <italic>Helicobacter</italic> spp. using CLUSTALW software (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/Tools/clustalw2/index.html">http://www.ebi.ac.uk/Tools/clustalw2/index.html</ext-link>).</p></sec><sec id="s4f"><title>Chemokine assays</title><p>CXC chemokine (Cxcl1, Cxcl2 or CXCL8) levels were determined from culture supernatants using cytokine enzyme-linked immunosorbent assay (ELISA) kits from either R&D Systems (Minneapolis, MN) or BD Pharmingen (CA).</p></sec><sec id="s4g"><title>Statistical analysis</title><p>Data were analyzed using the Student's t-test and Mann-Whitney test, as appropriate. Differences in data values were considered significant for <italic>P</italic>≤0.05.</p></sec></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pone.0005396.s001"><label>Figure S1</label><caption><p>Invasion efficiency of enterohepatic <italic>Helicobacter</italic> spp in HEK293 cells. The invasion efficiency of each bacterium was determined by the gentamycin protection assay <xref ref-type="bibr" rid="pone.0005396-Viala1">[26]</xref>. The values are expressed as the proportions (in percent) of internalized bacteria to the total numbers in the inocula added to cells. (n = 2 independent experiments for <italic>H. muridarum</italic> and <italic>H. bilis</italic>; n = 1 for <italic>H. hepaticus</italic>.)</p><p>(1.96 MB DOC)</p></caption><media xlink:href="pone.0005396.s001.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0005396.s002"><label>Figure S2</label><caption><p>m-IC<sub>cl2</sub> epithelial cells express Nod1 mRNA. RT-PCR detection of Nod1 (CARD4) mRNA expression in unstimulated m-IC<sub>cl2</sub> epithelial cells (1) and in cells co-cultured for 18 h with either (2) <italic>H. muridarum</italic> or (3) mouse TNF. RNA samples were standardized by performing PCR with oligonucleotides specific for β-actin (ACTB) (see <xref ref-type="sec" rid="s4">Materials and Methods</xref>). Amplicons (303 bp) from murine Nod1 (CARD4) were amplified using the following oligonucleotides: <named-content content-type="gene">5′-AGGAGGCCAACAGACGCC-3′</named-content> and <named-content content-type="gene">5′-CTGACCTAGAGGGTATCG-3′</named-content>.</p><p>(2.59 MB TIF)</p></caption><media xlink:href="pone.0005396.s002.tif" mimetype="image" mime-subtype="tiff"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0005396.s003"><label>Figure S3</label><caption><p>H. muridarum LPS does not induce NOD1 signaling in HEK293 cells. NF-κB responses of unstimulated HEK293 cells (control), unstimulated cells that had been transfected with a NOD1-expressing construct (control+NOD1), or in cells co-transfected with the NOD1-expressing construct as well as either <italic>H. pylori</italic> peptidoglycan (Hp PG+NOD1) or <italic>H. muridarum</italic> LPS (Hm LPS+NOD1). Data correspond to the mean±SEM (triplicate determinations) and are representative of two independent experiments. Statistical differences were observed between control cells and those stimulated with Hp PG+NOD1 (*, P<0.05).</p><p>(7.83 MB TIF)</p></caption><media xlink:href="pone.0005396.s003.tif" mimetype="image" mime-subtype="tiff"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>Dr. A. Vandewalle (Hôpital Xavier Bichat, Paris, France) is gratefully acknowledged for supplying the m-IC<sub>cl2</sub> epithelial cell line. Drs. A. Mansell (Monash Institute for Medical Research, Monash University, Clayton, Australia) and D. 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This work was funded by the Association Francois Aupetit (RLF); the Institut Pasteur, Paris (PTR 94 project No. 80944 to DJP and RLF); the Association pour la Recherche sur le Cancer (ARC project no. 4428 to RLF); the National Health and Medical Research Council (project grant no. 334127 to RLF); and the Health Research Board, Ireland (APM). DJP is an International Research Scholar of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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