Survey of small intestinal and systemic immune responses following murine Arcobacter butzleri infection

Background Arcobacter (A.) butzleri has been described as causative agent for sporadic cases of human gastroenteritis with abdominal pain and acute or prolonged watery diarrhea. In vitro studies revealed distinct adhesive, invasive and cytotoxic properties of A. butzleri. Information about the underlying immunopathological mechanisms of infection in vivo, however, are scarce. The aim of this study was to investigate the immunopathological properties of two different A. butzleri strains in a well-established murine infection model. Results Gnotobiotic IL-10 −/− mice, in which the intestinal microbiota was depleted by broad-spectrum antibiotic treatment, were perorally infected with two different A. butzleri strains isolated from a diseased patient (CCUG 30485) or fresh chicken meat (C1), respectively. Eventhough bacteria of either strain could stably colonize the intestinal tract at day 6 and day 16 postinfection (p.i.), mice did not exert infection induced symptoms such as diarrhea or wasting. In small intestines of infected mice, however, increased numbers of apoptotic cells could be detected at day 16, but not day 6 following infection with either strain. A strain-dependent influx of distinct immune cell populations such as T and B cells as well as of regulatory T cells could be observed upon A. butzleri infection which was accompanied by increased small intestinal concentrations of pro-inflammatory cytokines such as TNF, IFN-γ, MCP-1 and IL-6. Remarkably, inflammatory responses following A. butzleri infection were not restricted to the intestinal tract, given that the CCUG 30485 strain induced systemic immune responses as indicated by increased IFN-γ concentrations in spleens at day 6, but not day 16 following infection. Conclusion Upon peroral infection A. butzleri stably colonized the intestinal tract of gnotobiotic IL-10 −/− mice. The dynamics of distinct local and systemic inflammatory responses could be observed in a strain-dependent fashion pointing towards an immunopathogenic potential of A. butzleri in vivo. These results indicate that gnotobiotic IL-10 −/− mice are well suited to further investigate the molecular mechanisms underlying arcobacteriosis in vivo

Arcobacter butzleri Induce Colonic, Extra-Intestinal and Systemic Inflammatory Responses in Gnotobiotic IL-10 Deficient Mice in a Strain-Dependent Manner

BACKGROUND: The immunopathological impact of human Arcobacter (A.) infections is under current debate. Episodes of gastroenteritis with abdominal pain and acute or prolonged watery diarrhea were reported for A. butzleri infected patients. Whereas adhesive, invasive and cytotoxic capacities have been described for A. butzleri in vitro, only limited information is available about the immunopathogenic potential and mechanisms of infection in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Gnotobiotic IL-10-/- mice were generated by broad-spectrum antibiotic treatment and perorally infected with the A. butzleri strains CCUG 30485 and C1 shown to be invasive in cell culture assays. Bacterial colonization capacities, clinical conditions, intestinal, extra-intestinal and systemic immune responses were monitored at day six and 16 postinfection (p.i.). Despite stable intestinal A. butzleri colonization at high loads, gnotobiotic IL-10-/- mice were virtually unaffected and did not display any overt symptoms at either time point. Notably, A. butzleri infection induced apoptosis of colonic epithelial cells which was paralleled by increased abundance of proliferating cells. Furthermore A. butzleri infection caused a significant increase of distinct immune cell populations such as T and B cells, regulatory T cells, macrophages and monocytes in the colon which was accompanied by elevated colonic TNF, IFN-γ, nitric oxide (NO), IL-6, IL-12p70 and MCP-1 concentrations. Strikingly, A. butzleri induced extra-intestinal and systemic immune responses as indicated by higher NO concentrations in kidney and increased TNF, IFN-γ, IL-12p70 and IL-6 levels in serum samples of infected as compared to naive mice. Overall, inflammatory responses could be observed earlier in the course of infection by the CCUG 30485 as compared to the C1 strain. CONCLUSION/SIGNIFICANCE: Peroral A. butzleri infection induced not only intestinal but also extra-intestinal and systemic immune responses in gnotobiotic IL-10-/- mice in a strain-dependent manner. These findings point towards an immunopathogenic potential of A. butzleri in vertebrate hosts

Charakterisierung des Virulenzpotenzials von Arcobacter butzleri

Arcobacter (A.) spp. is a gram-negative, motile and spiral-shaped bacterium belonging together with Campylobacter (C.) and Sulfurospirillum to the family of Campylobacteraceae. At present, 18 Arcobacter species have been characterized with A. butzleri as the most important and predominant species associated with human diseases, such as gastroenteritis, bacteremia or septicaemia. A. butzleri have been rated a serious hazard to human health by the International Commission on Microbiological Specifications for Foods in 2002. The bacteria have become more important in public health due to their presence in different sources such as animals including various food products of animal origin and water. However, the most frequent source of human A. butzleri infection is contaminated undercooked poultry meat and water. In few studies several authors demonstrated that A. butzleri is the fourth most common Campylobacterales species recovered from patients suffering from. Reported clinical signs of an A. butzleri infection are diarrhea associated with abdominal pain, nausea and vomiting or more rarely fever. Compared to C. jejuni, A. butzleri caused more watery and persistent, but less acute and bloody diarrhea. As human infections with A. butzleri are not routinely investigated, the relevance of this pathogen could not be determined so far. However, a few case reports and some outbreak data on human infection with A. butzleri are available indicating that A. butzleri is a truly human pathogen. Although some progress has been made over the past decade, the knowledge about the pathogenic mechanisms and immune host responses of A. butzleri is still scarce. Several putative virulence determinants homologous to C. jejuni, were identified in the genome sequence of A. butzleri RM4018, while other virulence-associated genes of C. jejuni are missing. Results from phenotypic assays revealed adhesive, invasive and cytotoxic capabilities of A. butzleri on several cell lines in vitro. The barrier dysfunction caused by A. butzleri infection in monolayers of the human colon cell line HT-29/B6 highlights potential mechanisms by which diarrhea can be induced in human. This work focused on the investigation of virulence mechanisms of A. butzleri including its interactions with intestinal epithelial cells in vitro. Chapter 1 provides a literature review that emphasises the relevance of A. butzleri in human and veterinary public health. In the first study (Chapter 2), the presence of virulence genes homologous to those of C. jejuni and other enteric pathogens was investigated in several A. butzleri strains. Further the adhesive and invasive abilities of several A. butzleri strains on two different human intestinal epithelial cell lines (HT-29 and Caco-2) were demonstrated. No correlation was observed between putative virulence gene patterns and adhesive or invasive phenotypes with the tested cell lines; also the putative functional domains of CiaB, CadF and Cj1349 in the amino acid sequences showed no correlation with the different adhesive and invasive phenotypes. The aim of the second study (Chapter 3) was to obtain additional information on the pathogenicity and the pathomechanisms of A. butzleri strains. A. butzleri was investigated on two further epithelial cell lines to characterize different epithelial cell interactions and strain-specific pathomechanisms. The ability of A. butzleri strains for adhesion, invasion and cytotoxicity in human (HT-29/B6) and porcine (IPEC-J2) intestinal epithelial cell lines could be demonstrated. These A. butzleri strains were able to influence the transepithelial electrical resistance. The A. butzleri strain specific pathomechanisms has been observed with the human colon cell line HT-29/B6. Furthermore, A. butzleri induced systemic immune response in gnotobiotic IL-10 deficient mice in a strain-dependent manner (Chapter 4). Taken all data together, these findings emphasize the enteric pathogenic potential and strain-specific pathomechanisms of A. butzleri.Arcobacter (A.) sind gram-negative, bewegliche und spiralförmige Bakterien, die zusammen mit Campylobacter und Sulfurospirillum der Familie Campylobacteraceae angehören. Die Gattung umfasst 18 Spezies, wobei A. butzleri die wichtigste bzw. am häufigsten vorkommende Spezies darstellt, die mit Erkrankungen des Menschen wie Gastroenteritis, Bakteriämie oder Septikämie assoziiert wird. A. butzleri wurde bereits 2002 von der ICMSF zur erheblichen Gefahr für die menschliche Gesundheit eingestuft. Dieses Bakterium hat auf Grund seines Vorkommens in verschiedenen Quellen wie im Tier einschließlich zahlreicher Nahrungsmittel tierischen Ursprungs und Wasser erheblich an Bedeutung gewonnen. Die Hauptquelle für humane A. butzleri- Infektionen sind nicht ausreichend gegartes Geflügelfleisch und Wasser. Einige Studien zeigten, dass A. butzleri der vierthäufigste Campylobacterales-Erreger ist, der aus Fäzes von Patienten mit Durchfall isoliert wurde. Die Symptome einer A. butzleri-Infektion sind Durchfälle mit begleitenden Bauchkrämpfen, Übelkeit und Erbrechen oder seltener Fieber. Im Vergleich zu C. jejuni verursacht A. butzleri eher wässrigen und bis zu zwei Monaten anhaltenden, aber weniger akuten und blutigen Durchfall. Da A. butzleri-Infektionen nicht routinemäßig untersucht werden, kann die Relevanz dieses Erregers noch nicht eingeschätzt werden. Einige Fallberichte und A. butzleri-Ausbrüche belegen, dass A. butzleri ein echter Krankheitserreger des Menschen ist. Trotz gewisser Fortschritte im letzten Jahrzehnt existiert nur geringes Wissen über Pathogenitätsmechanismen von A. butzleri und die Immunantwort des Wirts. Miller et al. (2007) identifizierten in der Genomsequenz von A. butzleri RM 4018 mehrere putative Virulenzdeterminanten, die homolog zu C. jejuni sind, während andere Virulenz-assoziierten Gene von C. jejuni nicht existieren. Ergebnisse aus phänotypischen Untersuchungen zeigen adhäsive, invasive und zytotoxische Eigenschaften von A. butzleri auf mehreren Zelllinien in vitro. A. butzleri ist in der Lage Barriere-Funktionsstörungen in HT-29/B6 Monolayern hervorzurufen, welche potentielle Mechanismen sind, durch die Durchfall bei Menschen induziert werden könnte. Diese Arbeit widmet sich der Untersuchung einzelner Virulenzmechanismen von A. butzleri, einschließlich ihrer Interaktion mit intestinalen Epithelzellen in vitro. Kapitel 1 gibt eine Literaturübersicht, die die Relevanz von A. butzleri in der Human- und Veterinärmedizin unterstreicht. In der ersten Studie (Kapitel 2) wird die Verbreitung der 10 putativen Virulenzgene in A. butzleri-Stämmen beschrieben. Weiterhin werden die adhäsiven und invasiven Fähigkeiten ausgewählter A. butzleri-Stämme auf die zwei verschiedenen humanen Darmepithel-Zelllinien (HT-29 und Caco-2) gezeigt. Es wurde keine Korrelation zwischen dem putativen Virulenzgenmuster und den adhäsiven und invasiven Eigenschaften auf den getesteten Zelllinien beobachtet; auch die Aminosäuresequenzen der putativen funktionellen Domänen von CiaB, CadF und Cj1349 zeigten keine Korrelation zu den unterschiedlichen adhäsiven und invasiven Eigenschaften der untersuchten Stämme. Das Ziel der zweiten Studie (Kapitel 3) war es, zusätzliche Informationen zur Virulenz und Pathogenitätsmechanismen der A. butzleri-Stämme zu erhalten. A. butzleri wurde auf zwei weiteren Epithel-Zelllinien getestet, um verschiedenen epitheliale Zell-Interaktionen und stammspezifische Pathogenitätsmechanismen zu charakterisieren. Die Adhäsion, Invasion und Zytotoxizität von A. butzleri wurde bei humanen (HT-29/B6) und porzinen (IPEC-J2) Darmepithelzelllinien untersucht. Weiterhin waren diese A. butzleri- Stämme fähig, den transepithelialen elektrischen Widerstand zu beeinflussen. Eine stamm-abhängige Immunantwort konnte nach einer A. butzleri-Infektion in gnotobiotisch IL-10 defizienten Mäusen festgestellt werden (Kapitel 4). Zusammengefasst unterstreichen diese Daten das pathogene Potential und die stamm-spezifischen Pathogenitätsmechanismen von A. butzleri

Fecal shedding of <i>A</i>. <i>butzleri</i> strains in orally infected gnotobiotic IL-10^-/- mice.

<p>Gnotobiotic IL-10^−/− mice were generated by antibiotic treatment and perorally colonized either with (<b>A</b>) <i>A</i>. <i>butzleri</i> strain CCUG 30485 or (<b>B</b>) strain C1. <i>A</i>. <i>butzleri</i> loads were determined in fecal samples (CFU, colony forming units per gram) over 16 days postinfection (p.i.) by culture. Numbers of analysed animals are given in parentheses. Medians (black bars) are indicated. Data were pooled from three independent experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139402#pone.0139402.s001" target="_blank">S1 Table</a>).</p

Nitric oxide secretion in renal <i>ex vivo</i> biopsies of <i>A</i>. <i>butzleri</i> infected gnotobiotic IL-10^-/- mice.

<p>Gnotobiotic IL-10^−/− mice were generated by antibiotic treatment and orally infected either with <i>A</i>. <i>butzleri</i> strain CCUG 30485 (circles) or strain C1 (squares). Uninfected (naive) gnotobiotic IL-10^−/− mice served as negative controls (black diamonds). Concentrations of nitric oxide (NO) were determined in supernatans of <i>ex vivo</i> kidney biopsies at day six p.i. (filled symbols) and day 16 p.i. (open symbols). Numbers of analyzed animals are given in parentheses. Medians (black bars) and significance levels as determined by the Mann-Whitney U test are indicated. Data were pooled from three independent experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139402#pone.0139402.s005" target="_blank">S5 Table</a>).</p

Colonic pro-inflammatory mediator responses following <i>A</i>. <i>butzleri</i> infection of gnotobiotic IL-10^-/- mice.

<p>Gnotobiotic IL-10^−/− mice were generated by antibiotic treatment and perorally infected either with <i>A</i>. <i>butzleri</i> strain CCUG 30485 (circles) or strain C1 (squares). Uninfected (naive) gnotobiotic IL-10^−/− mice served as negative controls (black diamonds). Concentrations of <b>(A)</b> TNF, <b>(B)</b> IFN-γ, <b>(C)</b> nitric oxide (NO), <b>(D)</b> IL-6, <b>(E)</b> IL-12p70 and <b>(F)</b> MCP-1 were determined in supernatans of <i>ex vivo</i> colonic biopsies at day six p.i. (filled symbols) and day 16 p.i. (open symbols). Numbers of analyzed animals are given in parentheses. Medians (black bars) and significance levels as determined by the Mann-Whitney U test are indicated. Data were pooled from three independent experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139402#pone.0139402.s004" target="_blank">S4 Table</a>).</p

Colonic immune cell responses following <i>A</i>. <i>butzleri</i> infection of gnotobiotic IL-10^-/- mice.

<p>Gnotobiotic IL-10^−/− mice were generated by antibiotic treatment and perorally infected either with <i>A</i>. <i>butzleri</i> strain CCUG 30485 (circles) or strain C1 (squares). Uninfected gnotobiotic IL-10^−/− mice served as negative controls (black diamonds). The average number of cells positive for <b>(A)</b> CD3 (T lymphocytes), <b>(B)</b> FOXP3 (regulatory T cells, Tregs), <b>(C)</b> B220 (B Lymphocytes) and <b>(D)</b> F4/80 (macrophages and monocytes) from at least six high power fields (HPF, 400 x magnification) per animal were determined microscopically in immunohistochemically stained colonic paraffin sections derived from mice at day six p.i. (filled symbols) and day 16 p.i. (open symbols). Numbers of analyzed animals are given in parentheses. Medians (black bars) and significance levels as determined by the Mann-Whitney U test are indicated. Data were pooled from three independent experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139402#pone.0139402.s003" target="_blank">S3 Table</a>).</p

Systemic pro-inflammatory cytokine responses in <i>A</i>. <i>butzleri</i> infected gnotobiotic IL-10^-/- mice.

<p>Gnotobiotic IL-10^−/− mice were generated by antibiotic treatment and orally infected either with <i>A</i>. <i>butzleri</i> strain CCUG 30485 (circles) or strain C1 (squares). Uninfected gnotobiotic IL-10^−/− mice served as negative controls (black diamonds). Concentrations of <b>(A)</b> IFN-γ, <b>(B)</b> IL-12p70, <b>(C)</b> TNF and <b>(D)</b> IL-6 were determined in serum samples at day 6 p.i. (filled symbols) and day 16 p.i. (open symbols). Numbers of analyzed animals are given in parentheses. Medians (black bars) and significance levels as determined by the Mann-Whitney U test are indicated. Data were pooled from three independent experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139402#pone.0139402.s006" target="_blank">S6 Table</a>).</p

Headache in idiopathic/genetic epilepsy: Cluster analysis in a large cohort

Objective The link between headache and epilepsy is more prominent in patients with idiopathic/genetic epilepsy (I/GE). We aimed to investigate the prevalence of headache and to cluster patients with regard to their headache and epilepsy features. Methods Patients aged 6-40 years, with a definite diagnosis of I/GE, were consecutively enrolled. The patients were interviewed using standardized epilepsy and headache questionnaires, and their headache characteristics were investigated by experts in headache. Demographic and clinical variables were analyzed, and patients were clustered according to their epilepsy and headache characteristics using an unsupervised K-means algorithm. Results Among 809 patients, 508 (62.8%) reported having any type of headache; 87.4% had interictal headache, and 41.2% had migraine. Cluster analysis revealed two distinct groups for both adults and children/adolescents. In adults, subjects having a family history of headache, >= 5 headache attacks, duration of headache >= 24 months, headaches lasting >= 1 h, and visual analog scale scores > 5 were grouped in one cluster, and subjects with juvenile myoclonic epilepsy (JME), myoclonic seizures, and generalized tonic-clonic seizures (GTCS) were clustered in this group (Cluster 1). Self-limited epilepsy with centrotemporal spikes and epilepsy with GTCS alone were clustered in Cluster 2 with the opposite characteristics. For children/adolescents, the same features as in adult Cluster 1 were clustered in a separate group, except for the presence of JME syndrome and GTCS alone as a seizure type. Focal seizures were clustered in another group with the opposite characteristics. In the entire group, the model revealed an additional cluster, including patients with the syndrome of GTCS alone (50.51%), with >= 5 attacks, headache lasting >4 h, and throbbing headache; 65.66% of patients had a family history of headache in this third cluster (n = 99). Significance Patients with I/GE can be clustered into distinct groups according to headache features along with seizures. Our findings may help in management and planning for future studies