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1.  Toll-Like Receptor-4 is Essential for Arcobacter Butzleri-Induced Colonic and Systemic Immune Responses in Gnotobiotic IL-10–/– Mice 
Arcobacter butzleri causes sporadic cases of gastroenteritis, but the underlying immunopathological mechanisms of infection are unknown. We have recently demonstrated that A. butzleri-infected gnotobiotic IL-10–/– mice were clinically unaffected but exhibited intestinal and systemic inflammatory immune responses. For the first time, we here investigated the role of Toll-like receptor (TLR)-4, the main receptor for lipopolysaccharide and lipooligosaccharide of Gram-negative bacteria, in murine arcobacteriosis. Gnotobiotic TLR-4/IL-10-double deficient (TLR-4–/– IL-10–/–) and IL-10–/– control mice generated by broad-spectrum antibiotics were perorally infected with A. butzleri. Until day 16 postinfection, mice of either genotype were stably colonized with the pathogen, but fecal bacterial loads were approximately 0.5–2.0 log lower in TLR-4–/– IL-10–/– as compared to IL-10–/– mice. A. butzleri-infected TLR-4–/– IL-10–/– mice displayed less pronounced colonic apoptosis accompanied by lower numbers of macrophages and monocytes, T lymphocytes, regulatory T-cells, and B lymphocytes within the colonic mucosa and lamina propria as compared to IL-10–/– mice. Furthermore, colonic concentrations of nitric oxide, TNF, IL-6, MCP-1, and, remarkably, IFN-γ and IL-12p70 serum levels were lower in A. butzleri-infected TLR-4–/– IL-10–/– versus IL-10–/– mice. In conclusion, TLR-4 is involved in mediating murine A. butzleri infection. Further studies are needed to investigate the molecular mechanisms underlying Arcobacter–host interactions in more detail.
PMCID: PMC4681360  PMID: 26716021
Arcobacter butzleri; Toll-like receptor-4; lipopolysaccharide; lipooligosaccharide; gnotobiotic IL-10–/–; mice; pro-inflammatory immune responses; systemic immune responses; colon; apoptosis; innate and adaptive immunity
2.  Toll-Like Receptor-4 Dependent Small Intestinal Immune Responses Following Murine Arcobacter Butzleri Infection 
Sporadic cases of gastroenteritis have been attributed to Arcobacter butzleri infection, but information about the underlying immunopathological mechanisms is scarce. We have recently shown that experimental A. butzleri infection induces intestinal, extraintestinal and systemic immune responses in gnotobiotic IL-10–/– mice. The aim of the present study was to investigate the immunopathological role of Toll-like Receptor-4, the receptor for lipopolysaccharide and lipooligosaccharide of Gram-negative bacteria, during murine A. butzleri infection. To address this, gnotobiotic IL-10–/– mice lacking TLR-4 were generated by broad-spectrum antibiotic treatment and perorally infected with two different A. butzleri strains isolated from a patient (CCUG 30485) or fresh chicken meat (C1), respectively. Bacteria of either strain stably colonized the ilea of mice irrespective of their genotype at days 6 and 16 postinfection. As compared to IL-10–/– control animals, TLR-4–/– IL-10–/– mice were protected from A. butzleri-induced ileal apoptosis, from ileal influx of adaptive immune cells including T lymphocytes, regulatory T-cells and B lymphocytes, and from increased ileal IFN-γ secretion. Given that TLR-4-signaling is essential for A. butzleri-induced intestinal inflammation, we conclude that bacterial lipooligosaccharide or lipopolysaccharide compounds aggravate intestinal inflammation and may thus represent major virulence factors of Arcobacter. Future studies need to further unravel the molecular mechanisms of TLR-4-mediated A. butzleri-host interactions.
PMCID: PMC4681361  PMID: 26716022
Arcobacter butzleri; Toll-like Receptor-4; lipopolysaccharide; lipooligosaccharide; pro-inflammatory immune responses; small intestine; spleen; apoptosis; innate immunity; host-pathogen interactions
3.  The Role of Gelatinases in Campylobacter Jejuni Infection of Gnotobiotic Mice 
Matrix metalloproteinases (MMP)-2 and -9 (also referred to gelatinases-A and -B, respectively) are upregulated in the inflamed gut of mice and men. We recently demonstrated that synthetic gelatinase blockage reduced large intestinal pro-inflammatory immune responses and apoptosis following murine Campylobacter (C.) jejuni infection. In order to address which gelatinase mediates C. jejuni-induced immune responses, gnotobiotic MMP-2–/–, MMP-9–/–, and wildtype (WT) mice were generated by broadspectrum antibiotic treatment and perorally infected with C. jejuni strain 81-176. The pathogen stably colonized the murine intestinal tract irrespective of the genotype but did not translocate to extra-intestinal compartments. At days 8 and 14 postinfection (p.i.), less pronounced colonic histopathological changes were observed in infected MMP-2–/– mice, less distinct epithelial apoptosis, but more epithelial proliferation in both MMP-2–/– and MMP-9–/– mice, as compared to WT controls. Reduced immune responses in gelatinase-deficient mice were characterized by lower numbers of effector as well as innate and adaptive immune cells within the colonic mucosa and lamina propria. The expression of IL-22, IL-18, IL-17A, and IL-1β mRNA was higher in the colon of MMP-2–/– as compared to WT mice. In conclusion, both MMP-2 and MMP-9 are differentially involved in mediating C. jejuni-induced intestinal immunopathology.
PMCID: PMC4681353  PMID: 26716014
Campylobacter jejuni; in vivo infection model; gnotobiotic mice; matrix metalloproteinases; gelatinases; pro-inflammatory immune responses; translocation; IL-22; IL-18; apoptosis
4.  Survey of small intestinal and systemic immune responses following murine Arcobacter butzleri infection 
Gut Pathogens  2015;7:28.
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.
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.
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.
PMCID: PMC4610047  PMID: 26483849
Arcobacter butzleri; Strain differences; Pro-inflammatory immune responses; Extra-intestinal sequelae; Systemic immune responses; Small intestine; Spleen; Apoptosis; Regenerating cells; Innate and adaptive immunity
5.  Arcobacter butzleri Induce Colonic, Extra-Intestinal and Systemic Inflammatory Responses in Gnotobiotic IL-10 Deficient Mice in a Strain-Dependent Manner 
PLoS ONE  2015;10(9):e0139402.
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.
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.
PMCID: PMC4584000  PMID: 26406497
6.  Matrix Metalloproteinase-2 Mediates Intestinal Immunopathogenesis in Campylobacter Jejuni-Infected Infant Mice 
Increased levels of the matrix metalloproteinases (MMPs)-2 and -9 (also referred to gelatinase-A and -B, respectively) can be detected in the inflamed gut. We have recently shown that synthetic gelatinase blockage reduces colonic apoptosis and pro-inflammatory immune responses following murine Campylobacter (C.) jejuni infection. In order to dissect whether MMP-2 and/or MMP-9 is involved in mediating C. jejuni-induced immune responses, infant MMP-2–/–, MMP-9–/–, and wildtype (WT) mice were perorally infected with the C. jejuni strain B2 immediately after weaning. Whereas, at day 2 postinfection (p.i.), fecal C. jejuni B2 loads were comparable in mice of either genotype, mice expelled the pathogen from the intestinal tract until day 4 p.i. Six days p.i., colonic MMP-2 but not MMP-9 mRNA was upregulated in WT mice. Remarkably, infected MMP-2–/– mice exhibited less frequent abundance of blood in feces, less distinct colonic histopathology and apoptosis, lower numbers of effector as well as innate and adaptive immune cells within the colonic mucosa, and higher colonic IL-22 mRNA levels as compared to infected WT mice. In conclusion, these results point towards an important role of MMP-2 in mediating C. jejuni-induced intestinal immunopathogenesis.
PMCID: PMC4598886  PMID: 26495129
Campylobacter jejuni; infant mice; matrix metalloproteinase-2; gelatinases; pro-inflammatory immune responses; intestinal microbiota; IL-22; IL23; IL-18; apoptosis
7.  Anti-Inflammatory Properties of NAP in Acute Toxoplasma Gondii-Induced Ileitis in Mice 
The octapeptide NAP has been shown to exert neuroprotective properties. Here, we investigated potential anti-inflammatory effects of NAP in an acute ileitis model. To address this, C57BL/6j mice were perorally infected with Toxoplasma gondii (day 0). Within 1 week postinfection (p.i.), placebo (PLC)-treated mice developed acute ileitis due to Th1-type immune responses. Mice that were subjected to intraperitoneal NAP treatment from day 1 until day 6 p.i., however, developed less distinct macroscopic and microscopic disease as indicated by less body weight loss, less distinct histopathological ileal changes, and lower ileal apoptotic, but higher proliferating cell numbers, less abundance of neutrophils, macrophages, monocytes, and T lymphocytes, but higher numbers of regulatory T cells in the ileal mucosa and lamina propria, and lower concentrations of pro-inflammatory mediators in the ilea as compared to PLC controls at day 7 p.i. Remarkably, NAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments including liver and spleen. Strikingly, lower MCP-1, TNF, and IL-12p70 serum concentrations in NAP as compared to PLC-treated mice at day 7 p.i. indicate a pronounced systemic anti-inflammatory effect of NAP in acute ileitis. These findings provide first evidence for NAP as a potential novel treatment option in intestinal inflammation.
PMCID: PMC4598889  PMID: 26495132
NAP; ADNP; Toxoplasma gondii; acute ileitis; Th1-type immunopathology; anti-inflammatory effects; immunomodulatory properties; gut–brain axis
8.  The Goblet Cell Protein Clca1 (Alias mClca3 or Gob-5) Is Not Required for Intestinal Mucus Synthesis, Structure and Barrier Function in Naive or DSS-Challenged Mice 
PLoS ONE  2015;10(7):e0131991.
The secreted, goblet cell-derived protein Clca1 (chloride channel regulator, calcium-activated-1) has been linked to diseases with mucus overproduction, including asthma and cystic fibrosis. In the intestine Clca1 is found in the mucus with an abundance and expression pattern similar to Muc2, the major structural mucus component. We hypothesized that Clca1 is required for the synthesis, structure or barrier function of intestinal mucus and therefore compared wild type and Clca1-deficient mice under naive and at various time points of DSS (dextran sodium sulfate)-challenged conditions. The mucus phenotype in Clca1-deficient compared to wild type mice was systematically characterized by assessment of the mucus protein composition using proteomics, immunofluorescence and expression analysis of selected mucin genes on mRNA level. Mucus barrier integrity was assessed in-vivo by analysis of bacterial penetration into the mucus and translocation into sentinel organs combined analysis of the fecal microbiota and ex-vivo by assessment of mucus penetrability using beads. All of these assays revealed no relevant differences between wild type and Clca1-deficient mice under steady state or DSS-challenged conditions in mouse colon. Clca1 is not required for mucus synthesis, structure and barrier function in the murine colon.
PMCID: PMC4498832  PMID: 26162072
9.  Saponins increase susceptibility of vancomycin-resistant enterococci to antibiotic compounds 
The resistance of commensal bacteria to first and second line antibiotics has reached an alarming level in many parts of the world and endangers the effective treatment of infectious diseases. In this study, the influence of the plant-derived natural saponins glycyrrhizic acid, β-aescin, α-hederin, hederacoside C, and primulic acid 1 on the susceptibility of vancomycin-resistant enterococci (VRE) against antibiotics of clinical relevance was investigated in 20 clinical isolates. Furthermore, the antibacterial properties of saponins under study against VRE were determined in vitro. Results reveal that the susceptibility of VRE against gentamicin, teicoplanin, and daptomycin was enhanced in the presence of the saponin glycyrrhizic acid. Most importantly, glycyrrhizic acid (1 mg/ml) diminished the minimal inhibitory concentration (MIC) of gentamicin in gentamicin low-level intrinsic resistant VRE from 2 – >8 mg/l to ≤ 0.125–1 mg/l. The adding of β-aescin, α-hederin, hederacoside C, and primulic acid 1 to the antibiotics under study showed, compared to glycyrrhizic acid, less influence on the antibiotic potency. Only glycyrrhizic acid (1 mg/ml) and α‑hederin (0.2 mg/ml) showed weak antibacterial properties against the clinical isolates. Our study points towards a therapeutic potential of saponins in the coapplication with antibiotics for bacterial infections.
PMCID: PMC4271817  PMID: 25544893
combination effect; daptomycin; gentamicin; glycyrrhizic acid; saponins; teicoplanin; vancomycin-resistant enterococci
10.  Composition of Intestinal Microbiota in Immune-Deficient Mice Kept in Three Different Housing Conditions 
PLoS ONE  2014;9(11):e113406.
Abundance of commensals constituting the intestinal microbiota (IM) affects the immune system and predisposes to a variety of diseases, including intestinal infections, cancer, inflammatory and metabolic disorders. Housing conditions determine the IM and can hence influence the immune system. We analyzed how both variables affect the IM of four immune-compromized mouse lines kept under different housing conditions.
Methodology/Principal Findings
We investigated the IM composition in mice by quantitative 16S rRNA RT-PCR analysis of the main fecal bacterial groups (Enterobacteriaceae, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella (BP) spp., Clostridium leptum and coccoides groups). Mice were homozygous (HO) or heterozygous (HE) for a targeted inactivating mutation of either the IFN-γ Receptor (R), IFN-γ, Rag1 or IL-4 genes. Overall, differences in IM composition were subtle. However, in the SPF-barrier, total eubacterial loads were higher in Rag1 HE versus Rag1 HO mice as well as in IFN-γR HE versus IFN-γR HO and WT animals. Although absent in WT mice, bifidobacterial loads were higher in HO and HE IFN-γ and Rag1 as well as IL-4 HO mice. Furthermore, BP was slightly lower in HO and HE IFN-γR and IFN-γ mice as well as in IL-4 HO mice as compared to WT controls. Interestingly, IM compositions were comparable in WT mice when kept in individual ventilated cages (IVC) or open cages (OC). IFN-γ HO and HE mice, however, had higher enterobacteria and BP loads, but lacked bifidobacteria when kept in OC versus IVC, as was the case in HO and HE Rag1 mice. In addition, Rag1 HO mice harbored higher clostridial loads when housed in OC as compared to IVC. Unexpectedly, lactobacilli levels were higher in IFN-γR mice when kept in OC versus IVC.
Housing-dependent and immune-deficiency mediated changes in intestinal microbiota composition were rather subtle but may nevertheless impact immunopathology in experimental models.
PMCID: PMC4234647  PMID: 25401702
11.  Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae 
PLoS ONE  2014;9(9):e108389.
The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.
Methodology/Principal Findings
Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.
Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.
PMCID: PMC4169633  PMID: 25238233
12.  Nucleotide-Oligomerization-Domain-2 Affects Commensal Gut Microbiota Composition and Intracerebral Immunopathology in Acute Toxoplasma gondii Induced Murine Ileitis 
PLoS ONE  2014;9(8):e105120.
Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far.
Methodology/Principal Findings
Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra-intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals.
NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner.
PMCID: PMC4139296  PMID: 25141224
13.  Helicobacter pylori Induced Gastric Immunopathology Is Associated with Distinct Microbiota Changes in the Large Intestines of Long-Term Infected Mongolian Gerbils 
PLoS ONE  2014;9(6):e100362.
Gastrointestinal (GI) inflammation in mice and men are frequently accompanied by distinct changes of the GI microbiota composition at sites of inflammation. Helicobacter (H.) pylori infection results in gastric immunopathology accompanied by colonization of stomachs with bacterial species, which are usually restricted to the lower intestine. Potential microbiota shifts distal to the inflammatory process following long-term H. pylori infection, however, have not been studied so far.
Methodology/Principal Findings
For the first time, we investigated microbiota changes along the entire GI tract of Mongolian gerbils after 14 months of infection with H. pylori B8 wildtype (WT) or its isogenic ΔcagY mutant (MUT) strain which is defective in the type IV secretion system and thus unable to modulate specific host pathways. Comprehensive cultural analyses revealed that severe gastric diseases such as atrophic pangastritis and precancerous transformations were accompanied by elevated luminal loads of E. coli and enterococci in the caecum and together with Bacteroides/Prevotella spp. in the colon of H. pylori WT, but not MUT infected gerbils as compared to naïve animals. Strikingly, molecular analyses revealed that Akkermansia, an uncultivable species involved in mucus degradation, was exclusively abundant in large intestines of H. pylori WT, but not MUT infected nor naïve gerbils.
Taken together, long-term infection of Mongolian gerbils with a H. pylori WT strain displaying an intact type IV secretion system leads to distinct shifts of the microbiota composition in the distal uninflamed, but not proximal inflamed GI tract. Hence, H. pylori induced immunopathogenesis of the stomach, including hypochlorhydria and hypergastrinemia, might trigger large intestinal microbiota changes whereas the exact underlying mechanisms need to be further unraveled.
PMCID: PMC4062524  PMID: 24941045
14.  The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma gondii induced ileitis 
Gut Pathogens  2014;6:19.
Following peroral Toxoplasma (T.) gondii infection, susceptible mice develop acute ileitis due to a microbiota-dependent Th1 type immunopathology. Toll-like-receptor (TLR)-9 is known to recognize bacterial DNA and mediates intestinal inflammation, but its impact on intestinal microbiota composition and extra-intestinal sequelae following T. gondii infection has not yet been elucidated.
Methods and results
Seven days following peroral infection (p.i.) with 100 cysts of T. gondii ME49 strain, TLR-9-/- and wildtype (WT) mice suffered from comparable ileitis, whereas ileal parasitic loads as well as IFN-γ and nitric oxide levels were higher in TLR-9-/- compared to WT mice. Locally, TLR-9-/- mice exhibited increased ileal CD3+, but not FOXP3+ cell numbers at day 7 p.i.; in mesenteric lymph nodes IFN-γ-producing CD4+ cell numbers and TNF-α and IFN-γ concentrations were also increased in TLR-9-/- compared to WT mice. T. gondii DNA levels, however, did not differ in mice of either genotype. Differences in intestinal microbiota were rather subtle except for bifidobacteria that were virtually absent in both, naïve and T. gondii infected TLR-9-/-, but not WT mice. Extra-intestinally, TLR-9-/- mice displayed less distinct systemic immune responses as indicated by lower serum IL-6, and splenic TNF-α and IFN-γ levels as compared to WT mice despite higher translocation rates of intestinal bacteria to extra-intestinal compartments such as liver, spleen, kidney, and cardiac blood. Most importantly, brains were also affected in this inflammatory scenario as early as day 7 p.i. Remarkably, TLR-9-/- mice exhibited more pronounced inflammatory infiltrates with higher numbers of F4/80+ macrophages and microglia in the cortex and meninges as compared to WT mice, whereas T. gondii DNA levels did not differ.
We here show that TLR-9 is not required for the development of T. gondii induced ileitis but mediates distinct inflammatory changes in intestinal and extra-intestinal compartments including the brain.
PMCID: PMC4057803  PMID: 24932221
Toxoplasma gondii; Acute ileitis; TLR-9; Th1-type immunopathology; Extra-intestinal immune responses; Intracerebral inflammation; Pro-inflammatory cytokines; Intestinal microbiota composition; Bacterial translocation; Systemic inflammatory response; Bifidobacteria; FOXP3; Regulatory T cells; Gut-brain-axis
15.  The impact of serine protease HtrA in apoptosis, intestinal immune responses and extra-intestinal histopathology during Campylobacter jejuni infection of infant mice 
Gut Pathogens  2014;6:16.
Campylobacter jejuni has emerged as a leading cause of bacterial enterocolitis. The serine protease HtrA has been shown to be a pivotal, novel C. jejuni virulence factor involved in cell invasion and transmigration across polarised epithelial cells in vitro. However, the functional relevance of the htrA gene for the interaction of C. jejuni with the host immune system in the infant mouse infection model has not been investigated so far.
Here we studied the role of C. jejuni htrA during infection of 3-weeks-old infant mice. Immediately after weaning, conventional wild-type mice were perorally infected with the NCTC11168∆htrA mutant (∆htrA) or the parental wild-type strain. Approximately one third of infected infant mice suffered from bloody diarrhea until day 7 post infection (p.i.), whereas colonic histopathological changes were rather moderate but comparable between the two strains. Interestingly, parental, but not ∆htrA mutant infected mice, displayed a multifold increase of apoptotic cells in the colonic mucosa at day 7 p.i., which was paralleled by higher colonic levels of pro-inflammatory cytokines such as TNF-α and IFN-γ and the matrix-degrading enzyme matrixmetalloproteinase-2 (MMP-2). Furthermore, higher numbers of proliferating cells could be observed in the colon of ∆htrA infected mice as compared to the parental wild-type strain. Remarkably, as early as 7 days p.i. infant mice also exhibited inflammatory changes in extra-intestinal compartments such as liver, kidneys and lungs, which were less distinct in kidneys and lungs following ∆htrA versus parental strain infection. However, live C. jejuni bacteria could not be found in these organs, suggesting the induction of systemic effects during intestinal infection.
Upon C. jejuni ∆htrA strain infection of infant mice, intestinal and extra-intestinal pro-inflammatory immune responses were ameliorated in the infant mouse model system. Future studies will shed further light onto the molecular mechanisms of host-pathogen interactions.
PMCID: PMC4040118  PMID: 24883112
Cell invasion; Conventional infant mice; Ulcerative enterocolitis; Innate immunity; Host-pathogen-interaction; Apoptosis; Extra-intestinal immune responses; Hepatic; Renal; Pulmonal histopathology
16.  Small intestinal permeability in older adults 
Physiological Reports  2014;2(4):e00281.
It is not yet clear whether intestinal mucosal permeability changes with advancing age in humans. This question is of high importance for drug and nutrition approaches for older adults. Our main objective was to answer the question if small intestinal barrier integrity deteriorates with healthy aging. We conducted a cross‐sectional study including the pooled data of 215 nonsmoking healthy adults (93 female/122 male), 84 of whom were aged between 60 and 82 years. After a 12‐h fast, all participants ingested 10 g of lactulose and 5 g of mannitol. Urine was collected for 5 h afterwards and analyzed for test sugars. The permeability index (PI = lactulose/mannitol) was used to assess small intestinal permeability. Low‐grade inflammation defined by high‐sensitivity C‐reactive protein ≥1 mL/L and kidney function (estimated glomerular filtration rate) were determined in the older age group. The PI was similar in older compared to younger adults (P =0.887). However, the urinary recovery of lactulose and mannitol was lower in the older adults and this change was neither associated with urinary volume nor glomerular filtration rate. The PI was not significantly correlated with low‐grade inflammation or presence of noninsulin‐dependent type 2 diabetes. However, it significantly deteriorated in the copresence of both conditions compared to low‐grade inflammation alone (P =0.043) or type 2 diabetes alone (P =0.015). Small intestinal mucosal barrier does not deteriorate with age per se. But low‐grade inflammation coupled with minor disease challenges, such as type 2 diabetes, can compromise the small intestinal barrier.
Until now, it has not been clear if the small intestinal mucosal barrier deteriorates with age per se. We investigated the pooled data of 215 nonsmoking healthy adults, 84 of whom were aged between 60 and 82 years and found similar intestinal permeability results in all age classes. However, in participants with low‐grade inflammation coupled with type 2 diabetes the small intestinal integrity was compromised.
PMCID: PMC4001874  PMID: 24771689
Aging; cardiovascular risk; gut leakiness; small intestine; sugar test
17.  Impact of Campylobacter jejuni cj0268c Knockout Mutation on Intestinal Colonization, Translocation, and Induction of Immunopathology in Gnotobiotic IL-10 Deficient Mice 
PLoS ONE  2014;9(2):e90148.
Although Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden, the underlying molecular mechanisms of induced intestinal immunopathology are still not well understood. We have recently generated a C. jejuni mutant strain NCTC11168::cj0268c, which has been shown to be involved in cellular adhesion and invasion. The immunopathological impact of this gene, however, has not been investigated in vivo so far.
Methodology/Principal Findings
Gnotobiotic IL-10 deficient mice were generated by quintuple antibiotic treatment and perorally infected with C. jejuni mutant strain NCTC11168::cj0268c, its complemented version (NCTC11168::cj0268c-comp-cj0268c), or the parental strain NCTC11168. Kinetic analyses of fecal pathogen loads until day 6 post infection (p.i.) revealed that knockout of cj0268c did not compromise intestinal C. jejuni colonization capacities. Whereas animals irrespective of the analysed C. jejuni strain developed similar clinical symptoms of campylobacteriosis (i.e. enteritis), mice infected with the NCTC11168::cj0268c mutant strain displayed significant longer small as well as large intestinal lengths indicative for less distinct C. jejuni induced pathology when compared to infected control groups at day 6 p.i. This was further supported by significantly lower apoptotic and T cell numbers in the colonic mucosa and lamina propria, which were paralleled by lower intestinal IFN-γ and IL-6 concentrations at day 6 following knockout mutant NCTC11168::cj0268c as compared to parental strain infection. Remarkably, less intestinal immunopathology was accompanied by lower IFN-γ secretion in ex vivo biopsies taken from mesenteric lymphnodes of NCTC11168::cj0268c infected mice versus controls.
We here for the first time show that the cj0268c gene is involved in mediating C. jejuni induced immunopathogenesis in vivo. Future studies will provide further deep insights into the immunological and molecular interplays between C. jejuni and innate immunity in human campylobacteriosis.
PMCID: PMC3934979  PMID: 24587249
18.  The role of serine protease HtrA in acute ulcerative enterocolitis and extra-intestinal immune responses during Campylobacter jejuni infection of gnotobiotic IL-10 deficient mice 
Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden. C. jejuni can cross the intestinal epithelial barrier as visualized in biopsies derived from human patients and animal models, however, the underlying molecular mechanisms and associated immunopathology are still not well understood. We have recently shown that the secreted serine protease HtrA (high temperature requirement A) plays a key role in C. jejuni cellular invasion and transmigration across polarized epithelial cells in vitro. In the present in vivo study we investigated the role of HtrA during C. jejuni infection of mice. We used the gnotobiotic IL-10−/− mouse model to study campylobacteriosis following peroral infection with the C. jejuni wild-type (WT) strain NCTC11168 and the isogenic, non-polar NCTC11168ΔhtrA deletion mutant. Six days post infection (p.i.) with either strain mice harbored comparable intestinal C. jejuni loads, whereas ulcerative enterocolitis was less pronounced in mice infected with the ΔhtrA mutant strain. Moreover, ΔhtrA mutant infected mice displayed lower apoptotic cell numbers in the large intestinal mucosa, less colonic accumulation of neutrophils, macrophages and monocytes, lower large intestinal nitric oxide, IFN-γ, and IL-6 as well as lower TNF-α and IL-6 serum concentrations as compared to WT strain infected mice at day 6 p.i. Notably, immunopathological responses were not restricted to the intestinal tract given that liver and kidneys exhibited mild histopathological changes 6 days p.i. with either C. jejuni strain. We also found that hepatic and renal nitric oxide levels or renal TNF-α concentrations were lower in the ΔhtrA mutant as compared to WT strain infected mice. In conclusion, we show here that the C. jejuni HtrA protein plays a pivotal role in inducing host cell apoptosis and immunopathology during murine campylobacteriosis in the gut in vivo.
PMCID: PMC4050650  PMID: 24959425
ulcerative enterocolitis; colonization resistance; innate immunity; host-pathogen-interaction; bacterial translocation; intestinal immunopathology; extra-intestinal immune responses; systemic immune responses
19.  The Campylobacter jejuni Cj0268c Protein Is Required for Adhesion and Invasion In Vitro 
PLoS ONE  2013;8(11):e81069.
Adherence of Campylobacter jejuni to its particular host cells is mediated by several pathogen proteins. We screened a transposon-based mutant library of C. jejuni in order to identify clones with an invasion deficient phenotype towards Caco2 cells and detected a mutant with the transposon insertion in gene cj0268c. In vitro characterization of a generated non-random mutant, the mutant complemented with an intact copy of cj0268c and parental strain NCTC 11168 confirmed the relevance of Cj0268c in the invasion process, in particular regarding adherence to host cells. Whereas Cj0268c does not impact autoagglutination or motility of C. jejuni, heterologous expression in E. coli strain DH5α enhanced the potential of the complemented E. coli strain to adhere to Caco2 cells significantly and, thus, indicates that Cj0268c does not need to interact with other C. jejuni proteins to develop its adherence-mediating phenotype. Flow cytometric measurements of E. coli expressing Cj0268c indicate a localization of the protein in the periplasmic space with no access of its C-terminus to the bacterial surface. Since a respective knockout mutant possesses clearly reduced resistance to Triton X-100 treatment, Cj0268c contributes to the stability of the bacterial cell wall. Finally, we could show that the presence of cj0268c seems to be ubiquitous in isolates of C. jejuni and does not correlate with specific clonal groups regarding pathogenicity or pathogen metabolism.
PMCID: PMC3841222  PMID: 24303031
20.  Modification of Intestinal Microbiota and Its Consequences for Innate Immune Response in the Pathogenesis of Campylobacteriosis 
Campylobacter jejuni is the leading cause of bacterial food-borne gastroenteritis in the world, and thus one of the most important public health concerns. The initial stage in its pathogenesis after ingestion is to overcome colonization resistance that is maintained by the human intestinal microbiota. But how it overcomes colonization resistance is unknown. Recently developed humanized gnotobiotic mouse models have provided deeper insights into this initial stage and host's immune response. These studies have found that a fat-rich diet modifies the composition of the conventional intestinal microbiota by increasing the Firmicutes and Proteobacteria loads while reducing the Actinobacteria and Bacteroidetes loads creating an imbalance that exposes the intestinal epithelial cells to adherence. Upon adherence, deoxycholic acid stimulates C. jejuni to synthesize Campylobacter invasion antigens, which invade the epithelial cells. In response, NF-κB triggers the maturation of dendritic cells. Chemokines produced by the activated dendritic cells initiate the clearance of C. jejuni cells by inducing the actions of neutrophils, B-lymphocytes, and various subsets of T-cells. This immune response causes inflammation. This review focuses on the progress that has been made on understanding the relationship between intestinal microbiota shift, establishment of C. jejuni infection, and consequent immune response.
PMCID: PMC3845433  PMID: 24324507
21.  Small Intestinal Nematode Infection of Mice Is Associated with Increased Enterobacterial Loads alongside the Intestinal Tract 
PLoS ONE  2013;8(9):e74026.
Parasitic nematodes are potent modulators of immune reactivity in mice and men. Intestinal nematodes live in close contact with commensal gut bacteria, provoke biased Th2 immune responses upon infection, and subsequently lead to changes in gut physiology. We hypothesized that murine nematode infection is associated with distinct changes of the intestinal bacterial microbiota composition. We here studied intestinal inflammatory and immune responses in mice following infection with the hookworm Heligmosomoidespolygyrusbakeri and applied cultural and molecular techniques to quantitatively assess intestinal microbiota changes in the ileum, cecum and colon. At day 14 post nematode infection, mice harbored significantly higher numbers of γ-Proteobacteria/Enterobacteriaceae and members of the Bacteroides/Prevotella group in their cecum as compared to uninfected controls. Abundance of Gram-positive species such as Lactobacilli, Clostridia as well as the total bacterial load was not affected by worm infection. The altered microbiota composition was independent of the IL-4/-13 – STAT6 signaling axis, as infected IL-4Rα-/- mice showed a similar increase in enterobacterial loads. In conclusion, infection with an enteric nematode is accompanied by distinct intestinal microbiota changes towards higher abundance of gram-negative commensal species at the small intestinal site of infection (and inflammation), but also in the parasite-free large intestinal tract. Further studies should unravel the impact of nematode-induced microbiota changes in inflammatory bowel disease to allow for a better understanding of how theses parasites interfere with intestinal inflammation and bacterial communities in men.
PMCID: PMC3769368  PMID: 24040152
22.  Impact of metal ion homeostasis of genetically modified Escherichia coli Nissle 1917 and K12 (W3110) strains on colonization properties in the murine intestinal tract 
Metal ions are integral parts of pro- as well as eukaryotic cell homeostasis. Escherichia coli proved a valuable in vitro model organism to elucidate essential mechanisms involved in uptake, storage, and export of metal ions. Given that E. coli Nissle 1917 is able to overcome murine colonization resistance, we generated several E. coli Nissle 1917 mutants with defects in zinc, iron, copper, nickel, manganese homeostasis and performed a comprehensive survey of the impact of metal ion transport and homeostasis for E. coli colonization capacities within the murine intestinal tract. Seven days following peroral infection of conventional mice with E. coli Nissle 1917 strains exhibiting defined defects in zinc or iron uptake, the respective mutant and parental strains could be cultured at comparable, but low levels from the colonic lumen. We next reassociated gnotobiotic mice in which the microbiota responsible for colonization resistance was abrogated by broad-spectrum antibiotics with six different E. coli K12 (W3110) mutants. Seven days following peroral challenge, each mutant and parental strain stably colonized duodenum, ileum, and colon at comparable levels. Taken together, defects in zinc, iron, copper, nickel, and manganese homeostasis do not compromise colonization capacities of E. coli in the murine intestinal tract.
PMCID: PMC3832094  PMID: 24265943
bacterial colonization capacity; bacterial fitness; colonization resistance; copper homeostasis; E. coli K12 (W3110); E. coli Nissle 1917; enterobactin; gnotobiotic mice; iron homeostasis; manganese homeostasis; metal ion homeostasis and transport; metal ion mutant strains; periplasmic copper stress; zinc homeostasis
23.  Survey of extra-intestinal immune responses in asymptomatic long-term Campylobacter jejuni-infected mice 
Campylobacter jejuni is among the most frequently reported bacterial pathogens causing diarrhea in humans worldwide. We recently reported a murine infection model mimicking key features of human campylobacteriosis. Six days following oral C. jejuni infection immediately after weaning, infant mice developed acute enterocolitis resolving within 2 weeks. Thereafter, C. jejuni could still be isolated from the intestines of asymptomatic mice at low levels accompanied by distinct immune responses, both at intestinal and extra-intestinal locations. We here show that, at day 103 post infection (p.i.), long-term C. jejuni-infected mice exhibited higher numbers of T lymphocytes in liver, lung, kindneys, and cardiac muscle as compared to uninfected controls. In addition, B lymphocytes were slightly higher, but macrophage numbers were significantly lower in liver and lung of C. jejuni-infected versus naive mice. As compared to uninfected control animals, proliferating cells were significantly lower in liver, lung, kidneys, cardiac muscle, and spleen at day 103 p.i., whereas more apoptotic cells were abundant in the spleen with predominance in the red pulp. This study underlines that post-infectious, immunological sequelae at extra-intestinal locations are of importance even in asymptomatic long-term C. jejuni carriers and need to be further studied in order to unravel the underlying molecular mechanisms.
PMCID: PMC3832099  PMID: 24265935
apoptotic cells; B lymphocytes; Campylobacter jejuni; colonization resistance; extra-intestinal immune cell influx; host–pathogen interaction; infant mice; innate and adaptive immunity; intestinal microbiota; long-term C. jejuni infection; macrophages; proliferative cells; T lymphocytes
24.  Colonization resistance against genetically modified Escherichia coli K12 (W3110) strains is abrogated following broad-spectrum antibiotic treatment and acute ileitis 
Escherichia coli K12 (EcK12) is commonly used for gene technology purposes and regarded as a security strain due to its inability to adhere to epithelial cells. The conventional intestinal microbiota composition is critical for physiological colonization resistance against most bacterial species including pathogens. We were therefore interested whether intestinal colonization by a genetically modified EcK12 (W3110) strain carrying a chloramphenicol resistance cassette was facilitated following broad-spectrum antibiotic treatment eradicating the intestinal microbiota or induction of small intestinal inflammation accompanied by distinct microbiota shifts. Whereas conventional C57BL/6 and BALB/c mice had virtually expelled the EcK12 (W3110) strain within the first 3 days upon peroral infection, EcK12 (W3110) could establish within the small and large intestines of gnotobiotic mice generated by quintuple antibiotic treatment. Gnotobiotic mice perorally infected with EcK12 (W3110) plus fecal transplant from conventional donors harbored lower intestinal EcK12 (W3110) loads compared to animals challenged with EcK12 (W3110) alone. Furthermore, EcK12 (W3110) infection of conventional mice after but not before induction of ileitis resulted in stable colonization of ileum and colon by EcK12 (W3110). Taken together, broad-spectrum antibiotic treatment and intestinal inflammation compromise colonization resistance and thus facilitate colonization of the intestinal tract with genetically modified EcK12 security strains.
PMCID: PMC3832100  PMID: 24265942
acute small intestinal inflammation; broad-spectrum antibiotic treatment; colonization resistance; E. coli K12 (W3110); Escherichia coli; fecal transplantation; genetically modified security strains; gnotobiotic mice; intestinal microbiota; Toxoplasma gondii
25.  Inhibition of Helicobacter pylori urease activity in vivo by the synthetic nickel binding protein Hpn 
Helicobacter pylori infection is the most common cause of gastroduodenal ulcerations worldwide. Adaptation of H. pylori to the acidic environment is mediated by urease splitting urea into carbon dioxide and ammonia. Whereas neutralization of acid by ammonia is essential for gastric H. pylori colonization, the catalytic activity of urease is mediated by nickel ions. Therefore, nickel uptake and metabolism play key roles in H. pylori infection and urease is considered first line target for drug development and vaccination. Since nickel binding within H. pylori cells is mediated by the Histidine-rich protein designated Hpn, we investigated whether nickel binding by a synthetic Hpn is capable of abrogating urease activity of live H. pylori in liquid cultures. Supplementation of growth media with synthetic Hpn completely inhibited urease acitivity in live cells, indicating that H. pylori nickel uptake is effectively blocked by Hpn. Thus, nickel chelation by Hpn is stronger than nickel uptake of H. pylori offering therapeutic use of Hpn. Although the nickel binding of Hpn was confirmed by binding assays in vitro, its use in anti-H. pylori directed strategy will further need to be adapted to the gastric environment given that protons interfere with nickel binding and Hpn is degraded by pepsin.
PMCID: PMC3832076  PMID: 24265922
Helicobacter pylori, chronic gastritis, urease, nickel, histidine-rich protein

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