Anna Smoczek

Age-dependent TLR3 expression of the intestinal epithelium contributes to rotavirus susceptibility.

Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3−/−) or the adaptor molecule Trif (TrifLps2/Lps2) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection.

Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.

Norovirus triggered microbiota-driven mucosal inflammation in interleukin 10-deficient mice.

Background:Infection may trigger clinically overt mucosal inflammation in patients with predisposition for inflammatory bowel disease. However, the impact of particular enteropathogenic microorganisms is ill-defined. In this study, the influence of murine norovirus (MNV) infection on clinical, histopathological, and immunological features of mucosal inflammation in the IL10-deficient (Il10−/−) mouse model of inflammatory bowel disease was examined. Methods:C57BL/6J and C3H/HeJBir wild-type and Il10−/− mice kept under special pathogen-free conditions and devoid of clinical and histopathological signs of mucosal inflammation were monitored after MNV infection for structural and functional intestinal barrier changes by in situ MNV reverse transcription PCR, transgene reporter gene technology, histology, flux measurements, quantitative real-time PCR, immunohistology, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. In addition, the influence of the enteric microbiota was analyzed in MNV-infected germfree Il10−/− mice. Results:Although MNV-infected wild-type mice remained asymptomatic, mucosal inflammation was noted in previously healthy Il10−/− mice 2 to 4 weeks after infection. MNV-induced changes in Il10−/− mice included increased paracellular permeability indicated by increased mucosal mannitol flux, reduced gene expression of tight junction molecules, and an enhanced rate of epithelial apoptosis. MNV-induced reduction of tight junction protein expression and inflammatory lesions were absent in germfree Il10−/− mice, whereas epithelial apoptosis was still observed. Conclusions:Despite its subclinical course in wild-type animals, MNV causes epithelial barrier disruption in Il10−/− animals representing a potent colitogenic stimulus that largely depends on the presence of the enteric microbiota. MNV might thus trigger overt clinical disease in individuals with a nonsymptomatic predisposition for inflammatory bowel disease by impairment of the intestinal mucosa.

Comparative evaluation of establishing a human gut microbial community within rodent models

The structure of the human gut microbial community is determined by host genetics and environmental factors, where alterations in its structure have been associated with the onset of different diseases. Establishing a defined human gut microbial community within inbred rodent models provides a means to study microbial-related pathologies, however, an in-depth comparison of the established human gut microbiota in the different models is lacking. We compared the efficiency of establishing the bacterial component of a defined human microbial community within germ-free (GF) rats, GF mice and antibiotic-treated specific pathogen-free mice. Remarkable differences were observed between the different rodent models. While the majority of abundant human-donor bacterial phylotypes were established in the GF rats, only a subset was present in the GF mice. Despite the fact that members of the phylum Bacteriodetes were well established in all rodent models, mice enriched for phylotypes related to species of Bacteroides. In contrary to the efficiency of Clostridiales to populate the GF rat in relative proportions to that of the human-donor, members of Clostridia cluster IV only poorly colonize the mouse gut. Thus, the genetic background of the different recipient rodent systems (that is, rats and mice) strongly influences the nature of the populating human gut microbiota, determining each model’s biological suitability.

CpG Motifs of Bacterial DNA Exert Protective Effects in Mouse Models of IBD by Antigen-Independent Tolerance Induction

BACKGROUND & AIMS Prophylactic treatment of mice with CpG motifs of bacterial DNA protects from experimental inflammatory bowel disease, at least partly via induction of inhibitory T-cells. The aim of this study was to elucidate whether these CpG-dependent protective effects require presence of bacterial flora suggesting antigen-specific regulatory activity. METHODS Germ-free BALB/c and IL-10(-/-) mice were treated with CpG-oligodeoxynucleotides (ODN), control-ODN, or PBS. CD4(+)CD62L(+) cells of these mice were transferred into SCID recipients. CpG-ODN-treated germ-free IL-10(-/-) mice were transferred into colitogenic environment. Monoclonal antibodies were used to neutralize TGF-beta and IFN-alpha/beta during CpG-ODN treatment. CD4(+)CD62L(+) cells of donors were evaluated for cytokine secretion and FOXP3, PD-1, and CD25 expression. RESULTS Compared to PBS or control-ODN treatment, CpG-ODN application to germ-free donors led to decreased intestinal inflammation as indicated by histology, decreased proinflammatory cytokines, and increased IL-10 secretion. Protection was also observed after cotransfer of cells from PBS and CpG-ODN treated donors. Anti-TGF-beta and anti-INF-alpha/beta partly reversed the protective CpG-ODN effect. CpG-ODN-treated germ-free IL-10(-/-) mice transferred into colitogenic environment developed significantly less colitis than controls but not recipients of IL-10(-/-)CD4(+)CD62L(+)cells. CD4(+)CD62L(+)cells of CpG-treated germ-free animals displayed increased expression of regulatory markers. CONCLUSIONS Even without pre-existence of bacterial flora CpG-ODN exposition induces tolerance, indicating that CpG-ODN-induced regulatory T-cells are not bacterial antigen specific. TGF-beta and IFN-alpha/beta play major roles in induction of regulatory cells, and although IL10-independent mechanisms play a role in CpG-ODN protection, this cytokine likely is important for the effector mechanism of CpG-ODN-induced regulatory T-cells.

Strain-specific colitis susceptibility in IL10-deficient mice depends on complex gut microbiota–host interactions†

Background: Colitis susceptibility in Il10−/− mice depends on genetic background and microbiota composition. A major genetic locus mediating colitis susceptibility, Cdcs1, was transferred from susceptible C3Bir‐Il10−/− to resistant B6‐Il10−/− mice, resulting in susceptible congenic BC‐R3‐Il10−/− mice. The aim of this study was to determine the impact of microbiota on this differential colitis susceptibility using a Helicobacter hepaticus infection model. Methods: Parental C3Bir‐Il10−/−, B6‐Il10−/−, and congenic BC‐R3‐Il10−/− mice were inoculated with H. hepaticus and analyzed for inflammation. In parental Il10−/− mice, microbiota composition was determined by terminal restriction fragment length polymorphism (T‐RFLP) and quantitative polymerase chain reaction (qPCR). Results: Most severe inflammation was observed in C3Bir‐Il10−/− in the cecum, in BC‐R3‐Il10−/− in cecum and colon, and, unexpectedly, in B6‐Il10−/− in the colon. C3Bir‐Il10−/− and BC‐R3‐Il10−/− secreted significantly more interferon‐gamma (IFN&ggr;) and interleukin (IL)17 than B6‐Il10−/−. T‐RFLP analyses in C3Bir‐Il10−/− and B6‐Il10−/− mice revealed 1) a significant impact of H. hepaticus infection on species richness and diversity, and 2) strain differences in microbiota composition only after H. hepaticus infection. qPCR revealed higher numbers of Clostridia leptum and Bacteroides spp. in the cecum of infected C3Bir‐Il10−/− mice, and Lactobacillus spp. in B6‐Il10−/− mice. Conclusions: Cdcs1 modifies the response to H. hepaticus infection. However, this infection alone does not reflect the original response to a complex colitogenic biota. H. hepaticus‐induced inflammation altered intestinal microbiota in a mouse strain‐specific manner. Bacteroides spp. became more abundant in susceptible C3Bir‐Il10−/−, lactobacilli in B6‐Il10−/− mice. Therefore, both host immune response and differential compositional changes of microbiota play a role in strain‐specific colitis susceptibility in Il10−/− mice. (Inflamm Bowel Dis 2012;)

Klebsiella oxytoca : opportunistic infections in laboratory rodents

Opportunistic pathogens have become increasingly relevant as the causative agents of clinical disease and pathological lesions in laboratory animals. This study was conducted to evaluate the role of Klebsiella oxytoca as an opportunistic pathogen in laboratory rodents. Therefore, K. oxytoca-induced lesions were studied from 2004 to early 2006 in naturally infected rodent colonies maintained at The Jackson Laboratory (TJL), Bar Harbor, USA, the Animal Research Centre (Tierforschungszentrum, TFZ) of the University of Ulm, Germany and the Central Animal Facility (ZTM) of the Hannover Medical School, Germany. K. oxytoca infections were observed in substrains of C3H/HeJ mice, which carry the Tlr4Lps-d allele; in LEW.1AR1-iddm rats, the latter being prone to diabetes mellitus; in immunodeficient NMRI-Foxn1nu mice; and in mole voles, Ellobius lutescens. The main lesions observed were severe suppurative otitis media, urogenital tract infections and pneumonia. Bacteriological examination revealed K. oxytoca as monocultures in all cases. Clonality analysis performed on strains isolated at the ZTM and TFZ (serotyping, pulse field gel electrophoresis [PFGE], enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction, sequencing of 16S rRNA and rpoB genes) revealed that the majority of bacteria belonged to two clones, one in each facility, expressing the capsule type K55 (ZTM) or K72 (TFZ). Two strains, one isolated at the ZTM and one at the TFZ, showed different PFGE and ERIC pattern than all other isolates and both expressed capsule type K35. In conclusion, K. oxytoca is an opportunistic pathogen capable of inducing pathological lesions in different rodent species.

Sensitivity to Escherichia coli Nissle 1917 in mice is dependent on environment and genetic background

Escherichia coli Nissle 1917 (EcN) is a well‐characterized probiotic bacterium. Although genomic comparisons of EcN with the uropathogenic E. coli strain CFT073 revealed high degrees of similarity, EcN is generally considered a non‐pathogenic organism. However, as recent evidence suggests that EcN is capable of inducing inflammatory responses in host intestinal epithelial cells, we aimed to investigate potential pathogenic properties of EcN in an in vivo model using various germ‐free (GF) mouse strains. With the exception of C3H/HeJZtm mice, which carry a defective toll‐like receptor (TLR)4‐allele, no lesions were obvious in mice of different strains orally inoculated with EcN for 1 week, although organ cultures (blood, lung, mesenteric lymph node, pancreas, spleen, liver and kidney) tested positive to various degrees. C3H/HeJZtm mice inoculated with EcN became clinically ill and the majority died or had to be euthanized. Organs of all gnotobiotic C3H/HeJZtm mice were positive for EcN by culture; major histological findings were moderate to severe pyogranulomatous serositis, typhlitis and pancreatitis. Histological findings were corroborated by highly elevated tumour necrosis factor (TNF) serum levels. Lesions were not detected in specified pathogen free maintained C3H/HeJZtm mice, GF C3H/HeJ mice lacking the interleukin‐10 gene, or GF C3H/HeJZtm mice that were inoculated with E. coli K12 strain MG1655 as a control. In addition, mild histological lesions were detected in Ztm:NMRI mice 3 months after oral inoculation with EcN. This study shows that EcN is capable of displaying a virulent phenotype in GF C3H/HeJZtm mice. Whether this phenotype is linked to the bacterium’s probiotic nature should be the focus of further studies.

Efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics: An animal study in rabbits

Nanoporous silica layers are able to host molecules and release them over a certain period of time. These local drug delivery systems for antibiotics could be a new approach in the treatment of chronic otitis media. The aim of this study was to examine the efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics in vivo. Pseudomonas aeruginosa was inoculated into the middle ear of rabbits to induce an otitis media. The control group received coated Bioverit®II implants without antibiotics. Coated prostheses with loaded ciprofloxacin were implanted into the middle ears of the study group. After 1 week, the rabbits were sacrificed. The clinical examination as well as the microbiological and histological examinations of organs and middle ear irrigation revealed clear differences between the two groups. P. aeruginosa was detected in every middle ear of the control group and was almost completely eliminated in the study group. Organ examinations revealed the presence of P. aeruginosa in the control group and a prevention of a bacterial spread in the study group. The nanoporous silica layer as antibiotic delivery system showed convincing efficacy in induced pseudomonal otitis media in the rabbit.

Quantitative phenotyping of inflammatory bowel disease in the IL-10-deficient mouse by use of noninvasive magnetic resonance imaging.

Background:Although magnetic resonance imaging (MRI) is an increasingly used diagnostic tool in the assessment of inflammatory bowel disease (IBD) in humans, diagnosis and quantitation of intestinal inflammation in animal models of IBD still depends on ex vivo techniques. The aim of this study was to evaluate whether high-field MRI is suitable for the quantitative phenotyping of gut inflammation in a dextran sulfate sodium (DSS)-triggered interleukin (IL)10-deficient (IL-10−/−) mouse model of IBD, especially in longitudinal studies. Methods:Using colitis-susceptible and -resistant backgrounds, MRI and ex vivo analyses were applied to characterize this specific model, differentiating disease severity and time-dependent alterations. Colon wall thickness, cecum wall tissue intensity, spleen, and mesenteric lymph node (MLN) volumes were evaluated 1, 2, 4, and 12 weeks after disease onset by T2-weighted MRI. Ex vivo parameters included histology, spleen, and MLN weight and analysis of cytokine expression. Results:MRI and ex vivo determined parameters correlated well, revealing a mouse strain-specific colitis development over time with characteristics typical for the DSS model in the initial and for the IL-10−/− model in the chronic phase. To evaluate the use of high-field MRI for monitoring therapeutic studies, mice with a profound colitis were treated with IL-10-producing Saccharomyces boulardii and monitored by MRI. Conclusions:MRI can be utilized to quantify colitis development in the IL-10−/− model of IBD. Therefore, this noninvasive technique might be highly advantageous for an individual follow-up of colitis development in chronic models of IBD, facilitating the reduction of animal numbers in this kind of research.