Authors:Markus M. Heimesaat, Ursula Grundmann, Marie E. Alutis, André Fischer, and Stefan Bereswill
Host immune responses are crucial for combating enteropathogenic infections including Campylobacter jejuni. Within 1 week following peroral C. jejuni infection, secondary abiotic IL-10−/− mice develop severe immunopathological sequelae affecting the colon (ulcerative enterocolitis). In the present study, we addressed whether pathogen-induced pro-inflammatory immune responses could also be observed in the small intestines dependent on the innate receptor nucleotide-oligomerization-domain-protein 2 (Nod2). Within 7 days following peroral infection, C. jejuni stably colonized the gastrointestinal tract of both IL-10−/− mice lacking Nod2 (Nod2−/− IL-10−/−) and IL-10−/− controls displaying bloody diarrhea with similar frequencies. Numbers of apoptotic and regenerating epithelial cells increased in the small intestines of C. jejuni-infected mice of either genotype that were accompanied by elevated ileal T and B lymphocyte counts. Notably, ileal T cell numbers were higher in C. jejuni-infected Nod2−/− IL-10−/− as compared to IL-10−/− counterparts. Furthermore, multifold increased concentrations of pro-inflammatory cytokines including IFN-γ, TNF, and MCP-1 could be measured in small intestinal ex vivo biopsies derived from C. jejuni-infected mice of either genotype. In conclusion, C. jejuni-induced pro-inflammatory immune responses affected the small intestines of both Nod2−/− IL-10−/− and IL-10−/− mice, whereas ileal T lymphocyte numbers were even higher in the former.
Authors:Isabel Stephany-Brassesco, Stefan Bereswill, Markus M. Heimesaat, and Matthias F. Melzig
Antibiotic resistance of Streptococcus pneumoniae has risen to worrying levels in the past few decades worldwide, and subsequently, effective treatment of respiratory tract infections has become even more challenging. While the need to develop new strategies to combat bacterial infections is urgent, novel antibiotic compounds are no longer a priority of the pharmaceutical industry. However, resistance-modifying agents can alleviate the spread of antibiotic resistance and render existing antibiotics effective again. In the present study, we aimed to determine the combinatory antimicrobial effects of the commercial herbal product Cefabronchin® and antibiotic compounds, such as amoxicillin and clarithromycin, on 6 clinical isolates of S. pneumoniae. Therefore, the minimal inhibitory concentration (MIC) of each agent before and after adding Cefabronchin® at different concentrations was determined by applying the checkerboard method. Sub-inhibitory concentrations of the added Cefabronchin® were found to reduce the MIC down to between 3.4% and 29.2% of the amoxicillin MIC and down to between 10.4% and 45.8% of the clarithromycin MIC in all 6 strains. In conclusion, this study provides evidence for the improved antimicrobial effects of commonly used antibiotics in combination with Cefabronchin® in order to combat infections with antibiotic-resistant S. pneumoniae strains.
Authors:Eliane von Klitzing, Fulya Öz, Ira Ekmekciu, Ulrike Escher, Stefan Bereswill, and Markus M. Heimesaat
Secondary abiotic mice generated by broad-spectrum antibiotic treatment provide a valuable tool for association studies with microbiota derived from different vertebrate hosts. We here generated human microbiota-associated (hma) mice by human fecal microbiota transplantation of secondary abiotic mice and performed a comprehensive survey of the intestinal microbiota dynamics in offspring of hma mice over 18 weeks following weaning as compared to their mothers applying both cultural and molecular methods. Mice were maintained under standard hygienic conditions with open cages, handled under aseptic conditions, and fed autoclaved chow and water. Within 1 week post weaning, fecal loads of commensal enterobacteria and enterococci had decreased, whereas obligate anaerobic bacteria such as Bacteroides/Prevotella species and clostridia were stably colonizing the intestines of hma offspring at high loads. Lactobacilli numbers were successively increasing until 18 weeks post weaning in both hma offspring and mothers, whereas by then, bifidobacteria were virtually undetectable in the former only. Interestingly, fecal lactobacilli and bifidobacteria were higher in mothers as compared to their offspring at 5 and 18 weeks post weaning. We conclude that the intestinal microbiota composition changes in offspring of hma mice, but also their mothers over time particularly affecting aerobic and microaerobic species.
Authors:Markus M. Heimesaat, Gül Karadas, André Fischer, Ulf B. Göbel, Thomas Alter, Stefan Bereswill, and Greta Gölz
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 broadspectrum 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.
Authors:Greta Gölz, Gül Karadas, André Fischer, Ulf B. Göbel, Thomas Alter, Stefan Bereswill, and Markus M. Heimesaat
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.
Authors:Markus M. Heimesaat, Ursula Grundmann, Marie E. Alutis, André Fischer, Ulf B. Göbel, and Stefan Bereswill
Within 1 week following peroral Campylobacter jejuni infection, infant mice develop acute enteritis resolving thereafter. We here assessed colonic expression profiles of mediators belonging to the IL-23/IL-22/IL-18 axis and of matrix-degrading gelatinases MMP-2 and MMP-9 at day 6 post C. jejuni strain 81-176 infection. Whereas the pathogen readily colonized the intestines of infant IL-18−/− mice only, colonic mucin-2 mRNA, a pivotal mucus constituent, was downregulated in IL-22−/− mice and accompanied by increased expression of pro-inflammatory cytokines including IFN-γ, TNF, IL-17A, and IL-1β. Furthermore, in both naive and infected IL-22−/− mice, colonic expression of IL-23p19 and IL-18 was lower as compared to wildtype mice, whereas, conversely, colonic IL-22 mRNA levels were lower in IL-18−/− and colonic IL-18 expression lower in IL-23p19−/− as compared to wildtype mice. Moreover, colonic expression of MMP-2 and MMP-9 and their endogenous inhibitor TIMP-1 were lower in IL-22−/− as compared to wildtype mice at day 6 postinfection. In conclusion, mediators belonging of the IL-23/IL-22/IL-18 axis as well as the gelatinases MMP-2 and MMP-9 are involved in mediating campylobacteriosis of infant mice in a differentially regulated fashion.
Authors:Markus M. Heimesaat, Ursula Grundmann, Marie E. Alutis, André Fischer, and Stefan Bereswill
Host immune responses are pivotal for combating enteropathogenic infections. We here assessed the impact of the innate receptor nucleotide oligomerization domain protein 2 (NOD2) in murine Campylobacter jejuni-infection. Conventionally colonized IL-10−/− mice lacking NOD2 and IL-10−/− controls were perorally challenged with C. jejuni strain 81-176 and displayed comparable pathogenic colonization of intestines until day 14 postinfection (p.i.). Whereas overall intestinal microbiota compositions were comparable in naive mice, NOD2−/− IL-10−/− mice exhibited less fecal bifidobacteria and lactobacilli than IL-10−/− counterparts after infection. Interestingly, NOD2−/− IL-10−/− mice were clinically more compromised during the early phase of infection, whereas, conversely, IL-10−/− animals exhibited more frequently bloody feces lateron. While colonic apoptotic cell and T lymphocyte numbers were comparable in either C. jejuni-infected mice, B lymphocytes were lower in the colon of infected NOD2−/− IL-10−/− mice versus controls. At day 14 p.i., colonic TNF and IL-23p19 mRNA levels were upregulated in NOD2−/− IL-10−/− mice only. Translocation rates of intestinal commensals to mesenteric lymphnodes and extra-intestinal compartments including liver and kidney were comparable, whereas viable bacteria were more frequently detected in spleens derived from IL-10−/− as compared to NOD2−/− IL-10−/− mice. In conclusion, NOD2 is involved during C. jejuni infection in conventionally colonized IL-10−/− mice in a time-dependent manner.
Authors:Markus M. Heimesaat, Ulrike Escher, Anne Grunau, Ulrike Fiebiger, and Stefan Bereswill
Within 1 week following high-dose Toxoplasma gondii infection, mice develop lethal necrotizing ileitis. However, data from a subacute T. gondii-induced ileitis model are scarce. Therefore, mice harboring a human gut microbiota were perorally infected with one cyst of T. gondii. Within 9 days post-infection, the intestinal microbiota composition shifted towards higher loads of commensal enterobacteria and enterococci. Following T. gondii infection, mice were clinically only mildly affected, whereas ≈60% of mice displayed fecal blood and mild-to-moderate ileal histopathological changes. Intestinal inflammation was further characterized by increased apoptotic intestinal epithelial cells, which were accompanied by elevated proliferating gut epithelial cell numbers. As compared to naive controls, infected mice displayed elevated numbers of intestinal T lymphocytes and regulatory T-cells and increased pro-inflammatory mediator secretion. Remarkably, T. gondii-induced apoptotic and pro-inflammatory immune responses were not restricted to the gut, but could also be observed in extra-intestinal compartments including kidney, liver, and lung. Strikingly, low-dose T. gondii infection resulted in increased serum levels of pro- and anti-inflammatory cytokines. In conclusion, the here presented subacute ileitis model following peroral low-dose T. gondii infection of humanized mice allows for detailed investigations of the molecular mechanism underlying the “ménage à trois” of pathogens, human gut microbiota, and immunity.
Authors:Claudia Genger, Sigri Kløve, Soraya Mousavi, Stefan Bereswill, and Markus M. Heimesaat
The physiological colonization resistance exerted by the murine gut microbiota prevents conventional mice from Campylobacter jejuni infection. In the present study we addressed whether this also held true for Campylobacter coli. Following peroral application, C. coli as opposed to C. jejuni could stably establish within the gastrointestinal tract of conventionally colonized mice until 3 weeks post-challenge. Neither before nor after either Campylobacter application any changes in the gut microbiota composition could be observed. C. coli, but not C. jejuni challenge was associated with pronounced regenerative, but not apoptotic responses in colonic epithelia. At day 21 following C. coli versus C. jejuni application mice exhibited higher numbers of adaptive immune cells including T-lymphocytes and regulatory T-cells in the colonic mucosa and lamina propria that were accompanied by higher large intestinal interferon-γ (IFN-γ) concentrations in the former versus the latter but comparable to naive levels. Campylobacter application resulted in decreased splenic IFN-γ, tumor necrosis factor-α (TNF-α), and IL-6 concentrations, whereas IL-12p70 secretion was increased in the spleens at day 21 following C. coli application only. In either Campylobacter cohort decreased IL-10 concentrations could be measured in splenic and serum samples. In conclusion, the commensal gut microbiota prevents mice from C. jejuni, but not C. coli infection.
Authors:Markus M. Heimesaat, Marie E. Alutis, Ursula Grundmann, André Fischer, Ulf B. Göbel, and Stefan Bereswill
We have recently shown that, within 1 week following peroral Campylobacter jejuni infection, conventional infant mice develop self-limiting enteritis. We here investigated the role of IL-23, IL-22, and IL-18 during C. jejuni strain 81-176 infection of infant mice. The pathogen efficiently colonized the intestines of IL-18−/− mice only, but did not translocate to extra-intestinal compartments. At day 13 postinfection (p.i.), IL-22−/− mice displayed lower colonic epithelial apoptotic cell numbers as compared to wildtype mice, whereas, conversely, colonic proliferating cells increased in infected IL-22−/− and IL-18−/− mice. At day 6 p.i., increases in neutrophils, T and B lymphocytes were less pronounced in gene-deficient mice, whereas regulatory T cell numbers were lower in IL-23p19−/− and IL-22−/− as compared to wildtype mice, which was accompanied by increased colonic IL-10 levels in the latter. Until then, colonic pro-inflammatory cytokines including TNF, IFN-γ, IL-6, and MCP-1 increased in IL-23p19−/− mice, whereas IL-18−/− mice exhibited decreased cytokine levels and lower colonic numbers of T and B cell as well as of neutrophils, macrophages, and monocytes as compared to wildtype controls. In conclusion, IL-23, IL-22, and IL-18 are differentially involved in mediating C. jejuni-induced immunopathology of conventional infant mice.