Authors:Marie E. Alutis, Ursula Grundmann, André Fischer, Ulrike Hagen, Anja A. Kühl, Ulf B. Göbel, Stefan Bereswill, and Markus M. Heimesaat
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.
Authors:Markus M. Heimesaat, Gernot Reifenberger, Viktoria Vicena, Anita Illes, Gabriella Horvath, Andrea Tamas, Balazs D. Fulop, Stefan Bereswill, and Dora Reglodi
Pituitary adenylate cyclase activating polypetide (PACAP) constitutes a neuropeptide that is widely distributed in the host exerting essential cytoprotective properties, whereas PACAP−/− mice display increased susceptibility to distinct immunopathological conditions. The orchestrated interplay between the gut microbiota and the host is pivotal in immune homeostasis and resistance to disease. Potential pertubations of the intestinal microbiota in PACAP−/− mice, however, have not been addressed so far. For the first time, we performed a comprehensive survey of the intestinal microbiota composition in PACAP−/− and wildtype (WT) mice starting 2 weeks postpartum until 18 months of age applying quantitative culture-independent techniques. Fecal enterobacteria and enterococci were lower in PACAP−/− than WT mice aged 1 month and ≥6 months, respectively. Whereas Mouse Intestinal Bacteroides were slightly higher in PACAP−/− versus WT mice aged 1 and 6 months, this later in life held true for Bacteroides/Prevotella spp. (≥12 months) and lactobacilli (>15 months of age). Strikingly, health-beneficial bifidobacteria were virtually absent in the intestines of PACAP−/− mice, even when still breastfed. In conclusion, PACAP deficiency is accompanied by distinct changes in fecal microbiota composition with virtually absent bifidobacteria as a major hallmark that might be linked to increased susceptibility to disease.
Authors:Sascha Cording, Diana Fleissner, Markus M. Heimesaat, Stefan Bereswill, Christoph Loddenkemper, Satoshi Uematsu, Shizuo Akira, Alf Hamann, and Jochen Huehn
Compelling evidence demonstrates that intestinal commensal microbiota modulate conventional and regulatory T cell (Treg) responses that are required for effective host defence against pathogens and avoidance of autoimmunity and other immunopathologic conditions. Here, we investigated the contribution of the commensal microbiota and Toll-like receptor (TLR) signaling to homeostasis of Foxp3− conventional CD4+ T cells and Foxp3+ Tregs. Upon long-term antibiotics treatment, we observed a significant reduction of conventional CD4+ T cell proliferation in a systemic manner, whereas Foxp3+ Treg proliferation was locally impaired in gut-draining mesenteric lymph nodes and Peyer's patches. The proliferative response to microbial components was not mediated by TLRs as MyD88- and various TLR-deficient mice displayed normal or even increased conventional T cell and Foxp3+ Treg proliferation. Thus, commensal microbiota-derived stimuli support cycling of both conventional CD4+ T cells and Foxp3+ Tregs with TLR-mediated recognition of bacterial components not being the major mechanism controlling microbiota-driven T cell homeostasis.
Authors:Markus M. Heimesaat, Dennis Weschka, Sigri Kløve, Claudia Genger, Nina Biesemeier, Soraya Mousavi, and Stefan Bereswill
Non-antibiotic feed additives including competitive exclusion products have been shown effective in reducing pathogen loads including multi-drug resistant strains from the vertebrate gut. In the present study we surveyed the intestinal bacterial colonization properties, potential macroscopic and microscopic inflammatory sequelae and immune responses upon peroral application of the commercial competitive exclusion product Aviguard® to wildtype mice in which the gut microbiota had been depleted by antibiotic pre-treatment. Until four weeks following Aviguard® challenge, bacterial strains abundant in the probiotic suspension stably established within the murine intestines. Aviguard® application did neither induce any clinical signs nor gross macroscopic intestinal inflammatory sequelae, which also held true when assessing apoptotic and proliferative cell responses in colonic epithelia until day 28 post-challenge. Whereas numbers of colonic innate immune cell subsets such as macrophages and monocytes remained unaffected, peroral Aviguard® application to microbiota depleted mice was accompanied by decreases in colonic mucosal counts of adaptive immune cells such as T and B lymphocytes. In conclusion, peroral Aviguard® application results i.) in effective intestinal colonization within microbiota depleted mice, ii.) neither in macroscopic nor in microscopic inflammatory sequelae and iii.) in lower colonic mucosal T and B cell responses.
Authors:Andreas Kupz, André Fischer, Dietrich H. Nies, Gregor Grass, Ulf B. Göbel, Stefan Bereswill, and Markus M. Heimesaat
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.
Authors:Markus M. Heimesaat, Andreas Kupz, André Fischer, Dietrich H. Nies, Gregor Grass, Ulf B. Göbel, and Stefan Bereswill
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.
Authors:Markus M. Heimesaat, Lea-Maxie Haag, André Fischer, Bettina Otto, Anja A. Kühl, Ulf B. Göbel, and Stefan Bereswill
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.
Authors:Manja Boehm, Daniel Simson, Ulrike Escher, Anna-Maria Schmidt, Stefan Bereswill, Nicole Tegtmeyer, Steffen Backert, and Markus M. Heimesaat
Campylobacter jejuni is a major food-borne zoonotic pathogen, responsible for a large proportion of bacterial gastroenteritis cases, as well as Guillian-Barré and Miller-Fisher syndromes. During infection, tissue damage is mainly caused by bacteria invading epithelial cells and traversing the intestinal barrier. C. jejuni is able to enter the lamina propria and the bloodstream and may move into other organs, such as spleen, liver, or mesenteric lymph nodes. However, the involved molecular mechanisms are not fully understood. C. jejuni can transmigrate effectively across polarized intestinal epithelial cells mainly by the paracellular route using the serine protease high-temperature requirement A (HtrA). However, it appears that HtrA has a dual function, as it also acts as a chaperone, interacting with denatured or misfolded periplasmic proteins under stress conditions. Here, we review recent progress on the role of HtrA in C. jejuni pathogenesis. HtrA can be transported into the extracellular space and cleaves cell-to-cell junction factors, such as E-cadherin and probably others, disrupting the epithelial barrier and enabling paracellular transmigration of the bacteria. The secretion of HtrA is a newly discovered strategy also utilized by other pathogens. Thus, secreted HtrA proteases represent highly attractive targets for anti-bacterial treatment and may provide a suitable candidate for vaccine development.