Authors:Markus M. Heimesaat, Thomas Alter, Stefan Bereswill, and Greta Gölz
We have previously shown that Arcobacter butzleri induces intestinal, extra-intestinal, and systemic immune responses in perorally infected gnotobiotic IL-10−/− mice in a strain-dependent fashion. Here, we present a comprehensive survey of small and large intestinal expression profiles of inflammatory and regulatory mediators as well as of the matrix-degrading gelatinases MMP-2 and MMP-9 following murine A. butzleri infection. Gnotobiotic IL-10−/− mice were infected with A. butzleri strains CCUG 30485 or C1 of human and chicken origin, respectively. At day 6 following A. butzleri infection, mucin-2 mRNA, an integral part of the intestinal mucus layer, was downregulated in the colon, whereas TNF and IL-23p19 mRNA were upregulated in the ileum. Furthermore, IFN-γ, IL-17A, IL-1β, and IL-22 mRNA were upregulated in both colonic and ileal ex vivo biopsies at day 6 post strain CCUG 30485 infection. These changes were accompanied by downregulated colonic MMP-9 levels, whereas both MMP-2 and MMP-9 mRNA were upregulated in the ileum. In conclusion, these data indicate that A. butzleri infection induces changes in the expression of genes involved in pro-inflammatory and regulatory immune responses as well as in tissue degradation.
Authors:Greta Gölz, Thomas Alter, Stefan Bereswill, and Markus M. Heimesaat
We have previously shown that Arcobacter butzleri infection induces Toll-like receptor (TLR) -4 dependent immune responses in perorally infected gnotobiotic IL-10−/− mice. Here, we analyzed TLR-4-dependent expression of genes encoding inflammatory mediators and matrix-degrading gelatinases MMP-2 and -9 in the small and large intestines of gnotobiotic TLR-4-deficient IL-10−/− mice that were perorally infected with A. butzleri strains CCUG 30485 or C1, of human and chicken origin, respectively. At day 6 following A. butzleri infection, colonic mucin-2 mRNA, as integral part of the intestinal mucus layer, was downregulated in the colon, but not ileum, of IL-10−/− but not TLR-4−/− IL-10−/− mice. CCUG 30485 strain-infected TLR-4-deficient IL-10−/− mice displayed less distinctly upregulated IFN-γ, IL-17A, and IL-1β mRNA levels in ileum and colon, which was also true for colonic IL-22. These changes were accompanied by upregulated colonic MMP-2 and ileal MMP-9 mRNA exclusively in IL-10−/− mice. In conclusion, TLR-4 is essentially involved in A. butzleri mediated modulation of gene expression in the intestines of gnotobiotic IL-10−/− mice.
Authors:Eliane von Klitzing, Stefan Bereswill, and Markus M. Heimesaat
The World Health Organization has rated multidrug-resistant (MDR) Pseudomonas aeruginosa as a critical threat to human health. In the present study, we performed a survey of intestinal colonization, and local and systemic immune responses following peroral association of secondary abiotic mice with either a clinical MDR P. aeruginosa or a commensal murine Escherichia coli isolate. Depletion of the intestinal microbiota following antibiotic treatment facilitated stable intestinal colonization of both P. aeruginosa and E. coli that were neither associated with relevant clinical nor histopathological sequelae. Either stable bacterial colonization, however, resulted in distinct innate and adaptive immune cell responses in the intestines, whereas a pronounced increase in macrophages and monocytes could be observed in the small as well as large intestines upon P. aeruginosa challenge only, which also applied to colonic T lymphocytes. In addition, TNF secretion was exclusively elevated in large intestines of P. aeruginosa-colonized mice. Strikingly, association of secondary abiotic mice with MDR P. aeruginosa, but not commensal E. coli, resulted in pronounced systemic pro-inflammatory responses, whereas anti-inflammatory responses were dampened. Hence, intestinal carriage of MDR P. aeruginosa as compared to a mere commensal Gram-negative strain in otherwise healthy individuals results in distinct local and systemic pro-inflammatory sequelae.
Authors:Seher Nancy Bakal, Stefan Bereswill, and Markus M. Heimesaat
The progressive rise in multidrug-resistant (MDR) bacterial strains poses serious problems in the treatment of infectious diseases. While the number of newly developed antimicrobial compounds has greatly fallen, the resistance of pathogens against commonly prescribed drugs is further increasing. This rise in resistance illustrates the need for developing novel therapeutic and preventive antimicrobial options. The medicinal herb Nigella sativa and its derivatives constitute promising candidates. In a comprehensive literature survey (using the PubMed data base), we searched for publications on the antimicrobial effects of N. sativa particularly directed against MDR bacterial strains. In vitro studies published between 2000 and 2015 revealed that N. sativa exerted potent antibacterial effects against both Gram-positive and Gram-negative species including resistant strains. For instance, N. sativa inhibited the growth of bacteria causing significant gastrointestinal morbidity such as Salmonella, Helicobacter pylori, and Escherichia coli. However, Listeria monocytogenes and Pseudomonas aeruginosa displayed resistance against black cumin seed extracts. In conclusion, our literature survey revealed potent antimicrobial properties of N. sativa against MDR strains in vitro that should be further investigated in order to develop novel therapeutic perspectives for combating infectious diseases particularly caused by MDR strains.
Authors:Anne Grunau, Ulrike Escher, Stefan Bereswill, and Markus M. Heimesaat
The rising incidences of infections with multidrug-resistant (MDR) Gram-negative bacteria including Pseudomonas aeruginosa (PA) have gained increasing attention in medicine, but also in the general public and global health politics. The mechanisms underlying opportunistic pathogen—host interactions are unclear, however. To address this, we challenged secondary abiotic IL10−/− mice deficient for Toll-like receptor-4 (TLR4−/− × IL10−/−), the main receptor of the Gram-negative cell wall constituent lipopolysaccharide, with a clinical MDR PA isolate. Despite higher intestinal colonization densities, apoptotic colonic epithelial cell numbers were lower in TLR4−/− × IL10−/− mice as compared to IL10−/− controls at day 14 postinfection (p.i.), whereas proliferating/regenerating cells had increased in the latter only. Furthermore, PA-colonized TLR4−/− × IL10−/− mice displayed less distinct innate and adaptive immune cell responses in the colon as compared to IL10−/− counterparts that were accompanied by lower nitric oxide concentrations in mesenteric lymph nodes in the former at day 14 p.i. Conversely, splenic NO levels were higher in both naive and PA-colonized TLR4-deficient IL10−/− mice versus IL10−/− controls. Remarkably, intestinal MDR PA was able to translocate to extra-intestinal including systemic compartments of TLR4−/− × IL10−/− mice only. Hence, MDR PA-induced intestinal and systemic immune responses observed in secondary abiotic IL10−/− mice are TLR4-dependent.
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:Markus M. Heimesaat, R. Plickert, A. Fischer, U. B. Göbel, and S. Bereswill
Enterocolitis caused by Campylobacter jejuni represents an important socioeconomic burden worldwide. The host-specific intestinal microbiota is essential for maintaining colonization resistance (CR) against C. jejuni in conventional mice. Notably, CR is abrogated by shifts of the intestinal microbiota towards overgrowth with commensal E. coli during acute ileitis. Thus, we investigated whether oral transplantation (TX) of ileal microbiota derived from C. jejuni susceptible mice with acute ileitis overcomes CR of healthy conventional animals. Four days following ileitis microbiota TX or ileitis induction and right before C. jejuni infection, mice displayed comparable loads of main intestinal bacterial groups as shown by culture. Eight days following ileitis induction, but not ileal microbiota TX, however, C. jejuni could readily colonize the gastrointestinal tract of conventional mice and also translocate to extra-intestinal tissue sites such as mesenteric lymph nodes, spleen, liver, and blood within 4 days following oral infection. Of note, C. jejuni did not further deteriorate histopathology following ileitis induction. Lack of C. jejuni colonization in TX mice was accompanied by a decrease of commensal E. coli loads in the feces 4 days following C. jejuni infection. In summary, oral ileal microbiota TX from susceptible donors is not sufficient to abrogate murine CR against C. jejuni.
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.
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, André Fischer, Anja A. Kühl, Ulf B. Göbel, Illana Gozes, and Stefan Bereswill
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.