Julia-Stefanie Frick

A vegan or vegetarian diet substantially alters the human colonic faecal microbiota

Background/Objectives:Consisting of ∼1014 microbial cells, the intestinal microbiota represents the largest and the most complex microbial community inhabiting the human body. However, the influence of regular diets on the microbiota is widely unknown.Subjects/Methods:We examined faecal samples of vegetarians (n=144), vegans (n=105) and an equal number of control subjects consuming ordinary omnivorous diet who were matched for age and gender. We used classical bacteriological isolation, identification and enumeration of the main anaerobic and aerobic bacterial genera and computed absolute and relative numbers that were compared between groups.Results:Total counts of Bacteroides spp., Bifidobacterium spp., Escherichia coli and Enterobacteriaceae spp. were significantly lower (P=0.001, P=0.002, P=0.006 and P=0.008, respectively) in vegan samples than in controls, whereas others (E. coli biovars, Klebsiella spp., Enterobacter spp., other Enterobacteriaceae, Enterococcus spp., Lactobacillus spp., Citrobacter spp. and Clostridium spp.) were not. Subjects on a vegetarian diet ranked between vegans and controls. The total microbial count did not differ between the groups. In addition, subjects on a vegan or vegetarian diet showed significantly (P=0.0001) lower stool pH than did controls, and stool pH and counts of E. coli and Enterobacteriaceae were significantly correlated across all subgroups.Conclusions:Maintaining a strict vegan or vegetarian diet results in a significant shift in the microbiota while total cell numbers remain unaltered.

Bacteroides vulgatus protects against escherichia coli-induced colitis in gnotobiotic interleukin-2-deficient mice

BACKGROUND & AIMS The microflora plays a crucial role in inflammatory bowel diseases (IBDs). Specific pathogen-free (SPF), but not germ-free, interleukin (IL)-2-deficient (IL-2-/-) mice develop colitis. The colitogenicity of commensal bacteria was determined. METHODS Gnotobiotic IL-2-/- and IL-2+/+ mice were colonized with Escherichia coli mpk, Bacteroides vulgatus mpk, or both bacterial strains, or with E. coli strain Nissle 1917. DNA arrays were used to characterize E. coli mpk. Colitis was analyzed by histology and real-time reverse-transcription polymerase chain reaction (RT-PCR) for interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-10, and CD14 messenger RNA (mRNA) expression. Bacterial numbers in feces and bacterial localization in the colon was determined by culture and fluorescence in situ hybridization (FISH). RESULTS IL-2-/- but not IL-2+/+ mice monocolonized with E. coli mpk developed colitis, whereas mono-association with B. vulgatus mpk, or E. coli Nissle, or co-colonization with E. coli mpk and B. vulgatus mpk, did not induce colitis. DNA array experiments and cellular studies revealed that E. coli mpk is a nonpathogenic strain. FISH and culture methods revealed that the anticolitogenic effect of B. vulgatus mpk on E. coli mpk cannot be explained by a significant reduction in numbers of E. coli in the colon. E. coli mpk-induced colitis was associated with increased IFN-gamma, TNF-alpha, CD14, and IL-10 mRNA expression in the colon. CONCLUSIONS In IL-2-/- mice, B. vulgatus mpk protects against E. coli mpk-triggered colitis by an unknown mechanism. E. coli Nissle does not induce colitis. Various bacterial species common to the microflora differ in their ability to trigger IBD.

Contribution of Adenosine A2B Receptors to Inflammatory Parameters of Experimental Colitis

Inflammatory diseases influence tissue metabolism, significantly altering the profile of extracellular adenine nucleotides. A number of studies have suggested that adenosine (Ado) may function as an endogenously generated anti-inflammatory molecule. Given the central role of intestinal epithelial cells to the development of colitis, we hypothesized that specific Ado receptors would contribute to disease resolution in mucosal inflammation as modeled by dextran sodium sulfate (DSS) colitis. Initial profiling studies revealed that murine intestinal epithelial cells express predominantly the Ado A2B receptor (AA2BR) and to a lesser extent AA2AR. Guided by these results, we examined the contribution of AA2BR to colitis. Initial studies indicated that the severity of colitis was increased in Aa2br−/− mice relative to Aa2br+/+ controls, as reflected by increased weight loss, colonic shortening, and disease activity indices. Likewise, enteral administration of the selective AA2BR inhibitor PSB1115 to Aa2br+/+ mice resulted in a similar increase in severity of DSS colitis. Cytokine profiling of colonic tissue revealed specific deficiencies in IL-10 in Aa2br−/− mice relative to controls. Extensions of these findings in cultured human intestinal epithelial cells revealed that stable Ado analogs induce IL-10 mRNA and protein and that such increases can be blocked with PSB1115. Taken together, these studies indicate a central regulatory role for AA2BR-modulated IL-10 in the acute inflammatory phase of DSS colitis, thereby implicating AA2BR as an endogenously protective molecule expressed on intestinal epithelial cells.

Microbiota in pediatric inflammatory bowel disease.

OBJECTIVE To test the hypothesis that compared with controls, children with inflammatory bowel disease (IBD) exhibit differences in the relationships between gut microbiota and disease activity. STUDY DESIGN Children and adolescents (n = 69; median age, 14 years) with IBD and 25 healthy controls (median age, 14 years) were recruited for the study. The disease activity was determined according to the Pediatric Ulcerative Colitis Activity Index or the Pediatric Crohn Disease Activity Index. Cell counts of 9 bacterial groups and species in the fecal microbiota were monitored by real-time polymerase chain reaction analysis. RESULTS Although no major changes were observed in patients with ulcerative colitis, except for a decrease in bifidobacteria in the active state of IBD, children with active and inactive Crohns disease (CD) had lower numbers of Faecalibacterium prausnitzii and bifidobacteria (P <.05), and patients with active CD had higher numbers of Escherichia coli (P <.05). CONCLUSIONS The microbiota in children with CD is characterized by decreased numbers of F praunsitzii and increased numbers of E coli.

Nonpathogenic Bacteria Alleviating Atopic Dermatitis Inflammation Induce IL-10-Producing Dendritic Cells and Regulatory Tr1 Cells

The beneficial effects of nonpathogenic bacteria are increasingly being recognized. We reported in a placebo-controlled study with atopic dermatitis (AD) patients that cutaneous exposure to lysates of nonpathogenic bacteria alleviates skin inflammation. To now unravel underlying mechanisms, immune consequences of sensing nonpathogenic bacterium Vitreoscilla filiformis lysate (Vf) were characterized analyzing (1) differentiation of dendritic cells (DCs) and, consecutively, (2) effector functions of DCs and T helper (Th) cells in vitro and in a murine model of AD in NC/Nga mice in vivo. Topical treatment with Vf significantly reduced AD-like inflammation in NC/Nga mice. Importantly, cutaneous exposure to Vf in combination with the allergen FITC significantly also reduced subsequent allergen-induced dermatitis indicating active immune modulation. Indeed, innate sensing of Vf predominantly induced IL-10-producing DCs, which was dependent on Toll-like receptor 2 (TLR2) activation. Vf-induced IL-10+ DCs primed naive CD4+ T helper cells to become regulatory IFN-γ(low) IL-10(high) Tr1 (type 1 regulatory T) cells. These IL-10(high) Tr1 cells were also induced by Vf in vivo and strongly suppressed T effector cells and inflammation. In conclusion, we show that innate sensing of nonpathogenic bacteria by TLR2 induces tolerogenic DCs and regulatory Tr1 cells suppressing T effector cells and cutaneous inflammation. These findings indicate a promising therapeutic strategy for inflammatory skin diseases like AD.

Postinfectious irritable bowel syndrome: follow-up of a patient cohort of confirmed cases of bacterial infection with Salmonella or Campylobacter

Background  Gastrointestinal infections have been proposed to predict subsequent irritable bowel syndrome (IBS) but large‐scale infectious events are rare and long‐term data are missing.

Platelet-derived HMGB1 is a critical mediator of thrombosis

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Lactobacillus fermentum attenuates the proinflammatory effect of Yersinia enterocolitica on human epithelial cells.

Background: Lactobacilli represent a major component of the human microbiota. In this study we investigated whether and how Lactobacillus fermentum inhibits the proinflammatory responses of human epithelial cells on Yersinia enterocolitica infection. Methods: Human epithelial cells were exposed to Y. enterocolitica pYV− or L. fermentum or to both strains, combinations of heat‐killed L. fermentum or supernatant of L. fermentum cultures and Y. enterocolitica. The modulation of Y. enterocolitica induced IL‐8 levels in the culture supernatants was determined and activation of Rac, p38, and NF‐&kgr;B was investigated. Results: Exposure of human epithelial cells to L. fermentum does not induce NF‐&kgr;B activation and subsequent IL‐8 secretion in HeLa cells, whereas Y. enterocolitica induces NF‐&kgr;B activation and high levels of IL‐8. Viable L. fermentum, supernatant of L. fermentum cultures, but not heat‐killed L. fermentum, inhibited IL‐8 secretion of HeLa cells triggered by Y. enterocolitica. Lactobacillus fermentum‐exposed HeLa cells showed decreased Rac, p38, and NF‐&kgr;B activation after Y. enterocolitica infection. Treatment of L. fermentum supernatants with phospholipase C abolished the inhibitory effect, indicating that a secreted phospholipid mediates the antiinflammatory properties of L. fermentum. Adhesion to or invasion of Y. enterocolitica into epithelial cells was not altered by coincubation with L. fermentum. Conclusion: Our results lead to the conclusion that L. fermentum inhibits the Y. enterocolitica‐induced IL‐8 production by a possibly secreted phospholipid of <10 kDa molecular weight. These data suggest that L. fermentum may have probiotic properties modulating intestinal inflammatory responses and might offer new therapeutic strategies in the treatment of intestinal inflammatory diseases.

IL-6 and Maturation Govern TLR2 and TLR4 Induced TLR Agonist Tolerance and Cross-Tolerance in Dendritic Cells

Stimulation of naive mouse dendritic cells (DC) with LPS or Pam3CSK4 (P3C) induces production of TNF-α via TLR4- or TLR2-signaling. Although tolerance in macrophages has been studied in detail, we investigated the role of TLR agonist concentration and IL-6 for tolerance in DC. P3C- or LPS-primed DC were nonresponsive to P3C or LPS restimulation in terms of TNF-α but not IL-6 production. The mechanisms involved in tolerance were dependent on the concentration of the TLR ligand used for DC priming. DC primed with LPS or P3C at high concentrations developed a maturation dependent, IL-6 independent tolerance associated with inhibition of TLR signaling upstream of IκB as indicated by decreased IκB degradation. In contrast, priming of DC with LPS or P3C at low concentrations resulted in IL-6-dependent tolerance, which was abolished in IL-6 deficient DC, and was not accompanied by maturation of DC or by down-regulation of TLR2 or TLR4. In homotolerogenic DC primed with LPS or P3C at high concentrations, degradation of IκB upon restimulation with LPS or P3C was inhibited suggesting tolerance mechanism(s) upstream of IκB; in contrast, cross-tolerance in DC primed with LPS or P3C at low concentrations was not associated with reduced IκB degradation suggesting tolerance mechanisms downstream of IκB. Our data indicate that in naive DC TLR4- and TLR2-stimulation results in homo- and cross-tolerance; the mechanisms involved in tolerance depend on the concentration of the TLR agonist used for DC priming and are governed by IL-6 and maturation.

Immunomodulation by semi-mature dendritic cells: A novel role of Toll-like receptors and interleukin-6

Dendritic cells (DCs) are key players in activation of the adaptive immune system by their ability of antigen presentation to and priming of T cells. An increasing body of evidence suggests that DCs may also play an important role in induction of tolerance, predominantly by induction of regulatory T cells (T(reg)). More recently, data have been published on how Toll-like receptor (TLR) ligands and cytokines affect DC differentiation, and how DC subsets might be involved in immunoregulation and tolerance rather than in T cell activation. The most important features of tolerance-inducing DCs appear to be their maturation state and their cytokine secretion pattern. The following types of tolerance-inducing DCs have been reported: immature DCs (DCs(im)) or DCs in the steady state (DCs(st)), DCs(IL-10), semi-mature DCs(TNF-alpha), semi-mature DCs(IL-6). With this review article we would like to discuss the aforementioned types of tolerogenic DCs with a focus on semi-mature DCs(IL-6) and discuss their potential role in maintenance of (hepatic or intestinal) immune homeostasis and inflammatory diseases such as inflammatory bowel disease.