Pavel Rossmann

The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases

Metagenomic approaches are currently being used to decipher the genome of the microbiota (microbiome), and, in parallel, functional studies are being performed to analyze the effects of the microbiota on the host. Gnotobiological methods are an indispensable tool for studying the consequences of bacterial colonization. Animals used as models of human diseases can be maintained in sterile conditions (isolators used for germ-free rearing) and specifically colonized with defined microbes (including non-cultivable commensal bacteria). The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these models. Using this approach we demonstrated direct involvement of components of the microbiota in chronic intestinal inflammation and development of colonic neoplasia (i.e., using models of human inflammatory bowel disease and colorectal carcinoma). In contrast, a protective effect of microbiota colonization was demonstrated for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. Interestingly, the development of atherosclerosis in germ-free apolipoprotein E (ApoE)-deficient mice fed by a standard low-cholesterol diet is accelerated compared with conventionally reared animals. Mucosal induction of tolerance to allergen Bet v1 was not influenced by the presence or absence of microbiota. Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.

Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition

Commensal bacteria have been shown to modulate the host mucosal immune system. Here, we report that oral treatment of BALB/c mice with components from the commensal, Parabacteroides distasonis, significantly reduces the severity of intestinal inflammation in murine models of acute and chronic colitis induced by dextran sulphate sodium (DSS). The membranous fraction of P. distasonis (mPd) prevented DSS‐induced increases in several proinflammatory cytokines, increased mPd‐specific serum antibodies and stabilized the intestinal microbial ecology. The anti‐colitic effect of oral mPd was not observed in severe combined immunodeficient mice and probably involved induction of specific antibody responses and stabilization of the intestinal microbiota. Our results suggest that specific bacterial components derived from the commensal bacterium, P. distasonis, may be useful in the development of new therapeutic strategies for chronic inflammatory disorders such as inflammatory bowel disease.

Altered gut microbiota promotes colitis-associated cancer in IL-1 receptor-associated kinase M-deficient mice.

Background:Microbial sensing by Toll-like receptors (TLR) and its negative regulation have an important role in the pathogenesis of inflammation-related cancer. In this study, we investigated the role of negative regulation of Toll-like receptors signaling and gut microbiota in the development of colitis-associated cancer in mouse model. Methods:Colitis-associated cancer was induced by azoxymethane and dextran sodium sulfate in wild-type and in interleukin-1 receptor–associated kinase M (IRAK-M)–deficient mice with or without antibiotic (ATB) treatment. Local cytokine production was analyzed by multiplex cytokine assay or enzyme-linked immunosorbent assay, and regulatory T cells were analyzed by flow cytometry. Changes in microbiota composition during tumorigenesis were analyzed by pyrosequencing, and &bgr;-glucuronidase activity was measured in intestinal content by fluorescence assay. Results:ATB treatment of wild-type mice reduced the incidence and severity of tumors. Compared with nontreated mice, ATB-treated mice had significantly lower numbers of regulatory T cells in colon, altered gut microbiota composition, and decreased &bgr;-glucuronidase activity. However, the &bgr;-glucuronidase activity was not as low as in germ-free mice. IRAK-M–deficient mice not only developed invasive tumors, but ATB-induced decrease in &bgr;-glucuronidase activity did not rescue them from severe carcinogenesis phenotype. Furthermore, IRAK-M–deficient mice had significantly increased levels of proinflammatory cytokines in the tumor tissue. Conclusions:We conclude that gut microbiota promotes tumorigenesis by increasing the exposure of gut epithelium to carcinogens and that IRAK-M–negative regulation is essential for colon cancer resistance even in conditions of altered microbiota. Therefore, gut microbiota and its metabolic activity could be potential targets for colitis-associated cancer therapy.

Intestinal Microbiota Promotes Psoriasis-Like Skin Inflammation by Enhancing Th17 Response

Psoriasis is a chronic inflammatory skin disease in which Th17 cells play a crucial role. Since indigenous gut microbiota influences the development and reactivity of immune cells, we analyzed the link among microbiota, T cells and the formation of psoriatic lesions in the imiquimod-induced murine model of psoriasis. To explore the role of microbiota, we induced skin inflammation in germ-free (GF), broad-spectrum antibiotic (ATB)-treated or conventional (CV) BALB/c and C57BL/6 mice. We found that both mice reared in GF conditions for several generations and CV mice treated with ATB were more resistant to imiquimod-induced skin inflammation than CV mice. The ATB treatment dramatically changed the diversity of gut bacteria, which remained stable after subsequent imiquimod application; ATB treatment resulted in a substantial increase in the order Lactobacillales and a significant decrease in Coriobacteriales and Clostridiales. Moreover, as compared to CV mice, imiquimod induced a lower degree of local and systemic Th17 activation in both GF and ATB-treated mice. These findings suggest that gut microbiota control imiquimod-induced skin inflammation by altering the T cell response.

Detection of antigen in the coelomocytes of the earthworm, Eisenia foetida (Annelida)

Earthworms, Eisenia foetida, are able to respond to antigenic stimulation by the formation of the antigen-binding molecules by coelomocytes--the effector cells of annelids defence reactions. The ability to react with gold-labelled antigen was detected in agranular coelomocytes by electron microscopy. Furthermore, flow cytometry analysis used for quantitative evaluation of antigen binding showed significant increase of both antigen-binding cells and the amount of antigen bound per cell after stimulation. The antigen binding was inhibited by preincubation of cells with several similar proteins, although the most potent inhibitor was the immunizing antigen.

Detection of ICAM-1 in Experimentally Induced Colitis of ICAM-1-deficient and Wild-type Mice: An Immunohistochemical Study

Adhesion molecules (e.g. ICAM-1, CD 54) are known to be upregulated on activated vascular endothelial cells during inflammatory reactions. To study the role of ICAM-1 in intestinal inflammation in vivo, we induced acute experimental colitis in wild-type (C57BL/6) mice and ICAM-1-deficient mice, by feeding the animals with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. In the control strain the immunohistochemical staining showed a very pronounced endothelial upregulation of ICAM-1 after the DSS treatment observed in areas of inflammatory infiltrate, especially in venules or arterioles of the propria and submucosa, and partly in the mesocolon. DSS-fed ICAM-1-deficient mice showed no endothelial enhancement and only faint staining of venules or capillaries approaching that encountered in the control ICAM-1-deficient animals. Our data indicate that ICAM-1 may play a crucial role in the development of acute intestinal inflammation, consistent with our finding that ICAM-1 deficiency can obviate severe forms of experimentally induced colitis in mice.

Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains (Escherichia coli and Bacillus subtilis) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil.