Hana Kozakova

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.

Gut microbiota and lipopolysaccharide content of the diet influence development of regulatory T cells: studies in germ-free mice

BackgroundMammals are essentially born germ-free but the epithelial surfaces are promptly colonized by astounding numbers of bacteria soon after birth. The most extensive microbial community is harbored by the distal intestine. The gut microbiota outnumber ~10 times the total number of our somatic and germ cells. The host-microbiota relationship has evolved to become mutually beneficial. Studies in germ-free mice have shown that gut microbiota play a crucial role in the development of the immune system. The principal aim of the present study was to elucidate whether the presence of gut microbiota and the quality of a sterile diet containing various amounts of bacterial contaminants, measured by lipopolysaccharide (LPS) content, can influence maturation of the immune system in gnotobiotic mice.ResultsWe have found that the presence of gut microbiota and to a lesser extent also the LPS-rich sterile diet drive the expansion of B and T cells in Peyers patches and mesenteric lymph nodes. The most prominent was the expansion of CD4+ T cells including Foxp3-expressing T cells in mesenteric lymph nodes. Further, we have observed that both the presence of gut microbiota and the LPS-rich sterile diet influence in vitro cytokine profile of spleen cells. Both gut microbiota and LPS-rich diet increase the production of interleukin-12 and decrease the production of interleukin-4. In addition, the presence of gut microbiota increases the production of interleukin-10 and interferon-γ.ConclusionOur data clearly show that not only live gut microbiota but also microbial components (LPS) contained in sterile diet stimulate the development, expansion and function of the immune system. Finally, we would like to emphasize that the composition of diet should be regularly tested especially in all gnotobiotic models as the LPS content and other microbial components present in the diet may significantly alter the outcome of experiments.

Segmented filamentous bacteria in a defined bacterial cocktail induce intestinal inflammation in SCID mice reconstituted with CD45RBhigh CD4+ T cells

Background: The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RBhigh subset of CD4+T cells isolated from the spleen of normal BALB/c mice. Methods: A CD4+CD45RBhigh subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ‐free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combination Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen‐free (SPF) bacteria. Mice were evaluated 8–12 weeks after the cell transfer for clinical and morphological signs of inflammatory bowel disease (IBD). Results: After the transfer of the CD4+CD45RBhigh T‐cell subpopulation to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO‐1 (zona occludens). Conclusions: Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB. (Inflamm Bowel Dis 2007)

Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition

Microbiota and infant development Malnutrition in children is a persistent challenge that is not always remedied by improvements in nutrition. This is because a characteristic community of gut microbes seems to mediate some of the pathology. Human gut microbes can be transplanted effectively into germ-free mice to recapitulate their associated phenotypes. Using this model, Blanton et al. found that the microbiota of healthy children relieved the harmful effects on growth caused by the microbiota of malnourished children. In infant mammals, chronic undernutrition results in growth hormone resistance and stunting. In mice, Schwarzer et al. showed that strains of Lactobacillus plantarum in the gut microbiota sustained growth hormone activity via signaling pathways in the liver, thus overcoming growth hormone resistance. Together these studies reveal that specific beneficial microbes could potentially be exploited to resolve undernutrition syndromes. Science, this issue p. 10.1126/science.aad3311, p. 854 The gut microbiota supports the growth of juvenile mice via growth hormone signaling. In most animal species, juvenile growth is marked by an exponential gain in body weight and size. Here we show that the microbiota of infant mice sustains both weight gain and longitudinal growth when mice are fed a standard laboratory mouse diet or a nutritionally depleted diet. We found that the intestinal microbiota interacts with the somatotropic hormone axis to drive systemic growth. Using monocolonized mouse models, we showed that selected lactobacilli promoted juvenile growth in a strain-dependent manner that recapitulated the microbiotas effect on growth and the somatotropic axis. These findings show that the hosts microbiota supports juvenile growth. Moreover, we discovered that lactobacilli strains buffered the adverse effects of chronic undernutrition on the postnatal growth of germ-free mice.

Mucosal Immunity: Its Role in Defense and Allergy

The interface between the organism and the outside world, which is the site of exchange of nutrients, export of products and waste components, must be selectively permeable and at the same time, it must constitute a barrier equipped with local defense mechanisms against environmental threats (e.g. invading pathogens). The boundaries with the environment (mucosal and skin surfaces) are therefore covered with special epithelial layers which support this barrier function. The immune system, associated with mucosal surfaces covering the largest area of the body (200–300 m2), evolved mechanisms discriminating between harmless antigens and commensal microorganisms and dangerous pathogens. The innate mucosal immune system, represented by epithelial and other mucosal cells and their products, is able to recognize the conserved pathogenic patterns on microbes by pattern recognition receptors such as Toll-like receptors, CD14 and others. As documented in experimental gnotobiotic models, highly protective colonization of mucosal surfaces by commensals has an important stimulatory effect on postnatal development of immune responses, metabolic processes (e.g. nutrition) and other host activities; these local and systemic immune responses are later replaced by inhibition, i.e. by induction of mucosal (oral) tolerance. Characteristic features of mucosal immunity distinguishing it from systemic immunity are: strongly developed mechanisms of innate defense, the existence of characteristic populations of unique types of lymphocytes, colonization of the mucosal and exocrine glands by cells originating from the mucosal organized tissues (‘common mucosal system’) and preferential induction of inhibition of the responses to nondangerous antigens (mucosal tolerance). Many chronic diseases, including allergy, may occur as a result of genetically based or environmentally induced changes in mechanisms regulating mucosal immunity and tolerance; this leads to impaired mucosal barrier function, disturbed exclusion and increased penetration of microbial, food or airborne antigens into the circulation and consequently to exaggerated and generalized immune responses to mucosally occurring antigens, allergens, superantigens and mitogens.

Potential and opportunities for use of recombinant lactic acid bacteria in human health

Publisher Summary This chapter discusses the potential and future opportunities for the use of recombinant lactic acid bacteria in human health. It is now clear that sufficient advances in the genetics of lactic acid bacteria (LAB) have made it possible to construct safe LAB-based recombinant vaccines that are capable of eliciting protection against lethal challenge with toxin or a human pathogen in a relevant disease model. There are also opportunities to enhance the efficacy of LAB vaccines through increased antigen expression or through the combined delivery of multiple immunogens and specific adjuvants. Further insights may be gained through direct comparisons of LAB strains with different persistence and survival characteristics or immunostimulatory properties with different immunization routes or schedules against a selected target disease. Genetic engineering clearly has the potential to further optimize the survival characteristics of selected LAB, define optimal placement and dosage regimes in different clinical settings, and enhance their ability to deliver a pharmaceutical protein.

Involvement of Innate Immunity in the Development of Inflammatory and Autoimmune Diseases

Abstract: Initial events and effector mechanisms of most inflammatory and autoimmune diseases remain largely unknown. Dysfunction of the innate and adaptive immune systems associated with mucosae (the major interface between the organism and its environment, e.g., microbiota, food) can conceivably cause impairment of mucosal barrier function and development of localized or systemic inflammatory and autoimmune processes. Animal models help in elucidating the etiology and pathogenetic mechanisms of human diseases, such as the inflammatory bowel diseases, Crohns disease and ulcerative colitis, severe chronic diseases affecting the gut. To study the role of innate immunity and gut microbiota in intestinal inflammation, colitis was induced by dextran sulfate sodium (DSS) in mice with severe combined immunodeficiency (SCID). Conventionally reared (microflora‐colonized) SCID mice displayed severe inflammation like that seen in immunocompetent Balb/c mice, whereas only minor changes appeared in the intestinal mucosa of DSS‐fed gnotobiotic germ‐free SCID mice. The presence of microflora facilitates the inflammation in DSS‐induced colitis that develops in immunodeficient SCID mice, that is, in the absence of T and B lymphocytes. Celiac disease, a chronic autoimmune small bowel disorder, afflicts genetically susceptible individuals with wheat gluten intolerance. We showed that, in contrast with any other food proteins, wheat gliadin and its peptic fragments activate mouse macrophages and human monocytes to produce proinflammatory cytokines through the nuclear factor‐κB signaling pathway. Activation of innate immunity cells by food proteins or components from gut microbiota thus could participate in the impairment of intestinal mucosa and the development of intestinal and/or systemic inflammation.

Colonization of germ-free mice with a mixture of three lactobacillus strains enhances the integrity of gut mucosa and ameliorates allergic sensitization.

Increasing numbers of clinical trials and animal experiments have shown that probiotic bacteria are promising tools for allergy prevention. Here, we analyzed the immunomodulatory properties of three selected lactobacillus strains and the impact of their mixture on allergic sensitization to Bet v 1 using a gnotobiotic mouse model. We showed that Lactobacillus (L.) rhamnosus LOCK0900, L. rhamnosus LOCK0908 and L. casei LOCK0919 are recognized via Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptors and stimulate bone marrow-derived dendritic cells to produce cytokines in species- and strain-dependent manners. Colonization of germ-free (GF) mice with a mixture of all three strains (Lmix) improved the intestinal barrier by strengthening the apical junctional complexes of enterocytes and restoring the structures of microfilaments extending into the terminal web. Mice colonized with Lmix and sensitized to the Bet v 1 allergen showed significantly lower levels of allergen-specific IgE, IgG1 and IgG2a and an elevated total IgA level in the sera and intestinal lavages as well as an increased transforming growth factor (TGF)-β level compared with the sensitized GF mice. Splenocytes and mesenteric lymph node cells from the Lmix-colonized mice showed the significant upregulation of TGF-β after in vitro stimulation with Bet v 1. Our results show that Lmix colonization improved the gut epithelial barrier and reduced allergic sensitization to Bet v 1. Furthermore, these findings were accompanied by the increased production of circulating and secretory IgA and the regulatory cytokine TGF-β. Thus, this mixture of three lactobacillus strains shows potential for use in the prevention of increased gut permeability and the onset of allergies in humans.

Bifidobacterium longum CCM 7952 Promotes Epithelial Barrier Function and Prevents Acute DSS-Induced Colitis in Strictly Strain-Specific Manner

Background Reduced microbial diversity has been associated with inflammatory bowel disease (IBD) and probiotic bacteria have been proposed for its prevention and/or treatment. Nevertheless, comparative studies of strains of the same subspecies for specific health benefits are scarce. Here we compared two Bifidobacterium longum ssp. longum strains for their capacity to prevent experimental colitis. Methods Immunomodulatory properties of nine probiotic bifidobacteria were assessed by stimulation of murine splenocytes. The immune responses to B. longum ssp. longum CCM 7952 (Bl 7952) and CCDM 372 (Bl 372) were further characterized by stimulation of bone marrow-derived dendritic cell, HEK293/TLR2 or HEK293/NOD2 cells. A mouse model of dextran sulphate sodium (DSS)-induced colitis was used to compare their beneficial effects in vivo. Results The nine bifidobacteria exhibited strain-specific abilities to induce cytokine production. Bl 372 induced higher levels of both pro- and anti-inflammatory cytokines in spleen and dendritic cell cultures compared to Bl 7952. Both strains engaged TLR2 and contain ligands for NOD2. In a mouse model of DSS-induced colitis, Bl 7952, but not Bl 372, reduced clinical symptoms and preserved expression of tight junction proteins. Importantly, Bl 7952 improved intestinal barrier function as demonstrated by reduced FITC-dextran levels in serum. Conclusions We have shown that Bl 7952, but not Bl 372, protected mice from the development of experimental colitis. Our data suggest that although some immunomodulatory properties might be widespread among the genus Bifidobacterium, others may be rare and characteristic only for a specific strain. Therefore, careful selection might be crucial in providing beneficial outcome in clinical trials with probiotics in IBD.

Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization

To cite this article: Schwarzer M, Repa A, Daniel C, Schabussova I, Hrncir T, Pot B, Stepankova R, Hudcovic T, Pollak A, Tlaskalova‐Hogenova H, Wiedermann U, Kozakova H. Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T‐regulatory responses upon systemic sensitization. Allergy 2011; 66: 368–375.