Pre- and Neonatal Imprinting on Immunological Homeostasis and Epithelial Barrier Integrity by Escherichia coli Nissle 1917 Prevents Allergic Poly-Sensitization in Mice.

A steady rise in the number of poly-sensitized patients has increased the demand for effective prophylactic strategies against multi-sensitivities. Probiotic bacteria have been successfully used in clinics and experimental models to prevent allergic mono-sensitization. In the present study, we have investigated whether probiotic bacteria could prevent poly-sensitization by imprinting on the immune system early in life. We used two recombinant variants of probiotic Escherichia coli Nissle 1917 (EcN): i) EcN expressing birch and grass pollen, poly-allergen chimera construct (EcN-Chim), and ii) an "empty" EcN without allergen expression (EcN-Ctrl). Conventional mice (CV) were treated with either EcN-Chim or EcN-Ctrl in the last week of the gestation and lactation period. Gnotobiotic mice received one oral dose of either EcN-Chim or EcN-Ctrl before mating. The offspring from both models underwent systemic allergic poly-sensitization and intranasal challenge with recombinant birch and grass pollen allergens (rBet v 1, rPhl p 1, and rPhl p 5). In the CV setting, the colonization of offspring via treatment of mothers reduced allergic airway inflammation (AAI) in offspring compared to poly-sensitized controls. Similarly, in a gnotobiotic model, AAI was reduced in EcN-Chim and EcN-Ctrl mono-colonized offspring. However, allergy prevention was more pronounced in the EcN-Ctrl mono-colonized offspring as compared to EcN-Chim. Mono-colonization with EcN-Ctrl was associated with a shift toward mixed Th1/Treg immune responses, increased expression of TLR2 and TLR4 in the lung, and maintained levels of zonulin-1 in lung epithelial cells as compared to GF poly-sensitized and EcN-Chim mono-colonized mice. This study is the first one to establish the model of allergic poly-sensitization in gnotobiotic mice. Using two different settings, gnotobiotic and conventional mice, we demonstrated that an early life intervention with the EcN without expressing an allergen is a powerful strategy to prevent poly-sensitization later in life.

Oocyst-Derived Extract of Toxoplasma Gondii Serves as Potent Immunomodulator in a Mouse Model of Birch Pollen Allergy.

INTRODUCTION: Previously, we have shown that oral infection with Toxoplasma gondii oocysts prevented type I allergy in mice. Here we investigated whether the application of a T. gondii oocyst lysate antigen (OLA) could also reduce allergy development. BALB/c mice were immunised twice with OLA followed by sensitisation with the major birch pollen (BP) allergen Bet v 1 and an aerosol challenge with BP extract. METHODS: First, we tested OLA in vitro. Stimulation of splenocytes and bone marrow-derived dendritic cells (BMDC) with OLA led to the production of pro-inflammatory and regulatory cytokines such as IL-6, IFN-γ and IL-10. Moreover, BMDC exposed to OLA upregulated the maturation markers CD40, CD80, CD86, and MHCII. Furthermore, OLA was recognised by TLR2-transfected human embryonic kidney cells. RESULTS: Immunisation of mice with OLA induced high levels of Toxoplasma-specific IgG antibodies in sera along with increased production of IFN-γ and IL-10 in Toxoplasma-antigen restimulated splenocytes. OLA reduced allergic airway inflammation as manifested by significant reduction of eosinophils in bronchoalveolar fluids, decreased cellular infiltrates and mucus production in the lungs. Accordingly, Bet v 1-specific IgE was decreased in OLA-pretreated mice. The reduced allergic immune responses were accompanied by increased numbers of CD4+CD25highFoxp3+ regulatory T cells in spleens as well as by increased numbers of granulocytic myeloid-derived suppressor cells in lungs when compared to sensitised controls suggesting that these two cell populations might be involved in the suppression of the allergic immune responses. CONCLUSION: Our data demonstrate that pretreatment with the oocyst extract can exert anti-allergic effects comparable to T. gondii infection. Thus, the immunomodulatory properties of the parasite extract indicate that this extract and in the future defined molecules thereof might serve as immunomodulatory adjuvants in allergy treatment and prophylaxis.

Oesophagostomum dentatum extract modulates T cell-dependent immune responses to bystander antigens and prevents the development of allergy in mice.

One third of the human population is currently infected by one or more species of parasitic helminths. Certain helminths establish long-term chronic infections resulting in a modulation of the host's immune system with attenuated responsiveness to "bystander" antigens such as allergens or vaccines. In this study we investigated whether parasite-derived products suppress the development of allergic inflammation in a mouse model. We show that extract derived from adult male Oesophagostomum dentatum (eMOD) induced Th2 and regulatory responses in BALB/c mice. Stimulation of bone marrow-derived dendritic cells induced production of regulatory cytokines IL-10 and TGF-beta. In a mouse model of birch pollen allergy, co-administration of eMOD with sensitizing allergen Bet v 1 markedly reduced the production of allergen-specific antibodies in serum as well as IgE-dependent basophil degranulation. Furthermore, eMOD prevented the development of airway inflammation, as demonstrated by attenuation of bronchoalveolar lavages eosinophil influx, peribronchial inflammatory infiltrate, and mucus secretion in lungs and IL-4 and IL-5 levels in lung cell cultures. Reduced secretion of Th2-related cytokines by birch pollen-re-stimulated splenocytes and mesenteric lymph node cells was observed in eMOD-treated/sensitized and challenged mice in comparison to sensitized and challenged controls. The suppressive effects of eMOD were heat-stable. Immunization with model antigens in the presence of eMOD reduced production of antibodies to thymus-dependent but not to thymus-independent antigen, suggesting that suppression of the immune responses by eMOD was mediated by interference with antigen presenting cell or T helper cell function but did not directly suppress B cell function. In conclusion, we have shown that eMOD possesses immunomodulatory properties and that heat-stable factors in eMOD are responsible for the dramatic suppression of allergic responses in a mouse model of type I allergy. The identification and characterization of parasite-derived immune-modulating molecules might have potential for designing novel prophylactic/therapeutic strategies for immune-mediated diseases.

Prevention of birch pollen-related food allergy by mucosal treatment with multi-allergen-chimers in mice.

BACKGROUND: Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy. METHODOLOGY: BALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization. RESULTS: Intranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent ß-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-ß and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-ß, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer. CONCLUSION: Our results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy.

Perinatal maternal administration of Lactobacillus paracasei NCC 2461 prevents allergic inflammation in a mouse model of birch pollen allergy.

BACKGROUND: The hygiene hypothesis implies that microbial agents including probiotic bacteria may modulate foetal/neonatal immune programming and hence offer effective strategies for primary allergy prevention; however their mechanisms of action are poorly understood. We investigated whether oral administration of Lactobacillus paracasei NCC 2461 to mothers during gestation/lactation can protect against airway inflammation in offspring in a mouse model of birch pollen allergy, and examined the immune mechanisms involved. METHODS: BALB/c mice were treated daily with L. paracasei in drinking water or drinking water alone in the last week of gestation and during lactation. Their offspring were sensitized with recombinant Bet v 1, followed by aerosol challenge with birch pollen extract. RESULTS: Maternal exposure to L. paracasei prevented the development of airway inflammation in offspring, as demonstrated by attenuation of eosinophil influx in the lungs; reduction of IL-5 levels in bronchoalveolar lavage, and in lung and mediastinal lymph node cell cultures; and reduced peribronchial inflammatory infiltrate and mucus hypersecretion. While allergen-specific IgE and IgG antibody levels remained unchanged by the treatment, IL-4 and IL-5 production in spleen cell cultures were significantly reduced upon allergen stimulation in offspring of L. paracasei treated mice. Offspring of L. paracasei supplemented mothers had significantly reduced Bet v 1-specific as well as Concanavalin A-induced responses in spleen and mesenteric lymph node cell cultures, suggesting the modulation of both antigen-specific and mitogen-induced immune responses in offspring. These effects were associated with increased Foxp3 mRNA expression in the lungs and increased TGF-beta in serum. CONCLUSION: Our data show that in a mouse model of birch pollen allergy, perinatal administration of L. paracasei NCC 2461 to pregnant/lactating mothers protects against the development of airway inflammation in offspring by activating regulatory pathways, likely through TLR2/4 signalling.

Distinctive anti-allergy properties of two probiotic bacterial strains in a mouse model of allergic poly-sensitization.

We compared the immunomodulatory properties of Bifidobacterium longum NCC 3001 and Lactobacillus paracasei NCC 2461 in a mouse model of poly-sensitization to birch and grass pollen allergens. Mucosal application of both strains at the time of sensitization and challenge led to significant suppression of airway inflammation and down-regulated allergen-specific immune responses. In contrast, in the mice treated with probiotics prior to sensitization and challenge, only B. longum displayed protective effects. Our findings stress that the choice of probiotic strain and the timing of the application are crucial for tolerance induction. Furthermore, this is the first demonstration of anti-allergic effects of probiotic bacteria in poly-sensitized mice.

Immunoregulation by Toxoplasma gondii infection prevents allergic immune responses in mice.

Toxoplasma gondii is a ubiquitous intracellular parasite affecting most mammals including humans. In epidemiological studies, infection with T. gondii and allergy development have been postulated to be inversely related. Using a mouse model of birch pollen allergy we investigated whether infection with T. gondii influences allergic immune responses to birch pollen. BALB/c mice were infected with T. gondii oocysts either before or at the end of sensitisation with the major birch pollen allergen Bet v 1 and thereafter aerosol challenged with birch pollen extract. During the acute phase of infection, clinical signs correlated with increased levels of serum TNF-alpha, IL-6, IFN-gamma and anti-Toxoplasma-IgM. In the chronic phase, Toxoplasma-specific serum IgG, brain tissue cysts and high IFN-gamma production in spleen cell cultures were detected. Mice infected prior to allergic sensitisation produced significantly less allergen-specific IgE and IgG1, while IgG2a levels were markedly increased. IL-5 levels in spleen cell cultures and bronchoalveolar lavage fluid were significantly reduced, and airway inflammation was prevented in these mice. Notably, in mice infected at the end of the allergic sensitisation process, systemic and local immune responses to the allergen were markedly reduced. T.gondii infection was associated with up-regulation of Toll-like receptor 2 (TLR2), 4, 9 and 11, as well as T-bet (a differentiation factor for Th1 cells) mRNA expression in splenocytes; moreover, enhanced TGF-beta, IL-10 and Foxp3 mRNA expression in these cells suggested that regulatory mechanisms were involved in suppression of the allergic immune response. Kinetic studies confirmed the induction of Foxp3(+)CD4(+)CD25(+) regulatory T cells preferentially during the chronic phase of T. gondii infection. Our data demonstrate that T. gondii exhibits strong immunomodulating properties which lead to prevention of allergic immune responses and thereby support the hygiene hypothesis.