Oral probiotic bacterial administration suppressed allergic responses in an ovalbumin-induced allergy mouse model

This study investigated whether orally administered probiotic bacteria (Bifidobacterium bifidum and Lactobacillus casei) and a gram-negative bacterium (Escherichia coli) function as allergic immune modulators to prevent food allergy, according to the hygiene hypothesis. C3H/HeJ mice were sensitized with ovalbumin (OVA) and cholera toxin for 5 weeks. After sensitization, the OVA-induced mice that were not treated with bacteria had significantly increased levels of OVA-specific IgE, total IgE, and IgG1 in sera, as well as scab-covered tails. In comparison, groups treated with B. bifidum BGN4 (BGN4), L. casei 911 (L. casei), or Escherichia coli MC4100 (E. coli) had decreased levels of OVA-specific IgE, total IgE, and IgG1, and decreased levels of mast cell degranulation and tail scabs. OVA-specific IgA levels were decreased in BGN4- and L. casei-treated groups. In conclusion, administration of E. coli, BGN4, or L. casei decreased the OVA-induced allergy response. However, a normal increase in body weight was inhibited in the E. coli-treated mice and in the montreated mice groups during allergy sensitization. Thus, BGN4 and L. casei appear to be useful probiotic bacteria for the prevention of allergy.     

Oral tolerance revisited: prior oral tolerization abrogates cholera toxin-induced mucosal IgA responses

Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced IgG and IgE Ab- and Ag-specific CD4(+) T cell responses. It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract. To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed. OVA-specific serum IgG and IgA and mucosal IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS. Furthermore, when OVA-specific Ab-forming cells (AFCs) in both systemic and mucosa-associated tissues were examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased. Furthermore, marked reductions in OVA-specific CD4(+) T cell proliferative and cytokine responses in spleen and Peyer's patches were seen in mice given oral OVA but were unaffected in PBS-fed mice. We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance.     

Timing of Bifidobacterium Administration Influences the Development of Allergy to Ovalbumin in Mice

C3H/HeJ mice were sensitized with ovalbumin (OVA) and choleratoxin (CT) for 5 weeks, and then Bifidobacterium bifidum BGN4 was administered continuously for 7 weeks, starting 2 weeks before (pre-treatment group) and 2 weeks after (post-treatment group) the initial sensitization. After sensitization, the OVA-induced (sham group) mice showed growth inhibition and had scab-covered tails which was associated with serum levels of 9887±175 ng OVA-specific IgE/ml and 758±525 ng IgG1/ml. The sera of the pre-treatment group had 4805±245 ng OVA-specific IgE/ml and 193±87 ng IgG1/ml, as well as less severe tail symptoms. The sera of the post-treatment group had 5723±207 ng OVA-specific IgE/ml but the IgG1 and IgG2a levels were the same as those of the sham group. In spleen cultures, both pre-treatment and post-treatment increased the levels of IFN-γ but decreased the levels of IL-6 and IL-18. Taken together, the in vivo and in vitro results show that treatment with Bifidobacterium before OVA sensitization suppresses or modulates the allergic response more effectively than treatment with Bifidobacterium following OVA sensitization.     

Oral-tolerance induction in diet-induced obese mice

OBJECTIVE: It is known that immune functions are altered in various ways by obesity. However, changes in the intestinal immune system resulting from obesity remain poorly understood. Oral tolerance is a system that suppresses antigen specific immune responses to orally administrated antigens. The intestinal immune system is intimately associated with the oral tolerance system, that acts to prevent allergic and inflammatory diseases. In this study we investigated the effect of obesity on induction of oral tolerance to ovalbumin (OVA) in an animal model of obesity. RESEARCH METHODS AND PROCEDURES: Obese mice induced by a high fat diet and control mice were allowed free access for 3 days to a 1%-ovalbumin (OVA) solution in drinking water. After continuous feeding of the antigen, all the mice were immunized by two intraperitoneal injections of OVA administered 7 days apart. RESULTS: In the control mice, induction of oral tolerance caused an increase in antigen specific IgG1 levels and a decrease in IgG2a levels. In contrast, the IgG1/IgG2a ratio was reversed in obese mice. OVA-specific IL-2 production was suppressed by antigen feeding in both the control and obese mice; however, suppression of OVA-specific IL-10 was observed only in the control mice. Although OVA-specific IgA and IgM were not affected by antigen feeding, the obese groups of mice had significantly lower titers of antibodies. DISCUSSION: These findings suggest that obesity may affect induction of oral tolerance following antigen feeding and that these changes may be related to the inflammatory reaction.     

Suppression of oral tolerance by Lactococcus lactis in mice

Although oral ovabumin (OVA) administration suppressed the antibody (Ab) response in OVA-immunized mice, Lactococcus lactis increased OVA-specific IgG2a in these mice. L. lactis increased the casein-specific IgG level in NC/Nga mice fed on a casein diet. The percentage of CD4(+)CD25(+) cells was increased in DO11.10 mice orally given OVA, but this increase of CD4(+)CD25(+) cells were suppressed in L. lactis-fed DO11.10 mice.     

Topical superantigen exposure induces epidermal accumulation of CD8+ T cells, a mixed Th1/Th2-type dermatitis and vigorous production of IgE antibodies in the murine model of atopic dermatitis

Patients with atopic dermatitis (AD) have repeated cutaneous exposure to both environmental allergens and superantigen-producing strains of Staphylococcus aureus. We used a murine model of AD to investigate the role of staphylococcal enterotoxin B (SEB) in the modulation of allergen-induced skin inflammation. Mice were topically exposed to SEB, OVA, a combination of OVA and SEB (OVA/SEB), or PBS. Topical SEB and OVA/SEB exposure induced epidermal accumulation of CD8+ T cells and TCRVbeta8+ cells in contrast to OVA application, which induced a mainly dermal infiltration of CD4+ cells. SEB and OVA/SEB exposure elicited a mixed Th1/Th2-associated cytokine and chemokine expression profile within the skin. Restimulation of lymph node cells from OVA- and OVA/SEB-exposed mice with OVA elicited strong production of IL-13 protein, whereas substantial amounts of IFN-gamma protein were detected after SEB stimulation of cells derived from SEB- or OVA/SEB-exposed mice. Topical SEB treatment elicited vigorous production of SEB-specific IgE and IgG2a Abs and significantly increased the production of OVA-specific IgE and IgG2a Abs. The present study shows that topical exposure to SEB provokes epidermal accumulation of CD8+ T cells, a mixed Th2/Th1 type dermatitis and vigorous production of specific IgE and IgG2a Abs, which can be related to the chronic phase of atopic skin inflammation.     

Secondhand smoke induces allergic sensitization in mice

Epidemiological studies have suggested increased prevalence of atopy in children of maternal smokers. Although secondhand smoke or environmental tobacco smoke (ETS) has been shown to augment allergic responses, its role in atopic sensitization is still controversial. We studied whether ETS could initiate a Th2 response and thus induce primary allergic sensitization. Mice were exposed for 10 consecutive days to either 1% aerosolized OVA, ETS (5 cigarettes), or both ETS and OVA. C57BL/6 mice receiving both ETS and OVA developed OVA-specific IgE and IgG1, 12, 14, and 25 days after the initial exposure, whereas those receiving OVA alone did not. Thirty days after the initial challenge (20 days after its completion), mice were re-exposed to OVA. Bronchoalveolar lavage performed 24 h later revealed an influx of eosinophils in the group initially challenged with both ETS and OVA, but not in those exposed to ETS alone or OVA alone. Increases in IL-5, GM-CSF, and IL-2 were observed in bronchoalveolar lavage from this OVA/ETS-exposed group, whereas IFN-gamma levels were significantly inhibited. These results suggest that ETS can induce allergic sensitization to a normally harmless Ag, and they may explain why secondhand smoke is a major risk factor for the development of allergy in children.     

1alpha,25-dihydroxyvitamin D3 potentiates the beneficial effects of allergen immunotherapy in a mouse model of allergic asthma: role for IL-10 and TGF-beta

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a potent inhibitor of NF-kappaB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)(2)D(3) potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-beta are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 microg. Interestingly, coadministration of 10 ng of 1,25(OH)(2)D(3) with 100 microg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-beta and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-kappaB pathway using the selective IkappaB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-beta before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)(2)D(3) potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-beta play a crucial role in the effector phase of this mouse model.     

Establishment and characterization of a novel murine model for pollen allergy

Although there have been many studies revealing the mechanism and establishing the therapeutical method for allergic rhinitis, no suitable animal models for allergic rhinitis, especially for pollen allergy, are currently available. We therefore aimed in this study to develop a murine model producing IgE in response to an inhaled antigen without using any adjuvants. Ovalbumin (OVA)-specific T cell receptor transgenic mice (DO11.10) inhaled an OVA solution for one h, twice a week, for six weeks. The resulting increase of OVA-specific IgE in the serum was observed depending on the times of inhalation. Spleen cells from mice that had inhaled the antigen produced more IL-4 and less IFN-γ than those from the control mice in vitro. These results indicate that inhaled antigen enhanced the Th2-type responses and induced IgE production in a T cell-mediated manner. Our findings would contribute to studies on prevention and treatment of pollen allergy.     

Stimulation of respiratory immunity by oral administration of Lactococcus lactis

This work demostrates that nonrecombinant Lactococcus lactis NZ, administered by the oral route at the proper dose, is able to improve resistance against pneumococcal infection. Lactococcus lactis NZ oral administration was able to improve pathogen lung clearance, increased survival of infected mice, and reduced lung injuries. This effect was related to an upregulation of the respiratory innate and specific immune responses. Administration of L. lactis NZ improved production of bronchoalveolar lavage (BAL) fluid TNF-alpha, enhanced recruitment of neutrophils into the alveolar spaces, and induced a higher activation of BAL phagocytes compared with the control group. Lactococcus lactis NZ administered orally stimulated the IgA cycle, increased IgA+ cells in intestine and bronchus, and improved production of BAL IL-4 and IL-10 during infection. Moreover, mice treated with L. lactis NZ showed higher levels of BAL anti-pneumococcal IgA and IgG. Taking into consideration that orally administered L. lactis NZ stimulates both the innate and the specific immune responses in the respiratory tract and that bacterial and viral antigens have been efficiently produced in this strain, L. lactis NZ is an excellent candidate for the development of an effective pneumococcal oral vaccine.     

An essential role for IL-18 in CD8 T cell-mediated suppression of IgE responses

The ability of CD8 T cells to suppress IgE responses is well established. Previously, we demonstrated that CD8 T cells inhibit IgE responses via the induction of IL-12, which promotes Th1 and suppresses Th2 responses. In this study, we show that IL-18 also plays an essential role in IgE suppression. In vitro, IL-18 synergized with IL-12 to promote Th1/T cytotoxic 1 and inhibit Th2/T cytotoxic 2 differentiation. OVA-specific TCR transgenic (OT-I) CD8 cells induced both IL-12 and IL-18 when cultured with OVA(257-264) peptide-pulsed dendritic cells. In vivo, IL-18(-/-) mice exhibited higher IgE and IgG1 levels compared with wild-type mice after immunization with OVA/alum. Furthermore, adoptive transfer of CD8 T cells from OVA-primed mice suppressed IgE responses in OVA/alum-immunized mice, but not in IL-18(-/-) mice. IgE suppression in IL-18(-/-) mice was restored if CD8 T cells were coadoptively transferred with IL-18-competent wild-type bone marrow dendritic cell progenitors, demonstrating an essential role of IL-18 in CD8 T cell-mediated suppression of IgE responses. The data suggest that CD8 T cells induce IL-18 production during a cognate interaction with APCs that synergizes with IL-12 to promote immune deviation away from the allergic phenotype. Our data identify IL-18 induction as a potentially useful target in immunotherapy of allergic disease.     

Effect of orally administered non-viable Lactobacillus cells on murine humoral immune responses

BALB/c mice were immunized intraperitoneally with the food antigen ovalbumin (OVA) while they were fed with Lactobacillus GG heated killed cell preparation. The oral administration of Lactobacillus GG did not appear to modify the antigen-augmented serum IgE in the tested mice but significantly augmented serum OVA specific IgG in the tested mice fed with a diet containing 0.1% Lactobacillus GG as the non-viable cell preparation (P< 0.05). The fecal OVA specific IgA of the tested mice fed with nonviable Lactobacillus GG cells was also significantly elevated (P< 0.05) compared to those from OVA immunized mice. The spleen cells of mice fed with non-viable Lactobacillus GG cells secreted more IL-6 (P< 0.01). These results suggest that the non-viable Lactobacillus GG can augment the systemic and mucosal immune responses in a host animal favoring secretory IgA but not IgE in an adjuvant-like manner.     

双歧杆菌完整肽聚糖对食物过敏小鼠调节性T细胞影响的研究

目的以食物过敏小鼠为动物模型,通过灌喂双歧杆菌完整肽聚糖(Whole Peptidoglycan,WPG),观察其对调节性T细胞的作用。方法 4～6周龄SPF级无鸡蛋喂养BALB/c雌鼠随机分为3组,每组8只。取其中2组建立食物过敏模型,分别为OVA致敏阳性对照组、OVA激发后灌喂双歧杆菌WPG治疗组和生理盐水对照组。采用ELISA法检测各组血清中OVA特异性IgE、IL-10和TGF-β1水平;流式细胞术分析脾脏单个核细胞悬液中CD4+CD25+调节性T细胞的数量变化。结果 OVA组IL-10、TGF-β1水平显著高于对照组(P分别0.05和0.01);WPG组血清IL-10和TGF-β1水平显著低于OVA组(P0.05)。OVA组脾CD4+CD25+T淋巴细胞的百分比低于对照组(P0.05),WPG组与OVA组相比有升高趋势,差异有统计学意义(P0.05);OVA组脾Foxp3+CD4+CD25+调节性T细胞的百分比显著低于对照组(P0.01),WPG组与OVA组相比有升高趋势,差异有统计学意义(P0.01)。结论双歧杆菌WPG可以增加食物过敏小鼠CD4+CD25+调节性T细胞数量,刺激细胞因子IL-10和TGF-β分泌。     

IL-4 and IL-10 are essential for immunosuppression induced by high molecular weight proteins from Ascaris suum

The extract from Ascaris suum worms (Asc) impairs Th1 and Th2 responses to a non-related antigen, i.e. ovalbumin (OVA). Its suppressive capacity is due to high molecular weight components present in a gel filtration fraction (PI). This fraction is able to elicit IL-4 and IL-10 secretion. Interestingly enough, it induces anti-PI non-anaphylactic IgG1 synthesis through the action of IL-12/IFN-gamma. Here, we investigated the down-regulation of the immune response to OVA by PI in IL-12, IFN-gamma, IL-4 or IL-10 C57BL/6 knockout mice immunized with OVA+PI in adjuvant. OVA-induced delayed-type hypersensitivity (DTH) reactions, secretion of IL-2 and IFN-gamma, and IgG1, IgG2c and IgE antibody production were suppressed by PI in wild-type mice, as well as in IL-12- or IFN-gamma-deficient mice. In contrast, PI had no effect on anti-OVA IgE production and DTH, and induced only a partial suppression of IgG1 and IFN-gamma in IL-10(-/-) mice. The experiments also showed that IL-4 was involved in the PI-induced suppression of IgG2c antibodies and IL-2 secretion. Finally, down-regulation of IFN-gamma was not seen in mice lacking both IL-4 and IL-10, i.e. IL-4(-/-) mice treated with anti-IL-10 antibodies before immunization. These results exclude the participation of IL-12 and IFN-gamma in PI-induced immunosuppression, and highlight the essential role of IL-10 in the suppression of OVA-specific Th2-related parameters, as well as the cooperation between IL-10 and IL-4 in the suppression of Th1-related parameters.     

Effect of respiratory syncytial virus infection on the uptake of and immune response to other inhaled antigens

Groups of BALB/c mice were sham infected or inoculated intranasally (IN) with live RSV. From Day 4 to 8 after infection, the animals were exposed IN to ovalbumin (OVA) with or without alum adjuvant. At different intervals, levels of OVA concentration in serum, IgG-anti-OVA antibody activity in serum, and IgA-anti-OVA antibody activity in bronchial washings were determined, employing the ELISA technique. IgE-anti-OVA antibody titers in serum and bronchial washings were assessed by PCA. OVA concentrations in serum were significantly higher in RSV-infected animals compared to uninfected controls. The use of alum adjuvant also increased OVA uptake in uninfected animals but to a lesser extent than RSV infection. RSV-infected animals developed significantly higher OVA-specific antibody titers of IgG isotype in serum and IgA isotype in bronchial washings than the uninfected controls, while alum enhanced the immune response less markedly but still significantly in uninfected mice. An IgE antibody response to OVA in serum was demonstrable in 50% of RSV-infected mice immunized IN with OVA and alum, while all uninfected animals and RSV-infected animals immunized with OVA alone (without adjuvant) failed to develop a detectable IgE response. These findings suggest that infections with viral agents such as RSV may function as adjuvants for other antigens inhaled during acute respiratory infection. These observations may explain the alterations in the immune response to other antigens in patients with acute viral-induced bronchopulmonary diseases.     

Oral Intake of <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> NS8 Alleviates Ovalbumin-Induced Atopic Dermatitis in SKH-1 Hairless Mice

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease and is driven by strong type 2 immune responses. Lactobacillus helveticus NS8 (NS8), a probiotic strain isolated from Mongolian koumiss, has anti-inflammatory activities. Here, we evaluated the therapeutic potential of NS8 on AD-like skin lesions by using SKH-1 hairless mice that underwent three cycles of epicutaneous sensitization (EC) with ovalbumin (OVA). NS8 (5?×?10⁸ CFU/day) was orally administered to mice from 2 weeks before the first sensitization until the end of the study. NS8 attenuated the symptoms and pathological changes in the skin of AD mice. For example, NS8 reduced epidermal and dermal thickening and significantly restrained the infiltration of mast cells, eosinophils, and CD4⁺ T cells into the dermis. By analysing the Th1/Th2 cytokines produced in skin lesions, we found that NS8 significantly suppressed the expression of IL-4, IL-5, and IL-13 (P?<?0.05), while it had no discernible effect on the expression of IFN-γ. Systemically, NS-8 reduced the total IgE and OVA-specific IgE levels in serum (P?<?0.05). Our study demonstrates that oral administration of L. helveticus NS8 effectively alleviates AD severity in mice by suppressing the Th2 immune response. NS8 may be a promising candidate for prophylactic and therapeutic treatments of allergic diseases, such as AD.     

Silibinin attenuates antigen-specific IgE production through the modulation of Th1/Th2 balance in ovalbumin-sensitized BALB/c mice

The effect of silibinin on antigen-specific antibody production and T-cell cytokine expression was investigated. BALB/c mice were either left untreated or administered daily with vehicle (VH; saline) and/or silibinin (200 or 400 mg/kg) by gavage for 3 consecutive days prior to sensitization with ovalbumin (OVA). The antibody production in the serum and T-cell-derived cytokine expression by splenocytes were determined 7 days post OVA sensitization. Our results demonstrated that the production of OVA-specific serum IgE and total IgE was significantly attenuated by silibinin treatment, whereas OVA-specific IgG_2a was markedly enhanced. In parallel with the differential modulation of the production of IgG_2a and IgE, treatment of OVA-sensitized mice with silibinin markedly increased and decreased the production of IFN-γ and IL-4, respectively, by splenocytes cultured in the presence of OVA. Together, these results suggest that silibinin treatment polarizes the Th1/Th2 immune balance toward the Th1-dominant direction, which may be beneficial against IgE-mediated allergy.     

Selective blockade of NF-kappa B activity in airway immune cells inhibits the effector phase of experimental asthma

Knockout mice studies have revealed that NF-kappaB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-kappaB also plays a role in the effector phase of airway allergy and whether inhibiting NF-kappaB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-kappaB activity in the lungs during OVA challenge. Intratracheal administration of NF-kappaB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-kappaB activity in the airways upon OVA provocation. NF-kappaB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-kappaB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-kappaB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.     

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.