Maternal allergen immunisation to prevent sensitisation in offspring: Th2-polarising adjuvants are more efficient than a Th1-polarising adjuvant in mice

Background

Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period.

Results

Maternal immunization with OVA showed increased levels of FcγRIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-γ-secreting T cells and IL-4 and IL-12- secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced FcγRIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers.

Conclusion

Maternal immunization upregulates the inhibitory FcγRIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.     

In interleukin-4-deficient mice,alum not only generates T helper 1 responses equivalent to Freund's complete adjuvant,but continues to induce T helper 2 cytokine production

The role of interleukin (IL)-4 in the activity of two frequently used vaccine adjuvants, Freund's complete adjuvant (FCA) and the aluminum hydroxide gels (alum), was studied using the standard antigen ovalbumin (OVA) in IL-4 genedisrupted mice (IL-4 -/-). In the absence of adjuvant, there was an overall reduction in antibody production to OVA in IL-4 −/− mice and significantly greater amounts of interferon (IFN)-γ were produced following restimulation of splenocytes with antigen in vitro compared with immunocompetent controls (IL-4 +/+). FCA and alum boosted the immune response to OVA in both IL-4 −/− and IL-4 +/+ mice. In IL-4 +/+ mice, while FCA stimulated a wide-spectrum immunoglobulin response, including both Th1-associated IgG2a and Th2-associated IgG1, alum enhanced only Th2 antibody production and no OVA-specific IgG2a could be detected. In IL-4-deficient mice, however, not only was IgG2a production increased in all adjuvant-treated groups, but alum was as potent at stimulating this antibody subclass as FCA. Similarly, increased production in vitro by splenocytes of the Th1 cytokine IFN-γ, equivalent to that produced after inoculation with FCA/OVA, was only detected in IL-4 −/− mice inoculated with alum/OVA. There was no IgE production in IL-4 −/− mice and OVA-specific IgG1 production, although still at significant levels, was reduced compared with wild-type mice irrespective of the adjuvant used. However, although production of the Th2 cytokine IL-5 was totally inhibited in IL-4-deficient mice inoculated with FCA/OVA, there was no significant difference in IL-5 production between the two strains when alum was used as adjuvant.     

Adjuvant effect of zinc oxide on Th2 but not Th1 immune responses in mice

Background and aim: We investigated the effect of zinc oxide (ZnO) on Th1 and Th2 immune responses in mice.

Material and methods: Mice were intraperitoneally administered with ovalbumin (OVA) with or without varying doses of ZnO (day 0). On day 21, anti-OVA IgG, IgG2a, IgG1, and IgE antibodies in sera, OVA-specific proliferative responses of spleen cells, and production of Th1 cytokines including IFN-γ as well as Th2 cytokines such as IL-4 and IL-5 were measured.

Results: The results showed that administration of OVA with ZnO was followed by greater increases in anti-OVA IgG and the antigen-specific splenocyte proliferation compared to that of OVA alone. The production of anti-OVA IgG1 and IgE and secretion of IL-4 and IL-5 were markedly enhanced by ZnO. The enhancing effect of ZnO on these Th2 responses was as strong as aluminium hydroxide (Alum) that was widely used as an adjuvant. In contrast, treatment with OVA plus ZnO failed to affect production of anti-OVA IgG2a as well as IFN-γ. It was also observed that ZnO had a stimulating effect on the secretion of the proinflammatory cytokine IL-17 from a new lineage of effector Th cells.

Conclusion: These results suggest that ZnO appears to have an adjuvant effect on the immune system, especially Th2 but not Th1 immune responses.     

The adjuvant effect of particles-importance of genetic background and pre-sensitisation

BACKGROUND: We have previously reported that simple and well-characterised particles, such as polystyrene particles (PSP), have an IgE adjuvant effect in mice. The purpose of this study was to explore the importance of genetic background concerning the adjuvant effect of PSP in different strains of mice. METHODS: Inbred NIH/Ola, BALB/c and C3H/HeJ mice were given two intraperitoneal injections with either PSP plus OVA or OVA alone, and then an intraperitoneal challenge with OVA alone. NIH/Ola mice were also pre-sensitised to develop a weak or strong IgE response to OVA, and then given an intraperitoneal challenge with PSP plus OVA or OVA alone. Serum levels of total and allergen-specific IgE and IgG2a were measured. RESULTS: PSP had a specific IgE and IgG2a adjuvant effect in NIH/Ola mice but not in C3H/HeJ and BALB/c mice. Weakly pre-sensitised NIH/Ola mice showed the same response pattern as the naive NIH/Ola mice. In contrast, strongly pre-sensitised mice showed an antibody response pattern similar to that of high-responder BALB/c mice. CONCLUSIONS: Our results indicate that the allergen responder status, either genetic or induced, is of importance for the adjuvant effect from particles. The IgE and IgG2a adjuvant effect may depend on the genetically determined susceptibility of an individual to be influenced by exposure to the adjuvant. Therefore, the allergy-enhancing effect from particle pollution may differ between individuals.     

The imbalanced profile and clinical significance of T helper associated cytokines in bone marrow microenvironment of the patients with acute myeloid leukemia

Background

Immunological disorder has shown to be related to the pathogenesis of acute myeloid leukemia (AML). The microenvironment of AML is immunosuppressive, favoring the survival of malignant hematopoietic cells. However, the systematic research on AML abnormal immune microenvironment, especially the T helper (Th) cells imbalance, remains unsettled.

Design and methods

The levels of cytokines in bone marrow plasma including Th1-associated cytokine (IFN-γ), Th2-associated cytokine (IL-4), Th17-associated cytokines (IL-17, IL-6, TGF-β, and IL-21), regulatory T cell (Treg)-associated cytokines (IL-35 and IL-10) and Th22-associated cytokine (IL-22) were examined by enzyme-linked immunosorbent assay (ELISA) in AML patients and controls. The relative expression levels of IL-4, IL-10, and IL-21 mRNA were analyzed by real time polymerase chain reaction (PCR).

Results

Significant differences on cytokine levels tested were observed among the AML newly-diagnosed (ND) patients, AML patients in complete remission (CR) and controls except IL-21 and IL-35. In AML-ND group IFN-γ level was positively correlated with IL-21 or IL-22 level. Additionally, significant associations were observed between IL-17, IL-21 and some clinical characteristics.

Conclusion

Our results showed that many cytokines were abnormal in AML bone marrow microenvironment. The dysregulation of Th subsets cytokines is thought to contribute to the pathogenesis of AML.     

IL-10 controls Th2-type cytokine production and eosinophil infiltration in a mouse model of allergic airway inflammation

Interleukin-10 was originally described as a factor that inhibits cytokine production by murine Th1 clones. Recent studies have since shown that IL-10 can also downregulate Th2 clones and their production of IL-4 and IL-5. Because of its immuno-suppressive properties, IL-10 has been suggested as a potential therapy for allergic inflammation and asthma. However, the pathophysiological role of IL-10 in vivo has not been clearly elucidated. We investigated the effects of IL-10 administration on the production of IgE, cytokine and allergen-induced Th2 cytokine production as well as its effects on eosinophilic inflammation. We established GATA-3/TCR double transgenic (GATA-3/TCR-Tg) mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice; these mice were then sensitized using an intraperitoneal injection of OVA adsorbed to alum and challenged with the intratracheal instillation of an allergen. When GATA-3/TCR-Tg mice sensitized with OVA and alum were injected with C57-IL-10 cells before OVA inhalation, the levels of IL-5, IL-13, and IL-4 decreased by 40-85% and number of eosinophils decreased by 70% (P < 0.03) in the murine bronchoalveolar lavage fluid (BALF). These results suggest that IL-10 plays an important role downstream of the inflammatory cascade in the Th2 response to antigens and in the development of BALF eosinophilia and cytokine production in a murine model of asthma. These immunosuppressive properties in animal models indicate that IL-10 could be a potential clinical therapy for the treatment of allergic inflammation.     

Persistence of Entamoeba histolytica infection in CBA mice owes to intestinal IL-4 production and inhibition of protective IFN-γ

The mechanisms whereby certain mouse strains develop persistent intestinal infection with Entamoeba histolytica remain unclear. In this work, we characterized the kinetic pattern of cytokine responses during the course of natural infection in CBA mice and showed that intracecal amebic infection led to a rapid and sustained upregulation of Th2 (IL-4, IL-5, IL-13) and Th17 cytokine responses while Th1 cytokines, IL-12p35 and interferon (IFN)-γ, were suppressed. Depletion of IL-4 cleared infection by 14 days post-challenge, and this clearance correlated with and was mediated by IFN-γ. The protective role for IFN-γ was not strain-specific, as 129 background IFN-γR knockout mice exhibited a higher infection rate than their wild-type littermates. These studies indicate that IL-4 plays a critical pathogenic role in the persistence of E. histolytica infection through suppression of protective IFN-γ and provide a possible explanation for why certain humans spontaneously clear amebiasis while others progress to invasive disease.     

Suppression of Allergen‐Specific IgE in Offspring After Preconceptional Immunisation: Maternal,Paternal and Genetic Influences

Immunisation of female mice with the allergen ovalbumin (OVA) during pregnancy reduces the OVA‐specific IgE response in adult offspring. To approach primary prevention strategies for allergy, we investigated to what extent genetic, paternal and maternal factors influence this suppressive effect on allergic sensitisation in offspring and investigated the possibility of pregestational immunisation. Maternal allergen immunisation reduced OVA‐specific IgE levels in immunised offspring, even after maternal immunisation up to 8 weeks before conception without further allergen exposure. Immunisation of immunodeficient BALB/c severe combined immune deficiency (SCID) dams mated with wild type males did not lead to IgE suppression in offspring, indicating the importance of a functional maternal immune system. Immunisation of male mice before the relevant spermatogenesis did not cause antibody suppression in offspring. OVA‐specific IgG1, presumably of maternal origin, was present in naïve offspring only from immunised dams and was associated with suppressed IgE responses after offspring immunisation. The IgE‐suppressive effect of maternal immunisation was demonstrated in all three immunocompetent strains tested (NIH/OlaHsd, BALB/cA and C57BL/6 mice). In conclusion, suppression of allergen‐specific IgE production in offspring could not be induced by paternal immunisation, and genetic factors were of minor importance. In contrast, we demonstrate the necessity of maternal factors, possibly allergen‐specific IgG1, resulting from a functional adaptive immune response, for the IgE‐suppressive effect in offspring. These maternal factors could be induced by immunisation of female mice even before conception.     

Production of IFN-γ by CD4(+) T cells in response to malaria antigens is IL-2 dependent

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4(+) T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4(+) T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4(+) T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4(+) T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4(+) T cells. Further investigation revealed that host CD4(+) T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4(+) T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4(+) T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4(+) T cells.     

Administration of interleukin-12 exerts a therapeutic instead of a long-term preventive effect on mite Der p I allergen-induced animal model of airway inflammation

Interleukin-12 (IL-12) is a key cytokine, which promotes T helper type 1 (Th1) cell-mediated immunity and inhibits Th2-type responses. It has been previously shown that IL-12 administration during active immunization following a single allergen exposure can prevent antigen-induced increases in immunoglobulin E (IgE) formation, Th2 cytokine production and bronchoalveolar lavage (BAL) eosinophils in a murine model of allergic airway inflammation. Thus, these studies have now been extended and two IL-12 treatment protocols on this murine model were evaluated. Administration of IL-12 during the active immunization strikingly increased Der p I-specific serum IgG2a and transiently decreased the levels of IgG1 and IgE antibodies following multiple allergen challenges. Such early treatment of IL-12 down-regulated IL-5 production and modestly up-regulated interferon-gamma production but did not effect BAL eosinophilia. These results suggest that repeated exposure to antigen and IL-12 is necessary to maintain a persistent Th1-recall response. Furthermore, administration of IL-12 to actively immunized mice, in which Th2-associated responses were established, had a significant effect on IgG2a synthesis and a modest effect on IgE levels, also down-regulation of IL-5 production, and markedly increased interferon-gamma production and abolished recruitment of eosinophils. Therefore, these data indicate that IL-12 can inhibit antigen-induced eosinophil infiltration into airways, despite the existence of a Th2-associated response. Taken together, these studies suggest that IL-12 may be useful as an immunotherapeutic agent in the treatment of such pulmonary allergic disorders as bronchial asthma.     

Selective suppression of antigen-specific Th2 cells by continuous micro-dose oral tolerance

The effect on antigen (Ag)-specific Th2 response as well as IgE production of continuous oral administration of micro-doses of Ag was investigated. Transgenic (Tg) mice carrying the α β-T cell receptor (TCR) genes specific for ovalbumin (OVA) peptide fragment 323 – 339 were continuously fed with micro-doses of OVA (100 μg/day) for 14 days. Mice were first immunized by OVA in alum and pertussis toxin 7 days before the oral feeding and given a second immunization 1 day after the oral treatment. This feeding regimen tolerized Th2 but not Th1 responses as shown by decrease of Ag-driven cell proliferation and cytokine secretion of IL- 4 but not of IL-2 or IFN-γ as well as by the absence of Ag-specific antibody production of IgE and IgG1, but not of IgG2a or total IgG. Numbers of clonotype-specific TCR-high CD4-positive T cells in peripheral lymphoid tissues markedly decreased in the orally treated group but not in the control group. However, total numbers of CD4-positive T cells in thymus, spleen and lymph nodes were not affected by the oral treatment, indicating that tolerance induction in Th2 cells was mainly due to the down-regulation of TCR and not clonal deletion. The population of antigen-presenting cells expressing B7-2 (CD86) Ag on the surface was decreased in the spleen of the mice which underwent the feeding regimen. The present results suggest that Ag-specific low responsiveness in Th2 cells, which resulted in suppres sion of the Ag-specific IgE production, can be achieved by continuous feeding with microdoses of Ag.     

In the absence of IL-12, the induction of Th1-mediated protective immunity by the attenuated schistosome vaccine is impaired,revealing an alternative pathway with Th2-type characteristics

Vaccination of mice with irradiated Schistosoma mansoni larvae confers high levels of immunity which is mediated by Th1-type lymphocytes. To investigate a possible role for IL-12 in the induction of protection, we have compared the immune response of IL-12 p40-deficient (KO) mice and their C57BL/6 (WT) counterparts following vaccination. Cultured lymph node cells from KO mice had markedly altered cytokine profiles with significantly decreased production of IFN-γ increased IL-4. Correspondingly, KO mice had enhanced levels of IgE. After challenge, cells recovered from the lungs of KO mice secreted abundant IL-4 and IL-5 but little IFN-γ, while flow cytometric and histological analysis of lung cell populations recorded a very high proportion of eosinophils. The levels of protection in KO mice were substantially lower than in their WT counterparts, demonstrating the importance of IL-12 and Th1-mediated immune responses. This conclusion is reinforced by the administration of rIL-12 to KO mice immediately after vaccination which led to increased IFN-γ and the restoration of protective immunity. Nevertheless, the data also indicated that the limited levels of protection induced in KO mice occur via an IL-12-independent pathway, possibly mediated by Th2 cells.     

Suppressive versus stimulatory effects of allergen/cholera toxoid (CTB) conjugates depending on the nature of the allergen in a murine model of type I allergy.

Recent reports have demonstrated that feeding small amounts of antigen conjugated to the B subunit of cholera toxin (CTB) suppress immune responses in experimental models of certain Th1-based autoimmune diseases. We have established a model of aerosol sensitization leading to Th2-mediated allergic immune responses in BALB/c mice. In the present study two different antigens, the dietary antigen ovalbumin (OVA) and the inhalant allergen Bet v 1 (the major birch pollen allergen), chemically coupled to recombinant CTB were tested for their potential to influence Th2-like immune responses. Intranasal administration of OVA-CTB prior to sensitization with OVA led to a significant decrease of antigen-specific IgE antibody levels, but a marked increase of OVA-specific IgG2a antibodies as compared to non-pretreated, sensitized animals. Antigen-specific lympho-proliferative responses in vitro were reduced by 65% in the pretreated group; IL-5 and IL-4, but not IFN-gamma, production were markedly decreased in responder cells of lungs and spleens of nasally pretreated mice. In contrast, mucosal administration of rBet v 1-CTB conjugates prior to sensitization led to an up-regulation of allergen-specific IgE, IgG1 and IgG2a, increased in vitro lympho-proliferative responses as well as augmented production of IL-5, IL-4, IL-10 and IFN-gamma. Intranasal administration prior to sensitization of unconjugated allergens showed also contrasting effects: OVA could not significantly influence antigen-specific antibody or cytokine production, whereas intranasal pretreatment with unconjugated Bet v 1 suppressed allergen-specific immune responses in vivo and in vitro. These results demonstrated that the two antigens--in conjugated as in unconjugated form--had different effects on the Th2 immune responses. We therefore conclude that the tolerogenic or immunogenic properties of CTB--and probably also other antigen-delivery systems--strongly depend on the nature of the coupled antigen-allergen.     

Effect of sinomenine on collagen-induced arthritis in mice

The present study was designed to investigate the effect of sinomenine (SIN), an alkaloid extracted from sinomenium acutum on collagen-induced arthritis (CIA) in mice. For this investigation, mice were s.c. immunized with type II collagen (CII) emulsified with complete Freund's adjuvant (day 0). Varying doses of SIN were orally administered daily commencing on day 0 daily over a period of 55 days. The severity of arthritis was evaluated according to clinical score, the effect of SIN on immune responses were determined by measurement of proliferative responses of spleen cells, antibody levels in serum and cytokine assays. Anti-CII IgG2a and IFN-γ were measured as indicators of Th1 immune responses and anti-CII IgG1, IgE and IL-5 as those of Th2 responses. IL-10 and TGF-β were measured as indicators of T cell regulator responses. The results showed that treatment with SIN was followed by decreases in the incidence and severity of CIA, anti-CII IgG and the antigen-specific splenocyte proliferation. Production of all isotypes of antibodies including anti-CII IgG2a, IgG1 and IgE as well as secretion of cytokines such as IFN-γ and IL-5 were suppressed by SIN. In addition, SIN enhanced the secretion of TGF-β while it had no obvious effect on production of IL-10. These results suggest that the anti-arthritic effect of SIN may be related to the suppression of both Th1 and Th2 immune responses. TGF-β may at least in part contribute to the suppression of Th1 as well as Th2 immune responses.     

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study

Background

Burkholderia pseudomalleiis the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-γ response toB. pseudomalleibetween the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-γ in the relatively susceptible BALB/c micein vitro.

Results

Naïve BALB/c splenocytes were found to produce more IFN-γ in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-γ, while T cells and Gr-1^intermediatecells also contributed to the IFN-γ response. Although anti-Gr-1 depletion substantially reduced the IFN-γ response, this was not due to the contribution of Gr-1^high, Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-γ between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-γ response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-18 than C57BL/6 splenocytes after infection withB. pseudomallei.

Conclusion

Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-γ hyperproduction in BALB/c mice.     

The expression of murine eutaneous late phase reaction requires both IgE antibodies and CD4 T eells

Background Exposure of atopic patients to a specific allergen evokes an immediate response which is followed, in many cases, by a late phase reaction (LPR) some hours later. Here we have examined the immunological mechanisms required for the expression of cutaneous LPR in mice. Methods BALB/c mice were immunized by i.p. injection of ovalbumin (OVA) and alum actively or by i.v. injection of anti-OVA IgE monoclonal antibody (mAb) passively. After challenge by intradermal injection of OVA into ears, the changes in ear thickness, the number of eosinophils, and the levels of IL4 and IFN-γ protein at the site of antigen challenge were examined. Results Actively immunized mice developed a biphasic response at the site of OVA injection, while mice passively immunized with IgE anti-OVA mAb displayed a strong early response but no LPR. Cell transfer experiments using BALB/c nu/nu mice revealed that both OVA-specific IgE mAb and OVA-primed CD4 T cells were required to evoke LPR. Moreover, LPR was associated with increased levels of IL-4 production concomitant with reduced IFN-γ production and was abolished by pretreatment with anti-IL-4 neutralizing mAb. Conclusion It is suggested that murine cutaneous LPR against OVA is a type 2 inflammatory response in which both IgH antibodies and CD4 T cells play an obligatory role.