Mahasti S. Macedo

Bacterial Lipopolysaccharide Signaling Through Toll-Like Receptor 4 Suppresses Asthma-Like Responses Via Nitric Oxide Synthase 2 Activity

Asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. Microbial infections could prevent allergic responses by inducing the secretion of the type 1 cytokines, IL-12 and IFN-γ. In this study, we examined whether administration of bacterial LPS, a prototypic bacterial product that activates innate immune cells via the Toll-like receptor 4 (TLR4) could suppress early and late allergic responses in a murine model of asthma. We report that LPS administration suppresses the IgE-mediated and mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity. The suppression of asthma-like responses was not due to Th1 shift as it persisted in IL-12−/− or IFN-γ−/− mice. However, the suppressive effect of LPS was not observed in TLR4- or NO synthase 2-deficient mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses in vivo via the TLR4-dependent pathway that triggers NO synthase 2 activity.

Eosinophilic inflammation and airway hyper‐responsiveness are profoundly inhibited by a helminth (Ascaris suum) extract in a murine model of asthma

Background The increase of atopic disorders in developed countries has been associated with the decline of infectious diseases, including helminthic infections. We have already demonstrated that adult worm extracts from Ascaris suum (ASC) suppress the IgE antibody production against unrelated antigens.

Modulation of murine experimental asthma by Ascaris suum components.

Background We have recently isolated two distinct components from Ascaris suum adult worms with different effects on the immune system: the allergenic protein of A. suum (APAS‐3), which induces IgE antibody production, and suppressive protein of A. suum (PAS‐1), which inhibits humoral and cellular immune responses induced by unrelated antigens. In this study, we investigated the immunomodulatory effect of PAS‐1 on a murine model of asthma induced by APAS‐3.

Immunosuppressive components of Ascaris suum down‐regulate expression of costimulatory molecules and function of antigen‐presenting cells via an IL‐10‐mediated mechanism

High‐molecular‐weight components (PI) of Ascaris suum suppress both cell‐mediated and humoral responses against ovalbumin (OVA) via an IL‐4/IL‐10‐dependent mechanism. The aim of this work was to investigate the effect of PI on the ability of APC to activate T cells and the role of IL‐10 in this process. Flow cytometry analyses of MHC class II, CD80, CD86 and CD40 molecules on LN cells from mice immunized with OVA or OVA+PI showed that PI inhibits expression of these molecules on unfractionated cells and on purified CD11c+ cells. A low proliferative response was obtained when OVA‐specific TCR‐Tg T cells were incubated with CD11c+ cells from OVA+PI‐immunized mice pulsed with OVA, when compared to those incubated with cells from OVA‐immunized mice. Similar results were obtained using as APC CD11c+ cells from OVA‐immunized mice pulsed with OVA+PI, which also expressed less of the four markers. The inhibitory effect of PI on both the expression of costimulatory molecules and the induction of T cell proliferation was abolished in IL‐10‐deficient mice. Our data indicate that the potent immunosuppressive effect of A. suum extract components on the host immune system is primarily related to their property of down‐regulating the Ag‐presenting ability of DC via an IL‐10‐mediated mechanism.

Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.

Isolation of Ascaris suum components which suppress IgE antibody responses

An extract from adult Ascaris suum was fractionated on a Sephadex G-200 column and individual elution fractions assessed for their ability to induce or suppress an IgE antibody response in mice immunized simultaneously with a heterologous antigen (ovalbumin). The results showed that, whilst fractions eluted in the first peak suppressed the anti-ovalbumin IgE antibody production, those eluted in the third peak induced a significant anti-Ascaris IgE response. The mixture of the two kinds of fractions resulted in suppression of anti-Ascaris IgE antibodies. The apparent molecular weight (MW) of the first and third peaks was 530,000 and 29,000 daltons, respectively. After delipidation, the extract still retained most of its biological activities. SDS-PAGE of this material showed selective loss of high-MW components when compared to the whole extract. No bands were observed in SDS-PAGE of peak I in contrast to peak III, which displayed several bands. Immunoblots of all these samples showed at least 3 bands above MW 150,000, which reacted with anti-peak I antiserum, in the unfractionated extracts and in peak I, but not in peak III. When the extract was fractionated by affinity chromatography using anti-peak I antibodies as ligands, the immunogenic components were present in the effluent, whereas the suppressive components were recovered in the eluate.

PAS-1, a protein affinity purified from Ascaris suum worms, maintains the ability to modulate the immune response to a bystander antigen

Helminth infections and parasite components have potent immunomodulatory effects on a hosts immune system. In the present study, we investigated the effect of PAS‐1, a protein component of Ascaris suum adult worms recognized by a monoclonal antibody (MAIP‐1), on humoral and cell‐mediated responses to a bystander antigen (ovalbumin [OVA]). MAIP‐1 recognized only one of the three polypeptide chains of PAS‐1, but neutralized the suppressive effect of the whole worm extract on OVA‐specific antibody production. PAS‐1 inhibited antibody production against a T‐cell‐dependent, but not a T‐cell‐independent, antigen in a dose‐dependent way. IgM, IgG1, IgG2b, and also IgE and anaphylactic IgG1 levels were downregulated. In addition, PAS‐1 inhibited OVA‐specific delayed type hypersensitivity reactions in the footpad of mice, showing a potent immunosuppressive activity on both Th1 and Th2 responses that seems to be mediated by the induction of large amounts of IL‐10 and IL‐4. Indeed, PAS‐1‐specific spleen cells secreted sevenfold more IL‐10 and threefold more IL‐4 than OVA‐specific cells in response to in vitro restimulation with the respective antigens. In conclusion, we showed that PAS‐1, a single protein component from A. suum, maintains all its immunosuppressive properties.

PAS-1, a protein from Ascaris suum, modulates allergic inflammation via IL-10 and IFN-γ, but not IL-12

Helminths and their products have a profound immunomodulatory effect upon the inductive and effector phases of inflammatory responses, including allergy. We have demonstrated that PAS-1, a protein isolated from Ascaris suum worms, has an inhibitory effect on lung allergic inflammation due to its ability to down-regulate eosinophilic inflammation, Th2 cytokine release and IgE antibody production. Here, we investigated the role of IL-12, IFN-gamma and IL-10 in the PAS-1-induced inhibitory mechanism using a murine model of asthma. Wild type C57BL/6, IL-12(-/-), IFN-gamma(-/-) and IL-10(-/-) mice were immunized with PAS-1 and/or OVA and challenged with the same antigens intranasally. The suppressive effect of PAS-1 was demonstrated on the cellular influx into airways, with reduction of eosinophil number and eosinophil peroxidase activity in OVA+PAS-1-immunized wild type mice. This effect well correlated with a significant reduction in the levels of IL-4, IL-5, IL-13 and eotaxin in BAL fluid. Levels of IgE and IgG1 antibodies were also impaired in serum from these mice. The inhibitory activity of PAS-1 was also observed in IL-12(-/-) mice, but not in IFN-gamma(-/-) and IL-10(-/-) animals. These data show that IFN-gamma and IL-10, but not IL-12, play an important role in the PAS-1 modulatory effect.

Interference of methysergide, a specific 5‐hydroxytryptamine receptor antagonist, with airway chronic allergic inflammation and remodelling in a murine model of asthma

Background Airway remodelling encompasses the structural changes observed in asthmatic airways. Mast cells, through the release of histamine and 5‐hydroxytryptamine (serotonin), are implicated in early asthmatic reactions, bronchoconstriction and mucosal oedema, and in the development of bronchial hyperresponsiveness. However, the association between serotonin and remodelling processes in murine model of airways inflammation remains to be elucidated.

Modulation of the Induction of Lung and Airway Allergy in the Offspring of IFN-γ-Treated Mother Mice

Recent studies have highlighted the influence of fetal/maternal interactions on the development of asthma. Because IFN-γ reduces Th2-mediated allergic responses, we assessed its capacity to modulate asthma in the offspring when injected into mothers during pregnancy. IFN-γ was injected in CD1 female mice on day 6.5 of gestation. Immediately after birth, male newborns were housed in cages with interchanged mothers: the offspring from IFN-γ-treated mothers were breastfed by normal mothers (IFN/nor), and those from normal mothers were breastfed by IFN-γ-treated (Nor/IFN) or normal mothers (Nor/nor). Immediately after weaning, the spleen cells from IFN/nor and Nor/IFN mice produced less IL-4 and more IFN-γ than Nor/nor mice when stimulated with Con A. At the age of 6–7 wk, mice were immunized with OVA on days 0 and 7. From day 14 to 16, they were exposed to aerosolized OVA. The bronchoalveolar lavage fluid from Nor/nor mice showed eosinophilia, a large number of these cells being present in perivascular and peribronchial regions of lung tissues. IFN/nor or Nor/IFN mice showed greatly reduced eosinophil numbers in bronchoalveolar lavage fluid. In addition, lung sections from IFN/nor, but not Nor/IFN mice showed almost normal histology. In OVA-sensitized IFN/nor and Nor/IFN mice, the production of IFN-γ, IL-4, and IL-5 by spleen cells was significantly reduced as compared with cells from the OVA-sensitized Nor/nor group. IgE and anaphylactic IgG1 were also reduced in plasma of IFN/nor mice. In conclusion, the presence of IFN-γ during pregnancy confers to the fetus a protection against allergenic provocations in the adult life.