Allergic asthma is a dysregulation of the immune system which leads to the development of Th2 responses to innocuous antigens (allergens). Some infections and microbial components can re-direct the immune response toward the Th1 response, or induce regulatory T cells to suppress the Th2 response, thereby inhibiting the development of allergic asthma. Since Acinetobacter baumannii infection can modulate lung cellular and cytokine responses, we studied the effect of A. baumannii in modulating airway eosinophilia in a mouse model of allergic asthma. Ovalbumin (OVA)-sensitized mice were treated with live A. baumannii or phosphate buffered saline (PBS), then intranasally challenged with OVA. Compared to PBS, A. baumannii treatment significantly reduced pulmonary Th2 cytokine and chemokine responses to OVA challenge. More importantly, the airway inflammation in A. baumannii-treated mice was strongly suppressed, as seen by the significant reduction of the proportion and the total number of eosinophils in the bronchoalveolar lavage fluid. In addition, A. baumannii-treated mice diminished lung mucus overproduction and pathology. However, A. baumannii treatment did not significantly alter systemic immune responses to OVA. Serum OVA-specific IgE, IgG1 and IgG2a levels were comparable between A. baumannii- and PBS-treated mice, and tracheobronchial lymph node cells from both treatment groups produced similar levels of Th1 and Th2 cytokines in response to in vitro OVA stimulation. Moreover, it appears that TLR-4 and IFN-γ were not directly involved in the A. baumannii-induced suppression of airway eosinophilia. Our results suggest that A. baumannii inhibits allergic airway inflammation by direct suppression of local pulmonary Th2 cytokine responses to the allergen.
Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4+FoxP3+) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.
Mast cell numbers and allergen specific IgE are increased in the lungs of patients with allergic asthma and this can be reproduced in mouse models. The increased number of mast cells is likely due to recruitment of mast cell progenitors that mature in situ. We hypothesized that formation of IgE immune complexes in the lungs of sensitized mice increase the migration of mast cell progenitors to this organ. To study this, a model of allergic airway inflammation where mice were immunized with ovalbumin (OVA) in alum twice followed by three daily intranasal challenges of either OVA coupled to trinitrophenyl (TNP) alone or as immune complexes with IgE-anti-TNP, was used. Mast cell progenitors were quantified by a limiting dilution assay. IgE immune complex challenge of sensitized mice elicited three times more mast cell progenitors per lung than challenge with the same dose of antigen alone. This dose of antigen challenge alone did not increase the levels of mast cell progenitors compared to unchallenged mice. IgE immune complex challenge of sensitized mice also enhanced the frequency of mast cell progenitors per 106 mononuclear cells by 2.1-fold. The enhancement of lung mast cell progenitors by IgE immune complex challenge was lost in FcRγ deficient mice but not in CD23 deficient mice. Our data show that IgE immune complex challenge enhances the number of mast cell progenitors in the lung through activation of an Fc receptor associated with the FcRγ chain. This most likely takes place via activation of FcεRI, although activation via FcγRIV or a combination of the two receptors cannot be excluded. IgE immune complex-mediated enhancement of lung MCp numbers is a new reason to target IgE in therapies against allergic asthma.
Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined.
We sought to understand the cellular and molecular basis for suppression of established, IL-17-dependent allergic airways disease.
Mice were sensitized by airway instillations of ovalbumin (OVA) together with low levels of lipopolysaccharide. Leukocyte recruitment to the lung and AHR were assessed following daily challenges with aerosolized OVA. Flow cytometry and gene targeted mice were used to identify naturally-arising subsets of regulatory T cells (Tregs) and their cytokines required for the suppression of established allergic airway disease.
Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR declined, even though pulmonary levels of Th17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg subset expressing both Foxp3 and inducible co-stimulator (ICOS). These Tregs also expressed the regulatory cytokines, IL-10, TGF-beta and IL-35. Whereas IL-10 and TGF-beta were dispensable for suppression of airway hyperresponsiveness, IL-35 was required. Analysis of human ICOS+ Tregs revealed that they also selectively expressed IL-35.
IL-35 production by ICOS+ Tregs can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. The production of IL-35 by human ICOS+ Tregs suggests that targeting this pathway might be of therapeutic value for treating allergic asthma in humans.
Asthma; airway hyperresponsiveness; AHR; IL-17; Th17; Th2; IL-35; ICOS; ovalbumin
Although many studies show that pulmonary Surfactant Protein A (SP-A) functions in innate immunity, fewer studies have addressed its role in adaptive immunity and allergic hypersensitivity. We hypothesized that SP-A modulates the phenotype and prevalence of dendritic cells (DCs) and CD4+ T cells to inhibit Th2-associated inflammatory indices associated with allergen-induced inflammation. In an ovalbumin (OVA) model of allergic hypersensitivity, SP-A deficient (SP-A−/−) mice had greater eosinophilia, Th2-associated cytokine levels, and IgE levels compared to wild-type (WT) counterparts. Although both OVA-exposed groups had similar proportions of CD86+ DCs and Foxp3+ T regulatory cells, the SP-A−/− mice had elevated proportions of CD4+ activated (TA) and effector memory (TEM) T cells in their lungs compared to WT mice. Ex vivo recall stimulation of CD4+ T cell pools demonstrate that the cells from the SP-A−/− OVA mice had the greatest proliferative and IL-4 producing capacity, and this capability was attenuated with exogenous SP-A treatment. Additionally, tracking proliferation in vivo demonstrated that TA and TEM cells expand to the greatest extent in the lungs of SP-A−/− OVA mice. Taken together, our data suggest that SP-A influences the prevalence, types, and functions of CD4+ T cells in the lungs during allergic inflammation and that surfactant protein deficiency modifies the severity of inflammation in allergic hypersensitivity conditions like asthma.
Rodent; T cells; Allergy; Inflammation; Lung
Chronic helminth infections, such as schistosomes, are negatively associated with allergic disorders. Here, using B cell IL-10-deficient mice, Schistosoma mansoni-mediated protection against experimental ovalbumin-induced allergic airway inflammation (AAI) was shown to be specifically dependent on IL-10-producing B cells. To study the organs involved, we transferred B cells from lungs, mesenteric lymph nodes or spleen of OVA-infected mice to recipient OVA-sensitized mice, and showed that both lung and splenic B cells reduced AAI, but only splenic B cells in an IL-10-dependent manner. Although splenic B cell protection was accompanied by elevated levels of pulmonary FoxP3+ regulatory T cells, in vivo ablation of FoxP3+ T cells only moderately restored AAI, indicating an important role for the direct suppressory effect of regulatory B cells. Splenic marginal zone CD1d+ B cells proved to be the responsible splenic B cell subset as they produced high levels of IL-10 and induced FoxP3+ T cells in vitro. Indeed, transfer of CD1d+ MZ-depleted splenic B cells from infected mice restored AAI. Markedly, we found a similarly elevated population of CD1dhi B cells in peripheral blood of Schistosoma haematobium-infected Gabonese children compared to uninfected children and these cells produced elevated levels of IL-10. Importantly, the number of IL-10-producing CD1dhi B cells was reduced after anti-schistosome treatment. This study points out that in both mice and men schistosomes have the capacity to drive the development of IL-10-producing regulatory CD1dhi B cells and furthermore, these are instrumental in reducing experimental allergic inflammation in mice.
Apolipoprotein A-I (apoA-I) is a key component of high-density lipoproteins that mediates reverse cholesterol transport from cells and reduces vascular inflammation. We investigated whether endogenous apoA-I modulates ovalbumin (OVA)–induced airway inflammation in mice. We found that apoA-I expression was significantly reduced in the lungs of OVA-challenged, compared with saline-challenged, wild-type (WT) mice. Next, to investigate the role of endogenous apoA-I in the pathogenesis of OVA-induced airway inflammation, WT and apoA-I−/− mice were sensitized by intraperitoneal injections of OVA and aluminum hydroxide, followed by multiple nasal OVA challenges for 4 weeks. OVA-challenged apoA-I−/− mice exhibited a phenotype of increased airway neutrophils compared with WT mice, which could be rescued by an administration of a 5A apoA-I mimetic peptide. Multiple pathways promoted neutrophilic inflammation in OVA-challenged apoA-I−/− mice, including the up-regulated expression of (1) proinflammatory cytokines (IL-17A and TNF-α), (2) CXC chemokines (CXCL5), (3) vascular adhesion molecules (i.e., vascular cell adhesion molecule–1), and (4) granulocyte colony–stimulating factors (G-CSF). Because concentrations of G-CSF in bronchoalveolar lavage fluid (BALF) were markedly increased in OVA-challenged apoA-I−/− mice, we hypothesized that enhanced G-CSF expression may represent the predominant pathway mediating increased neutrophilic inflammation. This was confirmed by the intranasal administration of a neutralizing anti–G-CSF antibody, which significantly reduced BALF neutrophilia by 72% in OVA-challenged apoA-I−/− mice, compared with mice that received a control antibody. We conclude that endogenous apoA-I negatively regulates OVA-induced neutrophilic airway inflammation, primarily via a G-CSF–dependent mechanism. Furthermore, these findings suggest that apoA-I may play an important role in modulating the severity of neutrophilic airway inflammation in asthma.
airway inflammation; ovalbumin; neutrophil; apolipoprotein A-I; G-CSF
Allergen-induced imbalance of specific T regulatory (Treg) cells and T helper 2 cells plays a decisive role in the development of immune response against allergens.
To evaluate effects and potential mechanisms of DNA vaccine containing ovalbumin (OVA) and Fc fusion on allergic airway inflammation.
Bronchoalveolar lavage (BAL) levels of inflammatory mediators and leukocyte infiltration, expression of CD11c+CD80+ and CD11c+CD86+ co-stimulatory molecules in spleen dendritic cells (DCs), circulating CD4+ and CD8+ T cells, Foxp3+ in spleen CD4+ T cells and spleen CD4+ T cells were measured in OVA-sensitized and challenged animals pretreated with pcDNA, OVA-pcDNA, Fc-pcDNA, and OVA-Fc-pcDNA.
OVA-Sensitized and challenged mice developed airway inflammation and Th2 responses, and decreased the proliferation of peripheral CD4+and CD8+ T cells and the number of spleen Foxp3+ Treg. Those changes with increased INF-γ production and reduced OVA-specific IgE production were protected by the pretreatment with OVA-Fc-pcDNA.
DNA vaccine encoding both Fc and OVA showed more effective than DNA vaccine encoding Fc or OVA alone, through the balance of DCs and Treg.
Deficient suppression of T cell responses to allergen by CD4+CD25+ regulatory T cells has been observed in patients with allergic disease. Our current experiments used a mouse model of airway inflammation to examine the suppressive activity of allergen-specific CD4+CD25+ T cells in vivo. Transfer of ovalbumin (OVA) peptide–specific CD4+CD25+ T cells to OVA-sensitized mice reduced airway hyperreactivity (AHR), recruitment of eosinophils, and T helper type 2 (Th2) cytokine expression in the lung after allergen challenge. This suppression was dependent on interleukin (IL) 10 because increased lung expression of IL-10 was detected after transfer of CD4+CD25+ T cells, and regulation was reversed by anti–IL-10R antibody. However, suppression of AHR, airway inflammation, and increased expression of IL-10 were still observed when CD4+CD25+ T cells from IL-10 gene–deficient mice were transferred. Intracellular cytokine staining confirmed that transfer of CD4+CD25+ T cells induced IL-10 expression in recipient CD4+ T cells, but no increase in IL-10 expression was detected in airway macrophages, dendritic cells, or B cells. These data suggest that CD4+CD25+ T cells can suppress the Th2 cell–driven response to allergen in vivo by an IL-10–dependent mechanism but that IL-10 production by the regulatory T cells themselves is not required for such suppression.
Deficient suppression of T cell responses to allergen by CD4+CD25+ regulatory T cells has been observed in patients with allergic disease. Our current experiments used a mouse model of airway inflammation to examine the suppressive activity of allergen-specific CD4+CD25+ T cells in vivo. Transfer of ovalbumin (OVA) peptide-specific CD4+CD25+ T cells to OVA-sensitized mice reduced airway hyperreactivity (AHR), recruitment of eosinophils, and T helper type 2 (Th2) cytokine expression in the lung after allergen challenge. This suppression was dependent on interleukin (IL) 10 because increased lung expression of IL-10 was detected after transfer of CD4+CD25+ T cells, and regulation was reversed by anti-IL-10R antibody. However, suppression of AHR, airway inflammation, and increased expression of IL-10 were still observed when CD4+CD25+ T cells from IL-10 gene-deficient mice were transferred. Intracellular cytokine staining confirmed that transfer of CD4+CD25+ T cells induced IL-10 expression in recipient CD4+ T cells, but no increase in IL-10 expression was detected in airway macrophages, dendritic cells, or B cells. These data suggest that CD4+CD25+ T cells can suppress the Th2 cell-driven response to allergen in vivo by an IL-10-dependent mechanism but that IL-10 production by the regulatory T cells themselves is not required for such suppression.
Allergen-specific immunotherapy is a potential treatment for allergic diseases. We constructed an allergen–cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-encoding DNA vaccine, administered it directly to antigen-presenting cells (APCs), and investigated its ability and mechanisms to ameliorate allergic airway inflammation in an asthmatic mouse model. An allergen-CTLA-4 DNA plasmid (OVA-CTLA-4-pcDNA3.1) encoding an ovalbumin (OVA) and the mouse CTLA-4 extracellular domain was constructed and transfected into COS-7 cells to obtain the fusion protein OVA-CTLA-4, which was able to bind the B7 ligand on dendritic cells (DCs), and induced CD25+ Foxp3+ regulatory T (Treg) cells by the coculture of naive CD4+ T cells with DCs in vitro. In an animal study, BALB/c mice were sensitized and challenged with OVA to establish the asthmatic model. Vaccination with a high dose of OVA-CTLA-4-pcDNA3.1 significantly decreased interleukin-4 (IL-4) and IL-5 levels and eosinophil counts and prevented OVA-induced reduction of the gamma interferon level in the bronchoalveolar lavage fluid. In addition, these mice suffered less severe airway inflammation and had lower levels of OVA-specific IgE and IgG1 titers in serum. Also, high-dose OVA-CTLA-4-pcDNA3.1 vaccination inhibited the development of airway hyperreactivity and prevented OVA-induced reduction of the percentages of Foxp3+ Treg cells in the spleen. Our results indicate that a high dose of allergen-CTLA-4-encoding DNA vaccine was more effective in preventing an allergen-induced Th2-skewed immune response through the induction of Treg cells and may be a new alternative therapy for asthma.
The continual rise of asthma in industrialised countries stands in strong contrast to the situation in developing lands. According to the modified Hygiene Hypothesis, helminths play a major role in suppressing bystander immune responses to allergens, and both epidemiological and experimental studies suggest that the tropical parasitic trematode Schistosoma mansoni elicits such effects. The focus of this study was to investigate which developmental stages of schistosome infection confer suppression of allergic airway inflammation (AAI) using ovalbumin (OVA) as a model allergen. Moreover, we assessed the functional role and localization of infection-induced CD4+Foxp3+ regulatory T cells (Treg) in mediating such suppressive effects. Therefore, AAI was elicited using OVA/adjuvant sensitizations with subsequent OVA aerosolic challenge and was induced during various stages of infection, as well as after successful anti-helminthic treatment with praziquantel. The role of Treg was determined by specifically depleting Treg in a genetically modified mouse model (DEREG) during schistosome infection. Alterations in AAI were determined by cell infiltration levels into the bronchial system, OVA-specific IgE and Th2 type responses, airway hyper-sensitivity and lung pathology. Our results demonstrate that schistosome infection leads to a suppression of OVA-induced AAI when mice are challenged during the patent phase of infection: production of eggs by fecund female worms. Moreover, this ameliorating effect does not persist after anti-helminthic treatment, and depletion of Treg reverts suppression, resulting in aggravated AAI responses. This is most likely due to a delayed reconstitution of Treg in infected-depleted animals which have strong ongoing immune responses. In summary, we conclude that schistosome-mediated suppression of AAI requires the presence of viable eggs and infection-driven Treg cells. These data provide evidence that helminth derived products could be incorporated into treatment strategies that specifically target suppression of immune responses in AAI by inducing Treg cells.
Infections with schistosomes, such as S. mansoni, S. japonicum and S. haematobium, are considered a major public health concern. Morbidity arises through granulomatous responses to eggs that become trapped in infected tissues. Interestingly, schistosomes belong to the group of helminths that have been shown to reduce allergy or autoimmunity. Indeed, the evidence provided by epidemiological surveys and experimental animal models has been so overwhelming that such helminths are now included in the Hygiene Hypothesis. However, since helminths provoke immunological responses that are similar to those seen in allergy (increased eosinophilia and IgE) it is suggested that additional mechanisms dampen such allergic responses. Helminth-induced regulatory T cells (Treg) are considered a component of these modulatory networks. Using an allergic airway inflammation model, we have elucidated that schistosome-mediated protection requires patency, that is, active egg production from fecund female worms. In addition, protection was shown to be mediated by infection-induced Treg. Interestingly, in endemic countries it is usually individuals with strong patent infections that show reduced allergic prevalence. Thus, further research into the immunomodulatory capacity of schistosome-egg derived factors may elucidate novel drug candidates or enhance treatment strategies to reduce allergic responses on the cellular level.
Th2-dominated inflammatory response in the airway is an integral component in the pathogenesis of allergic asthma. Accumulating evidence supports the notion that the phosphoinositide 3-kinase (PI3K) pathway is involved in the process. We previously reported that SHIP-1, a negative regulator of the PI3K pathway, is essential in maintaining lung immunohomeostasis, potentially through regulation of innate immune cells. However, the function of SHIP-1 in adaptive immune response in the lung has not been defined. We sought to determine the role of SHIP-1 in adaptive immunity in response to aeroallergen stimulation in the airway.
SHIP-1 knockout (SHIP-1−/−) mice on BALB/c background were immunized with ovalbumin (OVA) plus aluminum hydroxide, a strong Th2-inducing immunization, and challenged with OVA. Airway and lung inflammation, immunoglobulin response, Th2 cytokine production and lymphocyte response were analyzed and compared with wild type mice. Even though there was mild spontaneous inflammation in the lung at baseline, SHIP-1−/− mice showed altered responses, including less cell infiltration around the airways but more in the parenchyma, less mucus production, decreased Th2 cytokine production, and diminished serum OVA-specific IgE, IgG1, but not IgG2a. Naïve and OVA sensitized SHIP-1−/− T cells produced a lower amount of IL-4. In vitro differentiated SHIP-1−/− Th2 cells produced less IL-4 compared to wild type Th2 cells upon T cell receptor stimulation.
These findings indicate that, in contrast to its role as a negative regulator in the innate immune cells, SHIP-1 acts as a positive regulator in Th2 cells in the adaptive immune response to aeroallergen. Thus any potential manipulation of SHIP-1 activity should be adjusted according to the specific immune response.
It is well established that female sex hormones have a pivotal role in inflammation. For instance, our group has previously reported that estradiol has proinflammatory actions during allergic lung response in animal models. Based on these findings, we have decided to further investigate whether T regulatory cells are affected by female sex hormones absence after ovariectomy. We evaluated by flow cytometry the frequencies of CD4+Foxp3+ T regulatory cells (Tregs) in central and peripheral lymphoid organs, such as the thymus, spleen and lymph nodes. Moreover, we have also used the murine model of allergic lung inflammation a to evaluate how female sex hormones would affect the immune response in vivo. To address that, ovariectomized or sham operated female Balb/c mice were sensitized or not with ovalbumin 7 and 14 days later and subsequently challenged twice by aerosolized ovalbumin on day 21. Besides the frequency of CD4+Foxp3+ T regulatory cells, we also measured the cytokines IL-4, IL-5, IL-10, IL-13 and IL-17 in the bronchoalveolar lavage from lungs of ovalbumine challenged groups. Our results demonstrate that the absence of female sex hormones after ovariectomy is able to increase the frequency of Tregs in the periphery. As we did not observe differences in the thymus-derived natural occurring Tregs, our data may indicate expansion or conversion of peripheral adaptive Tregs. In accordance with Treg suppressive activity, ovariectomized and ovalbumine-sensitized and challenged animals had significantly reduced lung inflammation. This was observed after cytokine analysis of lung explants showing significant reduction of pro-inflammatory cytokines, such as IL-4, IL-5, IL-13 and IL-17, associated to increased amount of IL-10. In summary, our data clearly demonstrates that OVA sensitization 7 days after ovariectomy culminates in reduced lung inflammation, which may be directly correlated with the expansion of Tregs in the periphery and further higher IL-10 secretion in the lungs.
Chemokine receptor (CCR) 5 is expressed on dendritic cells, macrophages, CD8 cells, memory CD4 T cells, and stromal cells, and is frequently used as a marker of T helper type 1 cells. Interventions that abrogate CCR5 or interfere with its ligand binding have been shown to alter T helper type 2–induced inflammatory responses. The role of CCR5 on allergic airway responses is not defined. CCR5-deficient (CCR5−/−) and wild-type (CCR5+/+) mice were sensitized and challenged with ovalbumin (OVA) and allergic airway responses were monitored 48 hours after the last OVA challenge. Cytokine levels in lung cell culture supernatants were also assessed. CCR5−/− mice showed significantly lower airway hyperresponsiveness (AHR) and lower numbers of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid compared with CCR5+/+ mice after sensitization and challenge. The levels of IL-4 and IL-13 in BAL fluid of CCR5−/− mice were lower than in CCR5+/+ mice. Decreased numbers of lung T cells were also detected in CCR5−/− mice after sensitization and challenge. Transfer of OVA-sensitized T cells from CCR5+/+, but not transfer of CCR5−/− cells, into CCR5−/− mice restored AHR and numbers of eosinophils in BAL fluid after OVA challenge. Accordingly, the numbers of airway-infiltrating donor T cells were significantly higher in the recipients of CCR5+/+ T cells. Taken together, these data suggest that CCR5 plays a pivotal role in allergen-induced AHR and airway inflammation, and that CCR5 expression on T cells is essential to the accumulation of these cells in the airways.
rodent; T cells; cytokines; chemokines; lung
As an E3 ubiquitin ligase and a molecular adaptor, Cbl-b controls the activation threshold of the antigen receptor and negatively regulates CD28 co-stimulation, functioning as an intrinsic mediator of T cell anergy that maintains tolerance. However, the role of Cbl-b in the airway immune response to aeroallergens is unclear.
To determine the contribution of Cbl-b in tolerance to aeroallergens, we examined ovalbumin (OVA)-induced lung inflammation in Cbl-b deficient mice.
Cbl-b-/- mice and wildtype (WT) C57BL/6 mice were sensitized and challenged with OVA intranasally, a procedure normally tolerated by WT mice. We analyzed lung histology, BAL total cell counts and differential, cytokines and chemokines in the airway, and cytokine response by lymphocytes after re-stimulation by OVA antigen.
Compared with WT mice, OVA challenged Cbl-b-/- mice showed significantly increased neutrophilic and eosinophilic infiltration in the lung and mucus hyperplasia. The serum levels of IgG2a and IgG1, but not IgE, were increased. The levels of inflammatory mediators IFN-γ, IL-10, IL-12, IL-13, IP-10, MCP-1, MIP-1α, Eotaxin, and RANTES, but not IL-17A or IL-6, were elevated in the airway of Cbl-b-/- mice. Lymphocytes from Cbl-b-/-mice released increased amount of IFN-γ, IL-10, IL-13, and IP-10 in response to OVA re-stimulation. However, no significant changes were noted in the CD4+CD25+ Treg cell populations in the lung tissues after OVA stimulation and there was no difference between WT and Cbl-b-/- mice.
These results demonstrate that Cbl-b deficiency leads to a breakdown of tolerance to OVA allergen in the murine airways, probably through increased activation of T effector cells, indicating that Cbl-b is a critical factor in maintaining lung homeostasis upon environmental exposure to aeroallergens.
Cbl-b; Ubiquitin E3 Ligase; Aeroallergen; Allergic inflammation; Asthma
Adiponectin is an adipose derived hormone that declines in obesity. We have previously shown that exogenous administration of adiponectin reduces allergic airways responses in mice. T-cadherin (T-cad; Cdh13) is a binding protein for the high molecular weight isoforms of adiponectin. To determine whether the beneficial effects of adiponectin on allergic airways responses require T-cad, we sensitized wildtype (WT), T-cadherin deficient (T-cad−/−) and adiponectin and T-cad bideficient mice to ovalbumin (OVA) and challenged the mice with aerosolized OVA or PBS. Compared to WT, T-cad−/− mice were protected against OVA-induced airway hyperresponsiveness, increases in BAL inflammatory cells, and induction of IL-13, IL-17, and eotaxin expression. Histological analysis of the lungs of OVA-challenged T-cad−/− versus WT mice indicated reduced inflammation around the airways, and reduced mucous cell hyperplasia. Combined adiponectin and T-cad deficiency reversed the effects of T-cad deficiency alone, indicating that the observed effects of T-cad deficiency require adiponectin. Compared to WT, serum adiponectin was markedly increased in T-cad−/− mice, likely because adiponectin that is normally sequestered by endothelial T-cad remains free in the circulation. In conclusion, T-cad does not mediate the protective effects of adiponectin. Instead, mice lacking T-cad have reduced allergic airways disease, likely because elevated serum adiponectin levels act on other adiponectin signaling pathways.
Rationale: Although there have been numerous studies on the development of allergen-induced inflammation, the mechanisms leading to resolution of inflammation remain poorly understood. This represents an important consideration because failure to resolve allergen driven inflammation potentially leads to irreversible airway remodeling, characteristic of chronic asthma.
Objectives: We investigated the resolution of allergic inflammation and identified the factors responsible.
Methods: BALB/c and C57BL/6 mice were sensitized to ovalbumin and challenged through the airways to induce allergic inflammation. Mice were analyzed at 24 hours and 7 days after the final challenge.
Measurements and Main Results: Airway hyperreactivity (AHR) and increased mucus production were present 7 days after the cessation of allergen challenge in BALB/c mice. Persisting AHR correlated with the continued presence of Th2 cells but not eosinophils in the lungs. The role of Th2 cells in maintaining AHR was confirmed using blocking antibodies against T1/ST2, IL-4, and IL-13 during the resolution period. Moreover, AHR in the “Th1 type” C57BL/6 mouse strain was resolved 1 week after allergen challenge, concomitant with clearance of Th2 cells from the lung. Expression of the T1/ST2 ligand, IL-33, also correlated with maintenance of AHR.
Conclusions: We have used blockade of Th2 function and strain differences to show for the first time that resolution of allergic inflammation and AHR may be dependent on the T1/ST2-IL-33 pathway and the presence of Th2 cells, suggesting they are necessary not only for the development of an allergic response but also for its maintenance.
Th2 cells; IL-13; IL-4
We recently reported that various environmental estrogens induce mast cell degranulation and enhance IgE-mediated release of allergic mediators in vitro.
We hypothesized that environmental estrogens would enhance allergic sensitization as well as bronchial inflammation and responsiveness. To test this hypothesis, we exposed fetal and neonatal mice to the common environmental estrogen bisphenol A (BPA) via maternal loading and assessed the pups’ response to allergic sensitization and bronchial challenge.
Female BALB/c mice received 10 μg/mL BPA in their drinking water from 1 week before impregnation to the end of the study. Neonatal mice were given a single 5 μg intraperitoneal dose of ovalbumin (OVA) with aluminum hydroxide on postnatal day 4 and 3% OVA by nebulization for 10 min on days 13, 14, and 15. Forty-eight hours after the last nebulization, we assessed serum IgE antibodies to OVA by enzyme-linked immunosorbent assay (ELISA) and airway inflammation and hyperresponsiveness by enumerating eosinophils in bronchoalveolar lavage fluid, whole-body barometric plethysmography, and a forced oscillation technique.
Neonates from BPA-exposed mothers responded to this “suboptimal” sensitization with higher serum IgE anti-OVA concentrations compared with those from unexposed mothers (p < 0.05), and eosinophilic inflammation in their airways was significantly greater. Airway responsiveness of the OVA-sensitized neonates from BPA-treated mothers was enhanced compared with those from unexposed mothers (p < 0.05).
Perinatal exposure to BPA enhances allergic sensitization and bronchial inflammation and responsiveness in a susceptible animal model of asthma.
airway hyperresponsiveness; asthma; bisphenol A; environmental estrogen; eosinophilia; experimental asthma; IgE; maternal exposure; perinatal sensitization
An increasing prevalence of allergic diseases, such as atopic dermatitis, allergic rhinitis and bronchial asthma, has been noted worldwide. Allergic asthma strongly correlates with airway inflammation caused by the unregulated production of cytokines secreted by allergen-specific type-2 T helper (Th2) cells. This study aims to explore the therapeutic effect of the airway gene transfer of IL-12, IL-10 and TGF-β on airway inflammation in a mouse model of allergic asthma.
BALB/c mice were sensitized to ovalbumin (OVA) by intraperitoneal injections with OVA and challenged by nebulized OVA. Different cytokine gene plasmids or non-coding vector plasmids were instilled daily into the trachea up to one day before the inhalatory OVA challenge phase.
Intratracheal administration of IL-10, IL-12 or TGF-β can efficiently inhibit antigen-induced airway hyper-responsiveness and is able to largely significantly lower the number of eosinophils and neutrophils in bronchoalveolar lavage fluid of ovalbumin (OVA) sensitized and challenged mice during the effector phase. Furthermore, the effect of IL-10 plasmids is more remarkable than any other cytokine gene plasmid. On the other hand, local administration of IL-4 gene plasmids before antigen challenge can induce severe airway hyper-responsiveness (AHR) and airway eosinophilia.
Our data demonstrated that anti- inflammatory cytokines, particularly IL-10, have the therapeutic potential for the alleviation of airway inflammation in murine model of asthma.
Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs).
Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling.
In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.
Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.
Mice sensitized to ovalbumin develop allergic airway disease (AAD) with short-term aerosol challenge; however, airway inflammation resolves with long-term aerosol challenge, referred to as local inhalational tolerance (LIT).
We sought to determine if resolution of airway inflammation correlated with increases in lymphocyte subsets in local lung compartments, including putative regulatory T cells.
At the AAD stage, total numbers of T and B lymphocytes in bronchoalveolar lavage (BAL) were significantly increased above controls; however, at LIT, T and B lymphocytes were significantly reduced compared to AAD. In the lung tissue, the only alteration was a significant increase in CD4+ CD25+ T cells at AAD. In the hilar lymph node (HLN), CD4+ and CD4+ CD25+ T cells were significantly increased at AAD and LIT. In addition, CD8+ T cells were significantly elevated in the HLN at LIT, and CD19+ B cells were significantly increased at AAD. Adoptive transfer of HLN lymphocytes to lymphopenic mice confirmed that AAD lymphocytes could induce airway inflammation in response to aerosol challenge, whereas LIT lymphocytes were unable to do so. Depletion of CD4+ CD25+ T cells in vivo resulted in exacerbation of inflammation at AAD and LIT. CD4+ CD25+ T cells in the HLN also displayed suppressive activity in vitro. Additionally, T cells expressing Foxp3 were increased in the BAL and HLN during LIT.
These results indicate that lymphocytes with regulatory functions are increased and sustained in local lung compartments at LIT and that their appearance correlates with the resolution of lung inflammation.
Adoptive transfer; Allergic airway inflammation; Foxp3; Lymphocytes; Mouse; Ovalbumin; Regulatory T cells
Pim kinases are a family of serine/threonine kinases whose activity can be induced by cytokines involved in allergy and asthma. These kinases play a role in cell survival and proliferation, but have not been examined, to the best of our knowledge, in the development of allergic disease. This study sought to determine the role of Pim1 kinase in the development of allergic airway responses. Mice were sensitized and challenged with antigen (primary challenge), or were sensitized, challenged, and rechallenged with allergen in a secondary model. To assess the role of Pim1 kinase, a small molecule inhibitor was administered orally after sensitization and during the challenge phase. Airway responsiveness to inhaled methacholine, airway and lung inflammation, cell composition, and cytokine concentrations were assessed. Lung Pim1 kinase concentrations were increased after ovalbumin sensitization and challenge. In the primary allergen challenge model, treatment with the Pim1 kinase inhibitor after sensitization and during airway challenges prevented the development of airway hyperresponsiveness, eosinophilic airway inflammation, and goblet cell metaplasia, and increased Th2 cytokine concentrations in bronchoalveolar fluid in a dose-dependent manner. These effects were also demonstrated after a secondary allergen challenge, where lung allergic disease was established before treatment. After treatment with the inhibitor, a significant reduction was evident in the number of CD4+ and CD8+ T cells and concentrations of cytokines in the airways. The inhibition of Pim1 kinase was effective in preventing the development of airway hyperresponsiveness, airway inflammation, and cytokine production in allergen-sensitized and allergen-challenged mice. These data identify the important role of Pim1 kinase in the full development of allergen-induced airway responses.
airway hyperresponsiveness; inflammation; Pim1 kinase; T cells
CD8+ T cells participate in airway hyperresponsiveness (AHR) and allergic pulmonary inflammation that are characteristics of asthma. CXCL10 by binding to CXCR3 expressed preferentially on activated CD8+ T cells, attracts T cells homing to the lung. We studied the contribution and limitation of CXCR3 to AHR and airway inflammation induced by ovalbumin (OVA) using CXCR3 knockout (KO) mice.
Mice were sensitized and challenged with OVA. Lung histopathological changes, AHR, cellular composition and levels of inflammatory mediators in bronchoalveolar lavage (BAL) fluid, and lungs at mRNA and protein levels, were compared between CXCR3 KO mice and wild type (WT) mice.
Compared with the WT controls, CXCR3 KO mice showed less OVA-induced infiltration of inflammatory cells around airways and vessels, and less mucus production. CXCR3 KO mice failed to develop significant AHR. They also demonstrated significantly fewer CD8+ T and CD4+ T cells in BAL fluid, lower levels of TNFα and IL-4 in lung tissue measured by real-time RT-PCR and in BAL fluid by ELISA, with significant elevation of IFNγ mRNA and protein expression levels.
We conclude that CXCR3 is crucial for AHR and airway inflammation by promoting recruitment of more CD8+ T cells, as well as CD4+ T cells, and initiating release of proinflammatory mediators following OVA sensitization and challenge. CXCR3 may represent a novel therapeutic target for asthma.
chemokine receptor; CXCR3; CD8+ T lymphocyte; airway inflammation; airway hyperresponsiveness
Active smoking increases asthma severity and is related to diminished treatment efficacy. Animal models in which inhalation of both allergen and mainstream cigarette smoke are combined can help us to understand the complex interaction between both agents. We have recently shown that, in allergic mice, the airway inflammation can be cleared by repeated allergen challenge, resulting in the establishment of a state of inhalational tolerance.
In this study, we assessed in vivo the impact of cigarette smoke on the efficacy and time course of this form of tolerance induction. We exposed sensitized mice to concurrent mainstream cigarette smoke and allergen (Ovalbumin- OVA) and measured the airway inflammation at different time points.
We first confirmed that aerosolized OVA administered for a prolonged time period (4–8 weeks) resulted in the establishment of tolerance. Concurrent OVA and smoke exposure for 2 weeks showed that tobacco smoke enhanced the Th-2 driven airway inflammation in the acute phase. In addition, the induction of the tolerance by repeated inhalational OVA challenge was delayed significantly by the tobacco smoke, since 4 weeks of concurrent exposure resulted in a more persistent eosinophilic airway inflammation, paralleled by a more mature dendritic cell phenotype. However, smoke exposure could not prevent the establishment of tolerance after 8 weeks of antigen exposure as shown by both histopathology (disappearance of the Th-2 driven inflammation) and by in vivo functional experiments. In these tolerized mice, some of the inflammatory responses to the smoke were even attenuated.
Cigarette smoke enhances acute allergic inflammation and delays, but does not abrogate the development of tolerance due to prolonged challenge with inhaled antigen in experimental asthma.