Treatment of experimental asthma by long-term gene therapy directed against IL-4 and IL-13

The clinical manifestations of allergic asthma are believed to result from a dysregulated, T helper 2 lymphocyte (Th2)-biased response to antigen. Although asthma symptoms can be controlled acutely, there is a need for a therapy that will address the underlying immune dysfunction and provide continuous control of chronic airway inflammation. The Th2-type cytokines, IL-13 and IL-4, have been demonstrated to play a crucial role in asthma pathogenesis and their selective neutralization results in the alleviation of asthmatic symptoms in mouse models. The activity of both of these cytokines can be inhibited by a mutant IL-4 protein, IL-4 receptor antagonist (IL-4RA), and thus, continual IL-4RA therapy might be beneficial in treatment of chronic asthma. To explore the potential utility of long-term gene therapy for the treatment of asthma we used a recombinant adeno-associated virus (AAV) vector to deliver and provide sustained expression of IL-4RA in vivo. We show that AAV-mediated delivery of IL-4RA to the airways of mice reduces airway hyperresponsiveness (AHR) and airway eosinophilia triggered by either IL-13 or IL-4. Furthermore, AAV-delivered IL-4RA, expressed either systemically or in the airways of mice following allergen sensitization, significantly inhibited development of airway eosinophilia and mucus production and reduced the levels of asthma-associated Th2 cytokines and AHR in the experimental mouse model of allergic asthma. Thus, gene therapy can be a potential therapeutic option to treat and control chronic airway inflammation and asthmatic symptoms.     

CD4+CD25+ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function

The role of natural CD4+CD25+ regulatory T (T reg) cells in the control of allergic asthma remains poorly understood. We explore the impact of T reg cell depletion on the allergic response in mice susceptible (A/J) or comparatively resistant (C3H) to the development of allergen-induced airway hyperresponsiveness (AHR). In C3H mice, anti-CD25-mediated T reg cell depletion before house dust mite treatment increased several features of the allergic diathesis (AHR, eosinophilia, and IgE), which was concomitant with elevated T helper type 2 (Th2) cytokine production. In similarly T reg cell-depleted A/J mice, we observed a moderate increase in airway eosinophilia but no effects on AHR, IgE levels, or Th2 cytokine synthesis. As our experiments suggested that T reg cell depletion in C3H mice before sensitization was sufficient to enhance the allergic phenotype, we characterized dendritic cells (DCs) in T reg cell-depleted C3H mice. T reg cell-depleted mice had increased numbers of pulmonary myeloid DCs with elevated expression of major histocompatibility complex class II, CD80, and CD86. Moreover, DCs from T reg cell-depleted mice demonstrated an increased capacity to stimulate T cell proliferation and Th2 cytokine production, which was concomitant with reduced IL-12 expression. These data suggest that resistance to allergen-driven AHR is mediated in part by CD4+CD25+ T reg cell suppression of DC activation and that the absence of this regulatory pathway contributes to susceptibility.     

Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice

Allergic asthma is characterized by airway hyperresponsiveness and pulmonary eosinophilia, and may be mediated by T helper (Th) lymphocytes expressing a Th2 cytokine pattern. Interleukin (IL) 12 suppresses the expression of Th2 cytokines and their associated responses, including eosinophilia, serum immunoglobulin E, and mucosal mastocytosis. We have previously shown in a murine model that antigen- induced increases in airway hyperresponsiveness and pulmonary eosinophilia are CD4+ T cell dependent. We used this model to determine the ability of IL-12 to prevent antigen-induced increases in airway hyperresponsiveness, bronchoalveolar lavage (BAL) eosinophils, and lung Th2 cytokine expression. Sensitized A/J mice developed airway hyperresponsiveness and increased numbers of BAL eosinophils and other inflammatory cells after single or repeated intratracheal challenges with sheep red blood cell antigen. Pulmonary mRNA and protein levels of the Th2 cytokines IL-4 and IL-5 were increased after antigen challenge. Administration of IL-12 (1 microgram/d x 5 d) at the time of a single antigen challenge abolished the airway hyperresponsiveness and pulmonary eosinophilia and promoted an increase in interferon (IFN) gamma and decreases in IL-4 and IL-5 expression. The effects of IL-12 were partially dependent on IFN-gamma, because concurrent treatment with IL-12 and anti-IFN-gamma monoclonal antibody partially reversed the inhibition of airway hyperresponsiveness and eosinophilia by IL-12. Treatment of mice with IL-12 at the time of a second antigen challenge also prevented airway hyperresponsiveness and significantly reduced numbers of BAL inflammatory cells, reflecting the ability of IL-12 to inhibit responses associated with ongoing antigen-induced pulmonary inflammation. These data show that antigen-induced airway hyperresponsiveness and inflammation can be blocked by IL-12, which suppresses Th2 cytokine expression. Local administration of IL-12 may provide a novel immunotherapy for the treatment of pulmonary allergic disorders such as atopic asthma.     

Lentiviral-mediated interleukin-4 and interleukin-13 RNA interference decrease airway inflammation and hyperresponsiveness

Interleukin (IL)-4 and IL-13 are two key cytokines released from activated T helper type 2 (Th2) cells and strongly associated with asthma and allergic disease. We applied silencing of the IL-4 and IL-13 gene expression by RNA interference delivered by a lentiviral vector to evaluate the therapeutic role of IL-4 and IL13 short hairpin RNAs (shRNAs) in a murine model of asthma. Mice were sensitized with ovalbumin (OVA), and one treatment of IL-4 and IL-13 shRNA lentiviral vector (Lenti-si-IL-4 and Lenti-si-IL-13) was instilled intratracheally 48?hr before challenge. After three challenges of OVA antigen, mice were assessed for airway inflammation and hyperresponsiveness. With infection of Lenti-si-IL-4 and Lenti-si-IL-13 in EL-4 cells, both RNA and protein expressions of IL-4 and IL-13 were obviously abrogated. Furthermore, intratracheal instillation of Lenti-si-IL-4 and Lenti-si-IL-13 in OVA-immunized mice resulted in a strong inhibition of local IL-4 and IL-13 cytokine release. Treatment with Lenti-si-IL-4 and Lenti-si-IL-13 successfully alleviated OVA-induced airway eosinophilia and Th2 cell cytokine release. Finally, to determine airway hyperresponsiveness by enhanced pause and pulmonary resistance in noninvasive and invasive body plethysmography, we found that administration of Lenti-si-IL-4 and Lenti-si-IL-13 markedly decreased airway hyperresponsiveness in OVA-immunized mice. These results suggest that inhibition of IL-4 and IL-13 gene expression by shRNA lentiviral vector markedly inhibits antigen-induced airway inflammation and hyperresponsiveness in mice.     

Construction of single-chain interleukin-12 DNA plasmid to treat airway hyperresponsiveness in an animal model of asthma.

Allergic asthma is strongly associated with the airway inflammation caused by the dysregulated production of cytokines secreted by the allergen-specific type-2 T helper (Th2) cells. Interleukin (IL)-12 is a heterodimeric cytokine, which strongly promotes the differentiation of naive CD4(+) T cells to the type-1 T helper (Th1) phenotype and suppresses the expression of Th2 cytokines. Therefore, immunotherapy with IL-12 has been suggested as a possible therapy for asthma. In previous studies, we developed a murine model of airway inflammation based on the purified, house dust-mite allergen Der p 1 (Dermatophagodies pteronyssinus) as a clinically relevant allergen. We hypothesized that the expression of IL-12 in the airway may represent an effective therapy for allergic airway diseases. In this study, we investigate whether the local transfer of the IL-12 gene to respiratory tissues modifies allergic inflammation and airway hyper-responsiveness (AHR) in our disease model. To enhance the in vivo delivery of the IL-12 gene, we expressed the murine single-chain IL-12 protein from a nonviral vector to which the two IL-12 subunits (p35 and p40) were linked by a 14- to 18-amino-acid linker. One of these single-chain IL-12s, containing an 18 amino-acid polypeptide linker, was stably expressed and had a high level of biological activity comparable to that of native IL-12 in vitro. In mice with Der p 1-induced asthma, the local administration of this IL-12 fusion gene into the lungs significantly prevented the development of AHR, abrogated airway eosinophilia, and inhibited type-2 cytokine production. These findings indicate that the local transfer of the single-chain IL-12 gene is effective in modulating pulmonary allergic responses and may be a convenient method for future applications of DNA vaccination.     

Blockade of CD49d (alpha4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma.

Immunized mice after inhalation of specific antigen have the following characteristic features of human asthma: airway eosinophilia, mucus and Th2 cytokine release, and hyperresponsiveness to methacholine. A model of late-phase allergic pulmonary inflammation in ovalbumin-sensitized mice was used to address the role of the alpha4 integrin (CD49d) in mediating the airway inflammation and hyperresponsiveness. Local, intrapulmonary blockade of CD49d by intranasal administration of CD49d mAb inhibited all signs of lung inflammation, IL-4 and IL-5 release, and hyperresponsiveness to methacholine. In contrast, CD49d blockade on circulating leukocytes by intraperitoneal CD49d mAb treatment only prevented the airway eosinophilia. In this asthma model, a CD49d-positive intrapulmonary leukocyte distinct from the eosinophil is the key effector cell of allergen-induced pulmonary inflammation and hyperresponsiveness.     

Adenovirus expressing Fas ligand gene decreases airway hyper-responsiveness and eosinophilia in a murine model of asthma

Allergic asthma is characterized by airway hyper-responsiveness (AHR) and cellular infiltration of the airway with predominantly eosinophils and Th2 cells. The normal resolution of inflammation in the lung occurs through the regulated removal of unneeded cells by Fas-Fas ligand-mediated apoptosis. Fas ligand (FasL) is a member of the tumor necrosis factor family, and when bound to Fas, it induces apoptosis of the cells. To examine the effect of the FasL gene on airway inflammation and immune effector cells in allergic asthma, recombinant adenovirus expressing murine FasL (Ad-FasL) was delivered intratracheally into ovalbumin (OVA)-immunized mice. We found that a single administration of Ad-FasL in OVA-immunized mice significantly alleviated AHR and eosinophilia by inducing the apoptosis of eosinophils and/or reducing eosinophil attractant factors, such as IL-5 and eotaxin levels. The number of infiltrated lymphocytes and Th2 cytokines, including IL-5 and IL-13, decreased in OVA-immunized mice by administration of Ad-FasL. KC and TNF-alpha production also decreased in Ad-FasL-treated OVA-immunized mice. These findings indicated that the administration of Ad-FasL to OVA-sensitized mice significantly suppressed pulmonary allergic responses. Although more studies are needed, these results suggested that Ad-FasL might be applied as an alternative therapy for allergic asthma.     

CCR3 is essential for skin eosinophilia and airway hyperresponsiveness in a murine model of allergic skin inflammation

The CC chemokine receptor 3 (CCR3) is expressed by eosinophils, mast cells, and Th2 cells. We used CCR3(-/-) mice to assess the role of CCR3 in a murine model of allergic skin inflammation induced by repeated epicutaneous sensitization with ovalbumin (OVA), and characterized by eosinophil skin infiltration, local expression of Th2 cytokines, and airway hyperresponsiveness (AHR) to inhaled antigen. Eosinophils and the eosinophil product major basic protein were absent from the skin of sham and OVA-sensitized CCR3(-/-) mice. Mast cell numbers and expression of IL-4 mRNA were normal in skin of CCR3(-/-) mice, suggesting that CCR3 is not important for infiltration of the skin by mast cells and Th2 cells. CCR3(-/-) mice produced normal levels of OVA-specific IgE, and their splenocytes secreted normal amounts of IL-4 and IL-5 following in vitro stimulation with OVA, indicating effective generation of systemic Th2 helper responses. Recruitment of eosinophils to lung parenchyma and bronchoalveolar lavage (BAL) fluid was severely impaired in CCR3(-/-) mice, which failed to develop AHR to methacholine following antigen inhalation. These results suggest that CCR3 plays an essential role in eosinophil recruitment to the skin and the lung and in the development of AHR.     

Critical role of IFN-alpha/beta and IFN-gamma in the regulation of airway inflammation

Immune balance, which is controlled by IFN-gamma-producing Th1 cells and IL-4-producing Th2 cells, plays a critical role in the regulation of airway inflammation. We have demonstrated that Th1 cells induced neutrophilia, airway hyperresponsiveness (AHR) but not mucus hypersecretion, while Th2 cells induced eosinophilia, AHR and mucus hypersecretion. Here, we indicated that IFN-gamma produced by Th1 cells accelerated neutrophilia and AHR but inhibited eosinophilia and mucus hypersecretion. We also demonstrated an important role of type 1 IFN-alpha/beta during inhibition of Th2-dependent airway inflammation by TLR-9-ligand, CpG-ODN. CpG-ODN-induced IFN-alpha/beta partially appeared to act against Th2 cells to inhibit the production of IL-4 and IL-13, which are key cytokines to regulate the activation and migration of Th2 cells in the lung.     

A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma

Complement component 5 (C5) has been described as either promoting or protecting against airway hyperresponsiveness (AHR) in experimental allergic asthma, suggesting pleomorphic effects of C5. Here we report that local pharmacological targeting of the C5a receptor (C5aR) prior to initial allergen sensitization in murine models of inhalation tolerance or allergic asthma resulted in either induction or marked enhancement of Th2-polarized immune responses, airway inflammation, and AHR. Importantly, C5aR-deficient mice exhibited a similar, increased allergic phenotype. Pulmonary allergen exposure in C5aR-targeted mice resulted in increased sensitization and accumulation of CD4+ CD69+ T cells associated with a marked increase in pulmonary myeloid, but not plasmacytoid, DC numbers. Pulmonary DCs from C5aR-targeted mice produced large amounts of CC chemokine ligand 17 (CCL17) and CCL22 ex vivo, suggesting a negative impact of C5aR signaling on pulmonary homing of Th2 cells. In contrast, C5aR targeting in sensitized mice led to suppressed airway inflammation and AHR but was still associated with enhanced production of Th2 effector cytokines. These data suggest a dual role for C5a in allergic asthma, i.e., protection from the development of maladaptive type 2 immune responses during allergen sensitization at the DC/T cell interface but enhancement of airway inflammation and AHR in an established inflammatory environment.     

Interleukin 18 acts on memory T helper cells type 1 to induce airway inflammation and hyperresponsiveness in a naive host mouse

Interleukin (IL)-18 was originally regarded to induce T helper cell (Th)1-related cytokines. In general, factors favoring interferon (IFN)-gamma production are believed to abolish allergic diseases. Thus, we tested the role of IL-18 in regulation of bronchial asthma. To avoid a background response of host-derived T cells, we administered memory type Th1 or Th2 cells into unsensitized mice and examined their role in induction of bronchial asthma. Administration of antigen (Ag) induced both airway inflammation and airway hyperresponsiveness (AHR) in mice receiving memory Th2 cells. In contrast, the same treatment induced only airway inflammation but not AHR in mice receiving memory Th1 cells. However, these mice developed striking AHR when they were coadministered with IL-18. Furthermore, mice having received IFN-gamma-expressing Th1 cells sorted from polarized Th1 cells developed severe airway inflammation and AHR after intranasal administration of Ag and IL-18. Thus, Th1 cells become harmful when they are stimulated with Ag and IL-18. Newly polarized Th1 cells and IFN-gamma-expressing Th1 cells, both of which express IL-18 receptor alpha chain strongly, produce IFN-gamma, IL-9, IL-13, granulocyte/macrophage colony-stimulating factor, tumor necrosis factor alpha, regulated on activation, normal T cell expressed and secreted, and macrophage inflammatory protein 1alpha upon stimulation with Ag, IL-2, and IL-18 in vitro. Thus, Ag and IL-18 stimulate memory Th1 cells to induce severe airway inflammation and AHR in the naive host.     

Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolized antigen in mice.

Our understanding of the pathogenesis of atopic dermatitis (AD) and its relationship to asthma remains incomplete. Herein, we describe a murine model of epicutaneous (EC) sensitization to the protein allergen, chicken egg albumin, ovalbumin (OVA), which results in a rise in total and OVA-specific serum IgE and leads to the development of a dermatitis characterized by infiltration of CD3(+) T cells, eosinophils, and neutrophils and by local expression of mRNA for the cytokines IL-4, IL-5, and interferon-gamma. A single exposure of the EC sensitized mice to aerosolized OVA induced eosinophilia in the bronchoalveolar lavage fluid and airway hyperresponsiveness to intravenous methacholine as assessed by measurement of pulmonary dynamic compliance (Cdyn). These results suggest a possible role for EC exposure to antigen in atopic dermatitis and in the development of allergic asthma.     

Allergen Uptake, Activation, and IL-23 Production by Pulmonary Myeloid DCs Drives Airway Hyperresponsiveness in Asthma-Susceptible Mice

Maladaptive, Th2-polarized inflammatory responses are integral to the pathogenesis of allergic asthma. As regulators of T cell activation, dendritic cells (DCs) are important mediators of allergic asthma, yet the precise signals which render endogenous DCs “pro-asthmatic”, and the extent to which these signals are regulated by the pulmonary environment and host genetics, remains unclear. Comparative phenotypic and functional analysis of pulmonary DC populations in mice susceptible (A/J), or resistant (C3H) to experimental asthma, revealed that susceptibility to airway hyperresponsiveness is associated with preferential myeloid DC (mDC) allergen uptake, and production of Th17-skewing cytokines (IL-6, IL-23), whereas resistance is associated with increased allergen uptake by plasmacytoid DCs. Surprisingly, adoptive transfer of syngeneic HDM-pulsed bone marrow derived mDCs (BMDCs) to the lungs of C3H mice markedly enhanced lung IL-17A production, and rendered them susceptible to allergen-driven airway hyperresponsiveness. Characterization of these BMDCs revealed levels of antigen uptake, and Th17 promoting cytokine production similar to that observed in pulmonary mDCs from susceptible A/J mice. Collectively these data demonstrate that the lung environment present in asthma-resistant mice promotes robust pDC allergen uptake, activation, and limits Th17-skewing cytokine production responsible for driving pathologic T cell responses central to the development of allergen-induced airway hyperresponsiveness.     

Ozone exposure in a mouse model induces airway hyperreactivity that requires the presence of natural killer T cells and IL-17

Exposure to ozone, which is a major component of air pollution, induces a form of asthma that occurs in the absence of adaptive immunity. Although ozone-induced asthma is characterized by airway neutrophilia, and not eosinophilia, it is nevertheless associated with airway hyperreactivity (AHR), which is a cardinal feature of asthma. Because AHR induced by allergens requires the presence of natural killer T (NKT) cells, we asked whether ozone-induced AHR had similar requirements. We found that repeated exposure of wild-type (WT) mice to ozone induced severe AHR associated with an increase in airway NKT cells, neutrophils, and macrophages. Surprisingly, NKT cell-deficient (CD1d(-/-) and Jalpha18(-/-)) mice failed to develop ozone-induced AHR. Further, treatment of WT mice with an anti-CD1d mAb blocked NKT cell activation and prevented ozone-induced AHR. Moreover, ozone-induced, but not allergen-induced, AHR was associated with NKT cells producing interleukin (IL)-17, and failed to occur in IL-17(-/-) mice nor in WT mice treated with anti-IL-17 mAb. Thus, ozone exposure induces AHR that requires the presence of NKT cells and IL-17 production. Because NKT cells are required for the development of two very disparate forms of AHR (ozone- and allergen-induced), our results strongly suggest that NKT cells mediate a unifying pathogenic mechanism for several distinct forms of asthma, and represent a unique target for effective asthma therapy.     

The absence of interleukin 9 does not affect the development of allergen-induced pulmonary inflammation nor airway hyperreactivity.

Interleukin (IL)-9 is a pleiotropic cytokine secreted by T helper (Th)2 cells and has been proposed as a candidate gene for asthma and allergy. We have used mice genetically deficient in IL-9 to determine the role of this cytokine in the pathophysiologic features of the allergic pulmonary response-airway hyperreactivity (AHR) and eosinophilia. We have demonstrated that IL-9 is not required for the development of a robust Th2 response to allergen in sensitized mice. IL-9 knockout mice developed a similar degree of eosinophilic inflammation and AHR to their wild-type littermates. Goblet cell hyperplasia and immunoglobulin (Ig) E production were also unaffected by the lack of IL-9. Moreover, levels of bronchoalveolar lavage (BAL) IL-4, IL-5, and IL-13 were comparable between wild-type and knockout mice. These findings indicate that IL-9 is not obligatory for the development of eosinophilia and AHR, and imply that other Th2 cytokines can act in a compensatory fashion.     

Helicobacter pylori infection prevents allergic asthma in mouse models through the induction of regulatory T cells

Atopic asthma is a chronic disease of the airways that has taken on epidemic proportions in the industrialized world. The increase in asthma rates has been linked epidemiologically to the rapid disappearance of Helicobacter pylori, a bacterial pathogen that persistently colonizes the human stomach, from Western societies. In this study, we have utilized mouse models of allergic airway disease induced by ovalbumin or house dust mite allergen to experimentally examine a possible inverse correlation between H. pylori and asthma. H. pylori infection efficiently protected mice from airway hyperresponsiveness, tissue inflammation, and goblet cell metaplasia, which are hallmarks of asthma, and prevented allergen-induced pulmonary and bronchoalveolar infiltration with eosinophils, Th2 cells, and Th17 cells. Protection against asthma was most robust in mice infected neonatally and was abrogated by antibiotic eradication of H. pylori. Asthma protection was further associated with impaired maturation of lung-infiltrating dendritic cells and the accumulation of highly suppressive Tregs in the lungs. Systemic Treg depletion abolished asthma protection; conversely, the adoptive transfer of purified Treg populations was sufficient to transfer protection from infected donor mice to uninfected recipients. Our results thus provide experimental evidence for a beneficial effect of H. pylori colonization on the development of allergen-induced asthma.