Acellular pertussis vaccine protects against exacerbation of allergic asthma due to Bordetella pertussis in a murine model

The prevalence of asthma and allergic disease has increased in many countries, and there has been speculation that immunization promotes allergic sensitization. Bordetella pertussis infection exacerbates allergic asthmatic responses. We investigated whether acellular pertussis vaccine (Pa) enhanced or prevented B. pertussis-induced exacerbation of allergic asthma. Groups of mice were immunized with Pa, infected with B. pertussis, and/or sensitized to ovalbumin. Immunological, pathological, and physiological changes were measured to assess the impact of immunization on immune deviation and airway function. We demonstrate that immunization did not enhance ovalbumin-specific serum immunoglobulin E production. Histopathological examination revealed that immunization reduced the severity of airway pathology associated with sensitization in the context of infection and decreased bronchial hyperreactivity upon methacholine exposure of infected and sensitized mice. These data demonstrate unequivocally the benefit of Pa immunization to health and justify selection of Pa in mass vaccination protocols. In the absence of infection, the Pa used in this study enhanced the interleukin-10 (IL-10) and IL-13 responses and influenced airway hyperresponsiveness to sensitizing antigen; however, these data do not suggest that Pa contributes to childhood asthma overall. On the contrary, wild-type virulent B. pertussis is still circulating in most countries, and our data suggest that the major influence of Pa is to protect against the powerful exacerbation of asthma-like pathology induced by B. pertussis.     

Whole-cell pertussis vaccine protects against Bordetella pertussis exacerbation of allergic asthma

The prevalence of asthma and allergic disease has increased in many countries and there has been speculation that immunization promotes allergic sensitization. Bordetella pertussis infection exacerbates allergic asthmatic responses. We investigated whether whole-cell pertussis vaccine (Pw) enhanced or prevented B. pertussis induced exacerbation of allergic asthma. Groups of mice were immunized with Pw, infected with B. pertussis and/or sensitized to ovalbumin. Immunological, pathological and physiological changes were measured to assess the impact of Pw immunization on immune deviation and airway function. Pw immunization modulated ovalbumin-specific serum IgE production, and reduced local and systemic IL-13 and other cytokine responses to sensitizing allergen. Histopathological examination revealed Pw immunization reduced the severity of airway pathology and decreased bronchial hyperreactivity to methacholine exposure. Pw does not enhance airway IL-13 and consequently does not enhance but protects against the exacerbation of allergic responses. We find no evidence of Pw contributing to allergic asthma, but rather provide evidence of a mechanism whereby whole-cell pertussis vaccination has a protective role.     

Attenuated Bordetella pertussis vaccine strain BPZE1 modulates allergen‐induced immunity and prevents allergic pulmonary pathology in a murine model

Background Virulent Bordetella pertussis, the causative agent of whooping cough, exacerbates allergic airway inflammation in a murine model of ovalbumin (OVA) sensitization. A live genetically attenuated B. pertussis mucosal vaccine, BPZE1, has been developed that evokes full protection against virulent challenge in mice but the effect of this attenuated strain on the development of allergic responses is unknown. Objective To assess the influence of attenuated B. pertussis BPZE1 on OVA priming in a murine model of allergic airway inflammation. Methods Mice were challenged with virulent or attenuated strains of B. pertussis, and sensitized to allergen (OVA) at the peak of bacterial carriage. Subsequently, airway pathology, local inflammation and OVA‐specific immunity were examined. Results In contrast to virulent B. pertussis, live BPZE1 did not exacerbate but reduced the airway pathology associated with allergen sensitization. BPZE1 immunization before allergen sensitization did not have an adjuvant effect on allergen specific IgE but resulted in a statistically significant decrease in airway inflammation in tissue and bronchoalveolar lavage fluid. BPZE1 significantly reduced the levels of OVA‐driven IL‐4, IL‐5 and IL‐13 but induced a significant increase in IFN‐γ in response to OVA re‐stimulation. Conclusions These data demonstrate that, unlike virulent strains, the candidate attenuated B. pertussis vaccine BPZE1 does not exacerbate allergen‐driven airway pathology. BPZE1 may represent an attractive T‐helper type 1 promoting vaccine candidate for eradication of whooping cough that is unlikely to promote atopic disease.     

Effects of anti-inflammatory therapies on recurrent and low-grade respiratory syncytial virus infections in a murine model of asthma.

BACKGROUND: Recurrent and subclinical viral respiratory tract infections could immunologically exacerbate allergic airway inflammation. However, the most appropriate treatment for virus-induced asthma exacerbation is yet to be established. The effects of glucocorticoids in virus-induced acute asthma are controversial. OBJECTIVE: To determine the effects of representative anti-inflammatory therapies for asthma--glucocorticoids and leukotriene receptor antagonists (LTRAs)--in mite allergen-sensitized and repeatedly low-grade respiratory syncytial virus (RSV)--infected mice. METHODS: Dermatophagoides farinae-sensitized mice were inoculated twice with low-grade RSV and subcutaneously injected with either a glucocorticoid or an LTRA for 4 consecutive days. Lung inflammation, cytokine profiles, LT production, and viral RNA in lung tissues were compared in 5 groups of 8 mice each: controls, D farinae allergen sensitized, D farinae sensitized and RSV infected, D farinae sensitized and RSV infected with dexamethasone, and D farinae sensitized and RSV infected with pranlukast, an LTRA. RESULTS: Allergic airway inflammation in D farinae mice was significantly enhanced by recurrent and low-grade RSV infections (RLRIs). The glucocorticoid attenuated allergic airway inflammation, which was associated with interleukin 5 (IL-5) and interferon-gamma (IFN-gamma) suppression in lung-draining lymph nodes without affecting viral quantity. The LTRA also attenuated allergic airway inflammation in D farinae-RSV mice with concomitant inhibition of IL-5 but not IFN-gamma. Dermatophagoides farinae allergen sensitization significantly increased LTs in the airway, whereas RLRIs did not further enhance LT production. CONCLUSIONS: Glucocorticoids and LTRAs significantly inhibit RLRI-induced exacerbation of allergic airway inflammation by distinct pathways. Dexamethasone suppressed nonspecific cytokines, whereas viral RNA did not increase via suppression of immunity. In contrast, pranlukast specifically inhibited IL-5 but not IFN-gamma.     

Prior Bordetella pertussis infection modulates allergen priming and the severity of airway pathology in a murine model of allergic asthma

BACKGROUND: It has been proposed that T helper (Th)2-driven immune deviation in early life can be countered by Th1 inducing childhood infections and that such counter-regulation can protect against allergic asthma. OBJECTIVE: To test whether Th1-inducing infection with Bordetella pertussis protects against allergic asthma using well-characterized murine models. METHODS: Groups of mice were sensitized to ovalbumin (OVA) in the presence or absence of B. pertussis, a well-characterized Th1 inducing respiratory infection. Immunological, pathological and physiological parameters were measured to assess the impact of infection on immune deviation and airway function. RESULTS: We demonstrate that OVA sensitization does not affect the development of B. pertussis-specific immune responses dominated by IgG2a and IFN-gamma and does not impair Th1-mediated clearance of airway infection. In contrast, B. pertussis infection at the time of sensitization modulated the response to OVA and significantly reduced total serum and OVA-specific IgE. The pattern of cytokine responses, in particular OVA-specific IL-5 responses in the spleen was also modulated. However, B. pertussis did not cause global suppression as IL-10 and IL-13 levels were enhanced in OVA-stimulated spleen cell cultures and in lavage fluid from infected co-sensitized mice. Histopathological examination revealed that B. pertussis infection prior to OVA sensitization resulted in increased inflammation of bronchiolar walls with accompanying hyperplasia and mucous metaplasia of lining epithelia. These pathological changes were accompanied by increased bronchial hyper-reactivity to methacholine exposure. CONCLUSION: Contrary to the above premise, a Th1 response induced by a common childhood infection does not protect against bronchial hyper-reactivity, but rather exacerbates the allergic asthmatic response, despite modulation of immune mediators.     

Allergic Airway Inflammation Decreases Lung Bacterial Burden following Acute Klebsiella pneumoniae Infection in a Neutrophil- and CCL8-Dependent Manner

The Th17 cytokines interleukin-17A (IL-17A), IL-17F, and IL-22 are critical for the lung immune response to a variety of bacterial pathogens, including Klebsiella pneumoniae. Th2 cytokine expression in the airways is a characteristic feature of asthma and allergic airway inflammation. The Th2 cytokines IL-4 and IL-13 diminish ex vivo and in vivo IL-17A protein expression by Th17 cells. To determine the effect of IL-4 and IL-13 on IL-17-dependent lung immune responses to acute bacterial infection, we developed a combined model in which allergic airway inflammation and lung IL-4 and IL-13 expression were induced by ovalbumin sensitization and challenge prior to acute lung infection with K. pneumoniae. We hypothesized that preexisting allergic airway inflammation decreases lung IL-17A expression and airway neutrophil recruitment in response to acute K. pneumoniae infection and thereby increases the lung K. pneumoniae burden. As hypothesized, we found that allergic airway inflammation decreased the number of K. pneumoniae-induced airway neutrophils and lung IL-17A, IL-17F, and IL-22 expression. Despite the marked reduction in postinfection airway neutrophilia and lung expression of Th17 cytokines, allergic airway inflammation significantly decreased the lung K. pneumoniae burden and postinfection mortality. We showed that the decreased lung K. pneumoniae burden was independent of IL-4, IL-5, and IL-17A and partially dependent on IL-13 and STAT6. Additionally, we demonstrated that the decreased lung K. pneumoniae burden associated with allergic airway inflammation was both neutrophil and CCL8 dependent. These findings suggest a novel role for CCL8 in lung antibacterial immunity against K. pneumoniae and suggest new mechanisms of orchestrating lung antibacterial immunity.     

Sustained protective immunity against Bordetella pertussis nasal colonization by intranasal immunization with a vaccine-adjuvant combination that induces IL-17-secreting TRM cells

Current acellular pertussis (aP) vaccines induce strong antibody and Th2 responses but fail to protect against nasal colonization and transmission of Bordetella pertussis. Furthermore, immunity wanes rapidly after immunization. We have developed a novel adjuvant combination (called LP-GMP), comprising c-di-GMP, an intracellular receptor stimulator of interferon genes (STING) agonist, and LP1569, a TLR2 agonist from B. pertussis, which synergistically induces production of IFN-β, IL-12 and IL-23, and maturation of dendritic cells. Parenteral immunization of mice with an experimental aP vaccine formulated with LP-GMP promoted Th1 and Th17 responses and conferred protection against lung infection with B. pertussis. Intranasal immunization with the same aP vaccine-induced potent B. pertussis-specific Th17 responses and IL-17-secreting respiratory tissue-resident memory (T_RM) CD4 T cells, and conferred a high level of protection against nasal colonization as well as lung infection, which was sustained for at least 10 months. Furthermore, long-term protection against nasal colonization with B. pertussis correlated with the number of IL-17-secreting T_RM cells in nasal tissue. Our study has identified an approach for inducing IL-17-secreting T_RM cells that sustain sterilizing immunity against nasal colonization of mice with B. pertussis, and could form the basis of a third generation pertussis vaccine for humans.     

Anisakis pegreffii-Induced Airway Hyperresponsiveness Is Mediated by Gamma Interferon in the Absence of Interleukin-4 Receptor Alpha Responsiveness

Infection with the fish parasite Anisakis following exposure to contaminated fish can lead to allergic reactions in humans. The present study examined the immunological mechanisms underlying the development of allergic airway inflammation in mice after different routes of sensitization to Anisakis. Wild-type and interleukin-4 receptor alpha (IL-4Rα)-deficient BALB/c mice were sensitized intraperitoneally with live or heat-killed Anisakis larvae or by intranasal administration of an Anisakis extract and were subsequently challenged intranasally with an Anisakis extract. Both routes of sensitization induced IL-4Rα-dependent allergic airway responses, whereas allergen-specific antibody responses developed only when mice were sensitized intraperitoneally. Intranasal sensitization induced airway hyperresponsiveness (AHR) in wild-type mice only, showing that AHR was IL-4/IL-13 dependent. Unexpectedly, infection with Anisakis larvae induced AHR in both wild-type and IL-4Rα-deficient mice. IL-4Rα-independent AHR was mediated by gamma interferon (IFN-γ), as evidenced by the fact that in vivo neutralization of IFN-γ abrogated AHR. Together, these results demonstrate that both infection with larvae and inhalational exposure to Anisakis proteins are potent routes of allergic sensitization to Anisakis, explaining food- and work-related allergies in humans. Importantly for diagnosis, allergic airway inflammation can be independent of detectable Anisakis-specific antibodies. Moreover, depending on the route of sensitization, AHR can be induced either by IL-4/IL-13 or by IFN-γ.While it is believed that many parasitic worms protect against allergy, the fish parasite Anisakis can induce acute gastroallergic or anaphylactic reactions in a subset of infected patients (4). Anisakis spp. are nematode parasites of marine mammals with larval stages that pass through several intermediate hosts. The infectious-stage larvae (L₃) are found worldwide in sea fish or cephalopods and can be accidentally ingested by humans (32). If ingested live due to consumption of raw or undercooked fish, Anisakis L₃ are able to parasitize humans, causing the zoonotic disease known as anisakiasis. This is usually an acute and transient infection, with the larvae dying within a few weeks, since the host environment does not permit development into adult worms (6). Within hours of being ingested, Anisakis L₃ penetrate the mucosal layers of the gastrointestinal tract, causing direct tissue damage that may lead to abdominal pain, nausea, and/or diarrhea. Furthermore, some patients develop an immunoglobulin E (IgE)-mediated “gastroallergic anisakiasis,” which presents with clinical manifestations ranging from urticaria to allergic reactions and life-threatening anaphylactic shock (5, 13, 14). To date, nine allergens from Anisakis simplex, some of which are cross-reactive to other seafood allergens, have been characterized on a molecular level (28). The importance of the problem is demonstrated by studies that have found Anisakis to be a leading cause of food allergy in Spain (2) and have found a higher prevalence of sensitization to Anisakis than to seafood among almost 5 million Japanese (24). Anisakis is also an important cause of occupational allergy in fish-processing workers, as shown in a recent epidemiological study by our group, including 578 workers from fish-processing factories in South Africa, in which Anisakis sensitization was associated with dermatitis and nonspecific bronchial hyperreactivity (37). Because sensitization to Anisakis was associated with increased fish consumption, we investigated the underlying immune mechanisms by infecting mice with Anisakis L₃ and subsequently challenging them orally with an Anisakis crude antigen extract. This induced striking allergic reactions, including airway inflammation and lung mucus hypersecretion, which were associated with T-helper 2 (Th2)/type 2 responses (37). Furthermore, mice exposed epicutaneously to Anisakis proteins developed protein contact dermatitis (36). Local skin pathology was interleukin-13 (IL-13) dependent, as evidenced by the fact that it was abolished in IL-13- and IL-4 receptor alpha (IL-4Rα)-deficient mice, whereas IL-4 was important for systemic allergic sensitization (36). Together, these studies show that sensitization by Anisakis infection and subsequent oral challenge with an Anisakis extract can cause allergic airway disease, while epicutaneous exposure to Anisakis proteins can lead to dermatitis, explaining the observations of human prevalence studies.However, it is still unclear whether live infection is needed for the development of allergic airway reactions or whether exposure to nematode-derived protein is sufficient (4). This is an important clinical question in view of the fact that several case and prevalence studies, including ours, indicate that sensitization by inhalation of Anisakis proteins might be an important cause of work-related allergies (1, 3, 7, 37, 39, 40). Aerosolized food allergens cause as much as 10% of asthma in the occupational environment (41), and Anisakis allergens aerosolized during fish cleaning, cooking, or fish meal production may therefore pose a risk for workers.In this study we aimed to determine whether sensitization through Anisakis infection is essential for the induction of allergic airway disease or if exposure to larval proteins or heat-killed larvae is sufficient to induce allergic airway reactions. In addition, we investigated the effects of IL-4/IL-13 signaling by using mice deficient in IL-4Rα, an important receptor chain in allergic airway disease through which both IL-4 and IL-13 signal (8). Wild-type and IL-4Rα^−/− mice were either sensitized intraperitoneally (i.p.) with live or heat-killed Anisakis L₃ or sensitized intranasally to an Anisakis extract and were subsequently challenged by intranasal administration of an Anisakis extract in order to mimic aerosolized exposure. All sensitization protocols induced symptoms of allergic airway disease, but allergen-specific antibody responses were present only when mice were sensitized intraperitoneally by live or heat-killed larvae. Interestingly, while the development of airway hyperresponsiveness (AHR) was dependent on IL-4Rα responsiveness when mice were sensitized intranasally, mice sensitized with live or heat-killed Anisakis L₃ developed AHR independently of IL-4Rα responsiveness. This IL-4Rα-independent AHR was shown to be dependent on gamma interferon (IFN-γ).     

Anti-IgE efficacy in murine asthma models is dependent on the method of allergen sensitization

BACKGROUND: Murine models used to delineate mechanisms and key mediators of asthma have yielded conflicting results and suggest that the dominant mechanism and mediators required for disease induction differ depending on the model and method of allergen sensitization used. OBJECTIVE: The goal of this study was to determine whether the mode of allergen sensitization influenced the role that IgE had in allergen-induced pulmonary eosinophilic inflammation. METHODS: Mice were exposed to dust mite extract in 2 models of allergic inflammation that differed in the method of sensitization. We compared sensitization by aerosol exposure with and without concomitant human respiratory syncytial virus infection with sensitization by means of systemic (intraperitoneal) exposure with adjuvant. After sensitization, animals were similarly challenged with aerosolized allergen. Animals were treated with anti-IgE mAb to deplete IgE and to determine its role in the induction of allergic inflammation and mucosa pathology in these models. RESULTS: Concomitant respiratory syncytial virus infection significantly enhanced allergen sensitization by aerosol exposure and exacerbated eosinophilic inflammation and airway mucosa pathology. Depletion of IgE in this model significantly reduced lung eosinophilic inflammation and airway mucosa pathology. However, in the model in which animals were sensitized by means of systemic allergen exposure with adjuvant, depletion of IgE had no ameliorative effect on lung inflammation or pathology. CONCLUSION: We demonstrated that the method of antigen sensitization can delineate the role of IgE in allergen-induced lung inflammation. In a murine model that more closely resembles ambient allergen exposure in human subjects, IgE had a critical role in the pathogenesis of allergic asthma and mucosa pathology. The results parallel the results reported with anti-IgE efficacy in allergic asthmatic human subjects.     

Modulation of Allergic Airway Inflammation by the Oral Pathogen Porphyromonas gingivalis

Accumulating evidence suggests that bacteria associated with periodontal disease may exert systemic immunomodulatory effects. Although the improvement in oral hygiene practices in recent decades correlates with the increased incidence of asthma in developed nations, it is not known whether diseases of the respiratory system might be influenced by the presence of oral pathogens. The present study sought to determine whether subcutaneous infection with the anaerobic oral pathogen Porphyromonas gingivalis exerts a regulatory effect on allergic airway inflammation. BALB/c mice sensitized and subsequently challenged with ovalbumin exhibited airway hyperresponsiveness to methacholine aerosol and increased airway inflammatory cell influx and Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13) content relative to those in nonallergic controls. Airway inflammatory cell and cytokine contents were significantly reduced by establishment of a subcutaneous infection with P. gingivalis prior to allergen sensitization, whereas serum levels of ovalbumin-specific IgE and airway responsiveness were not altered. Conversely, subcutaneous infection initiated after allergen sensitization did not alter inflammatory end points but did reduce airway responsiveness in spite of increased serum IgE levels. These data provide the first direct evidence of a regulatory effect of an oral pathogen on allergic airway inflammation and responsiveness. Furthermore, a temporal importance of the establishment of infection relative to allergen sensitization is demonstrated for allergic outcomes.A causative relationship between decreased microbial exposure and infection in recent decades and the concurrent increase in asthma prevalence in developed countries has been suggested and is thought to be attributable, at least in part, to a phenomenon known as the hygiene hypothesis (30). Originally put forth by Strachan (32), the hypothesis proposes that increased cleanliness of modern industrialized societies has resulted in decreased exposure to bacterial, viral, and other immunomodulatory organisms and their products, particularly in early life, and that this has in turn resulted in a loss of potentially protective effects of these exposures on the development of allergic diseases. Accumulating clinical and experimental evidence largely supports the hygiene hypothesis as it relates to asthma, although a consensus has not been reached. As reviewed recently (31), a variety of infections of a viral, bacterial, and parasitic nature influence the host immune response, such that regulation of the Th1-Th2 balance is modified to promote Th1 responses and impede Th2 responses, thereby reducing Th2-mediated allergic outcomes. However, this is likely a simplistic view of the effects of infections on immune system development and responses, and other factors, including host genetic makeup and timing of exposures to the infective agent relative to allergen exposure, undoubtedly contribute to the overall allergic phenotype.In addition to the influence of environmental exposure to microbes, the potential regulation of allergic diseases by the microflora of the host is receiving increased attention. Evidence suggests that the composition of the gastrointestinal microflora differs between individuals with and without allergy (reviewed in reference 25), and disruption of the normal gut microflora by antibiotic administration leads to allergic airway responses following allergen challenge in mice not previously sensitized to the allergen (24). Moreover, although similar benefits have not yet been demonstrated in humans, the oral administration of probiotic bacteria was recently shown to decrease allergic airway inflammation in mice (8, 9).As in the gut, the microenvironment of the oral cavity is complex and comprises hundreds of bacterial species. Porphyromonas gingivalis, a Gram-negative opportunistic periodontal pathogen, can initiate periodontal lesions in nonhuman primates when introduced into the periodontal microbiota (15) and is a major etiological agent in severe forms of periodontal disease such as chronic periodontitis (21). Interest in chronic oral infections and their potential role in adverse systemic health effects has been heightened by observations of positive associations between serum concentrations of antibodies to oral pathogens such as P. gingivalis and the incidence of cardiovascular diseases and renal dysfunction (3, 19, 28, 29). Recently, however, an inverse relationship between serum concentrations of antibodies to P. gingivalis and the prevalence of asthma, wheeze, and hay fever was observed in a representative sample of the population of the United States (2). Furthermore, a significant inverse association between periodontitis and the incidences of hay fever and allergy to house dust mites was reported for a northeast German population, with a borderline significant inverse association between periodontitis and asthma also observed (10). While limitations of these observational studies include potential recall bias pertaining to asthma symptoms and the inability to directly assess cause and effect, these findings nonetheless suggest a potential protective effect of infection with oral pathogens such as P. gingivalis on asthma pathogenesis.In order to examine the influence of oral pathogens on the development of allergic airway disease under controlled experimental conditions, the present study sought to determine whether infection with P. gingivalis modified allergic outcomes in a murine model of asthma. To accomplish this, a subcutaneous chamber model was employed wherein mice were subjected to a local infection with live P. gingivalis either before or after sensitization to allergen, and the effects of this infection on subsequent responses to allergen challenge were assessed. The results indicate that P. gingivalis infection exerts a modulatory effect on allergic airway responses and that this effect is dependent on the timing of infection relative to allergic sensitization.     

Dysregulation of alveolar macrophages unleashes dendritic cell–mediated mechanisms of allergic airway inflammation

Allergic asthma is a chronic inflammatory disorder characterized by eosinophilia and T helper type 2 (Th2) cell activation. However, little information is available on the mechanisms leading to this pathology. We previously showed that alveolar macrophages (AM) from rats with experimental asthma lose their ability to prevent asthma symptoms. To understand the implication of AM in lung immunity, we investigated the influence of AM sensitization status on lung dendritic cell (DC) activation induced by allergen challenge in vivo. Rat sensitized to ovalbumin developed airway inflammation (eosinophils and Th2 cells) and demonstrated myeloid DC (mDC) activation following allergen exposure. The replacement of AM of sensitized animals by AM from naive animals did not affect allergen-triggered eosinophilia but completely abolished lung mDC allergen capture and migration to the lymph nodes, as well as Th2 cell polarization. Moreover, immunosuppressive functions of naive AM occurred in conjunction with low engulfment of allergens but without variation of major histocompatibility complex II and CD23 expression. Interestingly, sensitized AM that were withdrawn from the inflammatory environment regained their immunosuppressive functions when transferred to sensitized rats. Thus, these are the first in vivo evidences showing that dysregulation of AM functions is sufficient to induce DC-triggered allergic response.     

Melatonin reduces airway inflammation in ovalbumin-induced asthma

Asthma is a common chronic inflammatory airway disease that is recognized as a major public health problem. In this study, we evaluated the effects of melatonin on allergic asthma using a murine model of ovalbumin (OVA)-induced allergic asthma and BEAS-2B cells. To induce allergic asthma, the mice were sensitized and airway-challenged with OVA. Melatonin was administered by intraperitoneal injection once per day at doses of 10 and 15 mg/kg from days 21 to 23 after the initial OVA sensitization. We investigated the effects of melatonin on proinflammatory cytokines and matrix metalloproteinase-9 (MMP-9) activity and expression in tumor necrosis factor (TNF)-α-stimulated BEAS-2B cells. The administration of melatonin significantly decreased the number of inflammatory cells, airway hyperresponsiveness, and immunoglobulin (Ig) E with reductions in interleukin (IL)-4, IL-5, and IL-13. Melatonin attenuated the airway inflammation and the mucus production in lung tissue and significantly suppressed elevated MMP-9 expression and activity induced by an OVA challenge. In TNF-α-stimulated BEAS-2B cells, treatment with melatonin significantly reduced the levels of proinflammatory cytokines and lowered the expression and activity of MMP-9. These results indicate that melatonin effectively suppressed allergic asthma induced by an OVA challenge. The results suggest a potential role for melatonin in treating asthma.     

Link between allergic asthma and airway mucosal infection suggested by proteinase-secreting household fungi

Active fungal proteinases are powerful allergens that induce experimental allergic lung disease strongly resembling atopic asthma, but the precise relationship between proteinases and asthma remains unknown. Here, we analyzed dust collected from the homes of asthmatic children for the presence and sources of active proteinases to further explore the relationship between active proteinases, atopy, and asthma. Active proteinases were present in all houses and many were derived from fungi, especially Aspergillus niger. Proteinase-active dust extracts were alone insufficient to initiate asthma-like disease in mice, but conidia of A. niger readily established a contained airway mucosal infection, allergic lung disease, and atopy to an innocuous bystander antigen. Proteinase produced by A. niger enhanced fungal clearance from lung and was required for robust allergic disease. Interleukin 13 (IL-13) and IL-5 were required for optimal clearance of lung fungal infection and eosinophils showed potent anti-fungal activity in vitro. Thus, asthma and atopy may both represent a protective response against contained airway infection due to ubiquitous proteinase-producing fungi.     

Amelioration of ovalbumin-induced allergic airway disease following Der p 1 peptide immunotherapy is not associated with induction of IL-35

In the present study, we show therapeutic amelioration of established ovalbumin (OVA)-induced allergic airway disease following house dust mite (HDM) peptide therapy. Mice were sensitized and challenged with OVA and HDM protein extract (Dermatophagoides species) to induce dual allergen sensitization and allergic airway disease. Treatment of allergic mice with peptides derived from the major allergen Der p 1 suppressed OVA-induced airway hyperresponsiveness, tissue eosinophilia, and goblet cell hyperplasia upon rechallenge with allergen. Peptide treatment also suppressed OVA-specific T-cell proliferation. Resolution of airway pathophysiology was associated with a reduction in recruitment, proliferation, and effector function of T_H2 cells and decreased interleukin (IL)-17⁺ T cells. Furthermore, peptide immunotherapy induced the regulatory cytokine IL-10 and increased the proportion of Fox p3⁺ cells among those expressing IL-10. Tolerance to OVA was not associated with increased IL-35. In conclusion, our results provide in vivo evidence for the creation of a tolerogenic environment following HDM peptide immunotherapy, leading to the therapeutic amelioration of established OVA-induced allergic airway disease.     

Intelectin contributes to allergen-induced IL-25, IL-33, and TSLP expression and type 2 response in asthma and atopic dermatitis

The epithelial and epidermal innate cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) have pivotal roles in the initiation of allergic inflammation in asthma and atopic dermatitis (AD). However, the mechanism by which the expression of these innate cytokines is regulated remains unclear. Intelectin (ITLN) is expressed in airway epithelial cells and promotes allergic airway inflammation. We hypothesized that ITLN is required for allergen-induced IL-25, IL-33, and TSLP expression. In two asthma models, Itln knockdown reduced allergen-induced increases in Il-25, Il-33, and Tslp and development of type 2 response, eosinophilic inflammation, mucus overproduction, and airway hyperresponsiveness. Itln knockdown also inhibited house dust mite (HDM)-induced early upregulation of Il-25, Il-33, and Tslp in a model solely inducing airway sensitization. Using human airway epithelial cells, we demonstrated that HDM-induced increases in ITLN led to phosphorylation of epidermal growth factor receptor and extracellular-signal regulated kinase, which were required for induction of IL-25, IL-33, and TSLP expression. In two AD models, Itln knockdown suppressed expression of Il-33, Tslp, and Th2 cytokines and eosinophilic inflammation. In humans, ITLN1 expression was significantly increased in asthmatic airways and in lesional skin of AD. We conclude that ITLN contributes to allergen-induced Il-25, Il-33, and Tslp expression in asthma and AD.     

Administration of Pigment Epithelium-Derived Factor Inhibits Airway Inflammation and Remodeling in Chronic OVA-Induced Mice via VEGF Suppression

Purpose

Pigment epithelium-derived factor (PEDF) is a recently discovered antiangiogenesis protein. PEDF possesses powerful anti-inflammatory, antioxidative, antiangiogenic, and antifibrosis properties. It has been reported that PEDF can regulate vascular endothelial growth factor (VEGF) expression. This study aimed to evaluate whether recombinant PEDF protein could attenuate allergic airway inflammation and airway remodeling via the negative regulation of VEGF using a murine model of chronic ovalbumin (OVA)-induced asthma and BEAS-2B human bronchial epithelial cells.

Methods

In an in vivo experiment, mice sensitized with OVA were chronically airway challenged with aerosolized 1% OVA solution for 8 weeks. Treated mice were given injections of recombinant PEDF protein (50 or 100 µg/kg body weight) via the tail vein. In an in vitro experiment, we investigated the effects of recombinant PEDF protein on VEGF release levels in BEAS-2B cells stimulated with IL-1β.

Results

Recombinant PEDF protein significantly inhibited eosinophilic airway inflammation, airway hyperresponsiveness, and airway remodeling, including goblet cell hyperplasia, subepithelial collagen deposition, and airway smooth muscle hypertrophy. In addition, recombinant PEDF protein suppressed the enhanced expression of VEGF protein in lung tissue and bronchoalveolar lavage fluid (BALF) in OVA-challenged chronically allergic mice. In the in vitro experiment, VEGF expression was increased after IL-1β stimulation. Pretreatment with 50 and 100 ng/mL of recombinant PEDF protein significantly attenuated the increase in VEGF release levels in a concentration-dependent manner in BEAS-2B cells stimulated by IL-1β.

Conclusions

These results suggest that recombinant PEDF protein may abolish the development of characteristic features of chronic allergic asthma via VEGF suppression, providing a potential treatment option for chronic airway inflammation diseases such as asthma.     

Severity of Allergic Airway Disease Due to House Dust Mite Allergen Is Not Increased after Clinical Recovery of Lung Infection with Chlamydia pneumoniae in Mice

Chlamydia pneumoniae is associated with chronic inflammatory lung diseases like bronchial asthma and chronic obstructive pulmonary disease. The existence of a causal link between allergic airway disease and C. pneumoniae is controversial. A mouse model was used to address the question of whether preceding C. pneumoniae lung infection and recovery modifies the outcome of experimental allergic asthma after subsequent sensitization with house dust mite (HDM) allergen. After intranasal infection, BALB/c mice suffered from pneumonia characterized by an increased clinical score, reduction of body weight, histopathology, and a bacterial load in the lungs. After 4 weeks, when infection had almost resolved clinically, HDM allergen sensitization was performed for another 4 weeks. Subsequently, mice were subjected to a methacholine hyperresponsiveness test and sacrificed for further analyses. As expected, after 8 weeks, C. pneumoniae-specific antibodies were detectable only in infected mice and the titer was significantly higher in the C. pneumoniae/HDM allergen-treated group than in the C. pneumoniae/NaCl group. Intriguingly, airway hyperresponsiveness and eosinophilia in bronchoalveolar lavage fluid were significantly lower in the C. pneumoniae/HDM allergen-treated group than in the mock/HDM allergen-treated group. We did observe a relationship between experimental asthma and chlamydial infection. Our results demonstrate an influence of sensitization to HDM allergen on the development of a humoral antibacterial response. However, our model demonstrates no increase in the severity of experimental asthma to HDM allergen as a physiological allergen after clinically resolved severe chlamydial lung infection. Our results rather suggest that allergic airway disease and concomitant cellular changes in mice are decreased following C. pneumoniae lung infection in this setting.     

Constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease

Deleterious responses to pathogens during infancy may contribute to infection and associated asthma. Chlamydia respiratory infections in early life are common causes of pneumonia and lead to reduced lung function and asthma. We investigated the role of interleukin-13 (IL-13) in promoting early-life Chlamydia respiratory infection, infection-induced airway hyperresponsiveness (AHR), and severe allergic airway disease (AAD). Infected infant Il13^−/− mice had reduced infection, inflammation, and mucus-secreting cell hyperplasia. Surprisingly, infection of wild-type (WT) mice did not increase IL-13 production but reduced IL-13Rα2 decoy receptor levels compared with sham-inoculated controls. Infection of WT but not Il13^−/− mice induced persistent AHR. Infection and associated pathology were restored in infected Il13^−/− mice by reconstitution with IL-13. Stat6^−/− mice were also largely protected. Neutralization of IL-13 during infection prevented subsequent infection-induced severe AAD. Thus, early-life Chlamydia respiratory infection reduces IL-13Rα2 production, which may enhance the effects of constitutive IL-13 and promote more severe infection, persistent AHR, and AAD.     

Inhibition of the IL-4/IL-13 receptor system prevents allergic sensitization without affecting established allergy in a mouse model for allergic asthma

BACKGROUND: IL-4 and IL-13 are considered as key regulators for the development of atopic disease. OBJECTIVE: This study addresses the therapeutic potential of an IL-4/IL-13 inhibitor on the basis of a mutated IL-4 variant (Q116D, Y119D) during allergic sensitization and in established disease in a murine asthma model with persistent airway pathologic condition. METHODS: BALB/c mice were sensitized with ovalbumin intranasally. Mice were treated with the IL-4/IL-13 inhibitor during the sensitization phase or alternatively after ovalbumin allergy was established. Specific antibodies were measured, and histologic lung sections were examined for goblet cell metaplasia. In addition, bronchoalveolar lavages were performed and checked for airway eosinophilia, IL-5 levels, and the number of IL-4 secreting CD4(+) T cells. Furthermore, airway responsiveness to inhaled methacholine was assessed. RESULTS: The inhibition of the IL-4/IL-13 system during allergic sensitization resulted in a dose-dependent reduction of ovalbumin-specific IgEs and inhibition of airway eosinophilia together with decreased IL-5 levels and decreased numbers of IL-4 secreting CD4(+) T cells. Moreover, goblet cell metaplasia and airway responsiveness to methacholine could be reduced significantly by the IL-4/IL-13 inhibitor. However, the inhibition of the IL-4/IL-13 system at various time points after allergy was established showed only little effect on all measured allergic parameters. CONCLUSION: Although the inhibition of the IL-4/IL-13 system can efficiently prevent the development of the allergic phenotype, these cytokines seem to play a minor role in established allergy. This is relevant for estimating the therapeutic effects of IL-4/IL-13 inhibitors in patients with allergic asthma.