Neutralization of both IL-1α/IL-1ß plays a major role in suppressing combined cigarette smoke/virus-induced pulmonary inflammation in mice.

Smoking is an important risk factor for the development of chronic obstructive pulmonary disease (COPD) and viral infections are believed to be major triggers of exacerbations, which periodically lead to a worsening of symptoms. The pro-inflammatory IL-1 family members IL-1α and IL-1ß are increased in COPD patients and might contribute to disease pathology. We investigated whether individual or combined inhibition of these cytokines reduced lung inflammation in cigarette smoke (CS)-exposed and H1N1-infected BALB/c mice. Animals were treated with individual or combined antibodies (Abs) directed against IL-1α, IL-1ß or IL-1R1. Cells in BAL fluid and cytokines/chemokines in lung homogenate were determined. The viral load was investigated. Blocking IL-1α had significant suppressive effects on total cells, neutrophils, and macrophages. Furthermore, it reduced KC levels significantly. Blocking of IL-1ß did not provide significant activity. In primary human bronchial epithelial air-liquid-interface cell cultures infected with H1N1, IL-1α Abs but not IL-1ß Abs reduced levels of TNF-α and IL-6. Concomitant usage of Abs against IL-1α/IL-1ß revealed strong effects in vivo and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1ß were significantly reduced and ICAM-1 and MUC5 A/C mRNA expression was attenuated. The viral load decreased significantly upon combined IL-1α/IL-1ß Ab treatment. Blocking the IL-1R1 provided significant effects on total cells, neutrophils and macrophages but was inferior compared to inhibiting both its soluble ligands IL-1α/IL-1ß. Our results suggest that combined inhibition of IL-1α/IL-1ß might be beneficial to reduce CS/H1N1-induced airway inflammation. Moreover, combined targeting of both IL-1α/IL-1ß might be more efficient compared to individual neutralization IL-1α or IL-1ß or inhibition of the IL-1R1.

Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke-Exposed Mice.

Viral infections trigger exacerbations in chronic obstructive pulmonary disease (COPD), and tiotropium, a M3 receptor antagonist, reduces exacerbations in patients by unknown mechanisms. In this report, we investigated whether tiotropium has anti-inflammatory effects in mice exposed to cigarette smoke (CS) and infected with influenza virus A/PR/8/34 (H1N1) or respiratory syncytial virus (RSV) and compared these effects with those of steroid fluticasone and PDE4-inhibitor roflumilast. Mice were exposed to CS; infected with H1N1 or RSV; and treated with tiotropium, fluticasone, or roflumilast. The amount of cells and cytokine levels in the airways, lung function, and viral load was determined. NCI-H292 cells were infected with H1N1 or RSV and treated with the drugs. In CS/H1N1-exposed mice, tiotropium reduced neutrophil and macrophage numbers and levels of interleukin-6 (IL-6) and interferon-γ (IFN-γ) in the airways and improved lung function. In contrast, fluticasone increased the loss of body weight; failed to reduce neutrophil or macrophage numbers; increased IL-6, KC, and tumor necrosis factor-α (TNF-α) in the lungs; and worsened lung function. Treatment with roflumilast reduced macrophage numbers, IL-6, and KC in the lungs but had no effect on neutrophil numbers or lung function. In CS/RSV-exposed mice, treatment with tiotropium, but not fluticasone or roflumilast, reduced neutrophil numbers and IL-6 and TNF-α levels in the lungs. Viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after fluticasone treatment, whereas tiotropium and roflumilast had no effect. In conclusion, tiotropium has anti-inflammatory effects on CS/virus-induced inflammation in mice that are superior to the effects of roflumilast and fluticasone. This finding might help to explain the observed reduction of exacerbation rates in COPD patients.

Modeling Pulmonary Disease Pathways Using Recombinant Adeno-Associated Virus 6.2.

Viral vectors have been applied successfully to generate disease-related animal models and to functionally characterize target genes in vivo. However, broader application is still limited by complex vector production, biosafety requirements, and vector-mediated immunogenic responses, possibly interfering with disease-relevant pathways. Here, we describe adeno-associated virus (AAV) variant 6.2 as an ideal vector for lung delivery in mice, overcoming most of the aforementioned limitations. In a proof-of-concept study using AAV6.2 vectors expressing IL-13 and transforming growth factor-ß1 (TGF-ß1), we were able to induce hallmarks of severe asthma and pulmonary fibrosis, respectively. Phenotypic characterization and deep sequencing analysis of the AAV-IL-13 asthma model revealed a characteristic disease signature. Furthermore, suitability of the model for compound testing was also demonstrated by pharmacological intervention studies using an anti-IL-13 antibody and dexamethasone. Similarly, the AAV-TGF-ß1 fibrosis model showed several disease-like pathophenotypes monitored by micro-computed tomography imaging and lung function measurement. Most importantly, analyses using stuffer control vectors demonstrated that in contrast to a common adenovirus-5 vector, AAV6.2 vectors did not induce any measurable inflammation and therefore carry a lower risk of altering relevant readouts. In conclusion, we propose AAV6.2 as an ideal vector system for the functional characterization of target genes in the context of pulmonary diseases in mice.

The absence of mrp4 has no effect on the recruitment of neutrophils and eosinophils into the lung after LPS, cigarette smoke or allergen challenge.

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4(-/-) and Mrp4(+/+) mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4(-/-) and Mrp4(+/+) mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4(+/+) or Mrp4(-/-) mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.

Probiotic Escherichia coli Nissle 1917 suppresses allergen-induced Th2 responses in the airways.

BACKGROUND: Recent clinical trials, epidemiological studies and animal experiments have suggested that probiotics may help suppress the development of allergic responses. OBJECTIVE: To investigate whether the application of the probiotic Escherichia coli strain Nissle 1917 (EcN) protects mice from developing ovalbumin (OVA)-specific T helper-2 responses in the airways. METHODS: OVA-specific Th2 responses were induced by 2 intraperitoneal (i.p.) injections with OVA/alum followed by 1 intranasal (i.n.) challenge with OVA. EcN was given orally during the entire sensitization and challenge period, together with OVA/alum during the i.p. sensitizations, or i.n. before or during the airway challenge with OVA. RESULTS: We found that when the bacteria were given together with OVA/alum airway eosinophilia, airway hyper-reactivity, goblet cell metaplasia and IL-5 levels in the bronchoalveolar lavage and mediastinal lymph node cell cultures were reduced. This effect was associated with increased numbers of IFN-gamma producing T helper-1 cells and IFN-gamma levels in the airways and strongly increased OVA-specific IgG(2a) titers in the serum. The suppressive effect on airway eosinophilia was dependent on IFN-gamma but not TLR-4. Applying EcN i.n. or orally did not reduce the development of allergen-specific Th2 responses. CONCLUSIONS: Our results suggest that EcN can inhibit the development of allergic responses when the bacteria are present at the site of Th2 cell priming and that this immunomodulatory effect is due to a shift from Th2 to Th1 response. The data support the hypothesis that probiotics may help reduce allergic responses and that EcN may also be used as adjuvant therapy to induce allergen-specific Th1 responses.

Development and characterisation of a novel and rapid lung eosinophil influx model in the rat.

Eosinophils play a major role in the development and severity of asthma. Robust and rapid preclinical animal models are desirable to profile novel therapeutics inhibiting the influx of eosinophils into the airways. To develop a rapid, airway eosinophil recruitment model in the rat, Brown-Norway (BN) rats were immunised with ovalbumin (OVA)/alum on day 0, 1 and 2 and challenged with OVA aerosol on day 5 and 6. On day 7 bronchoalveolar lavage fluid (BALF) was analysed for eosinophil numbers, eosinophil peroxidase (EPO) activity and cytokines. Lung sections were also examined. The immunised animals showed a strong selective influx of eosinophils into the airways correlating with enhanced EPO activity, Interleukin (IL-4), IL-5 and monocytes chemo attractant protein levels in the BALF in comparison to sham-sensitised rats. In addition the immunised rats developed goblet cell metaplasia in the lung and showed OVA specific IgG1 and IgE levels in the serum but no airway hyperreactivity after metacholine challenge. Airway inflammation was suppressed by applying the steroids Budesonide (intra tracheally) and Prednisolone (per orally), Roflumilast a phosphodiesterase-4 inhibitor, and the H1 receptor antagonists Epinastine and Ketotifen. Montelukast, a Leukotriene receptor antagonist and Chromoglycate, a mast cell stabiliser, had no effect in this model. In summary, in this novel preclinical rat model therapeutics expected to inhibit the development of airway eosinophilia can rapidly be tested.

Murine polyomavirus-like particles induce maturation of bone marrow-derived dendritic cells and proliferation of T cells.

We analysed the effects of murine polyomavirus-like particles (PLPs) on bone marrow-derived dendritic cells (BMDCs) and T cells in vitro. BMDCs activated with PLPs up-regulated CD40, CD80, CD86 and major histocompatibility complex (MHC) class II surface markers and produced proinflammatory cytokines. Chimeric PLPs [expressing the ovalbumin (OVA)-peptides OVA(257-264) or OVA(323-339)], but not wildtype PLPs, activated OVA-specific CD8 T cells and OVA-specific CD4 T cells, respectively, indicating both MHC class I and II presentation of the peptides by antigen-presenting cells. Our results suggest that PLPs may be used as vaccine adjuvants priming dendritic cells to induce potent T cell responses.

Helminth-derived products inhibit the development of allergic responses in mice.

RATIONALE: Epidemiological studies suggest that infections with helminths protect from the development of asthma. Supporting this view is our published finding that infection with Nippostrongylus brasiliensis decreased ovalbumin-induced Th2 responses in the lung of mice. OBJECTIVES: To evaluate if N. brasiliensis excretory-secretory products also prevent the development of asthma. METHODS: Mice were immunized with ovalbumin/alum intraperitoneally in the absence or presence of helminthic products and then challenged intranasally with ovalbumin. Six days later, we analyzed if the mice developed Th2 responses in the lung. MAIN RESULTS: The application of the helminthic products together with ovalbumin/alum during the sensitization period totally inhibited the development of eosinophilia and goblet cell metaplasia in the airways and also strongly reduced the development of airway hyperreactivity. Allergen-specific IgG1 and IgE serum levels were also strongly reduced. These findings correlated with decreased levels of IL-4 and IL-5 in the airways in product-treated animals. The suppressive effects on the development of allergic responses were independent of the presence of Toll-like receptors 2 and 4, IFN-gamma, and most important, IL-10. Interestingly, suppression was still observed when the helminthic products were heated or treated with proteinase K. Paradoxically, we found that strong helminth product-specific Th2 responses were induced in parallel with the inhibition of ovalbumin-specific responses. CONCLUSION: Our results suggest that helminths suppress the development of asthma by secreting substances that modulate allergic responses without affecting the generation of helminth-specific Th2 immunity. The identification of these products may lead to the design of novel therapeutic intervention strategies for the treatment of asthma.

Infection with Toxoplasma gondii reduces established and developing Th2 responses induced by Nippostrongylus brasiliensis infection.

Oral infection of C57BL/6 mice with 100 cysts of the protozoan parasite Toxoplasma gondii results in the development of small intestinal Th1-type immunopathology. In contrast, infection with intestinal helminths results in the development of protective Th2-type responses. We investigated whether infection with the helminth Nippostrongylus brasiliensis influences the development of T. gondii-induced Th1 responses and immunopathology in C57BL/6 mice infected with T. gondii. Prior as well as simultaneous infection of mice with N. brasiliensis did not alter the course of infection with 100 cysts of T. gondii. Coinfected mice produced high levels of interleukin-12 (IL-12) and gamma interferon (IFN-gamma), developed small intestinal immunopathology, and died at the same time as mice infected with T. gondii. Interestingly, local and systemic N. brasiliensis-induced Th2 responses, including IL-4 and IL-5 production by mesenteric lymph node and spleen cells and numbers of intestinal goblet cells and blood eosinophils, were markedly lower in coinfected than in N. brasiliensis-infected mice. Similar effects were seen when infection with 10 T. gondii cysts was administered following infection with N. brasiliensis. Infection of C57BL/6 mice with 10 T. gondii cysts prior to coinfection with N. brasiliensis inhibited the development of helminth-induced Th2 responses and was associated with higher and prolonged N. brasiliensis egg production. In contrast, oral administration of Toxoplasma lysate prior to N. brasiliensis infection had only a minor and short-lived effect on Th2 responses. Thus, N. brasiliensis-induced Th2 responses fail to alter T. gondii-induced Th1 responses and immunopathology, most likely because Th1 responses develop unchanged in C57BL/6 mice with a prior or simultaneous infection with N. brasiliensis. Our findings contribute to the understanding of immune regulation in coinfected animals and may assist in the design of immunotherapies for human Th1 and Th2 disorders.

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.

Influenza A virus infection inhibits the efficient recruitment of Th2 cells into the airways and the development of airway eosinophilia.

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.

Infection with influenza a virus leads to flu antigen-induced cutaneous anaphylaxis in mice.

It is well established, that viral infections may trigger urticaria or allergic asthma; however, as viral infections induce T helper 1 polarized responses, which lead to the inhibition of T helper 2 cell development, the opposite would be plausible. We wanted to investigate how viral infections may mediate allergic symptoms in a mouse model; therefore, we infected BALB/C mice with influenza A virus intranasally. Histologic analyses of lung sections and bronchoalveolar lavages were performed. In addition, cells from the mediastinal lymph nodes were restimulated in vitro to analyze which types of cytokines were induced by the flu infection. Furthermore, flu-specific antibody titers were determined and local anaphylaxis was measured after rechallenge with flu antigen. We found that airways inflammation consisted predominately of macrophages and lymphocytes, whereas only a few eosinophils were observed. interferon-gamma but no interleukin-4 and little interleukin-5 could be detected in the culture supernatants from in vitro restimulated T cells from the draining lymph nodes. The antibody response was characterized by high levels of virus-specific IgG2a, IgG2b, and IgG1 and, surprisingly, low levels of virus-specific IgE antibodies. Interestingly, flu-infected mice developed active and passive cutaneous anaphylaxis after rechallenge with flu-antigen. As the passive cutaneous anaphylaxis reaction persisted over 48 h and was significantly lower after passive transfer of the serum, which was IgE depleted, local anaphylaxis seemed to be mediated predominately by specific IgE antibodies. Taken together, our results demonstrate that mice infected with flu virus develop virus-specific mast cell degranulation in the skin. Our results may also have implications for the pathogenesis of urticaria or other atopic disorders in humans.