Edwardsiella tarda induces airways inflammation and production of autoantibodies against lung tissues through regulation of the IL-33-ST2 axis.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent chronic respiratory disease characterised by irreversible airways obstruction associated with chronic airways inflammation and remodelling, while the pathogenesis and the mechanistic differences between patients remain to be fully elucidated. We previously reported that alarmin cytokine IL-33 may contribute to the production of autoantibodies against respiratory epithelial cells. Here we expand the hypothesis that pulmonary autoimmune responses induced by airway microbiota also contribute to the progression of COPD. We focused on Edwardsiella tarda which we detected uniquely in the induced sputum of patients with acute exacerbations of COPD. Pernasal challenge of the airways of WT mice with supernatants of cultured E. tarda induced marked, elevated expression of IL-33 in the lung tissues. Immunisation of animals with supernatants of cultured E. tarda resulted in significantly elevated airways inflammation, the formation of tertiary lymphatic structures and significantly elevated proportions of T follicular helper T cells in the lung tissue and mediastinal lymph nodes. Interestingly, such challenge also induced production of IgG autoantibodies directed against lung tissue lysate, alveolar epithelial cell proteins and elastin fragment, while putrescine, one of metabolites generated by the bacterium, might play an important role in the autoantibody production. Furthermore, all of these effects were partly but significantly abrogated in mice with deletion of the IL-33 receptor ST2. Collectively, these data support the hypothesis that COPD is progressed at least partly by airways microbiota such as E. tarda initiating autoimmune attack of the airways epithelium mediated at least partly through the IL-33-ST2 axis.

Airways epithelial exposure to Streptococcus pneumoniae in the presence of the alarmin IL-33 induces a novel subset of pro-inflammatory ILC2s promoting a mixed inflammatory response.

BACKGROUND: We have previously shown that asthma-like airways inflammation may be induced by topical exposure to respiratory tract pathogens such as S. pneumoniae (SP) in concert with epithelial alarmins such as IL-33. Details of the pathogenesis of this murine surrogate remain however unexplored. METHODS: Airways inflammation was induced by repeated, intranasal exposure of Il-4-/-, Rag1-/- and Rag2-/-Il2rg-/- mice (in which B lymphocyte IgE switching, adaptive and innate immunity are respectively ablated) as well as wild type mice to inactivated SP, IL-33 or both. Airways pathological changes were analysed, and the subsets and functions of locally accumulated ILC2s investigated by single cell RNA sequencing and flow cytometry. RESULTS: In the presence of IL-33, repeated exposure of the airways to inactivated SP caused marked eosinophil- and neutrophil-rich inflammation and local accumulation of ILC2s, which was retained in the Il-4-/- and Rag1-/- deficient mice but abolished in the Rag2-/-Il2rg-/- mice, an effect partly reversed by adoptive transfer of ILC2s. Single cell sequencing analysis of ILC2s recruited following SP and IL-33 exposure revealed a Klrg1+Ly6a+subset, expressing particularly elevated quantities of the pro-inflammatory cytokine IL-6, type 2 cytokines (IL-5 and IL-13) and MHC class II molecules, promoting type 2 inflammation as well as involved in neutrophil-mediated inflammatory responses. CONCLUSION: Local accumulation of KLRG1+Ly6a+ ILC2s in the lung tissue is a critical aspect of the pathogenesis of airways eosinophilic and neutrophil-rich inflammation induced by repeated exposure to SP in the presence of the epithelial alarmin IL-33.

Bacterial antigens and asthma: a comparative study of common respiratory pathogenic bacteria.

Objective: In a previous study we have shown that, in the presence of interleukin (IL)-33, repeated, per-nasal challenge of murine airways with Streptococcus pneumoniae (S. pneumoniae) organisms induces human asthma-like airways inflammation. It is not clear, however, whether this effect is unique or manifest in response to other common respiratory pathogens.Methods: To explore this, airways of BALB/c mice were repeatedly challenged per-nasally with formaldehyde-inactivated bacterial bodies in the presence or absence of murine recombinant IL-33. Serum concentrations of S.pneumoniae, Moraxella catarrhalis (M.catarrhalis) and Haemophilus influenzae (H.influenzae) lysates-specific IgE were measured in patients with asthma and control subjects.Results: We showed that in the presence of IL-33, repeated, per-nasal airways exposure to the bodies of these bacteria induced airways hyperresponsiveness (AHR) in the experimental mice. This was accompanied by cellular infiltration into bronchoalveolar lavage fluid (BALF), eosinophilic infiltration and mucous hypertrophy of the lung tissue, with elevated local expression of some type 2 cytokines and elevated, specific IgG and IgE in the serum. The precise characteristics of the inflammation evoked by exposure to each bacterial species were distinguishable.Conclusions: These results suggest that in the certain circumstances, inhaled or commensal bacterial body antigens of both Gram-positive (S. pneumoniae) and Gram-negative (M. catarrhalis and H. influenzae) respiratory tract bacteria may initiate type 2 inflammation typical of asthma in the airways. In addition, we demonstrated that human asthmatic patients manifest elevated serum concentrations of M.catarrhalis- and H.influenzae-specific IgE.

Sphingosine-1-phosphate induces airway smooth muscle hyperresponsiveness and proliferation.

BACKGROUND: The emerging role of sphingosine-1-phosphate (S1P) in regulating smooth muscle functions has led to the exploration of the possibility that this sphingolipid could represent a potential therapeutic target in asthma and other lung diseases. Several studies in animal surrogates have suggested a role for S1P-mediated signaling in the regulation of airway smooth muscle (ASM) contraction, airway hyperresponsiveness, and airway remodeling, but evidence from human studies is lacking. OBJECTIVE: We sought to compare the responsiveness of the airways to S1P in healthy and asthmatic individuals in vivo, in isolated human airways ex vivo, and in murine airways dissected from healthy and house dust mite (HDM)-sensitized animals. METHODS: Airway responsiveness was measured by spirometry during inhalation challenges and by wire myography in airways isolated from human and mouse lungs. Thymidine incorporation and calcium mobilization assays were used to study human ASM cell responses. RESULTS: S1P did not induce contraction of airways isolated from healthy and HDM-exposed mice, nor in human airways. Similarly, there was no airway constriction observed in healthy and asthmatic subjects in response to increasing concentrations of inhaled S1P. However, a 30-minute exposure to S1P induced a significant concentration-dependent enhancement of airway reactivity to methacholine and to histamine in murine and human airways, respectively. HDM-sensitized mice demonstrated a significant increase in methacholine responsiveness, which was not further enhanced by S1P treatment. S1P also concentration-dependently enhanced proliferation of human ASM cells, an effect mediated through S1P receptor type 2, as shown by selective antagonism and S1P receptor type 2 small-interfering RNA knockdown. CONCLUSIONS: Our data suggest that S1P released locally into the airways may be involved in the regulation of ASM hyperresponsiveness and hyperplasia, defining a novel target for future therapies.

Topical therapy with negative allosteric modulators of the calcium-sensing receptor (calcilytics) for the management of asthma: the beginning of a new era?

In this review article we present the evidence to date supporting the role of the calcium-sensing receptor (CaSR) as a key, pluripotential molecular trigger for asthma and speculate on the likely benefits of topical therapy of asthma with negative allosteric modulators of the CaSR: calcilytics.

Early-life infection of the airways with Streptococcus pneumoniae exacerbates HDM-induced asthma in a murine model.

Respiratory tract infection early in life plays a significant role in the pathogenesis of asthma. In the present study we examine, using a murine surrogate, the effects of early life respiratory infection with Streptococcus pneumoniae (SP) on adult asthma induced by sensitisation and exposure to house dust mite (HDM) allergen. Mice (one week old) were infected with SP, then 3 weeks later sensitised to HDM emulsified with Al (OH)3 intraperitoneally and challenged intranasally with same allergen for up to a further 5 weeks to establish the asthma surrogate. Outcome measures were quantified using the FlexiVent apparatus, histology and immunohistology, ELISA and flow cytometry. The murine surrogates of asthma infected with SP early in life exhibited significantly more severe disease compared with the controls of mice without SP infection, as shown by airways responsiveness, inflammatory cellular infiltration of the airways, expression of markers of airways remodelling, serum concentrations of HDM-specific IgE and the concentrations of Th2-type cytokines and the numbers of activated Th2 and ILC2 cells in the lung tissues. These data are compatible with the hypothesis that early-life infection of the airways with SP exacerbates, at least in some individuals, subsequent HDM-induced allergic airways inflammation and associated asthma in adulthood in this murine surrogate.

Role of the IL-33/ST2 axis in cigarette smoke-induced airways remodelling in chronic obstructive pulmonary disease.

BACKGROUND: Efficient therapy and potential prophylaxis are confounded by current ignorance of the pathogenesis of airway remodelling and blockade in COPD. OBJECTIVE: To explore the role of the IL-33/ST2 axis in cigarette smoke (CS) exposure-induced airways remodelling. METHODS: C57BL/6, BALB/c and IL-1RL1 -/- mice exposed to CS were used to establish an animal surrogate of COPD (air-exposed=5~8, CS-exposed=6~12). Hallmarks of remodelling were measured in mice. Cigarette smoke extract (CSE)-induced proliferation and protein production in vitro by fibroblasts in the presence of anti-interleukin-33 (anti-IL-33) or hST2 antibodies were measured. Expression of IL-33 and ST2 and other remodelling hallmarks were measured, respectively, in bronchoalveolar lavage fluid (BALF) (controls=20, COPD=20), serum (controls=59, COPD=90) and lung tissue sections (controls=11, COPD=7) from patients with COPD and controls. RESULTS: Wild-type mice exposed to CS elevated expression of hallmarks of tissue remodelling in the lungs and also in the heart, spleen and kidneys, which were significantly abrogated in the IL-1RL1 -/- mice. Fibroblasts exposed to CSE, compared with control, exhibited early cellular translocation of IL-33, accompanied by proliferation and elevated protein synthesis, all inhabitable by blockade of IL-33/ST2 signalling. Expression of IL-33 and ST2 and hallmarks of tissue remodelling were significantly and proportionally elevated in BALF, serum and tissue samples from patients with COPD. CONCLUSIONS: Exposure to CS induces remodelling changes in multiple organs. The data support the hypothesis that CS-induced lung collagen deposition is at least partly a result of CS-induced IL-33 translocation and release from local fibroblasts.

IL-33 amplifies airways inflammation in a murine surrogate of asthma putatively via activation of dendritic cells.

Although contributions of IL-33 to pulmonary diseases, including asthma, have been well documented, the complexity of such regulation warrants additional exploration. To better understand the involvement of IL-33, we used a murine asthma surrogate based on sensitisation and challenge with dust mite extract in the presence/absence of IL-33. Murine models were established with Dermatophagoides farinae (Der f) to establish (1) the effect of co-administered rmIL-33; (2) the effect of prior glucocorticoid intervention; (3) the effect of IL-33 on challenge with sub-threshold dosage Der f. The effects of rmIL-33 on bone marrow-derived dendritic cells were explored in vitro. Mice challenged with Der f combined with IL-33 compared with diluent control evinced significantly more airways inflammation and local cytokine production which was less sensitive to inhibition by dexamethasone. IL-33 also induced airways hyperresponsiveness, eosinophilic inflammation and cytokine production in lung tissues of animals exposed to sub-threshold dosage of Der f. In vitro, IL-33-stimulated DCs showed a significantly elevated capacity to stimulate CD4+ T cell proliferation and cytokine production and were also significantly more resistant to dexamethasone-induced apoptosis. Our data suggest that IL-33 reduces the threshold for allergen-induced inflammation of the airways in acorticosteroid-resistant fashion possibly in part through acting on DCs, a phenomenon which may be relevant to the development of severe, corticosteroid-resistant airways obstruction in human asthmatic patients.

Repeated exposure to inactivated Streptococcus pneumoniae induces asthma-like pathological changes in mice in the presence of IL-33.

While environmental aeroallergens and epithelial alarmins such as IL-33 are firmly implicated in asthma, the possible role of Streptococcus pneumoniae (S. pneumoniae) antigens is less clear. To explore this, wild-type BALB/c mice were repeatedly challenged per-nasally with IL-33 and inactivated S. pneumoniae, either agent alone or diluent control. Some animals were rested then later re-challenged with inactivated S. pneumoniae alone. Serum concentrations of S. pneumoniae lysates-specific IgE were measured in patients with asthma and control subjects. Interestingly, in the presence of IL-33, repeated exposure to inactivated S. pneumoniae induced asthma-like pathological changes accompanied by a systemic adaptive immune response. Subsequent re-exposure of the sensitized animals to inactivated S. pneumoniae alone was able to induce such changes. The concentration of S. pneumoniae lysates-specific IgE was significantly elevated in the asthma patients. These data suggest that antigens derived from infectious microorganisms may participate in generating the mucosal inflammation which characterizes asthma.

Similarities and differences in the effects of sensitisation and challenge with Dermatophagoides farinae and Dermatophagoides pteronyssinus extracts in a murine asthma surrogate.

Patients with atopic asthma may become sensitised to the grain storage mite Dermatophagoides farinae (Der f), the house dust mite Dermatophagoides pteronyssinus (Der p) or both, but thus far little attention has been paid to date to possible variation in their pathophysiological effects. Here we present a side by side comparison of the effects of extracts of these two dust mites in a murine surrogate of atopic asthma. Compared with the Der p-challenged mice, however, the mice-challenged with Der f had favour changes in lung tissue elasticity and expression in matrix metalloproteinases in lung tissue, while the mice challenged with Der p showed more neutrophils infiltrating around the airway and stronger expression of steroid-resistant related cytokines in the lung tissue. Our data suggest that different dust mite crude extracts might lead different pathological characteristics, at least in murine models of asthma.

IL-33 induced airways inflammation is partially dependent on IL-9.

Asthma is an inflammatory disease of the airways and numerous cytokines contribute to this pathogenesis. It is shown that challenge of airways with IL-33 induces asthma-like pathological changes in mice, but the possible downstream cytokines in this process remain to be characterised. To explore this, we compared changes in the airways of wildtype (WT) and IL-9 deficient mice challenged with IL-33. In line with previous report, per-nasal challenge of WT mice with IL-33 significantly increased the responsiveness of the airways along with infiltration of inflammatory cells, goblet cell hyperplasia, collagen deposition and smooth muscle hypertrophy, and the expression of cytokines compared with control group. Surprisingly, all of these pathological changes were significantly attenuated in IL-9 deficient mice following identical IL-33 challenge. These data suggest that IL-9 is one downstream cytokine relevant to the effects of IL-33 in asthmatic airways and consequently a potential therapeutic target for the treatment of asthma.

Elevated Expression of IL-33 and TSLP in the Airways of Human Asthmatics In Vivo: A Potential Biomarker of Severe Refractory Disease.

The epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP), and IL-25 have been implicated in asthma pathogenesis because they promote Th2-type cytokine synthesis, but their expression is relatively poorly documented in "real-life" human asthma. Using bronchoalveolar lavage fluid (BALF), we measured airway concentrations of these mediators and compared them with those of Th1- and Th2-type cytokines, airway infiltration of neutrophils and eosinophils, and lung function in a large group of asthmatic patients with a range of disease severity (n = 70) and control subjects (n = 30). The median BALF concentrations of IL-33, TSLP, IL-4, IL-5, IL-13, and IL-12p70, but not IL-25, IL-2, or IFN-γ, were significantly elevated in asthmatics compared with controls (p < 0.05). The concentrations of IL-33 and TSLP, but not IL-25, correlated inversely with the lung function (forced expiratory volume in the first second) of asthmatics (IL-33: r = -0.488, p < 0.0001; TSLP: r = -0.565, p < 0.0001) independently of corticosteroid therapy. When divided according to disease severity and corticosteroid therapy, all subgroups of asthmatics had elevated median numbers of eosinophils in BALF, whereas the patients with more severe disease who were treated with corticosteroids had higher numbers of neutrophils compared with milder asthmatics not so treated and control subjects (p < 0.05). The data implicate TSLP and IL-33 in the pathogenesis of asthma that is characterized by persistent airway inflammation and impaired lung function despite intensive corticosteroid therapy, highlighting them as potential molecular targets.

Bronchial Allergen Challenge of Patients with Atopic Asthma Triggers an Alarmin (IL-33, TSLP, and IL-25) Response in the Airways Epithelium and Submucosa.

The alarmin cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) play a critical role in asthma pathogenesis by inducing mucosal Th2-type cytokine production. Although environmental exposure to aeroallergens has been proposed as an alarmin trigger in asthma, there has been no systematic parallel study of the effects of allergen exposure on the expression of these cytokines in the airways of human asthmatics. Using single and sequential double immunohistochemistry, we evaluated the numbers and phenotypes of IL-25-, IL-33-, and TSLP-immunoreactive cells in sections of bronchial biopsies from mild atopic asthmatics (n = 16) before and 24 h after allergen inhalational challenge. Allergen challenge highly increased expression of baseline immunoreactivity for IL-25, IL-33, and TSLP, both in the bronchial epithelium and submucosa (p < 0.001), to a degree that correlated with the extent of the late phase of airway obstruction. Aside from epithelial cells, the principal source of immunoreactivity for all three alarmins, TSLP, and IL-33 immunoreactivity colocalized principally with endothelial cells and mast cells, neutrophils, and fibroblasts, whereas IL-25 immunoreactivity colocalized principally with eosinophils as well as endothelial cells, mast cells, and fibroblasts. The data implicate that allergen challenge directly increases airway alarmin expression in atopic asthmatics to a degree correlating with increase late-phase airway obstruction, affirming these molecules as potential molecular targets for the inhibition of allergen-induced airway inflammation and obstruction.

Combined blockade of IL-25, IL-33 and TSLP mediates amplified inhibition of airway inflammation and remodelling in a murine model of asthma.

BACKGROUND AND OBJECTIVE: Isolated blockade of IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) has been shown to reduce airways inflammation and hyperresponsiveness in murine asthma model. The hypothesis that combined blockade of all three cytokines can accomplish this more effectively has never been addressed. METHODS: We studied a murine asthma model employing sensitization and challenge with ovalbumin (OVA) or saline control. To discern the effects of IL-33 blockade, we compared outcomes in strain identical, wild-type and IL-33 receptor (St2 -/- ) gene-deleted mice. We then examined, in the St2 -/- animals, the effects of additional, single or combined blockade of IL-25 and TSLP with blocking antibodies. Outcomes included airways reactivity, inflammatory cellular infiltration, epithelial cell metaplasia, deposition of fibrosis-related proteins, local Th2-type cytokine expression and total and specific serum IgE concentrations measured by ELISA and quantitative immunohistochemistry. RESULTS: St2 -/- gene deletion significantly reduced airways reactivity, inflammatory cellular infiltration, lung tissue expression of Th2 cytokines and fibrosis related proteins and serum total IgE in response to OVA sensitization and challenge. Additional administration of anti-IL-25 and anti-TSLP blocking antibodies to the St2 -/- mice further significantly reduced inflammation, Th2 cytokine expression, airways fibrosis and IgE production, while anti-TSLP alone reduced eosinophil infiltration and local IL-4 expression. The airways inflammatory cellular infiltrate and lung tissue expression of Th2 cytokine, but not fibrosis-related proteins were also reduced in the presence of isotype identical, control antibodies. CONCLUSION: Combined blockade of these three cytokines may better ameliorate airways pathological changes in this murine asthma model, with implications for human asthma.

IL-33 induces production of autoantibody against autologous respiratory epithelial cells: a potential mechanism for the pathogenesis of COPD.

The mechanisms underlying the chronic, progressive airways inflammation, remodelling and alveolar structural damage characteristic of human chronic obstructive pulmonary disease (COPD) remain unclear. In the present study, we address the hypothesis that these changes are at least in part mediated by respiratory epithelial alarmin (IL-33)-induced production of autoantibodies against airways epithelial cells. Mice immunized with homologous, syngeneic lung tissue lysate along with IL-33 administered directly to the respiratory tract or systemically produced IgG autoantibodies binding predominantly to their own alveolar type II epithelial cells, along with increased percentages of Tfh cells and B2 B-cells in their local, mediastinal lymph nodes. Consistent with its specificity for respiratory epithelial cells, this autoimmune inflammation was confined principally to the lung and not other organs such as the liver and kidney. Furthermore, the serum autoantibodies produced by the mice bound not only to murine, but also to human alveolar type II epithelial cells, suggesting specificity for common, cross-species determinants. Finally, concentrations of antibodies against both human and murine alveolar epithelial cells were significantly elevated in the serum of patients with COPD compared with those of control subjects. These data are consistent with the hypothesis that IL-33 contributes to the chronic, progressive airways obstruction, inflammation and alveolar destruction characteristic of phenotypes of COPD/emphysema through induction of autoantibodies against lung tissue, and particularly alveolar type II epithelial cells.

Perceptions of COPD patients of the proposed withdrawal of inhaled corticosteroids prescribed outside guidelines: A qualitative study.

Global Initiative for Chronic Obstructive Lung Disease guidelines support the prescription of fixed combination inhaled corticosteroids (ICS) and long-acting ß-agonists in symptomatic COPD patients with frequent or severe exacerbations, with the aim of preventing them. ICS are frequently also prescribed to COPD patients with mild or moderate airflow limitation, outside guidelines, with the risk of unwanted effects. No investigation to date has addressed the views of these milder COPD patients on ICS withdrawal. The objective is to assess the views of COPD patients with mild or moderate airflow limitation on the staged withdrawal of ICS prescribed outside guidelines. One-to-one semi-structured qualitative interviews exploring COPD patients' views about ICS use and their attitudes to proposed de-prescription were conducted. Interviews were audio-recorded and transcribed verbatim. Thematic analysis was completed. Seventeen eligible COPD patients were interviewed. Many participants were not aware they were using an ICS. None was aware that prevention of exacerbations was the indication for ICS therapy or the risk of associated side effects. Some were unconcerned by what they perceived as low individual risk. Others expressed fears of worsening symptoms on withdrawal. Most with mild or moderate airflow limitation would have been willing to attempt withdrawal or titration to a lower dosage of ICS if advised by their clinician, particularly if a reasoned explanation were offered. Attitudes in this study to discontinuing ICS use varied. Knowledge of the drug itself, the indications for its prescription in COPD and potential for side effects, was scant. The proposed withdrawal of ICS is likely to be challenging and requires detailed conversations between patients and respiratory healthcare professionals.

The effects of interleukin-33 on airways collagen deposition and matrix metalloproteinase expression in a murine surrogate of asthma.

It has been suggested that interleukin-33 (IL-33) plays an important role in the pathogenesis of asthma through a variety of pathways, but its role in airways fibrosis in asthma has not been fully elucidated. In the present study we evaluated changes in the expression of extracellular matrix proteins (ECMs) as well as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in an IL-33-induced, antigen-independent murine surrogate of asthma as well as a conventional surrogate employing per-nasal challenge of mice previously sensitized to produce an IgE response to ovalbumin (OVA). In addition, in in vitro experiments we explored the direct effects of IL-33 on the proliferation and function of murine fibroblasts. Per-nasal administration of IL-33 alone was sufficient to induce airways deposition of ECMs, including collagens I, III, V and fibronectin, to a degree comparable with that observed in the OVA-sensitized and challenged mice. These changes were associated with a local imbalance between the expression of extracellular MMPs and TIMPs. Per-nasal challenge of mice with IL-33 also induced elevated airways expression of connective tissue growth factor and fibroblast growth factor receptor 4, two key facilitators of local fibrosis, again to a degree compatible with that observed in OVA-sensitized and challenged mice. Deletion of the ST2 gene, which encodes the IL-33 receptor, abrogated these fibrotic changes in the airways in the OVA surrogate. In vitro, IL-33 significantly increased the proliferation and expression of collagen III by murine lung fibroblasts. These data suggest that direct exposure of murine airways to IL-33 is able to induce local fibrotic changes, at least partially through effects of signalling through the IL-33/ST2 axis on fibroblast function and local expression of MMPs and their inhibitors, and other fibrosis-related proteins.

Comprehensive attenuation of IL-25-induced airway hyperresponsiveness, inflammation and remodelling by the PI3K inhibitor LY294002.

BACKGROUND AND OBJECTIVE: Existing in vitro and in vivo studies suggest that both IL-25 and phosphoinositide 3-kinases (PI3Ks) exhibit broad effects on the functions of immune cells implicated in the pathogenesis of asthma. Whether the blockade of PI3K signalling directly inhibits the asthma relevant pathogenetic changes induced by IL-25 in an in vivo condition is still unclear. Using an established IL-25-induced murine model of asthma, we undertook a comprehensive evaluation of the effects of co-administered LY294002, a pharmacological pan-inhibitor of PI3K on IL-25-induced changes on this model, with particular regard to airway remodelling. METHODS: BALB/c mice were serially intranasally challenged with IL-25 according to an established protocol to induce airway inflammation, hyperresponsiveness (AHR) and remodelling. In an additional subgroup LY294002 was administered intranasally. Lung function and airway cytokine and chemokine concentrations and cellular infiltration and remodelling changes assessed by histology and immunohistochemistry were measured at specific time points. RESULTS: Intranasal administration of LY294002 significantly inhibited IL-25-induced AHR and recruitment of inflammatory cells into bronchoalveolar lavage fluid. LY294002 also attenuated IL-25-induced increased concentrations of cytokines and chemokines in lung tissue. Histological and immunohistochemical analysis showed that LY294002 also significantly inhibited IL-25-induced lung tissue eosinophilia, mucus production, collagen deposition, smooth muscle hypertrophy and angiogenesis. CONCLUSION: The PI3K pan-inhibitor LY294002 attenuated not only IL-25-induced asthma-like AHR and airway inflammation but also remodelling in this model, suggesting that PI3K is a major downstream messenger for IL-25 and that targeting this pathway might reduce asthma symptoms in the short term and airway remodelling in the longer term.