Research ArticleAsthma

Rhinovirus-induced IL-25 in asthma exacerbation drives type 2 immunity and allergic pulmonary inflammation

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Science Translational Medicine  01 Oct 2014:
Vol. 6, Issue 256, pp. 256ra134
DOI: 10.1126/scitranslmed.3009124

Abstract

Rhinoviruses (RVs), which are the most common cause of virally induced asthma exacerbations, account for much of the burden of asthma in terms of morbidity, mortality, and associated cost. Interleukin-25 (IL-25) activates type 2–driven inflammation and is therefore potentially important in virally induced asthma exacerbations. To investigate this, we examined whether RV-induced IL-25 could contribute to asthma exacerbations. RV-infected cultured asthmatic bronchial epithelial cells exhibited a heightened intrinsic capacity for IL-25 expression, which correlated with donor atopic status. In vivo human IL-25 expression was greater in asthmatics at baseline and during experimental RV infection. In addition, in mice, RV infection induced IL-25 expression and augmented allergen-induced IL-25. Blockade of the IL-25 receptor reduced many RV-induced exacerbation-specific responses including type 2 cytokine expression, mucus production, and recruitment of eosinophils, neutrophils, basophils, and T and non-T type 2 cells. Therefore, asthmatic epithelial cells have an increased intrinsic capacity for expression of a pro–type 2 cytokine in response to a viral infection, and IL-25 is a key mediator of RV-induced exacerbations of pulmonary inflammation.

INTRODUCTION

Asthma attacks (exacerbations) are the most clinically and economically important form of this disease. Respiratory viruses are the most common triggers of exacerbations, accounting for about 85% of cases, and rhinoviruses (RVs) represent the great majority of viruses detected (1). At present, our understanding of asthma is such that targeted therapies for exacerbations tailored to immunopathogenic mechanisms are lacking (2). What is clear is that allergen-driven type 2 immunity is central to the immunopathogenesis of allergic asthma. Many studies have detected increased type 2 cells (3, 4) and type 2 cytokines (5, 6) in asthma. Genetic studies have linked numerous type 2–associated genes with asthma [IL-4 (interleukin-4), IL-13, IL-4Rα, and STAT6 (signal transducer and activator of transcription 6)] (7, 8) and asthma exacerbations (IL-4 and IL-4Rα) (9, 10). We studied exacerbations in a human experimental RV infection model in allergic asthmatic and normal volunteers and demonstrated that asthmatics had more severe lower respiratory tract symptoms, reductions in lung function, and increases in bronchial hyperreactivity. Exacerbation severity was strongly correlated with bronchoalveolar lavage (BAL) CD4+ TH2 cell cytokine production and viral load (11). Mouse models of allergic asthma exacerbation have also demonstrated the capacity of RV infection to augment TH2 responses and associated features of allergic airway disease such as airway hyperreactivity (AHR) and mucus production (12, 13). Although TH2-mediated responses are clearly implicated in exacerbations, it is unclear how responses to RV infection interact with TH2 immunity to enhance disease in allergic asthma to cause exacerbations. Bronchial epithelium is likely to be critical for this interaction because it responds to both allergen (14) and RV infection (15) and has the capacity to produce cytokines that are potent activators of type 2 immunity such as IL-25 (16).

The IL-17 family member, IL-25 (IL-17E), has been identified as an initiator and regulator of type 2 immunity and plays a role in asthma pathogenesis (1719). Clinical studies have demonstrated increased IL-25 gene expression together with its receptor, IL-17RB, in tissue from patients with asthma (20) and atopic dermatitis (21), whereas eosinophils, mast cells, and the airway epithelium (17, 22) have been reported as sources within the lung. Blocking IL-25 in a mouse model of allergic airway disease before allergen sensitization and/or challenge resulted in a marked reduction in allergic inflammation, AHR, and type 2 cytokine (IL-5 and IL-13) production (18). IL-25 can augment type 2 cytokine production via activation of IL-17RB–expressing immune cells including TH2 cells (20) and type 2 innate lymphoid cells (ILC-2) (23).

Respiratory syncytial virus (RSV) infection has been shown to induce IL-25 in mouse lungs (24), but most asthma exacerbations are precipitated by RV infections, with only a small minority caused by RSV infections. The role of IL-25 in RV-induced asthma exacerbations is unknown. We therefore hypothesized that RV-induced IL-25 was required for the immune cascade, leading to increased type 2 immune responses and allergic airway inflammation during RV-induced asthma exacerbations.

We used human in vitro and in vivo RV infection studies using asthmatic and healthy volunteers to show that asthmatics have a greater capacity to express IL-25, which correlated with atopic status. Studies using mouse models confirmed that RV infection can induce lung IL-25 expression and augment allergen-driven IL-25 in an asthma exacerbation model characterized by increased type 2 cytokine production and recruitment of cells associated with type 2 immunity such as basophils (CD3, CD4, FcɛRI+, CD49b+, ST2+, and IL-4+) and IL-25 receptor–expressing non-T type 2 cells (CD3, CD4, CD49b, ICOS+, ST2+, and IL-17RB+) and TH2 cells (CD3+, CD4+, and IL-4+). Using an IL-25 receptor blocking antibody, we showed that RV-augmented IL-25 caused increased innate and adaptive type 2 immune responses and associated pulmonary allergic inflammation. We have identified that heightened IL-25 expression occurs during RV infection in asthma. Supporting mouse studies have defined a role for IL-25 in the immunopathogenesis of virally induced exacerbation of pulmonary allergic inflammation.

RESULTS

Asthmatic bronchial epithelial cells express increased IL-25 in response to RV infection in vitro

Human bronchial epithelial cells (BECs) were obtained from 10 atopic asthmatic and 10 nonatopic nonasthmatic healthy volunteers and infected with RV-1B in vitro (inclusion criteria described in table S1). There were no significant differences between uninfected (media control, M) asthmatic and healthy Il25 mRNA expression levels. Significant up-regulation of Il25 gene expression was detected in RV-infected asthmatic BECs at 8 hours after infection compared to mock-infected cells. About 10-fold greater Il25 mRNA levels in RV-infected BECs from asthmatic donors compared to those from healthy controls were observed. By 24 hours after infection, Il25 mRNA levels had returned to baseline (Fig. 1A). The peak of Il25 mRNA at 8 hours was followed by significantly increased IL-25 protein levels in the supernatant of RV-infected asthmatic BECs 24 hours after infection, whereas no significant increase over uninfected cells was observed with RV-infected healthy BECs at 8 or 24 hours after infection (Fig. 1B).

Fig. 1. Asthmatics expressed greater levels of IL-25 in response to RV infection in vitro and in vivo.

Cultured BECs obtained from 10 asthmatic and 10 healthy volunteers were infected with RV-1B (RV) or mock-infected (M). (A and B) Quantification of IL-25 (A) mRNA and (B) secreted protein levels at 8 and 24 hours post-infection (p.i.), as assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. (C) Correlation of IL-25 protein levels at 24 hours after infection with the number of positive SPTs in asthmatics. In another study, 28 asthmatic and 11 healthy human volunteers were experimentally infected with RV-16. (D) Baseline (BL) and peak IL-25 protein levels in the nasal mucosal fluid after RV-16 infection, quantified by Meso Scale Discovery (MSD) platform. Any value less than 10 pg/ml was given a value of zero. *P < 0.05 and **P < 0.01 as indicated (A and B), and *P < 0.05 and ***P < 0.001 baseline versus infection peak (D). All data represent means ± SEM qRT-PCR and ELISA data analyzed by one-way analysis of variance (ANOVA) with Bonferroni post-test. Baseline compared to peak infection IL-25 protein data were analyzed by Wilcoxon signed rank test. Correlations were assessed with linear regression and Spearman’s coefficient (r) value.

To assess if RV-induced IL-25 production by BECs in vitro was related to markers of allergy or disease severity, correlation analyses with serum immunoglobulin E (IgE) levels, skin prick test (SPT) positivity, FEV1 (forced expiratory volume in 1 s)/FVC (forced vital capacity) ratio, PC20, and the number of recorded asthma exacerbations per year were performed. Of these, increased RV-induced IL-25 expression was associated with sensitization to a greater number of common environmental allergens as indicated by positive wheal and flare responses in positive SPT scores (Fig. 1C), and there was a trend toward greater IL-25 levels in those with more frequent asthma exacerbations (r = 0.61, P = 0.067; table S2).

Experimental RV infection in vivo induces high levels of IL-25 expression in asthmatics

Next, we investigated IL-25 expression in the airways of 28 mild to moderate atopic asthmatic subjects and 11 nonasthmatic (healthy) subjects (inclusion criteria and baseline percentages of BAL eosinophils, neutrophils, and lymphocytes for all subjects are shown in table S3) during in vivo experimental RV-16 infection. Secreted IL-25 in the nasal mucosal fluid was sampled by the nasosorption technique before infection (baseline) and on several days up to 10 days after infection. Baseline IL-25 levels were higher in asthmatics, but this difference was not statistically significant (P = 0.220, Mann-Whitney test; n = 28 asthmatic and 11 healthy subjects). During RV infection, 61.0% (17 of 28) of asthmatics had increased levels of IL-25, and the increase was significant (P < 0.001, Wilcoxon rank sum test; n = 28). The nonasthmatic subjects also had a small increase in IL-25 during infection (P = 0.018, Wilcoxon rank sum test; n = 11), and peak levels of IL-25 during infection were much higher in asthmatics compared to healthy volunteers, but this difference was not statistically significant (P = 0.351, Mann-Whitney test; n = 28 and 11) (Fig. 1D).

RV infection augments IL-25 expression and allergic pulmonary inflammation

Having identified a link between RV infection, IL-25 expression, and disease severity in human asthmatic subjects, we moved to mouse models to identify potential mechanisms. We used a previously reported model of RV-exacerbated pulmonary allergic inflammation involving systemic ovalbumin (OVA) protein sensitization followed by intranasal OVA challenges and RV-1B infection (12). Sensitized mice were either challenged with OVA protein or sham-challenged with phosphate-buffered saline (PBS), once daily over three consecutive days. After the third OVA/PBS treatment, each group was either infected with RV-1B (RV) or mock-infected with UV (ultraviolet)–inactivated RV (UV). We measured inflammatory cells (macrophages, eosinophils, and lymphocytes) in the BAL to confirm viral-induced exacerbation of airway inflammation in the model (fig. S1). OVA-challenged groups (RV-OVA and UV-OVA) had elevated Il25 mRNA levels compared to nonallergic controls (RV-PBS and UV-PBS) (Fig. 2A). The combination of RV infection during allergic airway inflammation in RV-OVA mice further significantly enhanced Il25 gene expression by about 28-fold at 10 hours after infection compared to UV-OVA mice. Increased gene expression coincided with elevated lung protein, which was significant at day 1 after infection with a trend for prolonged increased expression observed at 2 and 4 days after infection (Fig. 2A). Although at significantly lower levels to that in allergen-challenged mice, we observed that RV infection alone (RV-PBS) induced IL-25 protein expression above that in unchallenged/uninfected (UV-PBS) negative controls, which was significant on day 4.

Fig. 2. RV infection increased pulmonary IL-25 expression, which was associated with increased type 2 cytokine production, basophil recruitment, and increased viral load in mice with allergic pulmonary inflammation.

OVA-sensitized mice were challenged intranasally with OVA or PBS before infection with RV-1B (RV-OVA or RV-PBS) or UV-inactivated RV-1B (UV-OVA or UV-PBS) (n = 5 per group). (A) Quantification of IL-25 mRNA and protein levels in lung tissue at the indicated time points after infection, as assessed by qRT-PCR and ELISA. (B) IL-25 protein expression in lung sections at day 2 after infection, as assessed by IHC. Black arrows indicate subepithelial IL-25+ inflammatory cells in OVA-challenged mice. Open arrows indicate areas of IL-25+ epithelium in RV-infected mice. Scale bar, 20 μm. Epithelial IL-25 staining intensity and number of IL-25+ inflammatory cells were measured and plotted. IHC data represent means ± SEM for three mice per treatment group. *P < 0.05 (unpaired t test) for RV-OVA versus indicated group. (C and D) Level of the type 2 cytokines IL-4, IL-5, and IL-13 protein levels in (C) BAL fluid and (D) lung homogenate by ELISA. (E) Total number of BAL and lung IL-4–expressing basophils at 1 day after infection, as assessed by flow cytometry. (F) Viral RNA in lung tissue for RV-infected mice, quantified by qRT-PCR. *P < 0.05, **P < 0.01, and ***P < 0.001 for RV-OVA versus UV-OVA, ##P < 0.01 for RV-PBS versus UV-PBS; ns, not significant. ELISA and qPCR results were analyzed by ANOVA, and differences between groups were identified using Bonferroni post-test.

To determine the cellular source of IL-25 in the lung during allergen challenge and RV infection, lung sections were taken at 2 days after infection for immunohistochemistry (IHC) analysis. Lung sections probed with an isotype control antibody instead of anti–IL-25 showed no IL-25 staining (fig. S2A). Epithelial expression of IL-25 was significantly higher in the lungs of RV-infected mice (RV-OVA and RV-PBS), whereas epithelium in large airways exhibited the strongest IL-25 expression. Bronchiolar and alveolar epithelium also expressed IL-25 during RV-1B infection (fig. S2B). Subepithelial IL-25+ inflammatory cells were frequently detectable in the lungs of OVA-challenged mice (RV-OVA and UV-OVA) and, when quantitated, were significantly increased in RV-OVA mice compared to non–allergen-challenged groups (Fig. 2B). These data are consistent with maximal IL-25 gene and protein observed in RV-OVA mice linked to combined expression by BECs and infiltrating immune cells.

Exacerbated IL-25 was associated with increased pulmonary type 2 cytokine expression. IL-4, IL-5, and IL-13 were only detectable after allergen challenge and RV-1B infection, with significantly enhanced levels of IL-4 and IL-13 at 10 hours and IL-5 in the BAL fluid at 24 hours after infection as compared to UV-OVA mice (Fig. 2C). Type 2 cytokines in lung tissue exhibited a prolonged expression profile similar to IL-25 with increased IL-4 and IL-5 levels evident at day 4 after infection (Fig. 2D). Basophils are a key component of allergic responses, present in asthmatic lungs and a source of type 2 cytokines. Basophils were identified as CD3CD4FcɛRI+CD49b+ and ST2+ cells (fig. S3). IL-4+ basophils peaked at 1 day after infection in OVA-challenged groups, with very few detected in unchallenged groups, and RV-1B infection resulted in significantly greater accumulation of IL-4+ basophils in the airways of OVA-challenged mice (Fig. 2E). Asthmatics are susceptible to more severe viral infections (25), and our model replicated this with increased viral loads detected in mice with allergic airway inflammation (RV-OVA) compared to nonallergic, infected mice (RV-PBS) (Fig. 2F).

RV induces accumulation of IL-17RB+ T and non-T type 2 cells

Using the same mouse model, we detected IL-25 receptor (IL-17RB+)–expressing cells in total leukocyte preparations from lung tissue and BAL (representative flow plots for each shown in Fig. 3A). We observed that virus infection exacerbated IL-17RB+ lung cells at early (day 1) and late (day 7) times after infection of OVA-challenged mice. Significantly increased IL-17RB+ BAL cells in this group were observed at day 1 only (Fig. 3A). To determine whether IL-25 receptor–expressing TH2 cells contributed to this response, we measured CD3+, CD4+, and IL-4+ T cells (fig. S4). At day 1, IL-4–expressing TH2 cells constituted 5% of the total IL-17RB+ signal in the lung. By day 7, TH2 cells accounted for about 30% of IL-17RB+ lung cells and were significantly higher in virally exacerbated mice. At day 1, BAL IL-17RB+ TH2 cells were exacerbated in RV-infected, OVA-challenged mice. Similar to the lung, IL-4+ TH2 cells accounted for about 5% of BAL IL-17RB+ cells at day 1, rising to about 30% by day 7 (Fig. 3B). Excluding T cells, which express ICOS, and natural killer (NK) cells (CD3, CD4, DX5) and selecting ICOS+ and IL-33 receptor–expressing (ST2+) cells allowed us to identify non-T type 2 cells (fig. S5). These cells were significantly more abundant in the lungs of RV-OVA–treated mice at day 1 compared to other groups (Fig. 4A). About 10% of these cells were IL-17RB+, with significantly greater numbers observed in the lung tissue and airways of virally infected, allergic mice compared to noninfected allergic controls at day 1 (Fig. 4B). T o determine whether these cells were likely to include ILC-2, we analyzed ICOS expression by lung and mediastinal lymph node leukocytes after allergen challenge and observed that almost all (>90%) ICOS+ cells were either T cells or ILC-2 (fig. S6).

Fig. 3. RV-induced accumulation of IL-25–responsive cells in mice with allergic pulmonary inflammation.

OVA-sensitized mice were challenged intranasally with OVA or PBS before infection with RV-1B (RV-OVA or RV-PBS) or UV-inactivated RV-1B (UV-OVA or UV-PBS) (n = 5 per group). (A) Representative flow plots of total IL-17RB+ leukocytes in lung and BAL and enumerated at days 1 and 7. (B) IL-4–expressing IL-17RB+ CD4+ T cells at 1 and 7 days after infection in lung and BAL measured by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. All data analyzed by ANOVA represent means ± SEM and are representative of two to three studies, and differences between groups were identified using Bonferroni post-test.

Fig. 4. RV-induced accumulation of IL-25-responsive non-T type 2 cells in mice with allergic pulmonary inflammation.

OVA-sensitized mice were challenged intranasally with OVA or PBS before infection with RV-1B (RV-OVA or RV-PBS) or UV-inactivated RV-1B (UV-OVA or UV-PBS) (n = 5 per group). (A and B) Representative flow cytometry plots and total BAL and lung numbers of (A) non-T/non-NK (from CD3, CD4, and DX5 gated cells) ICOS+/ST2+ cells and (B) IL-17RB+ non-T type 2 cells at 1 and 7 days after infection. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. All data analyzed by ANOVA represent means ± SEM and are representative of two to three studies, and differences between groups were identified using Bonferroni post-test.

IL-25 is necessary for RV-exacerbated allergic pulmonary inflammation

We used an IL-25 receptor blocking mouse monoclonal antibody [α-IL-17RB, clone D9.2 (23)] to define the role of IL-25 in the exacerbated inflammatory responses observed in OVA-challenged, RV-infected mice. Mice were treated with antibody after OVA challenges before and during RV-1B infection to specifically target virally augmented IL-25 and assess IL-25 regulation of inflammatory mediators. Blocking the IL-25 receptor reversed the RV-exacerbated expression of IL-4, IL-5, and IL-13 to levels similar to allergen-challenged, uninfected mice (Fig. 5A). IL-6 has also been associated with type 2 immune responses (26). Consistent with this, we observed that OVA challenge alone induced IL-6 protein expression, which was markedly increased (10-fold) by RV infection. However, in contrast to prototypic type 2 cytokines (IL-4, IL-5, and IL-13), IL-25R blockade further increased IL-6 expression in RV-exacerbated mice (Fig. 5B). RV-enhanced expression of the eosinophil-recruiting chemokines CCL11 and CCL24 in allergen-challenged mice was partially reduced with IL-17RB blocking, and a smaller reduction in CCL24 levels was also observed in UV-OVA–treated mice (Fig. 5C).

Fig. 5. Blocking IL-25 signaling attenuated OVA-induced and RV-exacerbated expression of type 2 mediators in mice with allergic pulmonary inflammation.

OVA-sensitized mice were challenged intranasally with OVA or PBS followed by intraperitoneal injection of anti–IL-17RB blocking antibody or isotype control (Ig) 4 hours before and 3 and 5 days after infection with RV-1B or UV-inactivated RV-1B (n = 5 per group). (A) Type 2 cytokines IL-4, IL-5, and IL-13 at 8 hours after infection. (B) Proinflammatory cytokine IL-6 at 8 hours after infection. (C) Eosinophil-recruiting chemokines CCL11 at 8 hours after infection and CCL24 at 24 hours after infection. (D) Type 2–associated epithelial-derived cytokines IL-25, IL-33, and TSLP in lung homogenate supernatant at 24 hours after infection. (E) Total serum IgE levels at 7 days after infection by ELISA. (F) Viral RNA in lung homogenate at 10 hours after infection, as assessed by qRT-PCR. (G) MUC5ac protein in the BAL fluid at 7 days after infection. All protein mediators in BAL and lung homogenate were assessed by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. All data analyzed by ANOVA represent means ± SEM and are representative of two to three studies, and differences between groups were identified using Bonferroni post-test.

Like IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) are type 2 immunity-promoting cytokines. For RV-OVA mice, IL-25 protein levels in lung tissue were reduced to UV-OVA levels with IL-17RB blocking by 24 hours after infection (Fig. 5D). Lung protein levels of IL-33 and TSLP were also reduced; however, this effect was observed in all OVA-challenged groups regardless of RV infection (Fig. 5D). Increased IgE contributes to heightened allergen-driven inflammation. Allergen-challenged RV-infected mice had the highest levels of serum IgE at 7 days after infection, and a trend for reduction was observed with IL-17RB blocking; however, the differences observed were not statistically significant (Fig. 5E). In addition to effector functions that drive allergic inflammation, type 2 cytokines can interfere with antiviral responses (27). It was possible that the increased viral loads observed in RV-infected allergic mice were due to IL-25–augmented type 2 responses suppressing antiviral immunity. In support of this, we observed decreased levels of viral RNA with inhibition of IL-25 signaling (Fig. 5F). MUC5ac protein is a principal constituent of respiratory mucus and is induced by type 2 cytokines and associated with asthma exacerbations (28). Virally enhanced MUC5ac was blocked by anti–IL-17RB, such that expression was restricted to levels similar to allergen-challenged mice (Fig. 5G). These data identify the role of virus-induced IL-25 in the exacerbation of allergen-driven cellular inflammation and type 2 mediator expression and effector function.

IL-17RB blockade ablates viral recruitment of innate and adaptive type 2 cell populations

We next assessed the impact of IL-17RB blockade on RV-exacerbated cellular pulmonary inflammation. Exacerbated airway neutrophil, eosinophil, and lymphocyte responses are features of RV-induced asthma exacerbations. Blocking IL-17RB significantly reduced airway neutrophilia in RV-OVA–treated mice and inhibited the RV-exacerbated airway eosinophil and lymphocyte responses to levels that were not statistically different to that observed in UV-OVA–treated mice (Fig. 6A). IL-4+ basophil numbers were also significantly reduced by α-IL-17RB in the exacerbated airways of RV-OVA mice (Fig. 6B).

Fig. 6. Blocking IL-25 signaling ablated RV-exacerbated type 2 leukocytic airway inflammation.

OVA-sensitized mice were challenged intranasally with OVA or PBS followed by intraperitoneal injection of anti–IL-17RB blocking antibody or isotype control (Ig) 4 hours before and 3 and 5 days after infection with RV-1B or UV–RV-1B (n = 5 per group). (A) Total number of BAL neutrophils and eosinophils at day 1 after infection and lymphocytes at day 7 after infection, as assessed by differential cell counts. (B to D) Total numbers of BAL (B) IL-4+ basophils 1 day after infection, (C) non-T (from CD3, CD4, and DX5 gated cells) ICOS+/ST2+ cells at 8 hours after infection, and (D) IL-4+ CD4+ T cells at 7 days after infection, as assessed by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. All data analyzed by ANOVA represent means ± SEM and are representative of two to three studies, and differences between groups were identified using Bonferroni post-test.

The α-IL-17RB antibody used to block the IL-25 receptor was the same clone used in previous experiments to detect IL-17RB expression by flow cytometry, and as a result, we were unable to measure IL-17RB expression in these experiments. However, we detected non-T type 2 cells by gating on CD4 cells that coexpressed ICOS and ST2. As early as 8 hours, we observed a significant decrease in virally exacerbated numbers of these cells after IL-25 receptor blocking (Fig. 6C). Similar results were observed later at day 7 for IL-4–expressing CD4+ T (TH2) cells (Fig. 6D). These data demonstrate the role of RV-enhanced IL-25 signaling in the exacerbation of both early and late innate and adaptive type 2 cellular responses in mice with allergic pulmonary inflammation.

DISCUSSION

Most studies investigating IL-25 have demonstrated expression in response to type 2–inducing stimuli such as parasitic helminth infections and allergen. No study to date has undertaken a detailed analysis of IL-25 protein expression in vivo during respiratory virus infection despite the role viruses play in allergic diseases such as asthma. More than 160 genetically distinct RV types belonging to three species (A, B, and C) are now recognized (29). We used species A viruses (RV-16 and RV-1B) for these studies because these have been shown to be more frequently associated with lower respiratory tract disease compared to viruses belonging to species B. RV C viruses have been detected at a similar rate to species A viruses during severe respiratory disease. RV C viruses were not used in this study because these viruses cannot be grown and purified using conventional virological techniques (30).

Using a human infection model, we were able to show that RV was capable of inducing IL-25 in the upper respiratory tract. Nasal mucosa protein analyses revealed that 20 of 22 asthmatic subjects had detectably increased IL-25 during experimental RV infection. Viral induction of IL-25 was clearly less robust in healthy subjects. Baseline and infection peak levels were also significantly higher in asthma, indicating that although RV infection could induce IL-25 expression in healthy and asthmatic subjects, asthmatic airways had a significantly increased propensity to produce this cytokine. To investigate if greater IL-25 expression was a consequence of the in vivo environment characteristic of asthma such as increased type 2 immunity or atopy, we conducted in vitro studies using serially passaged BECs from healthy and asthmatic donors. RV-infected asthmatic BECs had greater intrinsic capacity for IL-25 gene and protein expression compared to nonasthmatic cells. To further assess this possibility, we related IL-25 induction by BECs to markers of allergy or of asthma severity. IL-25 levels were significantly related to SPT positivity, and there was a trend toward greater IL-25 levels in those with more frequent asthma exacerbations. Because we only had 10 subjects with asthma in this part of the study, we feel that the lack of statistically significant correlations of IL-25 with markers of true asthma severity, rather than SPTs, which are a marker of allergy severity, likely relates to the small number of subjects studied. Although several studies using this in vitro RV infection model have identified deficiencies in expression of antiviral molecules (3133), this is the first report of increased expression of a pro–type 2 cytokine by virally infected cultured asthmatic BECs. The fact that increased expression was observed directly in vivo and in vitro raises the possibility that both extrinsic (such as immune) and intrinsic (such as epigenetic) factors are present in asthma and are involved in the heightened IL-25 response.

Treatment of mice with recombinant IL-25 induced type 2 cytokine expression by T and non-T cell populations (34). Forced expression of IL-25 by transgenic mice has been reported to enhance allergen-induced type 2 responses and pulmonary allergic inflammation (35). The OVA mouse model is designed to investigate type 2–driven allergic asthma, which represents at least 50% of adult asthmatics based on gene expression studies of BECs (8). The mouse model is different to human asthma in that the mice are systemically sensitized to a model allergen that is not a common aeroallergen in humans. However, it does provide a model system in which to investigate the interaction of type 2 immunity and RV infection in vivo, which we believe is mechanistically important in the pathogenesis of exacerbations for many asthmatics. To begin to better understand the role of IL-25 during viral exacerbations, we examined IL-25 receptor–expressing cell populations. We examined an early (day 1 after infection) time point for acute responses at the peak of infection and a later time point (day 7 after infection) to determine the duration of the response. At both times, we observed significantly increased numbers of IL-17RB–expressing lung leukocytes in virally exacerbated mice. At day 1, IL-4–expressing T cells accounted for about 5% of the IL-17RB–expressing leukocytes. By day 7, these cells represented about 30% of the IL-25–responsive cells in the lung, suggesting that TH2 cells were more important for later responses mediated by IL-25, potentially as a consequence of ongoing amplification of type 2 immune cascade. Although our focus was IL-4 expression as a marker of type 2 T cell responses, it is possible that IL-17RB+ CD4+ T cells expressing other type 2 cytokines such as IL-5 and IL-13 were also contributing to virally exacerbated type 2 cytokine production.

We also observed that non-T type 2 cells expressing IL-25 receptor were associated with virally exacerbated pulmonary inflammation. Previous studies have demonstrated that ILC-2 cells do not express any conventional lineage markers, but do express ICOS and the IL-33 receptor (ST2) (36, 37). To investigate the role of non-T type 2 cells, we identified a population of cells that were not T cells (CD3, CD4) and expressed ICOS and ST2. Although we did not use an exhaustive lineage panel to identify ILC-2 cells, the fact that ICOS is expressed almost exclusively on T cells (particularly TH2) (3840) and ILC-2 cells meant that we could approximate the number of ILC-2 cells by identifying the CD3, CD4, and ICOS+ ST2+ population. In our model, about 10% of these cells coexpressed the IL-25 receptor at day 1 and were significantly increased during viral exacerbation. We have previously reported that repeated dosing with recombinant IL-25 protein expands ILC-2 numbers in naïve mice (23). Here, RV-induced IL-25 did not induce similar levels of non-T type 2 cell expansion (most likely because the amount of recombinant IL-25 administered in previous studies far exceeded that produced in vivo in our studies). We did observe evidence of expansion as the number of lung leukocytes expressing IL-25 receptor on day 7 was double that observed on day 1. We have observed that RV-1B infection during allergic airway inflammation augments expression of leukocyte-recruiting chemokines such as CCL5, CCL17, and CCL22 (15). This provides a possible mechanism for augmented recruitment of ILC-2s and later TH2 cells, which we report here to express the IL-25 receptor and are sensitive to IL-25 receptor blockade. Thus, we propose that virally increased pulmonary IL-25 can promote survival, activation, and up-regulated type 2 cytokine expression by these cells, which are central to asthma exacerbation pathogenesis.

Although further studies are needed to identify all IL-25 receptor–expressing cells, we provide good evidence that RV-augmented IL-25 influences multiple T and non-T type 2 cellular responses. Other studies have begun to investigate mechanisms linking antiviral immunity and type 2 responses via deletion of key antiviral mechanisms. Depletion of NK cells and associated loss of interferon-γ (IFN-γ) expression lead to emergence of type 2 responses including IL-25 production during RSV infection (22). Neonatal mice lacking TLR7 infected with pneumonia virus of mice also exhibited exaggerated type 2 responses, which included prolonged IL-25 protein in the BAL (41). In both paramyxovirus infection models, viral induction of IL-25 was dependent on suppression of antiviral immunity. If you consider that asthma is characterized by chronic activation of type 2 immunity and therefore represents a state of antiviral immune suppression, then our observation of increased RV load associated with increased IL-25 and type 2 immune responses (which was reversed with blocking IL-25 receptor) is consistent with the two studies mentioned above. That is, there is critical balance between type 2 and antiviral immunity, which underpins the immunopathogenesis of virally exacerbated allergic diseases.

Blocking IL-25 had the dual effect of suppressing type 2 responses and viral infection, new evidence that IL-25 and type 2 immunity have two important immune-pathogenic roles: promote allergic inflammation and interfere with antiviral responses. This paradigm has important implications for allergic diseases that are exacerbated by viral infection such as asthma. Targeting type 2–promoting cytokines such as IL-25 has the potential to simultaneously suppress allergic inflammation and promote antiviral immunity. This paradigm dictates that therapies that either promote IFN production or inhibit type 2 responses could have therapeutic benefit. Both have recently been reported by separate human asthma clinical studies observing reduced exacerbation frequency or severity with either antibody-mediated blocking of TH2 cytokines (42, 43) or treatment with IFN-β (Synairgen, press release 2013).

This study has a number of limitations and caveats. The in vitro studies used submerged BEC monolayers, which are not differentiated. Air-liquid interface (ALI)–differentiated cell cultures contain fully differentiated ciliated epithelial cells, which may provide better insight into the interaction of RV with the asthmatic airway. It will be interesting to investigate whether cytokines differentially expressed by submerged culture asthmatic epithelial cells are similarly dysregulated in ALI cultures during RV infection. The use of ALI cultures might also enable RV-C viruses to be studied to determine whether common pathogenic mechanisms such as modulation of type 2 immunity by IL-25 are important across RV species. We showed infection-augmented IL-25 was associated with increased numbers of basophils and T and non-T type 2 cells, all of which potentially contributed to increased type 2 cytokine expression. Additional studies involving specific blockade or depletion of these cell populations will determine how IL-25 regulates pulmonary type 2 cytokine expression. The antibody that we used for these studies blocks IL-17RB. This receptor specifically binds both IL-17B and IL-17E/IL-25; thus, there is a possibility that certain effects observed could be mediated through blocking IL-17B binding to IL-17BR, as well as through blocking IL-17E/IL-25 binding to IL-17RB. Members of our laboratory have been unable to identify a robust IL-17B bioassay in which we could assess the effect of the anti–IL-17BR antibody on IL-17B function in vitro or in vivo.

Our studies identify blockade of IL-25 or its receptor as an attractive target for therapeutic development for asthma exacerbations. This approach should potentially be more effective than the above approaches targeting single TH2 cytokines, because anti–IL-25 should block induction of all TH2 cytokines. We believe that such therapies should urgently be investigated in the human experimental RV-induced asthma exacerbation model to determine whether RV-induced asthma exacerbations are indeed affected as our data suggest they should be. Further, because RSV infection has also been shown to induce IL-25 (24), it is likely that anti–IL-25 therapy would affect asthma exacerbations induced by other virus types, as well as those precipitated by RV infections, thus making this a broadly applicable potential therapy.

In summary, we provide substantial new insight into the biological activity of IL-25. Our data support a mechanism whereby RV up-regulates IL-25 expression by susceptible asthmatic epithelial cells. Augmented IL-25 can potently amplify the type 2 immune cascade involving activation of IL-25 receptor–expressing non-T type 2 cells and TH2 cells. This study also provides evidence to support therapies that target virally induced mediators such as IL-25 that are upstream of type 2 cytokine expression.

MATERIALS AND METHODS

Study design

The study objectives were to use human and mouse in vivo models of RV infection in asthma to investigate virus-induced and augmented IL-25 expression in allergic airways and its role in the exacerbation of type 2 immune responses, and identify IL-25 as a potential therapeutic target. Human subjects from whom BECs were obtained and those who took part in the experimental in vivo RV infection study were either healthy or asthmatic volunteers. Clinical characteristics of volunteers recruited by strict inclusion/exclusion criteria are summarized in tables S1 and S3.

For all work, human and mouse samples were blinded to the scientists assessing/quantifying the results (for example, counting stained cells from cytoslides). All controlled mouse experiments were replicated as indicated. Five mice per experimental group permitted reliable statistical analysis of data by two-way ANOVA, in addition to complying with the UK Home Office regulations to reduce total numbers of animals used where possible. The time points were selected on the basis of preliminary studies. No data were excluded (including outliers).

Viruses

RV-16 for the human challenge study was obtained from clinical isolates as previously described (11, 44), and RV-1B and RV-16 (American Type Culture Collection) were grown and titrated in HeLa cells (European Collection of Cell Cultures) by standard methods. For in vivo use in mice, virus was purified as previously described (12). Virus was inactivated by exposure to UV light at 1200 mJ/cm2 for 30 min.

RV infection of human BECs

BECs were obtained from 10 moderate atopic asthmatics [according to the BTS (British Thoracic Society) and GINA (Global Initiative for Asthma) guidelines (45, 46)] and 10 nonatopic nonasthmatic healthy volunteers. Recorded clinical characteristics of the volunteers are summarized in table S1. In brief, a subsection of the bronchial wall was scraped 5 to 10 times to obtain BECs that were seeded into flasks in BEGM with supplements according to the suppliers’ recommended protocol (Clonetics). At passages 2 and 3, cells were seeded into 12-well plates. At 80% confluency, cells were infected with RV-1B (MOI 2) or treated with PBS as a control for 1 hour with shaking, after which medium was replaced and supernatants and lysates were harvested at the relevant time points after infection. All subjects gave written informed consent, and the St. Mary’s Hospital Ethics Committee approved the study. Bronchoscopies were carried out at St. Mary’s Hospital, London, in accordance with standard guidelines (11).

Human experimental RV infection

Twenty-eight mild and moderate atopic asthmatics and 11 nonasthmatic healthy volunteers, baseline clinical characteristics summarized in table S3, were infected with RV-16 (100 TCID50) diluted in 250 μl of 0.9% saline, via an atomizer into both nostrils. Soluble mediators in the fluid lining the nasal mucosa were sampled with the nasosorption technique before infection (baseline) and at days 2, 3, 4, 5, 7, and 10 after inoculation. All subjects gave written informed consent, and the St. Mary’s Hospital Ethics Committee approved the study. Bronchoscopies were carried out at St. Mary’s Hospital, London, in accordance with standard guidelines (11).

Mouse model of RV-induced exacerbation of allergic airway inflammation

All experiments involving mice were in accordance with legislation outlined by the Home Office. We purchased 6- to 8-week-old female BALB/c mice (Charles River). The model used was previously described (12). In brief, mice were sensitized intraperitoneally with 50 μg of OVA (Calbiochem) and 2 mg of aluminum hydroxide in 200 μl of PBS on day −13. Lightly anesthetized (isoflurane) mice were challenged intranasally with 50 μg of OVA in 30 μl of PBS or PBS alone on days −2, −1, and 0, immediately followed by infection with 50 μl (2 × 106 TCID50) of RV-1B or UV–RV-1B on day 0. Mice were sacrificed at various times after infection for endpoint analyses. To block RV-induced IL-25 signaling in the mouse model of RV-induced exacerbation of allergic airway inflammation, 0.5 mg of anti-mouse IL-17RB antibody (clone D9.2) [provided by A. McKenzie, MRC Laboratory of Molecular Biology (LMB)] was administered intraperitoneally in 200 μl of PBS 4 hours before infection and on 3 and 5 days after infection. As a control, 0.5 mg of anti–c-Myc mouse IgG1 isotype control antibody (clone 9e10.2) (also provided by A. McKenzie, MRC LMB) was used. Airway cells were isolated from the BAL fluid after brief centrifugation, followed by red blood cell lysis using ACK (ammonium-chloride-potassium) buffer, washing, and resuspension in 1 ml of RPMI. Cells were stained with Quick-Diff (Reagena) for differential cell counts.

Flow cytometry

Mouse lungs were crudely dissociated using the gentleMACS tissue dissociator (Miltenyi Biotec) and digested upon incubation at 37°C in RPMI containing collagenase type XI (1 mg/ml) and bovine pancreatic DNase (deoxyribonuclease) type IV (80 U/ml) (both Sigma-Aldrich). A single-cell suspension was attained upon further gentleMACS dissociation, followed by treatment with ACK lysis buffer and filtration through a 100-μm cell strainer. For IL-4 expression analysis by intracellular cytokine staining (ICS), BAL and lung cells were stimulated for 4 hours with PMA (phorbol 12-myristate 13-acetate) (50 ng/ml) and ionomycin (500 ng/ml) (both Sigma-Aldrich) in the presence of monensin (BD GolgiStop, BD Biosciences). Staining with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit identified dead cells (Invitrogen). Cells were incubated with Fc Block (BD Biosciences) before staining for surface markers using anti-mouse antibodies for CD3 (500A2, eBioscience), CD4 (RM4-5, BioLegend), CD49b (DX5, BioLegend), FcɛR1 (MAR-1, eBioscience), ICOS (C398.4 A, BioLegend), T1/ST2 (DJB, MD Biosciences), and IL-17RB (D9.2, obtained from A. McKenzie, MRC LMB). ICS was performed by standard techniques and according to the manufacturer’s instructions (BD Biosciences Fix/Perm kit), and cells were stained with anti-mouse IL-4 (11B11, BioLegend) antibody. Data were collected on a BD Biosciences Fortessa flow cytometer and analyzed with FlowJo software (Tree Star).

Ragweed administration and identification of ICOS+ cells

Wild-type C57BL/6 mice were lightly anesthetized with isoflurane and immunized with 50 μl of short ragweed pollen (100 μg per dose of protein, GREER Laboratories) intranasally for four consecutive days. Mice were euthanized 24 hours after final administration, and tissues were collected for analysis. Lung tissues were chopped into small pieces and then incubated with collagenase D (720 μg/ml) (Amersham, Bucks) for 1 hour. Lung and mediastinal lymph node tissues were mechanically passed through 70-μm cell strainers to obtain single-cell suspensions. Tissue cell suspensions were incubated with anti–Fc receptor blocking antibody (anti-CD16/32, eBioscience, 14-0161-85) and then stained with the following antibody panel: CD19-PerCP-Cy5.5 (eBioscience, 45-0193-82), ICOS–Alexa Fluor 647 (BioLegend, 313516), CD4–Alexa Fluor 700 (eBioscience, 56-0041-80), Fixable Viability Dye eFluor 780 (eBioscience, 65-0865-14), CD8–Brilliant Violet 421 (BioLegend, 100738), and Lineage-PE/Cy7 [CD3 (eBioscience, 25-0031-82), CD11b (eBioscience, 25-0112-82), CD11c (eBioscience, 25-0114-82), FcɛR1 (eBioscience, 25-5898-82), Gr-1 (eBioscience, 25-5931-82), NK1.1 (eBioscience, 25-5941-82), Ter-119 (eBioscience, 25-5921-82)].

Protein quantification

IL-25 in the supernatant of BEC cultures was measured with a human IL-25 ELISA developed in-house by Novartis. IL-25 sampled from the nasal mucosal fluid of experimentally infected human volunteers was analyzed by MSD platform, with the limit of detection at 10 pg/ml. Mouse IFN-α, IFN-β, IFN-λ2/3, IL-4, IL-5, IL-6, IL-13, CCL11, and CCL24 in the BAL fluid and IL-25, IL-33, and TSLP in lung homogenate supernatant were quantified by ELISA (R&D Systems) according to the manufacturer’s specifications. IgE in mouse serum was quantified using the BD OptEIA Mouse IgE Set (BD Biosciences) according to the manufacturer’s specifications.

Immunohistochemistry

Single IHC staining on formalin-fixed and paraffin-embedded mouse lung sections was performed as previously described (15). Formalin-fixed, paraffin-embedded lungs were deparaffinized, and antigen unmasking was carried out by immersing sections in citrate buffer. Sections were then covered with 3% hydrogen peroxidase and left for 5 min before washing with PBS. Cell membranes were permeabilized with 0.1% saponin in PBS, and nonspecific staining was blocked with 5% rabbit serum. Sections were incubated with rat anti-mouse IL-25 antibody (60 μg/ml) (clone 35b) (BioLegend) or a rat IgG isotype control (Sigma-Aldrich). Sections were then washed and incubated with horseradish peroxidase–labeled rabbit anti-rat secondary antibody (Dako) for 1 hour. After a further wash with PBS, sections were incubated with ABC reagent (Vector ABC Kit), and the reaction was visualized with chromogen-fast 3,3′-diaminobenzidine (DAB) as a chromogenic substrate. Slides were counterstained with hematoxylin to provide nuclear and morphological detail, and mounted in DPX Mountant (Sigma-Aldrich). The sections of whole lungs (right + left) were stained for IL-25. All large airways (second and third generations) were scored for epithelial positivity. The average score from all airways was used to express epithelial IL-25 staining intensity for each lung, which was scored semiquantitatively from 0 to 1.5 (0 = negative, 0.5 = weak staining, 1 = moderate staining, and 1.5 = strong staining). The lengths of five airways (one airway per lobe of second- and third-generation airways) were measured and counted for numbers of subepithelial inflammatory cells expressing IL-25. Infiltrating IL-25+ inflammatory cells in the airway lamina propria were quantified and expressed as number of positive inflammatory cells per millimeter length of the reticular basement membrane. All counts on histology sections were performed by one investigator blinded to the treatment protocol.

mRNA quantification

Total RNA was extracted from BECs (RNeasy Kit, Qiagen), and 2 μg was reverse-transcribed for cDNA (complementary DNA) synthesis (Omniscript RT Kit, Qiagen). Total RNA was also extracted from mouse apical lung lobes stored in RNAlater (Qiagen), followed by cDNA synthesis as above. Quantitative PCR was performed with specific primers and probes for each gene and QuantiTect Probe PCR master mix (Qiagen). Mouse IL-25 primers and probe sequences were as follows: sense 5′-CACACCCACCACGCAGAAT-3′ 300 nm, antisense 5′-CAACTCATAGCTCCAAGGAGAGATG-3′ 300 nm, and probe 5′-FAM-CCAGCAAGGATGGCCCCCTCA-TAMRA-3′ 100 nm. Human IL-25 primers and probe sequences were as follows: sense 5′-GAGATATGAGTTGGACAGAGACTTGAA-3′ 300 nm, antisense 5′-CCATGTGGGAGCCTGTCTGTA-3′ 300 nm, and probe 5′-FAM-CTCCCCCAGGACCTGTACCACGC-TAMRA-3′ 100 nm. RV-1B genomic RNA primes and probe sequences were as follows: sense 5′-GTGAAGAGCCSCRTGTGCT-3′ 50 nm, antisense 5′-GCTSCAGGGTTAAGGTTAGCC-3′ 300 nm, and probe 5′-FAM-TGAGTCCTCCGGCCCCTGAATG-TAMRA-3′ 100 nM. An ABI 7500 TaqMan (Applied Biosystems) was used to analyze PCRs. Each gene was normalized to 18S rRNA (ribosomal RNA), was quantified using a standard curve generated by amplification of plasmid DNA, and is expressed as copies per microliter of cDNA reaction.

Statistical analysis

Human data were analyzed by one-way ANOVA using the Kruskal-Wallis test with a 95% confidence interval with Dunn’s multiple comparison test. Correlations were assessed with linear regression and Spearman’s coefficient (r) value. All data from mouse studies are presented as means ± SEM. Studies were conducted with five to six mice per group, and data are representative of at least two independent experiments unless indicated as combined experiments. Where groups were greater than two and/or multiple comparisons were analyzed, results were analyzed by ANOVA and differences between groups were identified using Bonferroni post-test with 95% confidence using GraphPad Prism 6 software. When only two groups were analyzed and one condition was variable, a two-tailed unpaired t test was used to compare groups.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/6/256/256ra134/DC1

Fig. S1. Airway leukocyte infiltration in a model of RV-induced allergic airways disease.

Fig. S2. RV infection induces expression by epithelial cells in large airways, bronchioles, and alveoli.

Fig. S3. Representative flow plots demonstrating the gating strategy for IL-4+ basophils in whole-lung tissue at day 1 after infection.

Fig. S4. Representative flow plots of IL-4+ CD4+ T cells in whole-lung tissue in a model of RV-induced allergic airways disease at day 7 after infection.

Fig. S5. Representative flow plots demonstrating the gating strategy for non-T type 2 cells in whole-lung tissue at day 7 after infection.

Fig. S6. ICOS+ populations in the lung and mediastinal lymph nodes (MedLN) after allergen challenge.

Table S1. Clinical characteristics of volunteers from whom BECs were obtained.

Table S2. Summary of correlations between clinical parameters and IL-25 protein levels 24 hours after RV-1B infection or media treatment of cultured BECs from atopic asthmatics (n = 10).

Table S3. Baseline demographic and clinical characteristics of volunteers for the human experimental RV infection study.

REFERENCES AND NOTES

  1. Acknowledgments: We thank R. Beavil at the MRC Protein Purification (Kings College London, UK) unit for providing purified anti–IL-17RB and isotype control antibodies. Funding: This work was supported by MRC grant numbers G0601236 and G1100168, European Research Council (ERC) FP7 grant 233015, a Chair from Asthma UK (CH11SJ), MRC Centre grant G1000758, National Institute for Health Research Biomedical Research Centre (NIHR BRC) grant P26095, Predicta FP7 Collaborative Project grant 260895, and the NIHR BRC at Imperial College London. Novartis Institute for Biomedical Research funded the IL-25 nasosorption assays. Author contributions: N.W.B., S.L.J., J.B., A.J., A.M., D.J.C., M.R.E., and R.P.W. devised and/or conducted mouse studies, analyzed the data, and prepared the manuscript. S.L.J. devised and J.D.R.M. conducted the human BEC study and provided associated data. S.L.J. devised and D.J.J. conducted the human experimental infection study and analyzed and provided associated data. J.Z. conducted the IHC analyses. Y.M.C. and Y.Y.H. provided flow cytometric analyses. B.S., M.E., and J.W. provided human IL-25 protein data. All authors revised and approved the final version of the manuscript. Competing interests: The authors declare that they have no competing interests.
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