International Immunopharmacology 21 (2014) 156–162
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Adenovirus expressing IFN-λ1 (IL-29) attenuates allergic airway inflammation and airway hyperreactivity in experimental asthma Yan Li a, Qiaoyan Gao a, Xianli Yuan a, Mi Zhou a, Xiao Peng a, Xiaojin Liu b, Xiaoxuan Zheng b, Damo Xu a,b,c,⁎, Mingcai Li a,b,⁎⁎ a b c
Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Immunology, Ningbo University School of Medicine, Ningbo 315211, China Institute of Inflammation and Immune Diseases, Shantou University Medical College, Shantou 515041, China Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK
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Article history: Received 21 November 2013 Received in revised form 18 March 2014 Accepted 23 April 2014 Available online 9 May 2014 Keywords: IFN-λ1 (IL-29) Th2 cells Eosinophils Hyperresponsiveness Airway inflammation
a b s t r a c t Background: Asthma is thought to result from the generation of T helper type 2 (Th2) responses, leading to bronchial inflammation. IFN-λ1 (also known as IL-29) is a recently described member of the IFN-λ family and has been shown to decrease production of Th2 cytokines in vitro. However, the role and mechanism of IFN-λ1 in asthma remain unknown. Objectives: The aim of this study was to clarify the importance of IFN-λ1 in allergen-induced airway hyperresponsiveness (AHR) and inflammation. Methods: We used a murine model for ovalbumin (OVA)-induced asthma to examine the effect of intranasal delivery of recombinant adenovirus expressing human IFN-λ1 (Ad-hIFN-λ1) on AHR and allergic airway inflammation. Results: Intranasal instillation of Ad-hIFN-λ1 before airway antigen challenge in OVA-immunized mice significantly decreased the severity of AHR and numbers of eosinophils and levels of IL-4, IL-5, and IL-13, but not IL-10 and IFN-γ; both in vivo, in the bronchoalveolar lavage fluid and in vitro, following stimulation of lymphocytes from spleens with OVA, compared with administration of a control virus (Ad-mock). Furthermore, Ad-hIFN-λ1 treatment inhibited serum IgE secretion and increased numbers of splenic CD4+CD25+FOXP3 + Treg cells. Histological studies showed that Ad-hIFN-λ1 attenuated OVA-induced lung tissue eosinophilia. Conclusions: These results demonstrate that delivery of the Ad-hIFN-λ1 can mitigate allergic airway inflammation in experimental asthma. The potent immunoregulatory action of IFN-λ1 may offer a novel therapeutic approach to treat allergic asthma. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Allergic asthma is a chronic disorder characterized by eosinophilic airway inflammation, mucus hypersecretion, high levels of T-helper type 2 (Th2) cytokines in bronchoalveolar lavage fluid (BALF), high levels of serum immunoglobulin (Ig) E, increasing airway hyperresponsiveness (AHR), and variable airway obstruction [1]. The process of airway inflammation involves various types of cells, such as eosinophils, mast cells, T lymphocytes, and dendritic cells (DCs) [1,2]. Various methods have been suggested to control the airway inflammation in patients. However, these approaches have limitations in terms of partial effectiveness or serious side effects in long term use [3,4]. Therefore, novel therapeutic approaches that allow a more effective treatment of allergic asthma are urgently desirable. ⁎ Correspondence to: D. Xu, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK. Tel./fax: +86 574 8760 9893. ⁎⁎ Correspondence to: M. Li, Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Immunology, Ningbo University School of Medicine, Ningbo 315211, China. Tel./fax: +86 574 8760 9893. E-mail addresses: [email protected] (D. Xu), [email protected] (M. Li).
http://dx.doi.org/10.1016/j.intimp.2014.04.022 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Interferon (IFN)-λ, including IFN-λ1/as known as interleukin (IL)-29, IFN-λ2/IL-28A, and IFN-λ3/IL-28B, is a newly described group of cytokines distantly related to the type I IFNs and IL-10 family members [5,6]. IFN-λs are now collectively referred to as type III IFNs. In addition to their antiviral and antiproliferative activities, IFN-λs exert immunomodulatory effects that overlap type I IFNs in innate and adaptive arms of the immune system [7]. Several reports have shown that IFN-λ1 plays an important role in the Th2 responses. Gallagher and his colleagues [8] revealed that IFN-λ1 inhibits the production of IL-13 by T cells in an IFN-γ-independent manner, which is mediated in part via monocyte-derived DCs. Furthermore, IFN-λ1 decreases IL-4 and IL-5 production, but its effects are not as consistent as those seen with IL13. Thus, IFN-λ1 appears to be an inhibitor of human Th2 responses whose action is directed primarily toward IL-13, but may also affect Th2 responses in general without invoking complementary elevation of IFN-γ [9]. The ability of IFN-λ1 to influence Th2 cytokine (IL-4, IL-5, and IL-13) secretion may be of direct relevance to the pathogenesis and treatment of asthma [10]. Bullens et al. [11] demonstrated that sputum IFN-λ1 mRNA correlates negatively with asthma symptoms in steroid-naive patients and is significantly higher in steroid-treated
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patients. Therefore, IFN-λ1 could have an immunoprotective role in the lower airways. Conversely, Miller et al. [12] have shown that IFN-λ1 is associated with exacerbation of asthma caused by human rhinoviruses (HRV) and IFN-λ1 levels increase with worsening acute asthma symptoms during HRV infections. Koltsida et al. [13] recently reported IFN-λ2 (IL-28A) modulates lung DC function to promote Th1 cell differentiation in vivo and suppress Th2-mediated responses in experimental allergic asthma. Based on these, it was proposed that IFN-λ1 could be considered as a target for asthma treatment. It's clear that more studies are necessary to determine whether IFN-λ1 should be blocked or promoted in asthma. To investigate the therapeutic potential of IFN-λ1 in allergic asthma, we evaluated the effects of recombinant adenovirus expressing human IFN-λ1 (Ad-hIFN-λ1) on pulmonary inflammation in a murine experimental model of asthma. Our results suggest that intranasal (i.n.) delivery of Ad-hIFN-λ1 attenuates eosinophilic airway inflammation and decreases the severity of AHR by down-regulation of antigen-driven Th2mediated responses and induction of CD4+CD25+Foxp3 + regulatory T (Treg) cells. Therefore replacement or augmentation of IFN-λ1 production could represent a new approach to treatment or prevention of allergic asthma. 2. Materials and methods 2.1. Animals 8- to 10-week-old female BALB/c mice were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China) and maintained under a specific pathogenfree facility at the Experimental Animal Center of Ningbo University. An ovalbumin (OVA)-free diet and water were supplied ad libitum. All animal studies were approved by the Institutional Animal Care and Use Committee of the Ningbo University. 2.2. Sensitization and antigen challenge BALB/c mice were sensitized and challenged as described previously with a slight modification [14]. Briefly, mice were sensitized on days 0 and 14 by intraperitoneal (i.p.) injection of 20 μg of OVA (Grade V; Sigma-Aldrich, St. Louis, MO) emulsified in 2 mg of aluminum hydroxide gel (alum, Sigma) in a total volume of 200 μL. On days 28, 29, and 30 after the initial sensitization, the mice were challenged for 30 min daily with an aerosol of 1% (wt/vol) OVA in sterile phosphate-buffered saline (PBS) (or with PBS as a control) using an ultrasonic nebulizer (Yuyue, Jiangsu, China). Twenty-four hours after the last exposure to OVA or PBS, mice were anesthetized and connected to a computercontrolled small animal ventilator (flexiVent, SCIREQ, Montreal, Canada), and AHR to intravenous methacholine (Mch) (Sigma) was measured as pulmonary resistance (RL) as described previously [15]. BALF, serum, and tissues were obtained for further analyses. 2.3. Adenoviral vectors and administration of Ad-hIFN-λ1 Recombinant adenoviral vector was constructed using the Ad-Easy system that has been described previously [16]. Briefly, the hIFN-λ1 cDNA [17] was inserted into a recombinant adenoviral shuttle vector (pAdTrack-CMV) and cotransformed into Escherichia coli BJ5183 cells with an adenoviral backbone plasmid (pAdEasy-1). Ad-mock was identical with Ad-hIFN-λ1 structurally, but contained no transgene in the expression cassette. The linearized recombinant plasmid was transfected into the adenovirus packaging 293 cell line. Viruses were purified from infected cells 48 h after infection by three freeze–thaw cycles followed by successive banding on cesium chloride density gradient centrifugation. The purified virus was dialyzed and stored at −80 °C until needed. Viral titers were measured by standard end-point dilution assay using
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the 293 cells. Furthermore, hIFN-λ1 expression was confirmed by measuring the culture supernatants of infected 293 cells (data not shown). Mice were anesthetized and held supine at an angle of 45° with pressure applied to the lower mandible to immobilize the tongue and prevent swallowing. Twenty-four hours before the first challenge with OVA (day 27), Ad-hIFN-λ1 (1 × 109 pfu) was introduced to the nasal planum using a micropipet. Control mice received the same dose of control virus (Ad-mock). 2.4. Bronchoalveolar lavage (BAL) BAL sample collection, total cell counts, and cell differential counts were performed as previously described [14]. Briefly, mice were anesthetized with a sodium pentobarbitone, the lungs were lavaged three times with 1 mL normal saline. Total cell and cell differential counts were counted using a hemocytometer and Diff-Quik stain (Fisher Scientific, Pittsburgh, PA), respectively. The supernatants of BALF were stored at −80 °C for cytokine analysis. 2.5. Histopathology Lung tissue was fixed in Carnoy's solution and embedded in paraffin using standard methods. Four-micrometer thick sections were stained with hematoxylin and eosin (H&E) to detect cellular infiltration, and periodic acid-Schiff/Alcian blue (PAS/AB) to detect mucus-secreting goblet cells. 2.6. Enzyme-linked immunosorbent assay (ELISA) Serum levels of OVA-specific IgE were measured by ELISA as previously described [14]. Total IgE was measured and compared with a known mouse IgE standard (BD PharMingen, San Diego, CA). Levels of murine IL-4, IL-5, IL-10, IL-13, and IFN-γ in BALF and culture supernatants were measured by ELISA (Biosource, Camarillo, CA) according to the manufacturer's instructions. Quantifications of human IFN-λ1 in BALF supernatants were evaluated using commercially available ELISA (R&D Systems, Minneapolis, MN). 2.7. Flow cytometry Single cell suspensions from spleens were resuspended at 1 × 106 cells/mL and stained with FITC-labeled anti-CD4 and PE-labeled anti-CD25 mAbs (BioLegend, San Diego, CA). For Foxp3 expression, cells were stained for surface markers and then fixed, permeabilized, and stained with APC-labeled anti-Foxp3 (eBioscience, San Diego, CA) as recommended by the manufacturer. Flow cytometry was performed with a FACSCalibur (Becton Dickinson, Mountain View, CA, USA) using CellQuest Software. 2.8. In vitro antigen-induced cytokine production by splenocytes Cells (3 × 106 in a 24-well plate) isolated from spleen were restimulated in vitro in the presence or absence of 100 μg/mL OVA. Supernatants were harvested after 4 days and IL-4, IL-5, IL-10, and IL-13 levels were determined by ELISA as described above. 2.9. Statistical analysis Data are presented as mean ± SEM. Significant differences among groups were identified by ANOVA. Individual comparisons between groups were confirmed by the student-t test. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). A P value of less than 0.05 was considered statistically significant.
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3. Results 3.1. hIFN-λ1 expression in BALF after i.n. delivery of Ad-hIFN-λ1 To elucidate the effect of hIFN-λ1 administration on airway inflammation in OVA-sensitized mice, we applied Ad-hIFN-λ1 for gene transfer to induce a high level of hIFN-λ1 transgenic expression in the mouse lung. We examined the kinetics of the hIFN-λ1 expression after a single dose i.n. delivery of Ad-hIFN-λ1. BALF samples were collected at the indicated times after the i.n. instillation of Ad-hIFN-λ1 or Ad-mock. We found that the level of hIFN-λ1 started to increase at day 1, peaked at day 3 after the i.n. delivery of Ad-hIFN-λ1, and gradually decreased thereafter. However, it still remained high even 10 days after administration of Ad-hIFN-λ1 (Fig. 1). In contrast, there was no detectable hIFN-λ1 protein in the Ad-mock-treated mice. Of note, at the dose of 1 × 109 pfu, there was no evidence of vascular congestion and hypersecretion of mucous in bronchioles as shown by lung H&E staining (data not shown).
mice relative to values in PBS-challenged controls. Interestingly, the levels of total and OVA-specific IgE were significantly lower in the group of mice that were Ad-hIFN-λ1-treated compared to mice that were OVA-challenged only. Treatment with Ad-mock after OVAchallenge did not affect the levels of total and OVA-specific IgE. Histological analysis revealed typical pathologic features of asthmalike inflammation in the OVA-sensitized and challenged mice stained with H&E. OVA-exposed mice increased the numbers of eosinophils and lymphocytes in the lung tissue compared with the PBS control. Ad-hIFN-λ1 treatment reduced significantly the numbers of eosinophils and lymphocytes in the peribronchial and alveolar regions (Fig. 2E). Furthermore, OVA-exposed mice showed goblet cell hyperplasia and mucus overproduction in the airways, which was markedly reduced by Ad-hIFN-λ1 treatment, as shown with PAS/AB staining (Fig. 2E). In contrast, treatment with Ad-mock had no effect on the appearance of eosinophils and lymphocytes in lung tissue or mucus hypersecretion. Taken together, these data suggest that hIFN-λ1 can inhibit inflammatory cell infiltration and goblet cell hyperplasia.
3.2. Ad-hIFN-λ1 attenuates allergic airway inflammation 3.3. Effect of Ad-hIFN-λ1 on in vivo cytokine production in BALF
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We assessed the impact of i.n. delivery of Ad-hIFN-λ1 on allergic airway hyperresponsiveness and inflammation in a murine experimental model of asthma (Fig. 2A). Antigen challenge of OVA-sensitized mice led to heightened AHR, as determined by increased airway resistance in response to doses of intravenous methacholine (Fig. 2B). Similarly, the airway resistance was increased in OVA-sensitized and OVAchallenged mice pretreated with Ad-mock. However, BALB/c mice that were sensitized and challenged with PBS revealed no increase in airway responsiveness. In contrast, pretreatment with Ad-hIFN-λ1 decreased significantly AHR as compared with Ad-mock- and OVA-treated groups (Fig. 2B). Total cell numbers in BALF were increased 24 h after the final antigen challenge in OVA-sensitized mice than in OVA-sensitized PBSchallenged mice. The increase of total cell numbers was associated with macrophage, eosinophils, lymphocytes, and neutrophils (Fig. 2C). As compared with Ad-mock, the treatment of OVA-sensitized and challenged mice with i.n. delivery of Ad-hIFN-λ1 reduced the increase in total cell numbers in BALF by two-thirds (P b 0.01). Specific decreases were observed in the number of eosinophils, lymphocytes, and neutrophils in BALF, compared with mice treated with Ad-mock (P b 0.05). These data suggest that hIFN-λ1 can modulate the inflammatory response by reducing the number of inflammatory cells in the airways of allergic mice. Total IgE and OVA-specific IgE levels were measured in sera taken from all mice at the time they were killed. Fig. 2D shows that serum levels of total and OVA-specific IgE were increased in OVA-challenged 60
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To determine the effect of Ad-hIFN-λ1 on airway inflammation, IL-4, IL-5, IL-10, IL-13, and IFN-γ concentrations in BALF were measured by ELISA 24 h after the last OVA challenge. Ad-hIFN-λ1 treatment attenuated the increase in IL-4, IL-5 and IL-13 observed following OVA challenge (P b 0.05) (Fig. 3). Treatment of OVA-exposed mice with Ad-mock had no apparent effect on the concentrations of IL-4, IL-5, and IL-13. The production of IL-10 and IFN-γ in BALF was not significantly changed by AdhIFN-λ1 treatment. 3.4. Effect of Ad-hIFN-λ1 on in vitro cytokine production by splenocytes To investigate the effect of Ad-hIFN-λ1 to OVA-treated mice on cytokine production, splenocytes from the different groups were cultured with or without OVA for 4 days. Fig. 4 shows the levels of IL-4, IL-5, IL10, and IL-13 measured in the culture supernatants resulted as expected in increased levels of these cytokines in response to OVA. Ad-hIFN-λ1 treatment markedly decreased IL-4, IL-5, and IL-13 and slightly increased IL-10 production (compare with Ad-mock). 3.5. Ad-hIFN-λ1 increases the number of regulatory T cells in mice spleen To investigate whether Ad-hIFN-λ1 induce Treg cells, we analyzed the expression of CD4, CD25 and Foxp3 on splenocytes of Ad-hIFN-λ1treated mice. Flow cytometric analysis revealed that the proportion of CD4+25+Foxp3 + Treg cells in CD4 + gated splenocyte populations was significantly elevated in Ad-hIFN-λ1-treated mice compared with the Ad-mock-treated groups (Fig. 5). Such increases in the percentage of CD4+CD25+Foxp3 + Treg cells were not observed in Ad-mocktreated or OVA-sensitized and challenged groups compared with the PBS control groups (Fig. 5).
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4. Discussion
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In this study, we demonstrated that the treatment of allergic mice with intranasal delivery of Ad-hIFN-λ1 before antigen challenge inhibited Th2 cytokine production, lung inflammatory cell infiltration, goblet cell hyperplasia, and mucus hypersecretion. Moreover, the serum levels of total and OVA-specific IgE were reduced after treatment with the Ad-hIFN-λ1. Transfer of the hIFN-λ1 gene also had a significant effect on established airway hyperreactivity. Although there are three genes encoding closely related but distinct human IFN-λs, the search of the mouse genome revealed the existence of only two intact mouse genes, representing mouse IFN-λ2 and IFN-λ3 gene orthologs [18]. The mouse IFN-λ1 gene ortholog contains a stop codon in the first exon and is therefore predicted not to encode an intact
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Days post-infection Fig. 1. hIFN-λ1 protein expression in BALF after i.n. treatment of Ad-hIFN-λ1. Groups of mice received a single i.n. delivery of 30 μL of either Ad-hIFN-λ1 or Ad-mock (1 × 109 pfu) on day 0. At different time points, BALF was collected from the mice and individually analyzed for the presence of hIFN-λ1 protein by a standard ELISA. The graph represents mean ± SEM from at least three different samples.
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Fig. 2. Ad-hIFN-λ1 treatment reduces allergic airway inflammation in a mouse model of asthma. (A) Protocol for inducing experimental asthma and administration of Ad-hIFN-λ1. Mice were sensitized by two i.p. injections of OVA/alum on days 0 and 14, and then received three consecutive days of aerosolized OVA challenge on days 28, 29, and 30. To assess the effect of hIFN-λ1 on airway inflammation, mice received i.n. instillation of the Ad-hIFN-λ1 (1 × 109 plaque-forming units (pfu)/mice) on day 27. Control mice received the same dose of control virus (Ad-mock) or PBS. On day 31, mice were killed, blood was collected, BAL was performed, and lungs were removed for histological analysis. (B) Effect of Ad-hIFN-λ1 on airway responsiveness to intravenous methacholine. RL was measured by means of forced oscillation. Data are representative of three separate experiments with similar results and expressed as the mean ± SEM (n = 6–10 per group). *Significant differences (P b 0.05) between Ad-hIFN-λ1 groups and Ad-mock groups. (C) Ad-hIFN-λ1 reduces inflammatory cell infiltration in BALF of OVA-sensitized/challenged mice. The number of total and differential cells of BALFs from PBS-sensitized and -challenged mice (PBS), OVA-sensitized/challenged mice (OVA), OVA-sensitized/challenged mice with the administration of Ad-mock and with the administration of Ad-hIFN-λ1 were determined 24 h after the last challenge. Data are representative of three separate experiments with similar results and expressed as the mean ± SEM (n = 6–10 per group). #Significant differences (P b 0.05) between sensitized/challenged control groups and sensitized/challenged Ad-hIFN-λ1 treatment groups. (D) Effect of Ad-hIFN-λ1 treatment on total and OVA-specific IgE in the serum of allergic mice. Levels of serum IgE were measured 24 h after the last OVA challenge. Data are representative of three separate experiments with similar results and expressed as the mean ± SEM (n = 6–10 per group). # Significant differences (P b 0.05) compared with OVA-challenge. (E) Ad-hIFN-λ1 reduces lung tissue eosinophilia and mucus secretion in OVA-sensitized/challenged mice. Lung tissue was fixed, sectioned at 4 μm thickness and detected with H&E staining for tissue eosinophilia or PAS/AB staining for goblet cells and mucus production by light microscopy (original magnification:×400). Lung tissues from PBS challenge, OVA challenge, Ad-mock control treatment and Ad-hIFN-λ1 treatment. Mice were processed 24 h after the last OVA challenge. A total of 6–10 mice in each group were analyzed and representative findings are shown.
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Fig. 3. Effect of Ad-hIFN-λ1 treatment on cytokine production in BALF. IL-4, IL-5, IL-10, IL-13, and IFN-γ concentrations in BALFs from PBS-sensitized/challenged mice (PBS), OVAsensitized/challenged mice (OVA), OVA-sensitized/challenged mice with the administration of Ad-mock and with the administration of Ad-hIFN-λ1 were assessed 24 h after the last challenge. Mean ± SEM of 6 mice per group representing data from three experiments are shown. #Significant differences (P b 0.05) between sensitized/challenged control groups and sensitized/challenged Ad-hIFN-λ1 treatment groups.
protein. In this study, we found that human IFN-λ1 could be expressed in mice after i.n. transfection of an Ad-hIFN-λ1. Furthermore, the expressed hIFN-λ1 could activate the antiviral proteins Mx1 and 2′, 5′-OAS in mice (data no shown). IFN usually has in vitro and in vivo antiviral activity in part via induction of protein kinase R, Mx protein, and 2′, 5′-OAS, which inhibit viral replication and degrade viral components. These findings demonstrate that i.n. delivery of cytokine genes is a simple and efficient method to introduce human cytokines in mice. Eosinophils are important effector cells in allergic diseases, but the mechanisms regulating their biological functions remain unclear. Our results showed that pulmonary eosinophilia was decreased by AdhIFN-λ1 treatment. On the other hand, increased mucus production by goblet cells in the airway epithelium is associated with airway inflammation and asthma. In the present study, treatment with the AdhIFN-λ1 reduced the increase in PAS/AB epithelial cells and mucus hypersecretion in the airways after antigen challenge. Th2 cytokines, T cells, and eosinophils are required to produce airway mucus accumulation and goblet cell degranulation [1,19]. Although a direct role of hIFNλ1 in these cells cannot be ruled out, the observed decrease in mucus production in Ad-IFN-λ1-treated mice lung tissue may be attributed to an indirect effect on goblet cells resulting from the combined effects of reduction in Th2 cytokine levels and eosinophilia in OVA-sensitized and challenged mice.
Th2 cytokines include IL-4, IL-5, and IL-13, whereas Th1 cytokines include IFN-γ. IL-4, IL-5, and IL-13 produced by Th2 cells have been postulated to have central roles in the initiation and maintenance of allergic inflammation [20]. The development of airway eosinophilia and allergen-induced AHR is also associated with increased levels of IL-4, IL-5, and IL-13 in BALF, consistent with development of a Th2-mediated allergic response. IL-4 regulates allergic inflammation by promoting Th2 cell differentiation, IgE synthesis, IgE receptor up-regulation, and mucus hypersecretion [21]. IL-5 promotes eosinophilic inflammation and infiltration into airways [22]. IL-13 promotes B cell differentiation and is capable of inducing isotype-switching in B cells to produce IgE [23]. Our present data showed that treatment with Ad-hIFN-λ1 significantly reduced the levels of IL-4, IL-5, and IL-13 in BALF from sensitized and challenged mice, thus indicating its effect in attenuating the inflammatory response. Our results are in agreement with previous reports demonstrating that IFN-λ1 is able to inhibit the production of IL-4, IL-5, and IL-13 by T cells in an IFN-γ-independent manner [8,9]. In addition, IL-4 and IL-13 are important in directing B cell growth, differentiation, and secretion of IgE [24]. We found that both serum levels of total and OVA-specific IgE were reduced by treatment with the Ad-hIFN-λ1 given before antigen challenge. Therefore, the observed reduction in serum IgE in our asthma model by hIFN-λ1 may be the result of inhibitory effects on B cell activation and reduced Th2 cytokine
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Fig. 4. Effect of Ad-hIFN-λ1 on cytokine production by cultured splenocytes. Splenocytes were prepared 24 h after the last OVA challenge, from PBS-sensitized/challenged mice (PBS), OVAsensitized/challenged mice (OVA), OVA-sensitized/challenged mice with the administration of Ad-mock and with the administration of Ad-hIFN-λ1. A total of 3 × 106 cells/well were cultured (in triplicates) and stimulated in the presence or absence of 100 μg/mL OVA for 4 days. Supernatants were removed and analyzed by ELISA, for IL-4, IL-5, IL-10, IL-13, and IFN-γ. Samples were assayed in triplicates. Data shown are mean ± SEM of two separate cultures from two independent experiments, with 6 mice/group. #Significant differences (P b 0.05) between sensitized/challenged control groups and sensitized/challenged Ad-hIFN-λ1 treatment groups.
release. The biological activities of IgE are mediated through highaffinity IgE receptors (FcεRI) on mast cells and basophils. Cross-linking of FcεRI initiates multiple signaling cascades leading to cellular degranulation and activation [25]. Regulatory T cells are key immune suppressive cells which mediate immune tolerance in autoimmune, inflammatory diseases and cancer. It has been identified that CD4+CD25+Foxp3 + Treg cells can potently suppress IgE production, directly or indirectly suppress effector cells of allergic inflammation, such as mast cells, basophils, and eosinophils, and contribute to remodeling in asthma [26]. In experimental models,
CD4+CD25+Foxp3 + Treg cells can reduce established lung eosinophilia, Th2 infiltration, and expression of IL-5 and IL-13, but have no effect on established airway hyperreactivity [27]. Mennechet and Uze [28] reported that IFN-λ1-treated DCs promote the generation of tolerogenic DCs and proliferation of CD4+0CD25+Foxp3 + Treg cells with contactdependent suppressive activity on T cell proliferation. However, whether in vivo IFN-λ1 could induce the generation of Treg cells has not been clarified. In this study, we demonstrate that treatment with Ad-hIFNλ1 significantly increases the proportion of CD4+CD25+Foxp3 +Treg cells in the spleen of OVA-sensitized and challenged mice. In conclusion, IFN-λ1 can alleviate some asthmatic symptoms, inhibit pulmonary eosinophilic inflammation, and decrease Th2 cytokine production in mice. Our data show that IFN-λ1-induced attenuation of the allergic airway response in mice is associated with increased numbers of CD4+CD25+Foxp3 + Treg cells in the spleen. With more studies, IFN-λ1 might be applied as an immunotherapeutic agent for the treatment of allergic diseases. Acknowledgments This study was supported by the grants from the National Natural Science Foundation of China (81070034 and 30671932), Zhejiang Provincial Natural Science Foundation of China (LY13H090014), Scientific Research Fund of Zhejiang Provincial Education Department (Y201120665), and sponsored by K.C. Wong Magna Fund in Ningbo University.
Fig. 5. Ad-hIFN-λ1 treatment increases splenic Treg cells in BALB/c mice. Spleen cells from PBS-sensitized/challenged mice (PBS), OVA-sensitized/challenged mice (OVA), and OVAsensitized/challenged mice with the administration of Ad-mock and with the administration of Ad-hIFN-λ1 mice were stained with specific antibodies to CD4, CD25, and Foxp3 for quantification of CD4+CD25+Foxp3 + Treg cells, and then analyzed using flow cytometry. #A significant increase (P b 0.05) in CD4+CD25+Foxp3 + Treg cell numbers was observed following treatment with Ad-hIFN-λ1 compared with the PBS, OVA or Ad-mock groups.
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[4] Holgate ST. Novel targets of therapy in asthma. Curr Opin Pulm Med 2009;15:63–71. [5] Kotenko SV, Gallagher G, Baurin VV, Lewis-Antes A, Shen M, Shah NK, et al. IFNlambdas mediate antiviral protection through a distinct class II cytokine receptor complex. Nat Immunol 2003;4:69–77. [6] Sheppard P, Kindsvogel W, Xu W, Henderson K, Schlutsmeyer S, Whitmore TE, et al. IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat Immunol 2003;4:63–8. [7] Li M, Liu X, Zhou Y, Su SB. Interferon-lambdas: the modulators of antivirus, antitumor, and immune responses. J Leukoc Biol 2009;86:23–32. [8] Jordan WJ, Eskdale J, Srinivas S, Pekarek V, Kelner D, Rodia M, et al. Human interferon lambda-1 (IFN-lambda1/IL-29) modulates the Th1/Th2 response. Genes Immun 2007;8:254–61. [9] Srinivas S, Dai J, Eskdale J, Gallagher GE, Megjugorac NJ, Gallagher G. Interferonlambda1 (interleukin-29) preferentially down-regulates interleukin-13 over other T helper type 2 cytokine responses in vitro. Immunology 2008;125:492–502. [10] Contoli M, Message SD, Laza-Stanca V, Edwards MR, Wark PA, Bartlett NW, et al. Role of deficient type III interferon-lambda production in asthma exacerbations. Nat Med 2006;12:1023–6. [11] Bullens DM, Decraene A, Dilissen E, Meyts I, De Boeck K, Dupont LJ, et al. Type III IFNlambda mRNA expression in sputum of adult and school-aged asthmatics. Clin Exp Allergy 2008;38:1459–67. [12] Miller EK, Hernandez JZ, Wimmenauer V, Shepherd BE, Hijano D, Libster R, et al. A mechanistic role for type III IFN-lambda1 in asthma exacerbations mediated by human rhinoviruses. Am J Respir Crit Care Med 2012;185:508–16. [13] Koltsida O, Hausding M, Stavropoulos A, Koch S, Tzelepis G, Ubel C, et al. IL-28A (IFNlambda2) modulates lung DC function to promote Th1 immune skewing and suppress allergic airway disease. EMBO Mol Med 2011;3:348–61. [14] Liu X, Li M, Wu Y, Zhou Y, Zeng L, Huang T. Anti-IL-33 antibody treatment inhibits airway inflammation in a murine model of allergic asthma. Biochem Biophys Res Commun 2009;386:181–5. [15] Shore SA, Terry RD, Flynt L, Xu A, Hug C. Adiponectin attenuates allergen-induced airway inflammation and hyperresponsiveness in mice. J Allergy Clin Immunol 2006;118:389–95.
[16] Wang CC, Fu CL, Yang YH, Lo YC, Wang LC, Chuang YH, et al. Adenovirus expressing interleukin-1 receptor antagonist alleviates allergic airway inflammation in a murine model of asthma. Gene Ther 2006;13:1414–21. [17] Li MC, Wang HY, Wang HY, Li T, He SH. Liposome-mediated IL-28 and IL-29 expression in A549 cells and anti-viral effect of IL-28 and IL-29 on WISH cells. Acta Pharmacol Sin 2006;27:453–9. [18] Bartlett NW, Buttigieg K, Kotenko SV, Smith GL. Murine interferon lambdas (type III interferons) exhibit potent antiviral activity in vivo in a poxvirus infection model. J Gen Virol 2005;86:1589–96. [19] Izuhara K, Ohta S, Shiraishi H, Suzuki S, Taniguchi K, Toda S, et al. The mechanism of mucus production in bronchial asthma. Curr Med Chem 2009;16:2867–75. [20] Finkelman FD, Hogan SP, Hershey GK, Rothenberg ME, Wills-Karp M. Importance of cytokines in murine allergic airway disease and human asthma. J Immunol 2010;184:1663–74. [21] Dabbagh K, Takeyama K, Lee HM, Ueki IF, Lausier JA, Nadel JA. IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo. J Immunol 1999;162:6233–7. [22] Busse WW, Ring J, Huss-Marp J, Kahn JE. A review of treatment with mepolizumab, an anti-IL-5 mAb, in hypereosinophilic syndromes and asthma. J Allergy Clin Immunol 2010;125:803–13. [23] Wynn TA. IL-13 effector functions. Annu Rev Immunol 2003;21:425–56. [24] Ozdemir C, Akdis M, Akdis CA. T-cell response to allergens. Chem Immunol Allergy 2010;95:22–44. [25] Hunt J, Bracher MG, Shi J, Fleury S, Dombrowicz D, Gould HJ, et al. Attenuation of IgE affinity for FcepsilonRI radically reduces the allergic response in vitro and in vivo. J Biol Chem 2008;283:29882–7. [26] Akdis M, Blaser K, Akdis CA. T regulatory cells in allergy: novel concepts in the pathogenesis, prevention, and treatment of allergic diseases. J Allergy Clin Immunol 2005;116:961–8 [quiz 9]. [27] Kearley J, Robinson DS, Lloyd CM. CD4+CD25+ regulatory T cells reverse established allergic airway inflammation and prevent airway remodeling. J Allergy Clin Immunol 2008;122:617–624 e6. [28] Mennechet FJ, Uze G. Interferon-lambda-treated dendritic cells specifically induce proliferation of FOXP3-expressing suppressor T cells. Blood 2006;107:4417–23.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Adenovirus expressing IFN-λ1 (IL-29) attenuates allergic airway inflammation and airway hyperreactivity in experimental asthma Yan Li a, Qiaoyan Gao a, Xianli Yuan a, Mi Zhou a, Xiao Peng a, Xiaojin Liu b, Xiaoxuan Zheng b, Damo Xu a,b,c,⁎, Mingcai Li a,b,⁎⁎ a b c
Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Immunology, Ningbo University School of Medicine, Ningbo 315211, China Institute of Inflammation and Immune Diseases, Shantou University Medical College, Shantou 515041, China Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK
a r t i c l e
i n f o
Article history: Received 21 November 2013 Received in revised form 18 March 2014 Accepted 23 April 2014 Available online 9 May 2014 Keywords: IFN-λ1 (IL-29) Th2 cells Eosinophils Hyperresponsiveness Airway inflammation
a b s t r a c t Background: Asthma is thought to result from the generation of T helper type 2 (Th2) responses, leading to bronchial inflammation. IFN-λ1 (also known as IL-29) is a recently described member of the IFN-λ family and has been shown to decrease production of Th2 cytokines in vitro. However, the role and mechanism of IFN-λ1 in asthma remain unknown. Objectives: The aim of this study was to clarify the importance of IFN-λ1 in allergen-induced airway hyperresponsiveness (AHR) and inflammation. Methods: We used a murine model for ovalbumin (OVA)-induced asthma to examine the effect of intranasal delivery of recombinant adenovirus expressing human IFN-λ1 (Ad-hIFN-λ1) on AHR and allergic airway inflammation. Results: Intranasal instillation of Ad-hIFN-λ1 before airway antigen challenge in OVA-immunized mice significantly decreased the severity of AHR and numbers of eosinophils and levels of IL-4, IL-5, and IL-13, but not IL-10 and IFN-γ; both in vivo, in the bronchoalveolar lavage fluid and in vitro, following stimulation of lymphocytes from spleens with OVA, compared with administration of a control virus (Ad-mock). Furthermore, Ad-hIFN-λ1 treatment inhibited serum IgE secretion and increased numbers of splenic CD4+CD25+FOXP3 + Treg cells. Histological studies showed that Ad-hIFN-λ1 attenuated OVA-induced lung tissue eosinophilia. Conclusions: These results demonstrate that delivery of the Ad-hIFN-λ1 can mitigate allergic airway inflammation in experimental asthma. The potent immunoregulatory action of IFN-λ1 may offer a novel therapeutic approach to treat allergic asthma. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Allergic asthma is a chronic disorder characterized by eosinophilic airway inflammation, mucus hypersecretion, high levels of T-helper type 2 (Th2) cytokines in bronchoalveolar lavage fluid (BALF), high levels of serum immunoglobulin (Ig) E, increasing airway hyperresponsiveness (AHR), and variable airway obstruction [1]. The process of airway inflammation involves various types of cells, such as eosinophils, mast cells, T lymphocytes, and dendritic cells (DCs) [1,2]. Various methods have been suggested to control the airway inflammation in patients. However, these approaches have limitations in terms of partial effectiveness or serious side effects in long term use [3,4]. Therefore, novel therapeutic approaches that allow a more effective treatment of allergic asthma are urgently desirable. ⁎ Correspondence to: D. Xu, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK. Tel./fax: +86 574 8760 9893. ⁎⁎ Correspondence to: M. Li, Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Immunology, Ningbo University School of Medicine, Ningbo 315211, China. Tel./fax: +86 574 8760 9893. E-mail addresses: [email protected] (D. Xu), [email protected] (M. Li).
http://dx.doi.org/10.1016/j.intimp.2014.04.022 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Interferon (IFN)-λ, including IFN-λ1/as known as interleukin (IL)-29, IFN-λ2/IL-28A, and IFN-λ3/IL-28B, is a newly described group of cytokines distantly related to the type I IFNs and IL-10 family members [5,6]. IFN-λs are now collectively referred to as type III IFNs. In addition to their antiviral and antiproliferative activities, IFN-λs exert immunomodulatory effects that overlap type I IFNs in innate and adaptive arms of the immune system [7]. Several reports have shown that IFN-λ1 plays an important role in the Th2 responses. Gallagher and his colleagues [8] revealed that IFN-λ1 inhibits the production of IL-13 by T cells in an IFN-γ-independent manner, which is mediated in part via monocyte-derived DCs. Furthermore, IFN-λ1 decreases IL-4 and IL-5 production, but its effects are not as consistent as those seen with IL13. Thus, IFN-λ1 appears to be an inhibitor of human Th2 responses whose action is directed primarily toward IL-13, but may also affect Th2 responses in general without invoking complementary elevation of IFN-γ [9]. The ability of IFN-λ1 to influence Th2 cytokine (IL-4, IL-5, and IL-13) secretion may be of direct relevance to the pathogenesis and treatment of asthma [10]. Bullens et al. [11] demonstrated that sputum IFN-λ1 mRNA correlates negatively with asthma symptoms in steroid-naive patients and is significantly higher in steroid-treated
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patients. Therefore, IFN-λ1 could have an immunoprotective role in the lower airways. Conversely, Miller et al. [12] have shown that IFN-λ1 is associated with exacerbation of asthma caused by human rhinoviruses (HRV) and IFN-λ1 levels increase with worsening acute asthma symptoms during HRV infections. Koltsida et al. [13] recently reported IFN-λ2 (IL-28A) modulates lung DC function to promote Th1 cell differentiation in vivo and suppress Th2-mediated responses in experimental allergic asthma. Based on these, it was proposed that IFN-λ1 could be considered as a target for asthma treatment. It's clear that more studies are necessary to determine whether IFN-λ1 should be blocked or promoted in asthma. To investigate the therapeutic potential of IFN-λ1 in allergic asthma, we evaluated the effects of recombinant adenovirus expressing human IFN-λ1 (Ad-hIFN-λ1) on pulmonary inflammation in a murine experimental model of asthma. Our results suggest that intranasal (i.n.) delivery of Ad-hIFN-λ1 attenuates eosinophilic airway inflammation and decreases the severity of AHR by down-regulation of antigen-driven Th2mediated responses and induction of CD4+CD25+Foxp3 + regulatory T (Treg) cells. Therefore replacement or augmentation of IFN-λ1 production could represent a new approach to treatment or prevention of allergic asthma. 2. Materials and methods 2.1. Animals 8- to 10-week-old female BALB/c mice were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China) and maintained under a specific pathogenfree facility at the Experimental Animal Center of Ningbo University. An ovalbumin (OVA)-free diet and water were supplied ad libitum. All animal studies were approved by the Institutional Animal Care and Use Committee of the Ningbo University. 2.2. Sensitization and antigen challenge BALB/c mice were sensitized and challenged as described previously with a slight modification [14]. Briefly, mice were sensitized on days 0 and 14 by intraperitoneal (i.p.) injection of 20 μg of OVA (Grade V; Sigma-Aldrich, St. Louis, MO) emulsified in 2 mg of aluminum hydroxide gel (alum, Sigma) in a total volume of 200 μL. On days 28, 29, and 30 after the initial sensitization, the mice were challenged for 30 min daily with an aerosol of 1% (wt/vol) OVA in sterile phosphate-buffered saline (PBS) (or with PBS as a control) using an ultrasonic nebulizer (Yuyue, Jiangsu, China). Twenty-four hours after the last exposure to OVA or PBS, mice were anesthetized and connected to a computercontrolled small animal ventilator (flexiVent, SCIREQ, Montreal, Canada), and AHR to intravenous methacholine (Mch) (Sigma) was measured as pulmonary resistance (RL) as described previously [15]. BALF, serum, and tissues were obtained for further analyses. 2.3. Adenoviral vectors and administration of Ad-hIFN-λ1 Recombinant adenoviral vector was constructed using the Ad-Easy system that has been described previously [16]. Briefly, the hIFN-λ1 cDNA [17] was inserted into a recombinant adenoviral shuttle vector (pAdTrack-CMV) and cotransformed into Escherichia coli BJ5183 cells with an adenoviral backbone plasmid (pAdEasy-1). Ad-mock was identical with Ad-hIFN-λ1 structurally, but contained no transgene in the expression cassette. The linearized recombinant plasmid was transfected into the adenovirus packaging 293 cell line. Viruses were purified from infected cells 48 h after infection by three freeze–thaw cycles followed by successive banding on cesium chloride density gradient centrifugation. The purified virus was dialyzed and stored at −80 °C until needed. Viral titers were measured by standard end-point dilution assay using
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the 293 cells. Furthermore, hIFN-λ1 expression was confirmed by measuring the culture supernatants of infected 293 cells (data not shown). Mice were anesthetized and held supine at an angle of 45° with pressure applied to the lower mandible to immobilize the tongue and prevent swallowing. Twenty-four hours before the first challenge with OVA (day 27), Ad-hIFN-λ1 (1 × 109 pfu) was introduced to the nasal planum using a micropipet. Control mice received the same dose of control virus (Ad-mock). 2.4. Bronchoalveolar lavage (BAL) BAL sample collection, total cell counts, and cell differential counts were performed as previously described [14]. Briefly, mice were anesthetized with a sodium pentobarbitone, the lungs were lavaged three times with 1 mL normal saline. Total cell and cell differential counts were counted using a hemocytometer and Diff-Quik stain (Fisher Scientific, Pittsburgh, PA), respectively. The supernatants of BALF were stored at −80 °C for cytokine analysis. 2.5. Histopathology Lung tissue was fixed in Carnoy's solution and embedded in paraffin using standard methods. Four-micrometer thick sections were stained with hematoxylin and eosin (H&E) to detect cellular infiltration, and periodic acid-Schiff/Alcian blue (PAS/AB) to detect mucus-secreting goblet cells. 2.6. Enzyme-linked immunosorbent assay (ELISA) Serum levels of OVA-specific IgE were measured by ELISA as previously described [14]. Total IgE was measured and compared with a known mouse IgE standard (BD PharMingen, San Diego, CA). Levels of murine IL-4, IL-5, IL-10, IL-13, and IFN-γ in BALF and culture supernatants were measured by ELISA (Biosource, Camarillo, CA) according to the manufacturer's instructions. Quantifications of human IFN-λ1 in BALF supernatants were evaluated using commercially available ELISA (R&D Systems, Minneapolis, MN). 2.7. Flow cytometry Single cell suspensions from spleens were resuspended at 1 × 106 cells/mL and stained with FITC-labeled anti-CD4 and PE-labeled anti-CD25 mAbs (BioLegend, San Diego, CA). For Foxp3 expression, cells were stained for surface markers and then fixed, permeabilized, and stained with APC-labeled anti-Foxp3 (eBioscience, San Diego, CA) as recommended by the manufacturer. Flow cytometry was performed with a FACSCalibur (Becton Dickinson, Mountain View, CA, USA) using CellQuest Software. 2.8. In vitro antigen-induced cytokine production by splenocytes Cells (3 × 106 in a 24-well plate) isolated from spleen were restimulated in vitro in the presence or absence of 100 μg/mL OVA. Supernatants were harvested after 4 days and IL-4, IL-5, IL-10, and IL-13 levels were determined by ELISA as described above. 2.9. Statistical analysis Data are presented as mean ± SEM. Significant differences among groups were identified by ANOVA. Individual comparisons between groups were confirmed by the student-t test. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). A P value of less than 0.05 was considered statistically significant.
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3. Results 3.1. hIFN-λ1 expression in BALF after i.n. delivery of Ad-hIFN-λ1 To elucidate the effect of hIFN-λ1 administration on airway inflammation in OVA-sensitized mice, we applied Ad-hIFN-λ1 for gene transfer to induce a high level of hIFN-λ1 transgenic expression in the mouse lung. We examined the kinetics of the hIFN-λ1 expression after a single dose i.n. delivery of Ad-hIFN-λ1. BALF samples were collected at the indicated times after the i.n. instillation of Ad-hIFN-λ1 or Ad-mock. We found that the level of hIFN-λ1 started to increase at day 1, peaked at day 3 after the i.n. delivery of Ad-hIFN-λ1, and gradually decreased thereafter. However, it still remained high even 10 days after administration of Ad-hIFN-λ1 (Fig. 1). In contrast, there was no detectable hIFN-λ1 protein in the Ad-mock-treated mice. Of note, at the dose of 1 × 109 pfu, there was no evidence of vascular congestion and hypersecretion of mucous in bronchioles as shown by lung H&E staining (data not shown).
mice relative to values in PBS-challenged controls. Interestingly, the levels of total and OVA-specific IgE were significantly lower in the group of mice that were Ad-hIFN-λ1-treated compared to mice that were OVA-challenged only. Treatment with Ad-mock after OVAchallenge did not affect the levels of total and OVA-specific IgE. Histological analysis revealed typical pathologic features of asthmalike inflammation in the OVA-sensitized and challenged mice stained with H&E. OVA-exposed mice increased the numbers of eosinophils and lymphocytes in the lung tissue compared with the PBS control. Ad-hIFN-λ1 treatment reduced significantly the numbers of eosinophils and lymphocytes in the peribronchial and alveolar regions (Fig. 2E). Furthermore, OVA-exposed mice showed goblet cell hyperplasia and mucus overproduction in the airways, which was markedly reduced by Ad-hIFN-λ1 treatment, as shown with PAS/AB staining (Fig. 2E). In contrast, treatment with Ad-mock had no effect on the appearance of eosinophils and lymphocytes in lung tissue or mucus hypersecretion. Taken together, these data suggest that hIFN-λ1 can inhibit inflammatory cell infiltration and goblet cell hyperplasia.
3.2. Ad-hIFN-λ1 attenuates allergic airway inflammation 3.3. Effect of Ad-hIFN-λ1 on in vivo cytokine production in BALF
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We assessed the impact of i.n. delivery of Ad-hIFN-λ1 on allergic airway hyperresponsiveness and inflammation in a murine experimental model of asthma (Fig. 2A). Antigen challenge of OVA-sensitized mice led to heightened AHR, as determined by increased airway resistance in response to doses of intravenous methacholine (Fig. 2B). Similarly, the airway resistance was increased in OVA-sensitized and OVAchallenged mice pretreated with Ad-mock. However, BALB/c mice that were sensitized and challenged with PBS revealed no increase in airway responsiveness. In contrast, pretreatment with Ad-hIFN-λ1 decreased significantly AHR as compared with Ad-mock- and OVA-treated groups (Fig. 2B). Total cell numbers in BALF were increased 24 h after the final antigen challenge in OVA-sensitized mice than in OVA-sensitized PBSchallenged mice. The increase of total cell numbers was associated with macrophage, eosinophils, lymphocytes, and neutrophils (Fig. 2C). As compared with Ad-mock, the treatment of OVA-sensitized and challenged mice with i.n. delivery of Ad-hIFN-λ1 reduced the increase in total cell numbers in BALF by two-thirds (P b 0.01). Specific decreases were observed in the number of eosinophils, lymphocytes, and neutrophils in BALF, compared with mice treated with Ad-mock (P b 0.05). These data suggest that hIFN-λ1 can modulate the inflammatory response by reducing the number of inflammatory cells in the airways of allergic mice. Total IgE and OVA-specific IgE levels were measured in sera taken from all mice at the time they were killed. Fig. 2D shows that serum levels of total and OVA-specific IgE were increased in OVA-challenged 60
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To determine the effect of Ad-hIFN-λ1 on airway inflammation, IL-4, IL-5, IL-10, IL-13, and IFN-γ concentrations in BALF were measured by ELISA 24 h after the last OVA challenge. Ad-hIFN-λ1 treatment attenuated the increase in IL-4, IL-5 and IL-13 observed following OVA challenge (P b 0.05) (Fig. 3). Treatment of OVA-exposed mice with Ad-mock had no apparent effect on the concentrations of IL-4, IL-5, and IL-13. The production of IL-10 and IFN-γ in BALF was not significantly changed by AdhIFN-λ1 treatment. 3.4. Effect of Ad-hIFN-λ1 on in vitro cytokine production by splenocytes To investigate the effect of Ad-hIFN-λ1 to OVA-treated mice on cytokine production, splenocytes from the different groups were cultured with or without OVA for 4 days. Fig. 4 shows the levels of IL-4, IL-5, IL10, and IL-13 measured in the culture supernatants resulted as expected in increased levels of these cytokines in response to OVA. Ad-hIFN-λ1 treatment markedly decreased IL-4, IL-5, and IL-13 and slightly increased IL-10 production (compare with Ad-mock). 3.5. Ad-hIFN-λ1 increases the number of regulatory T cells in mice spleen To investigate whether Ad-hIFN-λ1 induce Treg cells, we analyzed the expression of CD4, CD25 and Foxp3 on splenocytes of Ad-hIFN-λ1treated mice. Flow cytometric analysis revealed that the proportion of CD4+25+Foxp3 + Treg cells in CD4 + gated splenocyte populations was significantly elevated in Ad-hIFN-λ1-treated mice compared with the Ad-mock-treated groups (Fig. 5). Such increases in the percentage of CD4+CD25+Foxp3 + Treg cells were not observed in Ad-mocktreated or OVA-sensitized and challenged groups compared with the PBS control groups (Fig. 5).
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4. Discussion
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In this study, we demonstrated that the treatment of allergic mice with intranasal delivery of Ad-hIFN-λ1 before antigen challenge inhibited Th2 cytokine production, lung inflammatory cell infiltration, goblet cell hyperplasia, and mucus hypersecretion. Moreover, the serum levels of total and OVA-specific IgE were reduced after treatment with the Ad-hIFN-λ1. Transfer of the hIFN-λ1 gene also had a significant effect on established airway hyperreactivity. Although there are three genes encoding closely related but distinct human IFN-λs, the search of the mouse genome revealed the existence of only two intact mouse genes, representing mouse IFN-λ2 and IFN-λ3 gene orthologs [18]. The mouse IFN-λ1 gene ortholog contains a stop codon in the first exon and is therefore predicted not to encode an intact
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Fig. 2. Ad-hIFN-λ1 treatment reduces allergic airway inflammation in a mouse model of asthma. (A) Protocol for inducing experimental asthma and administration of Ad-hIFN-λ1. Mice were sensitized by two i.p. injections of OVA/alum on days 0 and 14, and then received three consecutive days of aerosolized OVA challenge on days 28, 29, and 30. To assess the effect of hIFN-λ1 on airway inflammation, mice received i.n. instillation of the Ad-hIFN-λ1 (1 × 109 plaque-forming units (pfu)/mice) on day 27. Control mice received the same dose of control virus (Ad-mock) or PBS. On day 31, mice were killed, blood was collected, BAL was performed, and lungs were removed for histological analysis. (B) Effect of Ad-hIFN-λ1 on airway responsiveness to intravenous methacholine. RL was measured by means of forced oscillation. Data are representative of three separate experiments with similar results and expressed as the mean ± SEM (n = 6–10 per group). *Significant differences (P b 0.05) between Ad-hIFN-λ1 groups and Ad-mock groups. (C) Ad-hIFN-λ1 reduces inflammatory cell infiltration in BALF of OVA-sensitized/challenged mice. The number of total and differential cells of BALFs from PBS-sensitized and -challenged mice (PBS), OVA-sensitized/challenged mice (OVA), OVA-sensitized/challenged mice with the administration of Ad-mock and with the administration of Ad-hIFN-λ1 were determined 24 h after the last challenge. Data are representative of three separate experiments with similar results and expressed as the mean ± SEM (n = 6–10 per group). #Significant differences (P b 0.05) between sensitized/challenged control groups and sensitized/challenged Ad-hIFN-λ1 treatment groups. (D) Effect of Ad-hIFN-λ1 treatment on total and OVA-specific IgE in the serum of allergic mice. Levels of serum IgE were measured 24 h after the last OVA challenge. Data are representative of three separate experiments with similar results and expressed as the mean ± SEM (n = 6–10 per group). # Significant differences (P b 0.05) compared with OVA-challenge. (E) Ad-hIFN-λ1 reduces lung tissue eosinophilia and mucus secretion in OVA-sensitized/challenged mice. Lung tissue was fixed, sectioned at 4 μm thickness and detected with H&E staining for tissue eosinophilia or PAS/AB staining for goblet cells and mucus production by light microscopy (original magnification:×400). Lung tissues from PBS challenge, OVA challenge, Ad-mock control treatment and Ad-hIFN-λ1 treatment. Mice were processed 24 h after the last OVA challenge. A total of 6–10 mice in each group were analyzed and representative findings are shown.
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protein. In this study, we found that human IFN-λ1 could be expressed in mice after i.n. transfection of an Ad-hIFN-λ1. Furthermore, the expressed hIFN-λ1 could activate the antiviral proteins Mx1 and 2′, 5′-OAS in mice (data no shown). IFN usually has in vitro and in vivo antiviral activity in part via induction of protein kinase R, Mx protein, and 2′, 5′-OAS, which inhibit viral replication and degrade viral components. These findings demonstrate that i.n. delivery of cytokine genes is a simple and efficient method to introduce human cytokines in mice. Eosinophils are important effector cells in allergic diseases, but the mechanisms regulating their biological functions remain unclear. Our results showed that pulmonary eosinophilia was decreased by AdhIFN-λ1 treatment. On the other hand, increased mucus production by goblet cells in the airway epithelium is associated with airway inflammation and asthma. In the present study, treatment with the AdhIFN-λ1 reduced the increase in PAS/AB epithelial cells and mucus hypersecretion in the airways after antigen challenge. Th2 cytokines, T cells, and eosinophils are required to produce airway mucus accumulation and goblet cell degranulation [1,19]. Although a direct role of hIFNλ1 in these cells cannot be ruled out, the observed decrease in mucus production in Ad-IFN-λ1-treated mice lung tissue may be attributed to an indirect effect on goblet cells resulting from the combined effects of reduction in Th2 cytokine levels and eosinophilia in OVA-sensitized and challenged mice.
Th2 cytokines include IL-4, IL-5, and IL-13, whereas Th1 cytokines include IFN-γ. IL-4, IL-5, and IL-13 produced by Th2 cells have been postulated to have central roles in the initiation and maintenance of allergic inflammation [20]. The development of airway eosinophilia and allergen-induced AHR is also associated with increased levels of IL-4, IL-5, and IL-13 in BALF, consistent with development of a Th2-mediated allergic response. IL-4 regulates allergic inflammation by promoting Th2 cell differentiation, IgE synthesis, IgE receptor up-regulation, and mucus hypersecretion [21]. IL-5 promotes eosinophilic inflammation and infiltration into airways [22]. IL-13 promotes B cell differentiation and is capable of inducing isotype-switching in B cells to produce IgE [23]. Our present data showed that treatment with Ad-hIFN-λ1 significantly reduced the levels of IL-4, IL-5, and IL-13 in BALF from sensitized and challenged mice, thus indicating its effect in attenuating the inflammatory response. Our results are in agreement with previous reports demonstrating that IFN-λ1 is able to inhibit the production of IL-4, IL-5, and IL-13 by T cells in an IFN-γ-independent manner [8,9]. In addition, IL-4 and IL-13 are important in directing B cell growth, differentiation, and secretion of IgE [24]. We found that both serum levels of total and OVA-specific IgE were reduced by treatment with the Ad-hIFN-λ1 given before antigen challenge. Therefore, the observed reduction in serum IgE in our asthma model by hIFN-λ1 may be the result of inhibitory effects on B cell activation and reduced Th2 cytokine
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Fig. 4. Effect of Ad-hIFN-λ1 on cytokine production by cultured splenocytes. Splenocytes were prepared 24 h after the last OVA challenge, from PBS-sensitized/challenged mice (PBS), OVAsensitized/challenged mice (OVA), OVA-sensitized/challenged mice with the administration of Ad-mock and with the administration of Ad-hIFN-λ1. A total of 3 × 106 cells/well were cultured (in triplicates) and stimulated in the presence or absence of 100 μg/mL OVA for 4 days. Supernatants were removed and analyzed by ELISA, for IL-4, IL-5, IL-10, IL-13, and IFN-γ. Samples were assayed in triplicates. Data shown are mean ± SEM of two separate cultures from two independent experiments, with 6 mice/group. #Significant differences (P b 0.05) between sensitized/challenged control groups and sensitized/challenged Ad-hIFN-λ1 treatment groups.
release. The biological activities of IgE are mediated through highaffinity IgE receptors (FcεRI) on mast cells and basophils. Cross-linking of FcεRI initiates multiple signaling cascades leading to cellular degranulation and activation [25]. Regulatory T cells are key immune suppressive cells which mediate immune tolerance in autoimmune, inflammatory diseases and cancer. It has been identified that CD4+CD25+Foxp3 + Treg cells can potently suppress IgE production, directly or indirectly suppress effector cells of allergic inflammation, such as mast cells, basophils, and eosinophils, and contribute to remodeling in asthma [26]. In experimental models,
CD4+CD25+Foxp3 + Treg cells can reduce established lung eosinophilia, Th2 infiltration, and expression of IL-5 and IL-13, but have no effect on established airway hyperreactivity [27]. Mennechet and Uze [28] reported that IFN-λ1-treated DCs promote the generation of tolerogenic DCs and proliferation of CD4+0CD25+Foxp3 + Treg cells with contactdependent suppressive activity on T cell proliferation. However, whether in vivo IFN-λ1 could induce the generation of Treg cells has not been clarified. In this study, we demonstrate that treatment with Ad-hIFNλ1 significantly increases the proportion of CD4+CD25+Foxp3 +Treg cells in the spleen of OVA-sensitized and challenged mice. In conclusion, IFN-λ1 can alleviate some asthmatic symptoms, inhibit pulmonary eosinophilic inflammation, and decrease Th2 cytokine production in mice. Our data show that IFN-λ1-induced attenuation of the allergic airway response in mice is associated with increased numbers of CD4+CD25+Foxp3 + Treg cells in the spleen. With more studies, IFN-λ1 might be applied as an immunotherapeutic agent for the treatment of allergic diseases. Acknowledgments This study was supported by the grants from the National Natural Science Foundation of China (81070034 and 30671932), Zhejiang Provincial Natural Science Foundation of China (LY13H090014), Scientific Research Fund of Zhejiang Provincial Education Department (Y201120665), and sponsored by K.C. Wong Magna Fund in Ningbo University.
Fig. 5. Ad-hIFN-λ1 treatment increases splenic Treg cells in BALB/c mice. Spleen cells from PBS-sensitized/challenged mice (PBS), OVA-sensitized/challenged mice (OVA), and OVAsensitized/challenged mice with the administration of Ad-mock and with the administration of Ad-hIFN-λ1 mice were stained with specific antibodies to CD4, CD25, and Foxp3 for quantification of CD4+CD25+Foxp3 + Treg cells, and then analyzed using flow cytometry. #A significant increase (P b 0.05) in CD4+CD25+Foxp3 + Treg cell numbers was observed following treatment with Ad-hIFN-λ1 compared with the PBS, OVA or Ad-mock groups.
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