OVA-induced Allergic Inflammation Research Articles

TSLP and IL-33 distinctively modulate the allergic response by differentially modulating Th2 cells and ILC2s

Abstract Alarmins are endogenous chemotactic or immune-activating proteins released after cell injury or immune activation, and these include thymic stromal lymphopoietin (TSLP) and IL-33. Mice lacking these cytokines exhibit defective type 2 responses, but there is controversy regarding the roles of these cytokines in allergic responses in vivo. Here, we demonstrate that the nature of the allergic response is dictated by the alarmin. Mice lacking IL-33 were resistant to pulmonary inflammation induced by papain, but they exhibited normal OVA-induced allergic inflammation. In contrast, mice lacking either TSLP or TSLPR had significantly lower pulmonary inflammation in response to OVA challenge but responded normally to papain. Moreover, while intranasal papain challenge rapidly induced the release of IL-33, OVA challenge predominantly released TSLP. Steady-state pulmonary ILC2s were activated in response to IL-33, and correspondingly, Il33 KO mice showed a similar defect in effector Th2 generation to that observed in animals lacking ILC2s. In contrast to IL-33, TSLP did not activate steady-state ILC2s. Instead, conditional deletion of TSLPR in T cells hampered Th2 cell generation in the OVA-induced asthma model, but these mice responded normally to papain. These data demonstrate that Th2 cells rely on IL-33-stimulated ILC2s, whereas in the OVA-induced asthma model, TSLP acts on Th2 cells to promote type 2 responses. Thus, TSLP and IL-33 are both alarmins, but they have distinctive effects on allergic responses based on their different release kinetics and on their differential actions on Th2 cells versus ILC2s. These findings indicate approaches to selectively target different allergic responses, with potential translational impact. This research was supported by the Intramural Research Programs of NIAID and NHLBI, NIH.

18\u03b2-Glycyrrhetinic acid suppresses allergic airway inflammation through NF-\u03baB and Nrf2/HO-1 signaling pathways in asthma mice

18β-Glycyrrhetinic acid (18β-GA), the main bioactive component of Glycyrrhizae Radix, is considered a promising anti-inflammatory and antioxidant agent. Here, we evaluated the anti-inflammatory and antioxidant effects of 18β-GA in an ovalbumin (OVA)-induced asthma mouse model, and examined the role of NF-κB and Nrf2/HO-1 signaling pathways. The histopathological changes of lung tissue in mouse were assessed by histochemical staining and counting of inflammatory cells. The levels of IgE and inflammatory cytokines in the bronchoalveolar lavage fluid of mice were detected by ELISA. In OVA-induced asthmatic mice, 18β-GA treatment can significantly improve lung function and reduce lung inflammation including infiltration of inflammatory cells. In addition, 18β-GA reduced the OVA-induced NF-κB phosphorylation in lungs of mice while increasing the expression of Nrf2 and HO-1. These results indicate that 18β-GA protects OVA-induced allergic inflammation of airway by inhibiting phosphorylation of NF-κB and enhancing the Nrf2/HO-1 pathway, and serves as a potential treatment option for allergic inflammation of airway.

Exploration of the effect of mixed probiotics on microbiota of allergic asthma mice

Co-relation between allergic asthma and microbiota varying with diet has been extensively investigated, implicating that oral supplement of alternative pharmaceuticals are potential for asthma control. Probiotics are of great concern due to its beneficial effects on the host, whereas the potential mechanisms and the optimal dose need to be further explored. In the present study, three different doses of mixed strains were given orally to mouse model of allergic asthma induced by ovalbumin (OVA). Continuous administration of mixed probiotics could alleviate OVA-induced allergic inflammation through reducing costimulatory molecules on the surface of dendritic cells (DCs) and increasing mucosal gut-primed Tregs induced by mesenteric CD103+DCs. Mixed probiotics-induced protective effect relates to gut microbiota instead of lung microbes. Microbial diversity and Bacteroidetes/Firmicutes (B/F) ratio are augmented upon probiotic strains. Interestingly, treatment with mixed strain lead to an increased levels of genus Lactobacillus. Lactobacillus genus-Operational Taxonomic Unit (OUT) analysis indicated that OTU9 may be the dominant strain from mixed probiotics providing protective effect. The low dose seems to be the best in the whole study. Our results provide new evidence for probiotic application in allergic diseases and support the idea that targeted gut microbiota will be an effective approach for allergic airway diseases.

Effect of γ-secretase inhibitor on middle ear ultrastructures in ovalbumin-mediated otitis media with effusion in rats

Objective: To study the effect of the inhibitor of Notch signaling pathway-γ-secretase inhibitor DAPT on the ultrastructures of middle ear in the ovalbumin (OVA)-mediated allergic OME in vivo. Methods: Male Sprague-Dawley (SD) rats, weighing 250-300 g, were completely and randomly divided into three groups (5 rats, 10 ears in each group):(1)Control group(2)OME group(3)OME+DAPT group. Rats in the OME group underwent systemic and local sensitization by intraperitoneal and intratympanic injection of ovalbumin to make the model of OVA-induced OME. Rats in the control group were sensitized with PBS. On the basis of establishing the OME model, OME+DAPT group were intraperitoneal injected with DAPT (10 mg/kg) for seven consecutive days and were administered before intratympanic injection of ovalbumin. After the model was successfully established, endoscopy,H&E staining and scanning electron microscopy were used to study the histology and mucous-ciliary ultrastructures of the non-ciliated and ciliated mucosa in the middle ear of each group. One-way ANOVA and Tukey methods were used for statistical analysis. Results: H&E staining showed that the three groups had statistically significant differences in submucosal thickness both in non-ciliated and ciliated regions (non-ciliated area:(6.83±1.47)μm, (38.58±9.57)μm, (32.17±11.89)μm, respectively. F=107.9;cilia area:(26.69±3.22)μm, (30.41±6.75)μm, (26.76±4.06)μm, respectively. F=5.62,both P<0.01). The thickness of the submucosa in the non-ciliated area and the cilia area of the OME group were significantly thicker than that of control group (F=42.08 and 4.40,both P<0.05); the thickness of the non-ciliated area and the ciliated area in OME+DAPT group were reduced compared to OME group(F=1.55 and 2.77,both P<0.05). Scanning electron microscopy showed that the array of cilia on the middle ear mucosa was disorderly arranged and inversed, this phenomenon was relieved in the OME+DAPT group. The number of goblet cells in the control group, OME group, and OME+DAPT group were 9.87±1.92; 15.67±5.77; 10.33±1.99 respectively and the difference between them was statistically significant (F=11.43, P<0.01). The number of goblet cells in the OME group were significantly higher than those in the control group (F=9.00,P<0.01) and the number of goblet cells in the OME+DAPT group were decreased compared to those of OME group (F=8.41, P<0.01). Conclusions: The study demonstrates the pathological changes of the ultrastructure in middle ear in OVA-induced OME and the effect of the γ-secretase inhibitor on it. In OME group, the cilia are disorderly arranged and inversed, the number of goblet cell is increased and they are swelled which suggest the hypersecretion of the mucus. DAPT can regulate OVA-induced allergic inflammation and relieve pathological changes of ultrastructure in middle ear mucociliary transport system through alleviating submucosal inflammation, reducing the hypersecretion of goblet cell and the morphological damage of cilia through the Notch signaling pathway.

The role of anti-elastin antibodies in a mouse model of asthma.

The role of anti-elastin antibody (Ab) in the lung is unclear, although they may be involved in chronic obstructive pulmonary disease (COPD). Recently, increased anti-elastin Ab levels were reported in asthma. To elucidate the role of anti-elastin Ab in asthma, we created a murine asthma model. Anti-elastin Ab in the airway was neutralized by intratracheal administration of elastin peptide, and the inhibitory effects of anti-elastin Ab on airway remodeling were evaluated. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14. After immunization, the mice received booster OVA via inhalation twice per week for 9 weeks, and bronchoalveolar lavage fluid (BALF) and lung tissues were evaluated. In lung tissues, airway remodeling occurred after 9 weeks of OVA sensitization. Peak levels of anti-elastin Ab and eosinophils in BALF were detected after 3 weeks of OVA sensitization. Anti-elastin Ab and eosinophil levels in BALF were significantly reduced after 3 weeks by the neutralization of anti-elastin Ab. Peak transforming growth factor-β1 levels in BALF were detected at 3 weeks after OVA sensitization and were significantly reduced by the neutralization of anti-elastin Ab. Airway remodeling in lung tissues was also significantly inhibited by the neutralization of anti-elastin Ab. In our murine asthma model, anti-elastin Ab was recruited to the airway by OVA-induced allergic inflammation. Airway remodeling was inhibited by the neutralization of anti-elastin Ab. Anti-elastin Ab may contribute to the progression of airway remodeling.

A 7-Amino Acid Peptide Mimic from Hepatitis C Virus Hypervariable Region 1 Inhibits Mouse Lung Th9 Cell Differentiation by Blocking CD81 Signaling during Allergic Lung Inflammation.

T helper (Th) cells orchestrate allergic lung inflammation in asthma pathogenesis. Th9 is a novel Th cell subset that mainly produces IL-9, a potent proinflammatory cytokine in asthma. A 7-amino acid peptide (7P) of the hypervariable region 1 (HVR1) of hepatitis C virus has been identified as an important regulator in the type 2 cytokine (IL-4, IL-5, and IL-13) immune response. However, it is unknown whether 7P regulates Th9 cell differentiation during ovalbumin- (OVA-) induced allergic lung inflammation. To address this, we studied wild-type mice treated with 7P and a control peptide in an in vivo mouse model of OVA-induced allergic inflammation and an in vitro cell model of Th9 differentiation, using flow cytometry, cytokine assays, and quantitative PCR. The binding of 7P to CD81 on naïve CD4+ T cells during lung Th9 differentiation was determined using CD81 overexpression and siRNA knockdown analyses. Administration of 7P significantly reduced Th9 cell differentiation after OVA sensitization and exposure. 7P also inhibited Th9 cell differentiation from naïve and memory CD4+ T cells in vitro. Furthermore, 7P inhibited the differentiation of human Th9 cells with high CD81 expression from naïve CD4+ T cells by blocking CD81 signaling. CD81 siRNA significantly reduced Th9 cell differentiation from naïve CD4+ T cells in vitro. Interestingly, CD81 overexpression in human naïve CD4+ T cells also enhanced Th9 development in vitro. These data indicate that 7P may be a good candidate for reducing IL-9 production in asthma.

MiR-146a Promotes Tolerogenic Properties of Dendritic Cells and through Targeting Notch1 Signaling

ABSTRACT Background MiR-146a has been shown to negatively regulate innate immune, inflammatory response and antiviral pathway, however, its role in the tolerogenic responses remains largely unknown. This study aimed to investigate the role of miR-146a in the OVA-induced allergic inflammation of dendritic cells (DCs). Methods Bone marrow-derived DCs (BMDCs) were treated with OVA (100 µg/ml) for 24 h. MiR-146a expressions were assessed by quantitative RT-PCR. BMDCs were transfected with miR-146a mimics or inhibitor. Cell surface markers were analyzed by flow cytometry. Cytokine levels were determined by ELISA assay. Mixed lymphocyte culture assay was adopted to assess CD4 + T-cell differentiation. The 3ʹ UTR luciferase reporter assay was utilized to determine the miRNA target sequence. Results OVA treatment significantly up-regulated miR-146a in BMDCs in a dose- and time-dependent manner. In the OVA-treated DCs, overexpression of miR-146a (mimics transfection) down-regulated the surface markers (CD80, CD86) and increased production of anti-inflammatory cytokines TGF-β1 and IL-10 but decreased pro-inflammatory cytokine IL-12. MiR-146a overexpression promoted immature DC to induce regulatory T cells (Treg) differentiation. By contrast, transfection of miR-146a inhibitor into DC exhibited the opposite trends. Notch1 was a direct target of miR-146a, and Notch1 knock-down induced similar effects as miR-146a mimics transfection in BMDCs. Moreover, the effect of miR-146a inhibitor on OVA-induced DC was attenuated by Notch1 knock-down. Conclusion miRNA-146a promoted tolerogenic properties of DCs, at least partially, through targeting Notch1 signaling.

Airborne Bacteria Enriched PM2.5 Enhances the Inflammation in an Allergic Adolescent Mouse Model Induced by Ovalbumin.

Air pollution events frequently occur in China during the winter. Most investigations of pollution studies have focused on the physical and chemical properties of PM2.5. Many of these studies have indicated that PM2.5 exacerbates asthma or eosinophil inflammation. However, few studies have evaluated the relationship between bacterial loads in PM2.5, and especially pathogenic bacteria and childhood asthma. Airborne PM2.5 samples from heavily polluted air were collected in Hangzhou, China between December 2014 and January 2015. PM2.5 and ovalbumin (OVA) were intratracheally administered twice in 4-week intervals to induce the allergic pulmonary inflammation in adolescent C57/BL6 mice. PM2.5 exposure caused neutrophilic alveolitis and bronchitis. In the presence of OVA, the levels of the Th2 cytokines IL-4, IL-12, and IL-17 were significantly increased in bronchoalveolar lavage fluids (BALF) after PM2.5 exposure, while eosinophil infiltration and mucin secretion were also induced. In addition to adjuvant effects on OVA-induced allergic inflammation, PM2.5 exposure also led to the maturation of dendritic cells. These results suggest that PM2.5 exposure may aggravate lung eosinophilia and that PM2.5-bound microbial can exacerbate allergic and inflammatory lung diseases.

Adeno-associated viral vector-mediated ATPase gene transfer alleviates asthmatic airway inflammation in OVA-sensitized mice model

Abstract Bronchial asthma is a chronic inflammatory disorder of the airway characterized by airway hyperresponsiveness (AHR), pulmonary eosinophilia, elevated serum IgE, and dysregulated T helper (Th) 2 cytokines. It is becoming urgent to identify target molecules for the preventive and therapeutic purpose. ATP accumulation in bronchoalveolar lavage fluid (BALF) of asthmatic subjects indicates that extracellular ATP and the downstream responses are involved. ATP is tightly regulated by CD39, a nucleoside triphosphate diphosphohydrolase (ATPase) encoded by entpd1. Upregulated BALF ATP levels and decreased CD39 RNA were accompanied with asthmatic cardinal features in ovalbumin (OVA)-sensitized mice. Recombinant adeno-associated virus (rAAV) has been developed to carry exogenous genes with safe and low toxic therapeutic potential. The different serotypes and tropisms of AAVs benefit tissue specific gene targeting. This study aims to determine whether lung-targeting rAAV carrying CD39 (AAV/CD39) is able to reduce extracellular ATP in BALF and inhibit the OVA-induced allergic inflammation in an asthmatic murine model. rAAV vectors were administrated into the lungs of mice 3 days before the first OVA exposure. The data indicate that ATP levels in BALF were downregulated accompanied with rescued CD39 expression. The administration of AAV/CD39 was able to suppress AHR, pulmonary eosinophilia, and the production of IL-5 and IL-13 from OVA-activated mediastinal lymph nodes (MLNs) cells. IL-33 in BALF was also reduced. These data indicate that the attenuation of asthmatic airway inflammation may affect innate lymphoid cells (ILC) 2 activation. Thus, ATP level could be a potential target for asthma gene therapy.

Vitexin inhibits inflammation in murine ovalbumin-induced allergic asthma

Vitexin is an important component of various medicinal plants frequently used to treat asthma, such as Crataegus spp., Vitex spp., Passiflora spp., and Echinodorus spp. However, there is no information about the vitexin potential as anti-asthmatic. The aim of the present work was to evaluate the anti-hypersensitive activity of vitexin in a murine ovalbumin (OVA)-induced allergic asthma model. Mice were sensitized to OVA by i.p. injection on days 1st and 10th, followed by a daily challenge with OVA using a nebulizer, from days 19th to 24th. Vitexin or dexamethasone were orally administered 1h before each OVA challenge. Vitexin attenuates migration induced by OVA-hypersensitivity of eosinophil, neutrophil, and mononuclear cells in bronchoalveolar lavage fluid (BALF). Histological analysis of the lungs shows that vitexin suppressed leukocyte infiltration, mucus production and pulmonary edema. Increases in Th2 cytokines in BALF in OVA-induced asthma is also attenuated by vitexin, as well as plasma levels of IgE. Overall, these results suggest that vitexin can suppress OVA-induced allergic inflammation in mice and provide a strong rationale for further developing vitexin as a candidate treatment for allergic hypersensitivity. These data corroborate the popular use of vitexin-rich plants for asthma treatment.

The recombinant fusion protein of cholera toxin B and neutrophil-activating protein expressed on Bacillus subtilis spore surface suppresses allergic inflammation in mice.

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) has been identified as a modulator with anti-Th2 inflammation activity, and cholera toxin B (CTB) is a mucosal adjuvant that can also induce antigen tolerance. In this study, we constructed a CTB-NAP fusion protein on the surface of Bacillus subtilis spore and evaluate the efficiency of oral administration of the recombinant CTB-NAP spores in preventing asthma in mice. Oral administration of recombinant CTB or CTB-NAP spores significantly decreased serum ovalbumin (OVA)-specific IgE (p<0.001) and increased fecal IgA (p<0.01) compared to the treatment with non-recombinant spores. Oral administration of recombinant CTB or CTB-NAP spores induced IL-10 and IFN-γ expression and reduced IL-4 levels in bronchoalveolar lavage fluid (BALF). Moreover, CTB and CTB-NAP spores reduced the eosinophils in BALF and inflammatory cell infiltration in the lungs. Furthermore, CD4+CD25+Foxp3+ Tregs in splenocytes were significantly increased in mice treated with recombinant CTB or CTB-NAP spores. The number of CD4+CD25+Foxp3+ Tregs caused by CTB-NAP was higher than that by CTB alone. Our study indicated that B. subtilis spores with surface expression of subunit CTB or CTB-NAP could inhibit OVA-induced allergic inflammation in mice. The attenuated inflammation was attributed to the induction of CD4+CD25+Foxp3+ Tregs and IgA. Moreover, the fusion protein CTB-NAP demonstrated a better efficiency than CTB alone in inhibiting the inflammation.

Immunologic regulatory effects of human umbilical cord blood-derived mesenchymal stem cells in a murine ovalbumin asthma model.

Mesenchymal stem cells (MSCs) have multiple immunomodulatory properties and hold therapeutic potential for inflammatory diseases. However, the therapeutic and immunologic effects of human umbilical cord blood-derived MSCs (huMSCs) remain largely unexamined for asthma. This study was to investigate the immunomodulatory properties of huMSCs in an ovalbumin (OVA)-induced murine asthma model. Mice were injected intraperitoneally with OVA and an aluminium hydroxide adjuvant. huMSCs were administered via the tail vein (5×105 cells/100uL) to female BALB/c mice prior to the initial OVA challenge. The effects of huMSCs were assessed by investigating airway hyperresponsiveness, histological changes, inflammatory cell numbers, serum allergen-specific antibodies, cytokine production in spleen, lung tissue, and bronchoalveolar lavage (BAL) fluid as well as expansion of regulatory T cells. Administration of huMSCs significantly reduced methacholine bronchial hyperresponsiveness and eosinophil counts in BAL cells. Similarly, there was a significant decrease in serum OVA-specific IgE and IgG1 levels along with Th2 cytokine production (IL-4, IL-5, and IL-13) in the lung and spleen tissues, whereas increased percentage of regulatory T cells was observed after treatment with huMSCs. Our results suggest that huMSC treatment reduces OVA-induced allergic inflammation, which could be mediated by regulatory T cells.

Rheb1 deletion in myeloid cells aggravates OVA-induced allergic inflammation in mice

The small GTPase ras homolog enriched in brain (Rheb) is a downstream target of tuberous sclerosis complex 1/2 (TSC1/2) and an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), the emerging essential modulator of M1/M2 balance in macrophages. However, the role and regulatory mechanisms of Rheb in macrophage polarization and allergic asthma are not known. In the present study, we utilized a mouse model with myeloid cell-specific deletion of the Rheb1 gene and an ovalbumin (OVA)-induced allergic asthma model to investigate the role of Rheb1 in allergic asthma and macrophage polarization. Increased activity of Rheb1 and mTORC1 was observed in myeloid cells of C57BL/6 mice with OVA-induced asthma. In an OVA-induced asthma model, Rheb1-KO mice demonstrated a more serious inflammatory response, more mucus production, enhanced airway hyper-responsiveness, and greater eosinophil numbers in bronchoalveolar lavage fluid (BALF). They also showed increased numbers of bone marrow macrophages and BALF myeloid cells, elevated M2 polarization and reduced M1 polarization of macrophages. Thus, we have established that Rheb1 is critical for the polarization of macrophages and inhibition of allergic asthma. Deletion of Rheb1 enhances M2 polarization but decreases M1 polarization in alveolar macrophages, leading to the aggravation of OVA-induced allergic asthma.

Mahuang Fuzi Xixin Decoction Attenuates Th1 and Th2 Responses in the Treatment of Ovalbumin-Induced Allergic Inflammation in a Rat Model of Allergic Rhinitis.

Allergic rhinitis (AR) is one of the most common allergic diseases, which adversely affect patients' quality of life. Mahuang Fuzi Xixin decoction (MFXD) has been widely used to treat AR in clinics in Asian countries. This study investigated the effect and possible therapeutic mechanisms of MFXD in the treatment of AR. A Wistar rat model of ovalbumin- (OVA-) induced AR was established and then treated with three doses of MFXD; AR symptoms, serum total immunoglobulin E, histamine, histopathological features, and release and expression of factors related to type 1 helper T (Th1) and type 2 helper T (Th2) responses were analyzed. Our study demonstrated that MFXD has a good therapeutic effect on OVA-induced allergic inflammation in an AR rat model as manifested in reduced frequencies of sneezing and nasal scratching and in reduced serum levels of total IgE and HIS. In addition, MFXD regulates imbalance in Th1/Th2 cells caused by AR by simultaneously attenuating Th1 and Th2 responses, such as by reducing the serum levels of IFN-γ and IL-4 and mRNA expression levels of IFN-γ, IL-4, GATA-3, and STAT-6. This study provided valuable information on the immunoregulatory effect of MFXD for the treatment of AR in future clinical studies.

Co-exposure to zymosan A and heat-inactivated Asian sand dust exacerbates ovalbumin-induced murine lung eosinophilia.

BackgroundEpidemiological studies have implicated Asian sand dust (ASD) in the increased prevalence of respiratory disorders, including asthma. It has been observed that fungal elements such as β-glucan can be adsorbed onto ASD. In the present study, the exacerbating effect of the combined exposure to zymosan A (ZymA) containing yeast β-glucan and heat-inactivated ASD on ovalbumin (OVA)-induced murine lung eosinophilia was investigated.MethodsBALB/c mice were repeatedly instilled intratracheally with one of eight immunogenic formulations consisting of various combinations of (1) ZymA, (2) ASD that was briefly heated to remove organic substances (H-ASD), and (3) OVA in normal saline, or each of the above alone. Pathologic changes, cytological alterations in bronchoalveolar lavage fluid (BALF), changes in inflammatory cytokines and chemokines in BALF, and OVA-specific IgE and IgG1 antibodies in serum were investigated.ResultsExposure to ZymA with or without OVA had no effect on most indicators of lung inflammation. Exposure to H-ASD with OVA increased the recruitment of inflammatory cells to the lungs and the serum levels of OVA-specific IgE and IgG1. The combination OVA + ZymA + H-ASD induced a marked recruitment of eosinophils and upregulation of T helper 2 (Th2) cytokines (interleukin [IL]-4 and IL-13), IL-6, eotaxin/CCL11, and monocyte chemotactic protein (MCP)-3/CCL7 in BALF and OVA-specific IgE in serum. This treatment also induced the most severe pathological changes in the lungs of mice. ZymA was found to boost the effects of H-ASD, thereby exacerbating the OVA-induced allergic inflammation, even though ZymA alone did not have such effect.ConclusionsThe results suggest that fungal elements such as β-1,3-glucan aggravate the allergic inflammation caused by ASD. Our findings may facilitate prophylaxis of some allergic diseases in Asia.

Adeno-associated viral vector-mediated anti-inflammasome gene transfer alleviates asthmatic airway inflammation in OVA-sensitized mice model

Abstract Bronchial asthma is a chronic inflammatory disorder of the airway characterized by airway hyperresponsiveness (AHR), pulmonary eosinophilia, elevated serum IgE, and dysregulated T helper (Th) 2 cytokines. It is becoming urgent to identify target molecules for the preventive and therapeutic purpose. ATP accumulation in bronchoalveolar lavage fluid (BALF) of asthmatic subjects indicates that extracellular ATP and the downstream responses are involved. ATP is an activator of NLRP3 inflammasomes and tightly regulated by CD39, a nucleoside triphosphate diphosphohydrolase encoded by entpd1. Upregulated BALF ATP levels and decreased CD39 RNA were accompanied with asthmatic cardinal features in ovalbumin (OVA)-sensitized mice. Recombinant adeno-associated virus (rAAV) has been developed to carry exogenous genes with safe and low toxic therapeutic potential. The different serotypes and tropisms of AAVs benefit tissue specific gene targeting. This study aims for determining whether lung-targeting rAAV carrying CD39 (AAV/CD39) is able to reduce extracellular ATP in BALF and inhibit the OVA-induced allergic inflammation in an asthmatic murine model. The mice were sensitized and challenged with OVA. rAAV vectors were administrated into the lungs of mice 3 days before the first OVA exposure. The data indicated that ATP levels in BALF were downregulated accompanied with rescued CD39 expression. The administration of AAV/CD39 was able to suppress AHR, pulmonary eosinophilia, and the production of IL-5 from OVA-activated mediastinal lymph nodes (MLNs) cells. The treatment of AAV/CD39 attenuated the asthmatic airway inflammation and AHR. Collectively, ATP could be a potential target for gene therapy on asthma.

CCR9 Is a Key Regulator of Early Phases of Allergic Airway Inflammation.

Airway inflammation is the most common hallmark of allergic asthma. Chemokine receptors involved in leukocyte recruitment are closely related to the pathology in asthma. CCR9 has been described as a homeostatic and inflammatory chemokine receptor, but its role and that of its ligand CCL25 during lung inflammation remain unknown. To investigate the role of CCR9 as a modulator of airway inflammation, we established an OVA-induced allergic inflammation model in CCR9-deficient mice. Here, we report the expression of CCR9 and CCL25 as early as 6 hours post-OVA challenge in eosinophils and T-lymphocytes. Moreover, in challenged CCR9-deficient mice, cell recruitment was impaired at peribronchial and perivenular levels. OVA-administration in CCR9-deficient mice leads to a less inflammatory cell recruitment, which modifies the expression of IL-10, CCL11, and CCL25 at 24 hours after OVA challenge. In contrast, the secretion of IL-4 and IL-5 was not affected in CCR9-deficient mice compared to WT mice. These results demonstrate for the first time that CCR9 and CCL25 expressions are induced in the early stages of airway inflammation and they have an important role modulating eosinophils and lymphocytes recruitment at the first stages of inflammatory process, suggesting that they might be a potential target to regulate inflammation in asthma.

Antiallergic Function of KR62980, a Peroxisome Proliferator-Activated Receptor-γ Agonist, in a Mouse Allergic Rhinitis Model.

PurposePeroxisome proliferator-activated receptor γ (PPAR-γ) has been shown to play an important role in the control of inflammatory responses acting on macrophages, mast cells, T cells and eosinophils. A novel PPAR-γ ligand, KR62980 have been recently focused on due to the lower undesirable effects than other PPAR-γ ligands such as rosiglitazone and pioglitazone. The present study was aimed to investigate the effects of KR62980 on nasal symptoms and immunopathological profiles in allergic nasal mucosa in murine allergic rhinitis model.MethodsBALB/c mice were sensitized and challenged intranasally with ovalbumin (OVA). KR62980 was administered intraperitoneally or orally 3 hours before each intranasal OVA challenge.ResultsAdministration of KR62980 significantly decreased the number of nasal rubbing, nasal sneezing, ova-specific IgE and total IgE in serum, secretion of Interleukin (IL)-4, IL-5, and IL-17 from the spleen and eosinophilic infiltration in the nasal mucosa. KR62980 decreased the expression of IL-4, IL-5 and IL-10 mRNAs in the nasal mucosal tissue, while, it elevated the level of IL-10 and IFN-γ in splenocyte culture. KR62980 seemed to decrease IL-17 level in local and systemic level even though it did not reach to statistical significance. The anti-inflammatory effect was more definite when the KR62980 was administered intraorally than intraperitoneally.ConclusionsA novel PPAR-γ ligand, KR62980 can attenuate OVA-induced allergic inflammation in mice mainly through modulation of Th2 cytokines. This finding suggests that PPAR-γ might have a role in the treatment of allergic rhinitis.

Immune Modulatory Effects of IL-22 On Allergen-Induced Pulmonary Inflammation

RationaleAllergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system.MethodsInducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined.ResultsIL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE.ConclusionsThese findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma. RationaleAllergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system. Allergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system. MethodsInducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined. Inducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined. ResultsIL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE. IL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE. ConclusionsThese findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma. These findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma.

A specific sphingosine kinase 1 inhibitor attenuates airway hyperresponsiveness and inflammation in a mast cell–dependent murine model of allergic asthma

Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined. We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice. Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs. S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.