Ovalbumin Group Research Articles

Fine Particulate Matter-Induced Exacerbation of Allergic Asthma via Activation of T-cell Immunoglobulin and Mucin Domain 1.

Background:Fine particulate matter (PM2.5) exacerbates airway inflammation and hyperreactivity in patients with asthma, but the mechanism remains unclear. The aim of this study was to observe the effects of prolonged exposure to high concentrations of PM2.5 on the pathology and airway hyperresponsiveness (AHR) of BALB/c mice undergoing sensitization and challenge with ovalbumin (OVA) and to observe the effects of apoptosis and T-cell immunoglobulin and mucin domain 1 (TIM-1) in this process.Methods:Forty female BALB/c mice were divided into four groups: control group, OVA group, OVA/PM group, and PM group (n = 10 in each group). Mice in the control group were exposed to filtered clean air. Mice in the OVA group were sensitized and challenged with OVA. Mice in the OVA/PM group were sensitized and challenged as in the OVA group and then exposed to PM2.5 for 4 h per day and 5 days per week for a total of 8 weeks using a nose-only “PM2.5 online enrichment system” in The Second Hospital of Hebei Medical University. Mice in the PM group were exposed to the PM2.5 online enrichment system only. AHR was detected. Bronchoalveolar lavage fluid (BALF) was collected for cell classification. The levels of interleukin-4 (IL-4), IL-5, and IL-33 in BALF were measured using enzyme-linked immunosorbent assay. Changes in histological structures were examined by light microscopy, and changes in ultramicrostructures were detected by electron microscopy. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay in the lung tissues. Western blotting and immunohistochemistry were utilized to analyze the expression of Bcl-2, Bax, and TIM-1 in the lungs.Results:The results showed that AHR in the OVA/PM group was significantly more severe than that in the OVA and PM groups (P < 0.05). AHR in the PM group was also considerably more severe than that in the control group (P < 0.05). The BALF of OVA/PM group (28.00 ± 6.08 vs. 12.33 ± 4.51, t = 4.631, P = 0.002) and PM group (29.00 ± 3.00 vs. 12.33 ± 4.51, t = 4.927, P = 0.001) had more lymphocytes than the BALF of the control group. The number of neutrophils in the BALF of the OVA/PM group (6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020) and PM group (6.67 ± 1.53 vs. 3.33 ± 1.53, t = 2.886, P = 0.020) was much higher than those in the BALF of OVA group (P < 0.05). TUNEL assays showed that the number of apoptotic cells in the OVA/PM group was significantly higher than that in the OVA group (Tunel immunohistochemical scores [IHS%], 1.20 ± 0.18 vs. 0.51 ± 0.03, t = 8.094, P < 0.001) and PM group (Tunel IHS%, 1.20 ± 0.18 vs. 0.51 ± 0.09, t = 8.094, P < 0.001), and that the number of apoptotic cells in the PM group was significantly higher than that in the control group (Tunel IHS%, 0.51 ± 0.09 vs. 0.26 ± 0.03, t = 2.894, P = 0.020). The concentrations of IL-4 (77.44 ± 11.19 vs. 48.02 ± 10.02 pg/ml, t = 4.595, P = 0.002) and IL-5 (15.65 ± 1.19 vs. 12.35 ± 0.95 pg/ml, t = 3.806, P = 0.005) and the Bax/Bcl-2 ratio (1.51 ± 0.18 vs. 0.48 ± 0.10, t = 9.654, P < 0.001) and TIM-1/β-actin ratio (0.78 ± 0.11 vs. 0.40 ± 0.06, t = 6.818, P < 0.001) in the OVA/PM group were increased compared to those in the OVA group. The concentrations of IL-4 (77.44 ± 11.19 vs. 41.47 ± 3.40 pg/ml, t = 5.617, P = 0.001) and IL-5 (15.65 ± 1.19 vs. 10.99 ± 1.40 pg/ml, t = 5.374, P = 0.001) and the Bax/Bcl-2 ratio (1.51 ± 0.18 vs. 0.97 ± 0.16, t = 5.000, P = 0.001) and TIM-1/β-actin ratio (0.78 ± 0.11 vs. 0.31 ± 0.06, t = 8.545, P < 0.001) in the OVA/PM group were increased compared to those in the PM group. The concentration of IL-4 (41.47 ± 3.40 vs. 25.46 ± 2.98 pg/ml, t = 2.501, P = 0.037) and the Bax/Bcl-2 ratio (0.97 ± 0.16 vs. 0.18 ± 0.03, t = 7.439, P < 0.001) and TIM-1/β-actin ratio (0.31 ± 0.06 vs. 0.02 ± 0.01, t = 5.109, P = 0.001) in the PM group were also higher than those in the control group.Conclusions:Exacerbated AHR associated with allergic asthma caused by PM2.5 is related to increased apoptosis and TIM-1 activation. These data might provide insights into therapeutic targets for the treatment of acute exacerbations of asthma induced by PM2.5.

Inhibitory effects and mechanism of Triticum aestivum on OVA-induced anaphylaxis in mice

Objective To investigate the inhibitory effects and mechanism of Triticum aestivum (TA) on anaphylaxis in ovalbumin (OVA)-sensitized mice. Methods Twenty-five female BALB/c mice were randomly divided into five groups (n=5), including control group, OVA group, 100 mg/kg TA group, 200 mg/kg TA group and DEX (dexamethasone) group. OVA (20 μg) and aluminum hydroxide (1 mg) were dissolved in 100 μl of PBS solution and injected into the abdominal cavity of each experimental mouse, while the mice in the control group were injected with 100 μl of 0.9% normal saline. All mice were given drinking water. The above processes were repeated two weeks later, and different concentrations of experimental drugs were diluted in 1% CMC (carboxymethyl cellulose) solution to feed mice. Allergic symptoms and behaviors of each mouse were scored in the process of feeding. On the 12th day after first sensitization, mice in the control and experimental groups were subcutaneously injected in the auricle with 20 μl of 0.9% saline solution and OVA, respectively. The thickness of each mouse′s auricle was measured 6 and 24 hours later. Histopathologic changes in auricle tissues were observed by hematoxylin-eosin(HE) staining 24 hours later. The mice were euthanized by cervical dislocation in the 6th week after first sensitization. Abdominal aorta serum samples were collected and tested for specific inflammatory cytokines (IgE, IgG1, TNF-α, IL-4) and anti-OVA antibodies (IgE, IgG1) by ELISA. ELISA and RT-PCR were used to detect the expression of IFN-γ, IL-4, IL-5, IL-12 and IL-13 after spleen lymphocytes were stimulated with different concentrations of drugs. Results Both the score of allergic symptoms and behavior and the thickness of the auricle of the OVA group were the highest among all groups (P<0.05). A TA dose-dependent decrease was found in both of the two parameters, which was confirmed by histopathological analysis. ELISA results showed that the plasma levels of IgE, IgG1, IL-4 and TNF-α in the experimental groups were significantly higher than those in the control group, but all of them were decreased in a TA dose-dependent manner. TA at the high concentration of 200 mg/kg was similar to DEX in inhibiting the expression of IgE and TNF-α. RT-PCR results showed that Th1 cytokines (IFN-y and IL-12) in the experimental groups increased significantly as compared with those in the control group (P<0.05). Expression of IFN-y and IL-12 was not significantly affected by TA at various concentrations, but markedly inhibited by DEX. The levels of Th2 cytokines (IL-4, IL-5 and IL-13) in the experiment groups were significantly higher than those in the control group (P<0.05). Compared with the OVA group, the levels of IL-4, IL-5 and IL-13 were significantly reduced in TA groups, especially in the TA 100 μg/ml group in which the expression of the three cytokines was decreased by about 60%, 18% and 20%, respectively. Conclusion TA helps to maintain immune balance by selectively inhibiting the expression of Th2 cytokines (IL-4, IL-5, IL-13), IgE and IgG and not influencing Th1 cytokines (IFN-γ and IL-12). It has an anti-allergic effect as it effectively suppresses allergic responses. This research provides both theoretical and experimental basis for further development of novel anti-anaphylactic treatment with TA. Key words: Triticum aestivum; Ovalbumin; Anaphylaxis; Th1 cell; Th2 cell

Azithromycin ameliorates OVA-induced airway remodeling in Balb/c mice via suppression of epithelial-to-mesenchymal transition

Azithromycin is a potent agent that prevents airway remodeling. In this study, we hypothesized that azithromycin (35 mg/kg orally) alleviated airway remodeling through suppression of epithelial-to-mesenchymal transition (EMT) via downregulation of transforming growth factor-beta 1 (TGF-β1)/receptor for activated C-kinase1 (RACK1)/snail in mice. An ovalbumin (OVA)-induced Balb/c mice airway allergic inflammatory model was used. Airway inflammation and remodeling were evaluated with hematoxylin and eosin (HE), periodic acid–Schiff (PAS), and Masson staining. E-cadherin and N-cadherin (molecular markers of EMT) were analyzed by immunofluorescence, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and western blotting; α-smooth muscle actin (α-SMA) was evaluated using immunohistochemistry (IHC), qRT-PCR, and western blotting; and expression of TGF-β1/RACK1/Snail in lungs was measured by qRT-PCR and western blotting. Our data showed that azithromycin significantly reduced inflammation score, peribronchial smooth muscle layer thickness, goblet cell metaplasia, and deposition of collage fibers (P < 0.05), and effectively suppressed airway EMT (upregulated E-cadherin level, and downregulated N-cadherin and α-SMA levels) compared with the OVA group (P < 0.05). Moreover, the increasing mRNA and protein expressions of TGF-β1 and RACK1 and mRNA level of Snail in lung tissue were all significantly decreased in azithromycin-treated mice (P < 0.05). Taken together, our results suggest that azithromycin has the greatest effects on reducing airway remodeling by inhibiting TGF-β1/RACK1/Snail signal and improving the EMT in airway epithelium.

Immune response elicited by graphene oxide-based nanovaccine

Objective To study the antigen-specific immune response induced by the graphene oxide (GO) in mice. Methods OVA-loaded GO nano-immunocomplexes (GO-OVA) were prepared by co-incubation of nano GO with model antigen ovalbumin (OVA). Nano GO was characterized by atomic force microscopy and laser particle size analyzer. The cytotoxicity of GO to mouse bone marrow dendritic cells (BMDCs) was detected by cell counting kit (CCK-8). The GO-OVA uptake of BMDCs were observed by fluorescent staining. C57BL/6 mice were divided into OVA group, aluminum adjuvant OVA (Al-OVA) group and GO-OVA group (6 mice in each group) by body weight for in vivo immunization. The levels of OVA-specific antibody IgG (total IgG, IgG1, and IgG2a) in serum of mice were detected by enzyme-linked immunosorbent assay (ELISA). The T lymphocyte subsets in spleen and inguinal lymph nodes of mice were detected by flow cytometry. Results The average particle size of the prepared nano GO was (294.34±4.68) nm, and the polydispersity coefficient was 0.208. Nano GO has less toxicity to mouse BMDCs. The results of in vitro experiments indicated that GO-OVA nanovaccine can be efficiently internalized by mouse BMDCs. The antigen-specific IgG antibodies induced by the GO-OVA was similar to that of aluminum adjuvant and the difference was not statistically significant (P>0.05), and the Th1-type response was predominant. The proportions of CD4+ and CD8+ T lymphocytes in the spleen and inguinal lymph nodes in GO-OVA group were significantly higher than those in OVA and Al-OVA groups, and the differences were statistically significant (all P<0.05). Conclusions GO-OVA nano-immunocomplexes can induce both humoral and cellular immune responses in mice, which provides basis for the development of novel vaccine vectors and adjuvants. Key words: Graphene oxide; Nanovaccine; Humoral immune response; Cellular immune response

MicroRNA-200a Affects the Proliferation of Airway Smooth Muscle Cells and Airway Remodeling by Targeting FOXC1 via the PI3K/AKT Signaling Pathway in Ovalbumin-Induced Asthmatic Mice

The etiology of asthma, which is a complicated disorder with various symptoms, including wheezing, coughing, and airflow obstruction, remains poorly understood. In addition, the effects of microRNAs (miRs) have not been explored. This study explored the effect of microRNA-200a (miR-200a) on airway smooth muscle cells (ASMCs) and airway remodeling in asthmatic mice. Furthermore, we speculated that miR-200a achieves its effect by targeting FOXC1 via the PI3K/AKT signaling pathway based on differentially expressed gene screening, target miR predictions and a bioinformatics analysis. Eighty mice were assigned to a saline group or an ovalbumin (OVA) group, and the OVA group was transfected with a series of inhibitors, activators, and siRNAs to test the established mouse model. Airway reactivity and the ratio of eosinophils (EOSs) to leukocytes were detected. An ELISA was adopted to measure the levels of interleukin (IL)-4, IL-6, IL-8, tumor necrosis factor (TNF)-α, and immunoglobulin E (IgE). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to determine the expression of FOXC1, PI3K, AKT, NF-κB, cyclin D1, TGF-β1 and p-AKT in ASMCs. Finally, CCK-8 assays were performed to detect cell proliferation and flow cytometry to detect apoptosis and cell cycle entry. The bioinformatics analysis indicated that miR-200a mediated the PI3K/AKT signaling pathway by targeting FOXC1. In addition, mouse models of asthma were established. An elevated expression of miR-200a, a decreased mRNA and protein expression of FOXC1, PI3K, AKT, NF-κB, cyclin D1 and TGF-β1, a decreased expression of p-AKT, suppressed cell proliferation, accelerated apoptosis, and an increased number of cells at the G0/G1 phase were observed following the upregulation of miR-200a and downregulation of FOXC1. The overexpression of miR-200a may downregulate FOXC1, thereby inhibiting the activation of the PI3K/AKT signaling pathway and ultimately suppressing ASMC proliferation and airway remodeling in asthmatic mice. This evidence supports the potential that miR-200a represents a new approach to treating asthma.

Preventive and therapeutic effects of vitamin D in a mouse model of allergic asthma.

Vitamin D produces an anti-allergic effect that prevents inflammation due to asthma. We investigated whether vitamin D has an anti-inflammatory effect on the sensitization and challenge stages of asthma development in a murine model. Mice were divided into the following five groups according to ovalbumin (OVA) and vitamin D (VD) administration: control group, OVA group, preventive VD group (VD injection before OVA sensitization), inhibitory VD group (VD injection after OVA challenge), and dual VD groups (VD injection before OVA sensitization and after OVA challenge). Each group was evaluated for airway hyperresponsiveness (AHR), cell counts, cytokines, total IgE, and OVA-specific IgE using bronchoalveolar lavage fluid (BALF). Cytokines in the lysate and eosinophils in the lung tissue were also evaluated. AHR occurred less in the groups to which VD was administered than in the OVA group. The eosinophils, neutrophils, IL-5 in BALF, IL-4, TGF-β, and eosinophils in lysate decreased with the administration of VD in the preventive, inhibitory, and dual VD groups compared with the OVA group. Although the lymphocytes, macrophages, IL-4 in BALF, and IL-5 in lysate decreased with administration of VD in the inhibitory and dual VD groups, they were not affected by preventive VD administration. These anti-allergic effects of VD were most noticeable with VD administration for dual (preventive and inhibitory) purposes. VD may produce preventive and inhibitory effects on the development and exacerbation of asthma in a murine model. These effects are most noticeable when VD is used for dual purposes.

Paeonol Ameliorates Ovalbumin-Induced Asthma through the Inhibition of TLR4/NF-κB and MAPK Signaling.

Asthma is a chronic inflammatory disease of the airways, with complex signaling pathways involved in its pathogenesis. It was reported that paeonol attenuated airway inflammation of ovalbumin (OVA)-induced mice. Therefore, it is of importance to further investigate the underlying mechanism. BALB/c mice were challenged with OVA for the asthma model, which was validated by the changed levels of IL-4, IFN-γ, and IgE. The elevation of IL-4 and the decreasing of IFN-γ were significantly in middle (p<0.05) or high (p<0.01) paeonol dose groups compared with OVA group. MIP-1β in bronchoalveolar lavage fluid (BALF) also decreased significantly in middle and high paeonol group compared with OVA group (p<0.01), which is similar to the change of its mRNA in lung tissues. Moreover, the inflammatory cells infiltration and collagen deposition were attenuated by paeonol and montelukast sodium via histology examination. At last the immune blot of the protein extracted from lung tissues demonstrated that paeonol decreased the expression of TLR4 and the nuclear translocation of NF-κB, as well as the phosphorylation levels of P38 and ERK in asthma model. In conclusion, paeonol ameliorated OVA-induced asthma through the TLR4/NF-κB and mitogen-activated protein kinase (MAPK) signaling.

Anti-inflammatory effect of wogonin on allergic responses in ovalbumin-induced allergic rhinitis in the mouse.

BackgroundWogonin is commonly used for the treatment of allergic diseases. However, neither its precise effect in preventing allergic rhinitis (AR) nor its mechanism of action are known.ObjectivesIn this study, the effect of wogonin on allergic responses in ovalbumin (OVA) induced AR was investigated in mice.MethodsBALB/c mice were sensitized with intraperitoneal (i.p.) OVA and then challenged intranasally with OVA. Wogonin (10 and 30 mg/kg) was given to the treatment groups, and the effect of wogonin on the release of allergic inflammatory mediators, specifically OVA-specific immunoglobulin E (IgE) and inflammatory cytokines, was explored. Eosinophil infiltration and the levels of interleukin (IL) 5 and IL-13 were measured by immunohistochemistry.ResultsIn mice with AR, wogonin decreased OVA-specific IgE levels in serum, and the levels of the cytokines IL-4, IL-5, IL-13, eotaxin, and RANTES in nasal lavage fluid. Serum levels of IL-4, IL-5, and IL-13 were lower in both groups of wogonin-pretreated mice than in the OVA group. A reduction in eosinophil infiltration of the nasal mucosa and inhibition of the expression of IL-5 and IL-13 were also noted in the treated groups.ConclusionWogonin induced antiallergic effects in a murine model of AR by decreasing the infiltration of eosinophils and levels of T-helper type 2 cytokines. Thus, wogonin merits consideration as a therapeutic agent for treating AR.

Intranasal immunization with phosphorylcholine suppresses allergic rhinitis in mice.

Intranasal immunization with phosphorylcholine (PC) is known to reduce immunoglobulin (Ig)E production. However, its effects on the occurrence of allergic rhinitis (AR) are unknown. This study was performed to evaluate the effects of PC-keyhole limpet hemocyanin (PC-KLH) and to examine the effects on the occurrence of AR in a murine model of AR. In vivo study using an animal model. Forty-five female BALB/c mice were divided into three groups; those pretreated with intranasal administration of PC-KLH followed by intraperitoneal sensitization and nasal challenge with ovalbumin (OVA) (group A), those untreated with PC-KLH followed by sensitization and nasal challenge with OVA (group B), and those untreated with PC-KLH or OVA as controls (group C). Nasal symptoms, allergic inflammation in the nasal mucosa, OVA specific IgE production, and cytokine profile were compared among those three groups. Dendritic cells (DCs) were isolated from splenic cells and PC-KLH-stimulated interleukin (IL)-12p40 production was measured. The mice pretreated with PC-KLH showed lower allergic nasal symptoms and inflammation compared to untreated mice. The levels of total IgE and OVA-specific IgE in serum, and IL-4 production by nasal and splenic CD4+ T cells were significantly reduced by PC-KLH pretreatment. Furthermore, IL-12p40 production by DCs was induced by PC-KLH in a dose-dependent manner. Intranasal administration of PC-KLH suppressed allergic inflammation in nasal mucosa and antigen-specific IgE production by downregulating Th2-type immune response. Intranasal immunization with PC might be useful to prevent AR and upper airway bacterial infection. NA. Laryngoscope, 128:E234-E240, 2018.

Protective effect of baicalin on the small intestine in rats with food allergy

AimsThe therapeutic effect of baicalin and its mechanism were explored. Materials and methodsA total of 30 Sprague Dawley (SD) rats were randomly divided into 3 groups of 10: ovalbumin group (OVA group), baicalin intervention group (HQ group), and saline-group (NC group). Serum OVA-IgE antibody levels were detected by enzyme-linked immunosorbent assay (ELISA); and diarrhea in rats was observed. Animals were sacrificed at week seven. Then, a 5-cm long duodenum beneath the Treitz ligament was collected from each rat, and was fixed, embedded, sliced and stained with toluidine blue to evaluate the integrity of mast cells. Next, pathological changes of the intestine were observed by hematoxylin and eosin (H&E) staining, and the ultrastructure of the intestinal mucosa was observed under a transmission electron microscope. Key findingsSerum OVA-sIgE level were significantly lower (at sixth week, OVA group: 12.86±1.35, HQ group: 3.47±0.51, F=117.05, P<0.01), the number of eosinophils significantly decreased (HQ group: 2.73±1.02, OVA group: 16.48±2.32, P<0.01), mast cell integrated rate was significantly increased (HQ group: 89.90±4.43, OVA group: 35.30±9.78, P<0.01) uniform small intestinal villi were observed, the organelles were basically normal, and lesions were significantly fewer in the HQ group, compared with the OVA group. SignificanceBaicalin can effectively reduce serum OVA-sIgE in rats with food allergy, increase mast cell integrated rate and alleviate intestinal pathological changes. Hence, baicalin has a good therapeutic effect on food allergy.

Mislocalization of Runt-related transcription factor 3 results in airway inflammation and airway hyper-responsiveness in a murine asthma model.

The Runt-related transcription factor (RUNX) gene family consists of three members, RUNX1, -2 and -3, which heterodimerize with a common protein, core-binding factor β, and contain the highly conserved Runt-homology domain. RUNX1 and -2 have essential roles in hematopoiesis and osteogenesis. Runx3 protein regulates cell lineage decisions in neurogenesis and thymopoiesis. The aim of the present study was to determine the expression features of the Runx3 protein in a murine asthma model. In vivo, Runx3 protein and mRNA were found to be almost equivalently expressed in the murine lung tissue of the control, ovalbumin (OVA) and genistein groups; however, the nuclear Runx3 protein was abated in lung tissue in OVA-immunized and challenged mice. Following treatment with genistein, which is a flavonoid previously demonstrated to decrease airway inflammation in asthma, the allergic airway inflammation and airway hyper-responsiveness were attenuated and the Runx3 protein tended to augment in the nucleus. These results were further determined in vitro. These results indicated that the mislocalization of Runx3 protein is a molecular mechanism of allergic inflammation and airway hyper-responsiveness in a murine asthma model.

Immunological evaluation of Gum kondagogu (Cochlospermum gossypium): A tree gum with potential applications in food and pharma industry

Gum kondagogu (KG) is a natural polysaccharide derived as an exudate from the bark of the tree Cochlospermum gossypium (Bixaceae family). The toxicological assessment of KG established that this gum to be nontoxic, while the immunological response to KG was yet to be explored, as the protein content of KG has been reported to be 5.0–6.3% (w/w). The current investigation evaluates immunogenic aspects of three grades of gum (KG-I, II and III) in a rodent model. The immunogenicity of KG was evaluated in comparison with ovalbumin, which is a known food allergen. As a measure of immunological response, delayed-type hypersensitivity (DTH) reaction, blood histamine levels and antibody (IgG) response were assessed. The DTH reaction of KG-III gum was found to be similar to that of ovalbumin, as measured by mean skin fold thickness of each ear sensitised with test and reference immunogens. KG-I and II did not elicit any DTH reaction. Statistically significant difference in histamine and IgG levels were observed between the control (PBS), ovalbumin and KG-III rat groups (p < 0.05). The experimental observations showed similar immune response between KG-III and ovalbumin in experimental groups sensitised with respective immunogens. Immune response to KG-III gum could be attributed to the impurities originating from the bark during the process of exudation. As there is no statistical significant immunological response to KG-I and II, these grades of gum may find potential applications in food and pharmaceutical industry.

Programmed vaccination may increase the prevalence of asthma and allergic diseases.

The prevalence of asthma and allergic diseases has risen in recent decades. The etiology of asthma and allergic diseases has not been entirely elucidated. In this study, we investigated the possibility that programmed vaccination in China may have a potential role in asthma and allergic diseases. In this animal model, newborn BALB/c mice were randomly divided into three groups: vaccine plus ovalbumin (OVA), OVA, and control. The mice of vaccine plus OVA only group were inoculated with vaccines by following the National Vaccines Inoculation Program in China. Mice of vaccine plus OVA and OVA only groups were sensitized and challenged with OVA. Airway hyperresponsiveness was assessed by lung function and serum interleukin (IL) 4 and interferon (IFN) γ were measured. The results of lung function showed that mice of the vaccine plus OVA group exhibited an increase in enhanced pause (Penh) compared with that in the OVA group at methacholine concentrations of 6.25 and 12.5 mg/mL (p < 0.05). Serum IL-4 in the vaccine plus OVA group was higher than that in the OVA group (p < 0.01). The serum IFN-γ level in the OVA group was lower than that in the control group (p < 0.01), and also lower than that in the vaccine plus OVA group (p < 0.05). The ratio of IFN-γ to IL-4 both in the OVA and vaccine plus OVA group was lower than that in the control group (p < 0.01). Results of our study indicated that programmed vaccination in China may have a potential role in the prevalence of asthma and allergic diseases by inducing T-helper 2 cytokine expression and may be responsible for the increasing prevalence of asthma and allergic diseases in China.

Critical roles of adenosine A2A receptor in regulating the balance of Treg/Th17 cells in allergic asthma.

Deficiency of Treg cells and hyperactivity of Th17 cells together are involved in the immunological pathogenesis of asthma. The adenosine A2A receptor (A2AR) plays a critical role in the increased Foxp3 expression of Treg cells and the decreased Th17 generation. The study aimed to investigate A2AR expression in peripheral blood and its regulatory effect on balance of Treg/Th17 cells in asthma. Thirty-one patients with chronic persistent asthma were recruited and divided into 18 intermittent to mild asthma patients, 13 moderate to severe asthma patients. A2AR, Foxp3, and ROR-γt mRNA expression levels in peripheral blood mononuclear cells (PBMCs) were measured by quantitative polymerase chain reaction (qPCR). TGF-β, IL-17, and IgE in plasma were detected with enzyme-linked immunosorbent assay (ELISA). Forty-two BALB/c mice were randomly, equally assigned to control group, ovalbumin (OVA) group and OVA + CGS (CGS21680, A2AR agonist) group. The infiltration of lung inflammation cells were evaluated by HE, A2AR, Foxp3, and ROR-γt mRNA in lung tissues measured by qPCR, TGF-β, IL-17, and IgE in plasma measured with ELISA, and IL-17 and TGF-β protein in lung tissues analyzed with immunohistochemical. Our results showed that expression A2AR mRNA in PBMCs was associated with asthma severity. Foxp3 mRNA, TGF-β, and FEV1%pred positively correlated with A2AR mRNA in asthma. ROR-γt mRNA and IL-17 negatively correlated with A2AR mRNA in asthma. CGS could promote Foxp3 mRNA expression, TGF-β, and improve lung function while inhibit ROR-γt mRNA expression, IL-17, and the infiltration of lung inflammation cells. A2AR could regulate the balance of Treg/Th17 cells in asthma.

Peripheral Tolerance Regulates RBC-specific CD4+ T Cells in a Novel Mouse

Abstract T cell tolerance mechanisms towards tissue-restricted antigens are well described and include deletion, anergy, or regulatory cells; however tolerance mechanisms against RBC-restricted antigens are poorly understood. Herein, we utilized the HOD mouse to investigate T cell tolerance to RBC-specific antigens. The HOD mouse expresses a fusion protein consisting of hen egg lysosyme (HEL), ovalbumin (OVA), and human blood group, Duffy driven by an RBC-specific promoter. The HOD mouse was crossed to TCR transgenic OTII mouse to generate HOD mice with autoreactive CD4+ T cells (OTII+HOD+). Young OTII+HOD+ mice do not have detectable autoantibodies, suggesting immune tolerance. Analysis of thymii 8 week-old mice revealed similar numbers of OTII CD4+ SP thymocytes between OTII+HOD+ and OTII+HOD−, suggesting that central tolerance plays little role in RBC specific CD4+ T cells. However, in the periphery, several tolerance mechanisms were identified in OTII+HOD+ mice. Autoreactive splenic CD4+ T cells in OTII+HOD+ mice underwent deletion, expressed higher levels of inhibitory markers PD-1, LAG3, CD5, and had higher numbers of CD25+Foxp3+ regulatory T cells, when compared to OTII+HOD−. Furthermore, CD4+ T cells from OTII+HOD+ had significantly reduced proliferation in response to cognate antigen, suggesting anergy or functional inactivation. Analysis of antigen presenting cells in OTII+HOD+ revealed higher frequencies of myeloid DC, known to express high levels of MHC II and propensity to activate CD4+ T cells. Finally, a break in tolerance was observed in 30% of OTII+HOD+ at 6 months, as indicated by anti-HOD antibodies. Future studies will focus on identifying defective tolerance mechanism that contributed to autoantibody genesis.

Oxidative Stress Impairs Glucocorticoids Sensitivity of Allergic Asthma Animal Model

SESSION TITLE: Asthma III SESSION TYPE: Original Investigation Poster PRESENTED ON: Saturday, April 16, 2016 at 11:45 AM - 12:45 PM PURPOSE: To observe the influence of ozone inhalation on the typical therapeutic effects of glucocorticoids and further to explore the involvements of p38MAPK activation and MKP-1 expression upon a mouse asthma model. METHODS: Mice were sensitized and challenged with ovalbumin (OVA), dexamethasone were administrated ip 1 hr before each challenge (day24,25,26). Ozone expsoure (1.0 ppm 2hr) were performed on day 23, 25 and 27, and SB239063 were injected 1hr before and 4hr after each exposure. Enhanced pause (Penh) were measured at day 28 and followed by bronchoalveolar lavage (BAL). Lung tissues were collected for analysis of expression of inflammatory genes, MKP-1 and p38MAPK. RESULTS: Ozone inhalation significantly increased the AHR and airway inflammation of OVA model. Dexamethasone significantly decreased the AHR and airway inflammation of mice from OVA model. These inhibitory effects were significantly decreased after the ozone exposure. Administration of SB239063 partly restored the impaired effects of dexamethasone, parallel with inhibitions of the AHR and airway inflammation, especially the lung expression of IL-17 mRNA, in mice of O3-exposed OVA model. The activation of p38MAPK was promoted by ozone inhalation and inhibited by SB239063. There was no striking effect of dexamethasone on activation of p38MAPK except in O3-exposed OVA model group. Dexamethasone increased the mRNA and protein expression of MKP-1 on lungs of mice only from OVA group. This effects were diminished by ozone inhalation regardless of SB239063 administration. O3 inhalation promoted the expression of MKP-1 on mRNA but not on protein level. CONCLUSIONS: Ozone inhalation disturbs the inhibitory effects of glucocorticoids on allergic airway inflammation. This disturbance may involve the activation of p38 MAPK, induction of Th17 cytokines and negative regulation on MKP-1 protein expression. CLINICAL IMPLICATIONS: Glucocorticoid resistance is a common and hard-to-treat problem in chronic severe asthmatic patients. This study explored the underlying involvements of p38 MAPK and MKP-1 in the oxidative stress induced steroids resistance in a mouse asthma model, which may contribute to the knowledge accumulation to help understand the underlying mechanisms of Glucocorticoid resistance. DISCLOSURE: The following authors have nothing to disclose: Aihua Bao, Xin Zhou No Product/Research Disclosure Information

Effect of carvacrol on various cytokines genes expression in splenocytes of asthmatic mice.

With regard to pharmacological effects of carvacrol on the respiratory system, its effect on cytokines genes expression in splenocytes of asthmatic mice was examined in this study. Splenocytes were isolated from non-sensitized (control group), sensitized mice to ovalbumin (OVA) (group S), and S animals treated with dexamethasone, and three concentrations of carvacrol. IL-4, IFN-γ, TGF-β, FOXP3, and IL-17 genes expression were carried out in cultured splenocytes using the real-time PCR method. Compared to the control group, IFN-γ and FOXP3 genes expression were significantly decreased (P<0.001 for both cases), but IL-4 and IL-17 genes expression were significantly increased in the S group (P<0.001 and P<0.05, respectively). IL-4 gene expression due to treatment of all concentrations of carvacrol, TGF-β gene expression due to its two higher concentrations, and IL-17 gene expression due to its high concentration were significantly decreased compared to group S (P<0.01 to P<0.001). IFN-γ gene expression was significantly increased due to last carvacrol concentration (300 µg/ml, P<0.01), and FOXP3 due to its two last concentrations (150 and 300 µg/ml, P<0.05 and P<0.001, respectively) in treated S splenocytes. Dexamethasone treatment of sensitized splenocytes only showed significant inhibitory effect on IL-4 and TGF-β genes expression (P<0.001 for both cases). These results showed the immunomodulatory effect of carvacrol indicating increased IFN-γ and FOXP3 but decreased IL-4, TGF-β, and IL-17 genes expression, which was more selective than the effect of dexamethasone in sensitized mice splenocytes, which indicates its possible therapeutic value in allergy, autoimmunity, and infectious diseases.

IκB kinase β inhibitor, IMD-0354, prevents allergic asthma in a mouse model through inhibition of CD4+ effector T cell responses in the lung-draining mediastinal lymph nodes

IκB kinase (IKK) is important for nuclear factor (NF)-κB activation under inflammatory conditions. It has been demonstrated that IMD-0354, i.e. a selective inhibitor of IKKβ, inhibited allergic inflammation in a mouse model of ovalbumin (OVA)-induced asthma. The present study attempts to shed light on the involvement of CD4+ effector (Teff) and regulatory (Treg) T cells in the anti-asthmatic action of IMD-0354. The animals were divided into three groups: vehicle treated, PBS-sensitized/challenged mice (PBS group); vehicle treated, OVA-sensitized/challenged mice (OVA group); and IMD-0354-treated, OVA-sensitized/challenged mice. The analyzed parameters included the absolute counts of Treg cells (Foxp3+CD25+CD4+), activated Teff cells (Foxp3−CD25+CD4+) and resting T cells (CD25−CD4+) in the mediastinal lymph nodes (MLNs), lungs and peripheral blood. Moreover, lung histopathology was performed to evaluate lung inflammation. It was found that the absolute number of cells in all studied subsets was considerably increased in the MLNs and lungs of mice from OVA group as compared to PBS group. All of these effects were fully prevented by treatment with IMD-0354. Histopathological examination showed that treatment with IMD-0354 protected the lungs from OVA-induced allergic airway inflammation. Our results indicate that IMD-0354 exerts anti-asthmatic action, at least partially, by blocking the activation and clonal expansion of CD4+ Teff cells in the MLNs, which, consequently, prevents infiltration of the lungs with activated CD4+ Teff cells. The beneficial effects of IMD-0354 in a mouse model of asthma are not mediated through increased recruitment of Treg cells into the MLNs and lungs and/or local generation of inducible Treg cells.

The Guinea Pig Sensitized by House Dust Mite: A Model of Experimental Cough Studies.

The guinea pig sensitized by ovalbumin is the most widely used model to study cough experimentally, as the neurophysiology of the vagus nerve in the guinea pig is closest to humans. Nonetheless, the choice of the antigen remains questionable, which influences the translation of results into clinical medicine. The present study seeks to develop an alternative model of cough study using house dust mite sensitization (HDM). Thirty guinea pigs were divided into the HDM group, ovalbumin (OVA) group, and control group based on their cough response to 0.4M citric acid. In the HDM group animals were sensitized by 0.25%HDM aerosol, which they inhaled for 5min over 5days, followed by inhalation of 0.5%HDM in the same protocol. Sensitization was confirmed by a skin test. Symptoms of allergic rhinitis were induced by intranasal application of 15μl 0.5%HDM and cough challenges with citric acid were performed. Airway resistance was measured in vivo by Pennock's method. We found that both HDM and OVA-sensitized groups showed a significantly enhanced nasal reactivity and cough response compared with controls. The airway resistance data did not show significant differences. We conclude that the HDM cough model replicates functional aspects of the OVA model, which may make it an alternative to the latter. However, the superiority of the HDM model for experimental cough studies remains to be further explored.

Effect of intranasal rosiglitazone on airway inflammation and remodeling in a murine model of chronic asthma.

Background/Aims:Asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Peroxisome proliferator-activated receptors have been reported to regulate inflammatory responses in many cells. In this study, we examined the effects of intranasal rosiglitazone on airway remodeling in a chronic asthma model.Methods:We developed a mouse model of airway remodeling, including smooth muscle thickening, in which ovalbumin (OVA)-sensitized mice were repeatedly exposed to intranasal OVA administration twice per week for 3 months. Mice were treated intranasally with rosiglitazone with or without an antagonist during OVA challenge. We determined airway inflammation and the degree of airway remodeling by smooth muscle actin area and collagen deposition.Results:Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation, compared with control mice. Additionally, the mice developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer. Administration of rosiglitazone intranasally inhibited the eosinophilic inflammation significantly, and, importantly, airway smooth muscle remodeling in mice chronically exposed to OVA. Expression of Toll-like receptor (TLR)-4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was increased in the OVA group and decreased in the rosiglitazone group. Co-treatment with GW9660 (a rosiglitazone antagonist) and rosiglitazone increased the expression of TLR-4 and NF-κB.Conclusions:These results suggest that intranasal administration of rosiglitazone can prevent not only air way inf lammation but also air way remodeling associated with chronic allergen challenge. This beneficial effect is mediated by inhibition of TLR-4 and NF-κB pathways.