Disruption of the mast cell carboxypeptidase A3 gene does not attenuate airway inflammation and hyperresponsiveness in two mouse models of asthma.

Mast cells are effector cells known to contribute to allergic airway disease. When activated, mast cells release a broad spectrum of inflammatory mediators, including the mast cell-specific protease carboxypeptidase A3 (CPA3). The expression of CPA3 in the airway epithelium and lumen of asthma patients has been associated with a Th2-driven airway inflammation. However, the role of CPA3 in asthma is unclear and therefore, the aim of this study was to investigate the impact of CPA3 for the development and severity of allergic airway inflammation using knockout mice with a deletion in the Cpa3 gene. We used the ovalbumin (OVA)- and house-dust mite (HDM) induced murine asthma models, and monitored development of allergic airway inflammation. In the OVA model, mice were sensitized with OVA intraperitoneally at seven time points and challenged intranasally (i.n.) with OVA three times. HDM-treated mice were challenged i.n. twice weekly for three weeks. Both asthma protocols resulted in elevated airway hyperresponsiveness, increased number of eosinophils in bronchoalveolar lavage fluid, increased peribronchial mast cell degranulation, goblet cell hyperplasia, thickening of airway smooth muscle layer, increased expression of IL-33 and increased production of allergen-specific IgE in allergen-exposed mice as compared to mocktreated mice. However, increased number of peribronchial mast cells was only seen in the HDM asthma model. The asthma-like responses in Cpa3-/- mice were similar as in wild type mice, regardless of the asthma protocol used. Our results demonstrated that the absence of a functional Cpa3 gene had no effect on several symptoms of asthma in two different mouse models. This suggest that CPA3 is dispensable for development of allergic airway inflammation in acute models of asthma in mice.

Endothelin-1-mediated Brainstem Glial Activation Produces Asthmatic Airway Vagal Hypertonia Via Enhanced ATP-P2X4 Receptor Signaling in Sprague-Dawley Rats.

The occurrence of major asthma symptoms is largely attributed to airway vagal hypertonia, of which the central mechanisms remain unclear. This study tests the hypotheses that endothelin-1-mediated brainstem glial activation produces asthmatic airway vagal hypertonia via enhanced action of adenosine 5'-triphosphate on neuronal purinergic P2X4 receptors. A rat model of asthma was prepared using ovalbumin. Airway vagal tone was evaluated by the recurrent laryngeal discharge and plethysmographic measurement of pulmonary function. The changes in the brainstem were examined using ELISA, Western blot, luciferin-luciferase, quantitative reverse transcription-polymerase chain reaction, enzyme activity assay and immunofluorescent staining, respectively. The results showed that in the medulla of rats, endothelin receptor type B and P2X4 receptors were primarily expressed in astrocytes and neurons, respectively, and both of which, along with endothelin-1 content, were significantly increased after ovalbumin sensitization. Ovalbumin sensitization significantly increased recurrent laryngeal discharge, which was blocked by acute intracisternal injection of P2X4 receptor antagonist 5-BDBD, knockdown of brainstem P2X4 receptors, and chronic intraperitoneal injection of endothelin receptor type B antagonist BQ788, respectively. Ovalbumin sensitization activated microglia and astrocytes and significantly decreased ecto-5'-nucleotidase activity in the medulla, and all of which, together with the increase of medullary P2X4 receptor expression and decrease of pulmonary function, were reversed by chronic BQ788 treatment. These results demonstrated that in rats, allergic airway challenge activates both microglia and astrocytes in the medulla via enhanced endothelin-1/endothelin receptor type B signaling, which subsequently causes airway vagal hypertonia via augmented adenosine 5'-triphosphate/P2X4 receptor signaling in central neurons of airway vagal reflex.

Probiotic lactic acid bacteria promote anti-tumor immunity through enhanced major histocompatibility complex class I-restricted antigen presentation machinery in dendritic cells.

Lactic acid bacteria (LAB) possess the ability to argument T cell activity through functional modification of antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages. Nevertheless, the precise mechanism underlying LAB-induced enhancement of antigen presentation in APCs remains incompletely understood. To address this question, we investigated the detailed mechanism underlying the enhancement of major histocompatibility complex (MHC) class I-restricted antigen presentation in DCs using a probiotic strain known as Lactococcus lactis subsp. Cremoris C60. We found that Heat-killed-C60 (HK-C60) facilitated the processing and presentation of ovalbumin (OVA) peptide antigen OVA257-264 (SIINFEKL) via H-2Kb in bone marrow-derived dendritic cells (BMDCs), leading to increased generation of effector CD8+ T cells both in vitro and in vivo. We also revealed that HK-C60 stimulation augmented the activity of 20S immunoproteasome (20SI) in BMDCs, thereby enhancing the MHC class I-restricted antigen presentation machinery. Furthermore, we assessed the impact of HK-C60 on CD8+ T cell activation in an OVA-expressing B16-F10 murine melanoma model. Oral administration of HK-C60 significantly attenuated tumor growth compared to control treatment. Enhanced Ag processing and presentation machineries in DCs from both Peyer's Patches (PPs) and lymph nodes (LNs) resulted in an increased tumor antigen specific CD8+ T cells. These findings shed new light on the role of LAB in MHC class-I restricted antigen presentation and activation of CD8+ T cells through functional modification of DCs.

[Effect and mechanism of Maxing Shigan Decoction on reducing inflammatory response in rats with cough variant asthma via TLR4/MyD88/NF-κB and p38 MAPK signaling pathways].

This study aims to investigate the effect and mechanism of Maxingshigan Decoction on inflammation in the rat model of cough variant asthma(CVA). The SPF-grade SD rats of 6-8 weeks were randomized into normal, model, Montelukast sodium, and low-, medium-, and high-dose Maxing Shigan Decoction groups, with 8 rats in each group. The CVA rat model was induced by ovalbumin(OVA) and aluminum hydroxide sensitization and ovalbumin stimulation. The normal group and model group were administrated with equal volume of normal saline by gavage, and other groups with corresponding drugs by gavage. After the experiment, the number of white blood cells in blood and the levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α) in the serum were measured. The lung tissue was stained with hematoxylin-eosin(HE). Western blot was employed to determine the protein levels of nuclear factor-κB(NF-κB), Toll-like receptor 4(TLR4), myeloid differentiation protein(MyD88), and mitogen-activated protein kinase(MAPK) in the lung tissue. Real-time PCR was carried out to measure the mRNA levels of TLR4 and MyD88 in the lung tissue. Compared with the normal group, the model group showed increased white blood cells, elevated IL-6 and TNF-α levels(P<0.01), lowered IL-10 level(P<0.01), up-regulated protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK(P<0.01) and mRNA levels of TLR4 and MyD88(P<0.01) in the lung tissue. HE staining showed obvious infiltration of inflammatory cells around the airway and cell disarrangement in the model group. Compared with the model group, Montelukast sodium and high-dose Maxing Shigan Decoction reduced the white blood cells, lowered the IL-6 and TNF-α levels(P<0.01), and elevated the IL-10 level(P<0.01). Moreover, they down-regulated the protein levels of TLR4, MyD88, p-p65/NF-κB p65, p-p38 MAPK/p38 MAPK in the lung tissue(P<0.01) and the mRNA levels of TLR4 and MyD88 in the lung tissue(P<0.01). HE staining showed that Montelukast sodium and high-dose Maxing Shigan Decoction reduced inflammatory cell infiltration and cell disarrangement. The number of white blood cells, the levels of IL-10 and TNF-α in the serum, the protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK, and the mRNA levels of TLR4 and MyD88 in the lung tissue showed no significant differences between the Montelukast sodium group and high-dose Maxing Shigan Decoction group. Maxing Shigan Decoction can inhibit airway inflammation in CVA rats by inhibiting the activation of TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.

Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L-1 in shake flasks using the OVA expression cassette containing a tac promoter (Ptac) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (PP5 combined with Ptac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L-1, respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L-1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

Intranasal administration of ceramide liposome suppresses allergic rhinitis by targeting CD300f in murine models.

Allergic rhinitis (AR) is caused by type I hypersensitivity reaction in the nasal tissues. The interaction between CD300f and its ligand ceramide suppresses immunoglobulin E (IgE)-mediated mast cell activation. However, whether CD300f inhibits the development of allergic rhinitis (AR) remains elusive. We aimed to investigate the roles of CD300f in the development of AR and the effectiveness of intranasal administration of ceramide liposomes on AR in murine models. We used ragweed pollen-induced AR models in mice. Notably, CD300f deficiency did not significantly influence the ragweed-specific IgE production, but increased the frequency of mast cell-dependent sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in our models. Similar results were also obtained for MCPT5-exprssing mast cell-specific loss of CD300f. Importantly, intranasal administration of ceramide liposomes reduced the frequency of sneezing as well as the numbers of degranulated mast cells and eosinophils in the nasal tissues in AR models. Thus, CD300f-ceramide interaction, predominantly in mast cells, alleviates the symptoms and progression of AR. Therefore, intranasal administration of ceramide liposomes may be a promising therapeutic approach against AR by targeting CD300f.

The contribution of the WNT pathway to the therapeutic effects of montelukast in experimental murine airway inflammation induced by ovalbumin and lipopolysaccharide.

The wingless/integrase-1 (WNT) pathway involved in the pathogenesis of inflammatory airway diseases has recently generated considerable research interest. Montelukast, a leukotriene receptor antagonist, provides therapeutic benefits in allergic asthma involving eosinophils. We aimed to investigate the role of the WNT pathway in the therapeutic actions of montelukast (MT) in a mixed type of allergic-acute airway inflammation model induced by ovalbumin (OVA) and lipopolysaccharide (LPS) in mice. Female mice were sensitized with intraperitoneal OVA-Al(OH)3 administration in the initiation phase and intranasal OVA followed by LPS administration in the challenge phase. The mice were divided into eight groups: control, asthmatic, and control/asthmatic treated with XAV939 (inhibitor of the canonical WNT pathway), LGK-974 (inhibitor of the secretion of WNT ligands), or MT at different doses. The inhibition of the WNT pathway prevented tracheal 5-HT and bradykinin hyperreactivity, while only the inhibition of the canonical WNT pathway partially reduced 5-HT and bradykinin contractions compared to the inflammation group. Therefore, MT treatment hindered 5-HT and bradykinin hyperreactivity associated with airway inflammation. Furthermore, MT prevented the increases in the phosphorylated GSK-3ß and WNT5A levels, which had been induced by airway inflammation, in a dose-dependent manner. Conversely, the MT application caused a further increase in the fibronectin levels, while there was no significant alteration in the phosphorylation of the Smad-2 levels in the isolated lungs of the mice. The MT treatment reversed the increase in the mRNA expression levels of interleukin-17A. An increase in eosinophil and neutrophil counts was observed in bronchoalveolar lavage fluid samples obtained from the mice in the inflammation group, which was hampered by the MT treatment. The inhibition of the WNT pathway did not alter inflammatory cytokine expression or cell infiltration. The WNT pathway mediated the therapeutic effects of MT due to the inhibition of GSK-3ß phosphorylation as well as the reduction of WNT5A levels in a murine airway inflammation model.

Electrospun Fiber Membrane with the Dual Affinity of Chelation and Covalent Interactions for the Efficient Enrichment of Glycoproteins.

As important biomarkers of many diseases, glycoproteins are of great significance to biomedical science. It is essential to develop efficient glycoprotein enrichment platforms and investigate their adsorption mechanism. In this work, a conspicuous enrichment strategy for glycoproteins was developed by using an electrospun fiber membrane wrapped with polydopamine (PDA) and modified with 3-aminophenylboronic acid and nickel ions, named PAN/DA@PDA@APBA/Ni. The enrichment characteristics of PAN/DA@PDA@APBA/Ni toward glycoproteins were explored through adsorption behavior. Thanks to the existence of two sites of interaction (metal ion chelation and boronate affinity), PAN/DA@PDA@APBA/Ni exhibited significant enrichment capacity for glycoproteins, ovalbumin (604.6 mg/g), and human immunoglobulin G (331.0 mg/g). The adsorption kinetic results of glycoprotein ovalbumin on PAN/DA@PDA@APBA/Ni conform to the pseudo-first-order kinetic model in the first adsorption stage, while the second half adsorption stage is more in line with the pseudo-second-order kinetic model. Moreover, the physical characteristics of PAN/DA@PDA@APBA/Ni and subsequent adsorption experiments on electrospun fiber modified with only phenylboronic acid or nickel ions both confirmed two sites of interaction (metal ion chelation and boronate affinity, respectively). Furthermore, a stepwise elution method with dual-affinity interaction was designed and successfully applied to enrich glycoproteins in real biological samples. This work provides an idea for sample pretreatment, especially for the design of dual-affinity materials in glycoproteins enrichment.

The glucocorticoid dexamethasone alleviates allergic inflammation through a mitogen-activated protein kinase phosphatase-1-dependent mechanism in mice.

Glucocorticoids are widely used in the treatment of allergic and inflammatory diseases. Glucocorticoids have a widespread action on gene expression resulting in their pharmacological actions and also an array of adverse effects which limit their clinical use. It remains, however, to be studied which target gene effects are essential for the anti-allergic activity of glucocorticoids. Mitogen-activated protein kinase phosphatase-1 (MKP-1) inhibits proinflammatory signalling by suppressing the activity of mitogen activated protein kinase (MAP kinase) pathways. MKP-1 is one of the anti-inflammatory genes whose expression is enhanced by glucocorticoids. In the present study, we aimed to investigate the role of MKP-1 in the therapeutic effects of the glucocorticoid dexamethasone in acute allergic reaction. The effects of dexamethasone were studied in wild-type and MKP-1 deficient mice. The mice were first sensitized to ovalbumin, and the allergic reaction was then induced by a subcutaneous ovalbumin injection in the hind paw. Inflammatory edema was quantified with plethysmometer and expression of inflammatory factors was measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Dexamethasone reduced the ovalbumin-induced paw edema at 1.5, 3 and 6 h time points in wild-type mice by 70%, 95% and 89%, respectively. The effect was largely abolished in MKP-1 deficient mice. Furthermore, dexamethasone significantly attenuated the expression of ovalbumin-induced inflammatory factors cyclooxygenase-2 (COX-2); inducible nitric oxide synthase (iNOS); interleukins (IL) 1ß, 6 and 13; C-C motif chemokine 11 (CCL-11); tumour necrosis factor (TNF) and thymic stromal lymphopoietin (TSLP) in wild-type mice by more than 40%. In contrast, in MKP-1 deficient mice dexamethasone had no effect or even enhanced the expression of these inflammatory factors. The results suggest that dexamethasone alleviates allergic inflammation through an MKP-1-dependent mechanism. The results also demonstrate MKP-1 as an important conveyor of the favourable glucocorticoid effects in ovalbumin-induced type I allergic reaction. Together with previous findings, the present study supports the concept of MKP-1 enhancing compounds as potential novel anti-inflammatory and anti-allergic drugs.

A novel quinoline with airway relaxant effects and anti-inflammatory properties.

BACKGROUND: In chronic pulmonary diseases characterized by inflammation and airway obstruction, such as asthma and COPD, there are unmet needs for improved treatment. Quinolines is a group of small heterocyclic compounds that have a broad range of pharmacological properties. Here, we investigated the airway relaxant and anti-inflammatory properties of a novel quinoline (RCD405). METHODS: The airway relaxant effect of RCD405 was examined in isolated airways from humans, dogs, rats and mice. Murine models of ovalbumin (OVA)-induced allergic asthma and LPS-induced airway inflammation were used to study the effects in vivo. RCD405 (10 mg/kg) or, for comparisons in selected studies, budesonide (3 mg/kg), were administered intratracheally 1 h prior to each challenge. Airway responsiveness was determined using methacholine provocation. Immune cell recruitment to bronchi was measured using flow cytometry and histological analyses were applied to investigate cell influx and goblet cell hyperplasia of the airways. Furthermore, production of cytokines and chemokines was measured using a multiplex immunoassay. The expression levels of asthma-related genes in murine lung tissue were determined by PCR. The involvement of NF-κB and metabolic activity was measured in the human monocytic cell line THP-1. RESULTS: RCD405 demonstrated a relaxant effect on carbachol precontracted airways in all four species investigated (potency ranking: human = rat > dog = mouse). The OVA-specific IgE and airway hyperresponsiveness (AHR) were significantly reduced by intratracheal treatment with RCD405, while no significant changes were observed for budesonide. In addition, administration of RCD405 to mice significantly decreased the expression of proinflammatory cytokines and chemokines as well as recruitment of immune cells to the lungs in both OVA- and LPS-induced airway inflammation, with a similar effect as for budesonide (in the OVA-model). However, the effect on gene expression of Il-4, IL-5 and Il-13 was more pronounced for RCD405 as compared to budesonide. Finally, in vitro, RCD405 reduced the LPS-induced NF-κB activation and by itself reduced cellular metabolism. CONCLUSIONS: RCD405 has airway relaxant effects, and it reduces AHR as well as airway inflammation in the models used, suggesting that it could be a clinically relevant compound to treat inflammatory airway diseases. Possible targets of this compound are complexes of mitochondrial oxidative phosphorylation, resulting in decreased metabolic activity of targeted cells as well as through pathways associated to NF-κB. However, further studies are needed to elucidate the mode of action.

NButGT Reinforces the Beneficial Effects of Epinephrine on Cardiac Mitochondrial Respiration, Lactatemia and Cardiac Output in Experimental Anaphylactic Shock.

Anaphylactic shock (AS) is the most severe form of acute systemic hypersensitivity reaction. Although epinephrine can restore patients' hemodynamics, it might also be harmful, supporting the need for adjuvant treatment. We therefore investigated whether NButGT, enhancing O-GlcNAcylation and showing beneficial effects in acute heart failure might improve AS therapy. Ovalbumin-sensitized rats were randomly allocated to six groups: control (CON), shock (AS), shock treated with NButGT alone before (AS+pre-Nbut) or after (AS+post-Nbut) AS onset, shock treated with epinephrine alone (AS+EPI) and shock group treated with combination of epinephrine and NButGT (AS+EPI+preNBut). Induction of shock was performed with an intravenous (IV) ovalbumin. Cardiac protein and cycling enzymes O-GlcNAcylation levels, mean arterial pressure (MAP), heart rate, cardiac output (CO), left ventricle shortening fraction (LVSF), mitochondrial respiration, and lactatemia were evaluated using Western blotting experiments, invasive arterial monitoring, echocardiography, mitochondrial oximetry and arterial blood samples. AS decreased MAP (-77%, p < 0.001), CO (-90%, p < 0.001) and LVSF (-30%, p < 0.05). Epinephrine improved these parameters and, in particular, rats did not die in 15 min. But, cardiac mitochondrial respiration remained impaired (complexes I + II -29%, p < 0.05 and II -40%, p < 0.001) with hyperlactatemia. NButGT pretreatment (AS+pre-Nbut) efficiently increased cardiac O-GlcNAcylation level as compared to the AS+post-Nbut group. Compared to epinephrine alone, the adjunction of NButGT significantly improved CO, LVSF and mitochondrial respiration. MAP was not significantly increased but lactatemia decreased more markedly. Pretreatment with NButGT increases O-GlcNAcylation of cardiac proteins and has an additive effect on epinephrine, improving cardiac output and mitochondrial respiration and decreasing blood lactate levels. This new therapy might be useful when the risk of AS cannot be avoided.

Immunomodulation in allergic rhinitis: Insights from Th2 cells and NLRP3/IL-18 pathway.

Allergic rhinitis (AR) is characterized by nasal symptoms such as rubbing and sneezing, often triggered by allergen exposure. The purpose of this study is to dissect the roles of NLRP3-mediated immune modulation and macrophage pyroptosis in modulating T cell differentiation within the context of ovalbumin (OVA)-induced AR in mice. OVA-induced AR was established in mice, evaluating nasal symptoms, macrophage infiltration, cytokine levels, and T cell differentiation. Manipulations using NLRP3-/-, ASC-/- mice, clodronate liposome treatment, and NLRP3 inhibitor MCC950 were performed to assess their impact on AR symptoms and immune responses. Following OVA stimulation, increased nasal symptoms were observed in the OVA group along with augmented GATA3 expression and elevated IL-4 and IL-1b levels, indicative of Th2 polarization and cellular pyroptosis involvement. NLRP3-/- and ASC-/- mice exhibited reduced CD3+ T cells post OVA induction, implicating cellular pyroptosis in AR. Macrophage depletion led to decreased IgE levels, highlighting their involvement in allergic responses. Further investigations revealed enhanced macrophage pyroptosis, influencing Th1/Th2 differentiation in AR models. IL-18 released through NLRP3-mediated pyroptosis induced Th2 differentiation, distinct from IL-1b. Additionally, MCC950 effectively mitigated AR symptoms by modulating Th2 responses and reducing macrophage infiltration. This comprehensive study unravels the pivotal role of NLRP3-mediated immune modulation and macrophage pyroptosis in Th1/Th2 balance regulation in OVA-induced AR. Targeting NLRP3 pathways with MCC950 emerged as a promising strategy to alleviate AR symptoms, providing insights for potential therapeutic interventions in AR management.

Wogonoside Ameliorates Airway Inflammation and Mucus Hypersecretion via NF-κB/STAT6 Signaling in Ovalbumin-Induced Murine Acute Asthma.

Asthma is recognized as a chronic respiratory illness characterized by airway inflammation and airway hyperresponsiveness. Wogonoside, a flavonoid glycoside, is reported to significantly alleviate the inflammation response and oxidative stress. Herein, this study aimed to investigate the therapeutic effect and underlying mechanism of wogonoside on airway inflammation and mucus hypersecretion in a murine asthma model and in human bronchial epithelial cells (16HBE). BALB/c mice were sensitized and challenged with ovalbumin (OVA). Pulmonary function and the number of cells in the bronchoalveolar lavage fluid (BALF) were examined. Pathological changes in lung tissue in each group were evaluated via hematoxylin and eosin and periodic acid-Schiff staining, and changes in levels of cytokines in BALF and of immunoglobulin E in serum were determined via an enzyme-linked immunosorbent assay. The expression of relevant genes in lung tissue was analyzed via real-time PCR. Western blotting and immunofluorescence were employed to detect the expression of relevant proteins in lung tissue and 16HBE cells. Treatment with 10 and 20 mg/kg wogonoside significantly attenuated the OVA-induced increase of inflammatory cell infiltration, mucus secretion, and goblet cell percentage and improved pulmonary function. Wogonoside treatment reduced the level of T-helper 2 cytokines including interleukin (IL)-4, IL-5, and IL-13 in BALF and of IgE in serum and decreased the mRNA levels of cytokines (IL-4, IL-5, IL-6, IL-13, and IL-1ß and tumor necrosis factor-α), chemokines (CCL-2, CCL-11, and CCL-24), and mucoproteins (MUC5AC, MUC5B, and GOB5) in lung tissues. The expression of MUC5AC and the phosphorylation of STAT6 and NF-κB p65 in lung tissues and 16HBE cells were significantly downregulated after wogonoside treatment. Thus, wogonoside treatment may effectively decrease airway inflammation, airway remodeling, and mucus hypersecretion via blocking NF-κB/STAT6 activation.

The protective effects of Arctiin in asthma by attenuating airway inflammation and inhibiting p38/NF-κB signaling.

Asthma is a common chronic inflammatory disease of the airways, which affects millions of people worldwide. Arctiin, a bioactive molecule derived from the traditional Chinese Burdock, has not been previously reported for its effects on asthma in infants. In this study, an asthma model was established in mice by stimulation with ovalbumin (OVA). Bronchoalveolar lavage (BALF) was collected from OVA-challenged mice and the cells were counted. Lung tissue was harvested for hematoxylin-eosin (HE) staining and measurement of Wet/Dry weight ratios. The expressions of proteins were detected using enzyme-linked immunosorbent assay (ELISA) and Western blots. The superoxide dismutase (SOD) activity in lung tissue was measured using a commercial kit. We found that Arctiin had beneficial effects on asthma treatment. Firstly, it attenuated OVA-challenged lung pathological alterations. Secondly, it ameliorated pro-inflammatory response by reducing the number of inflammatory cells and mitigating the imbalance of Th1/Th2 factors in the bronchoalveolar lavage (BALF) of OVA-challenged mice. Importantly, Arctiin ameliorated OVA-induced lung tissue impairment and improved lung function. Additionally, we observed that oxidative stress (OS) in the pulmonary tissue of OVA-challenged mice was ameliorated by Arctiin. Mechanistically, Arctiin prevented OVA-induced activation of p38 and nuclear factor-κB (NF-κB). Based on these findings, we conclude that Arctiin might serve as a promising agent for the treatment of asthma.

Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Antigen-Specific Stimulation of CD8+ T Cells by Murine Bone Marrow-Derived Dendritic Cells After Treatment with Nanoparticles.

The assessment of antigen presentation by dendritic cells and subsequent antigen-dependent activation of T lymphocytes is a critical step underlying the efficacy of nanoparticle-based therapeutic vaccines. Since nanoparticle physicochemical properties determine their interactions with the immune system, the early stages of nanotechnology-based vaccine development commonly involve optimizing the particles' properties to create a formulation with desired stability, antigen release, targeting of desired cell populations, and efficacy. To accelerate this process, in vitro models suitable for the rapid assessment of a novel vaccine candidate's efficacy are highly desirable. One such model is described in this protocol. Herein, nanoparticles are formulated to deliver a model antigen, SIINFEKL (OVA257-264), the immunodominant class I peptide derived from ovalbumin. These nanoparticles are added to the culture of murine bone marrow-derived dendritic cells, which are subsequently co-incubated with CD8+ T cells from OT-I transgenic mice. The efficient antigen presentation by dendritic cells results in the antigen-dependent proliferation of CD8+ T cells, which is detected by flow cytometry.

Detection of Antigen Presentation by Murine Bone Marrow-Derived Dendritic Cells After Treatment with Nanoparticles.

Nanoparticles are frequently considered in vaccine applications due to their ability to co-deliver multiple antigens and adjuvants to antigen-presenting cells. Some nanoparticles also have intrinsic adjuvant properties that further enhance their ability to stimulate immune cells. The delivery of tumor-specific antigens to antigen-presenting cells (APCs) with subsequent antigenic peptide presentation in the context of class I major histocompatibility complex (MHC-I) molecules represents an essential effort in developing nanotechnology-based cancer vaccines. Experimental models are, therefore, needed to gauge the efficiency of nanotechnology carriers in achieving peptide antigen delivery to APCs and presentation in the context of MHC-I. The assay described herein utilizes a model antigen ovalbumin and model APCs, murine bone marrow-derived dendritic cells. The 25-D1.16 antibody, specific to the ovalbumin (OVA) MHC-I peptide SIINFEKL, recognizes this peptide presented in the context of the murine H2-Kb class I MHC molecule, allowing the presentation of this antigen on APCs to be detected by flow cytometry after nanoparticle delivery.

Effects of increasing sensitizing doses of ovalbumin on airway hyperresponsiveness in asthmatic mice.

BACKGROUND: The dosage of ovalbumin (OVA) during the sensitization stage is considered a crucial factor in the development of airway hyperresponsiveness (AHR). However, the inconsistent dosages of sensitizing OVA used in current studies and the lack of research on their impact on AHR are notable limitations. METHODS: We examined the impact of increasing sensitizing doses of OVA in a murine asthma model, which entailed initial sensitization with OVA followed by repeated exposure to OVA aerosols. BALB/c mice were primed with doses of OVA (0, 10, 20, 50, and 100 µg) plus 1 mg Alum on Days 0 and 7, and were challenged with OVA aerosols (10 mg/mL for 30 min) between Days 14 and 17. Antigen-induced AHR to methacholine (MCh), as well as histological changes, eosinophilic infiltration, and epithelial injury were assessed. RESULTS: The result indicated that there are striking OVA dose-related differences in antigen-induced AHR to MCh. The most intense antigen-induced AHR to MCh was observed with sensitization at 50 µg, while weaker responses were seen at 10, 20, and 100 µg. Meanwhile, there was a significant increase in eosinophil count with sensitization at 50 µg. The changes of AHR were correlated with total cells count, lymphocytes count, eosinophils count, and basophils count in bronchoalveolar lavage fluid; however, it did not correlate with histological changes such as cellular infiltration into bronchovascular bundles and goblet cell hyperplasia of the bronchial epithelium. CONCLUSION: Overall, this study demonstrated that sensitization with 50 µg of OVA resulted in the most significant AHR compared to other dosages. These findings may offer valuable insights for future research on mouse asthma modeling protocols.

Lung Gene Expression Suggests Roles for Interferon-Stimulated Genes and Adenosine Deaminase Acting against RNA-1 in Pathologic Responses to Diisocyanate.

Mechanisms underlying methylene diphenyl diisocyanate (MDI) and other low molecular weight chemical-induced asthma are unclear and appear distinct from those of high molecular weight (HMW) allergen-induced asthma. We sought to elucidate molecular pathways that differentiate asthma-like pathogenic vs nonpathogenic responses to respiratory tract MDI exposure in a murine model. Lung gene expression differences in MDI exposed immune-sensitized and nonsensitized mice vs unexposed controls were measured by microarrays, and associated molecular pathways were identified through bioinformatic analyses and further compared with published studies of a prototypic HMW asthmagen (ovalbumin). Respiratory tract MDI exposure significantly altered lung gene expression in both nonsensitized and immune-sensitized mice, vs controls. Fifty-three gene transcripts were altered in all MDI exposed lung tissue vs controls, with levels up to 10-fold higher in immune-sensitized vs nonsensitized mice. Gene transcripts selectively increased in MDI exposed immune-sensitized animals were dominated by chitinases and chemokines and showed substantial overlap with those increased in ovalbumin-induced asthma. In contrast, MDI exposure of nonsensitized mice increased type I interferon stimulated genes (ISGs) in a pattern reflecting deficiency in adenosine deaminase acting against RNA (ADAR-1), an important regulator of innate, as well as "sterile" or autoimmunity triggered by tissue damage. Thus, MDI-induced changes in lung gene expression were identified that differentiate nonpathogenic innate responses in nonsensitized hosts from pathologic adaptive responses in immune-sensitized hosts. The data suggest that MDI alters unique biological pathways involving ISGs and ADAR-1, potentially explaining its unique immunogenicity/allergenicity.

miR-224-5p Attenuates Allergic Responses in Mice with Allergic Rhinitis by Modulating the Th1/Th2 Response.

Background: Allergic rhinitis (AR) is a common chronic respiratory disease that has become a global health problem. miRNAs play an important role in multiple immune and inflammatory diseases, including AR. In this work, the mechanism by which miR-224-5p regulates AR in vivo and in vitro was examined. Methods: Human nasal epithelial cells (HNEpCs) were used to establish an AR cell model induced by Der P1, and C57BL/6 mice were used to establish an AR animal model induced by OVA (ovalbumin). RT-qPCR was used to determine the level of miR-224-5p; western blot analysis was used to determine GATA3; ELISA was used to determine the levels of OVA-specific IgE, IFN-γ, IL-4, IL-5, and IL-13; flow cytometry was used to determine the differentiation of Th1 and Th2 cells; and HE and PAS staining was used to observe the histopathological alterations in the mouse nasal mucosa and spleen. Results: miR-224-5p was downregulated in nasal mucosa from mice with AR and an AR cell model. Overexpressed miR-224-5p can improve AR development and attenuate AR symptoms by regulating GATA3-mediated Th1/Th2 responses. Conclusion: miR-224-5p attenuates allergic reactions in mice with AR by regulating the Th1/Th2 response.