Experimental Model Of Allergic Asthma Research Articles

Modulation by Withania somnifera of stress-induced anxiogenesis and airway inflammation in rats.

Stress is an aversive stimulus which disrupts the biological milieu of the organism and a variety of emotional and environmental stressors are known to influence allergic and immunological disorders like bronchial asthma but the pharmacological basis of such interactions is not clearly defined. Withania somnifera (ashwagandha) is a potent anti-stress agent used widely in Indian traditional medicine and the present experimental study evaluated the effects of W.somnifera extract (WSE) on chronic stress-induced neurobehavioral and immunological responses in an experimental model of allergic asthma in rats. Wistar rats (200-250 g) were immunized and challenged with ovalbumin (OVA) and exposed to restraint stress (RS) and WSE treatments for 15days. Following this, anxiety behavior was assessed by the elevated plus maze (EPM) test, and blood and BAL fluid samples were collected for measuring of inflammatory/immune markers by ELISA and biochemical assay. The data of the various treatment groups were analyzed by ANOVA and Tukey's test. Restraint stress (RS) induced anxiogenic behavior in the (EPM) test in OVA immunized rats, and this was attenuated by WSE (200 and 400 mg/kg), in a dose related manner. Examination of blood and BAL fluid in these RS exposed rats also resulted in elevations in IgE, TNF-α andIL-4 levels, which were also attenuated by WSE pretreatments. Further, WSE pretreatment neutralized the such RS induced changes in oxidative stress markers viz. elevated MDA and reduced GSH levels. The data pharmacologically validates role of stress in asthma and suggests that adaptogens like WSE could be a potential complementary agent for reducing anxiety as well as airway inflammation by a multi-targeted and holistic approach. The study also highlights the significance of integration of traditional and modern medical concepts in such chronic disorders.

Modulation of Immunological, Biochemical, and Histopathological Changes of Airway Remodeling by Withania somnifera in an Experimental Model of Allergic Asthma in Rats.

Airway remodeling in asthma involves chronic inflammation associated with structural changes, which result in severe airflow limitation and very few therapeutic options. Thus, the present study was designed to experimentally evaluate the ameliorative effects of Withania somnifera (WS) root extract against Ovalbumin (OVA)-induced airway remodeling in a rat model of asthma. Wistar rats were immunized (i.p) and challenged (aerosol) with ovalbumin (OVA), and the effects of WS extract were investigated on the development and progress of airway remodeling by assessing immunological, biochemical, and histological changes in these rats. OVA-immunization and challenge in rats resulted in significant increases in the levels of IL-13, 8-OhdG, TGF-β, hydroxyproline, and periostin in bronchoalveolar lavage fluid (BALF) and serum/lung homogenate compared to normal control (saline only) rats, and these changes were attenuated after WS extract (200 and 400 mg/kg), as well as dexamethasone (DEX, 1 mg/kg) pretreatments. Further, WS extract attenuated histopathological changes and maintained lung integrity. In herb-drug interactions, sub-threshold doses of WS extract and DEX showed synergistic effects on all parameters studied as compared to either form of monotherapy. These results indicated that WS exerted significant protective effects against airway remodeling in the experimental model by modulating inflammatory and fibrotic cytokines, and could have the potential for developing a therapeutic alternative/adjunct for the treatment of airway remodeling of bronchial asthma.

Methyl P-Coumarate Ameliorates the Inflammatory Response in Activated-Airway Epithelial Cells and Mice with Allergic Asthma

Methyl p-coumarate (methyl p-hydroxycinnamate) (MH) is a natural compound found in a variety of plants. In the present study, we evaluated the ameliorative effects of MH on airway inflammation in an experimental model of allergic asthma (AA). In this in vitro study, MH was found to exert anti-inflammatory activity on PMA-stimulated A549 airway epithelial cells by suppressing the secretion of IL-6, IL-8, MCP-1, and ICAM-1. In addition, MH exerted an inhibitory effect not only on NF-κB (p-NF-κB and p-IκB) and AP-1 (p-c-Fos and p-c-Jun) activation but also on A549 cell and EOL-1 cell (eosinophil cell lines) adhesion. In LPS-stimulated RAW264.7 macrophages, MH had an inhibitory effect on TNF-α, IL-1β, IL-6, and MCP-1. The results from in vivo study revealed that the increases in eosinophils/Th2 cytokines/MCP-1 in the bronchoalveolar lavage fluid (BALF) and IgE in the serum of OVA-induced mice with AA were effectively inhibited by MH administration. MH also exerted a reductive effect on the immune cell influx, mucus secretion, and iNOS/COX-2 expression in the lungs of mice with AA. The effects of MH were accompanied by the inactivation of NF-κB. Collectively, the findings of the present study indicated that MH attenuates airway inflammation in mice with AA, suggesting its potential as an adjuvant in asthma therapy.

Airway inflammation induces anxiety-like behavior through neuroinflammatory, neurochemical, and neurometabolic changes in an allergic asthma model.

Allergic asthma is characterized by chronic airway inflammation and is constantly associated with anxiety disorder. Recent studies showed bidirectional interaction between the brain and the lungtissue. However, where and how the brain is affected in allergic asthma remains unclear. We aimed to investigate the neuroinflammatory, neurochemical, and neurometabolic alterations that lead to anxiety-like behavior in an experimental model of allergic asthma. Mice were submitted to an allergic asthma model induced by ovalbumin (OVA) and the control group received only Dulbecco's phosphate-buffered saline (DPBS). Our findings indicate that airway inflammation increases interleukin (IL) -9, IL-13, eotaxin, and IL-1β release and changes acetylcholinesterase (AChE) and Na+,K+-ATPase activities in the brain of mice. Furthermore, we demonstrate that a higher reactive oxygen species (ROS) formation and antioxidant defense alteration that leads to protein damage and mitochondrial dysfunction. Therefore, airway inflammation promotes a pro-inflammatory environment with an increase of BDNF expression in the brain of allergic asthma mice. These pro-inflammatory environments lead to an increase in glucose uptake in the limbic regions and to anxiety-like behavior that was observed through the elevated plus maze (EPM) test and downregulation of glucocorticoid receptor (GR). In conclusion, the present study revealed for the first time that airway inflammation induces neuroinflammatory, neurochemical, and neurometabolic changes within the brain that leads to anxiety-like behavior. Knowledge about mechanisms that lead to anxiety phenotype in asthma is a beneficial tool that can be used for the complete management and treatment of the disease.

Assessment of Allergen-Responsive Regulatory T Cells in Experimental Asthma Induced in Different Mouse Strains.

Background Regulatory T cells (Tregs) are important in regulating responses to innocuous antigens, such as allergens, by controlling the Th2 response, a mechanism that appears to be compromised in atopic asthmatic individuals. Different isogenic mouse strains also have distinct immunological responses and susceptibility to the experimental protocols used to develop lung allergic inflammation. In this work, we investigated the differences in the frequency of Treg cell subtypes among A/J, BALB/c, and C57BL/6, under normal conditions and following induction of allergic asthma with ovalbumin (OVA). Methods Subcutaneous sensitization followed by 4 consecutive intranasal OVA challenges induced asthma characteristic changes such as airway hyperreactivity, inflammation, and production of Th2 cytokines (IL-4, IL-13, IL-5, and IL-33) in the lungs of only A/J and BALB/c but not C57BL/6 strain and evaluated by invasive whole-body plethysmography, flow cytometry, and ELISA, respectively. Results A/J strain naturally showed a higher frequency of CD4+IL-10+ T cells in the lungs of naïve mice compared to the other strains, accompanied by higher frequencies of CD4+IL-4+ T cells. C57BL/6 mice did not develop lung inflammation and presented higher frequency of CD4+CD25+Foxp3+ Treg cells in the bronchoalveolar lavage fluid (BALF) after the allergen challenge. In in vitro settings, allergen-specific stimulation of mediastinal LN (mLN) cells from OVA-challenged animals induced higher frequency of CD4+IL-10+ Treg cells from A/J strain and CD4+CD25+Foxp3+ from C57BL/6. Conclusions The observed differences in the frequencies of Treg cell subtypes associated with the susceptibility of the animals to experimental asthma suggest that CD4+CD25+Foxp3+ and IL-10-producing CD4+ Treg cells may play different roles in asthma control. Similar to asthmatic individuals, the lack of an efficient regulatory response and susceptibility to the development of experimental asthma in A/J mice further suggests that this strain could be preferably chosen in experimental models of allergic asthma.

Hypoxic hUCMSC-derived extracellular vesicles attenuate allergic airway inflammation and airway remodeling in chronic asthma mice

BackgroundAs one of the main functional forms of mesenchymal stem cells (MSCs), MSC-derived extracellular vesicles (MSC-EVs) have shown an alternative therapeutic option in experimental models of allergic asthma. Oxygen concentration plays an important role in the self-renewal, proliferation, and EV release of MSCs and a recent study found that the anti-asthma effect of MSCs was enhanced by culture in hypoxic conditions. However, the potential of hypoxic MSC-derived EVs (Hypo-EVs) in asthma is still unknown.MethodsBALB/c female mice were sensitized and challenged with ovalbumin (OVA), and each group received PBS, normoxic human umbilical cord MSC-EVs (Nor-EVs), or Hypo-EVs weekly. After treatment, the animals were euthanized, and their lungs and bronchoalveolar lavage fluid (BALF) were collected. With the use of hematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Masson’s trichrome staining, enzyme-linked immune sorbent assay (ELISA), Western blot analysis, and real-time PCR, the inflammation and collagen fiber content of airways and lung parenchyma were investigated.ResultsHypoxic environment can promote human umbilical cord MSCs (hUCMSCs) to release more EVs. In OVA animals, the administration of Nor-EVs or Hypo-EVs significantly ameliorated the BALF total cells, eosinophils, and pro-inflammatory mediators (IL-4 and IL-13) in asthmatic mice. Moreover, Hypo-EVs were generally more potent than Nor-EVs in suppressing airway inflammation in asthmatic mice. Compared with Nor-EVs, Hypo-EVs further prevented mouse chronic allergic airway remodeling, concomitant with the decreased expression of pro-fibrogenic markers α-smooth muscle actin (α-SMA), collagen-1, and TGF-β1-p-smad2/3 signaling pathway. In vitro, Hypo-EVs decreased the expression of p-smad2/3, α-SMA, and collagen-1 in HLF-1 cells (human lung fibroblasts) stimulated by TGF-β1. In addition, we showed that miR-146a-5p was enriched in Hypo-EVs compared with that in Nor-EVs, and Hypo-EV administration unregulated the miR-146a-5p expression both in asthma mice lung tissues and in TGF-β1-treated HLF-1. More importantly, decreased miR-146a-5p expression in Hypo-EVs impaired Hypo-EV-mediated lung protection in OVA mice.ConclusionOur findings provided the first evidence that hypoxic hUCMSC-derived EVs attenuated allergic airway inflammation and airway remodeling in chronic asthma mice, potentially creating new avenues for the treatment of asthma.

Copaiba oil suppresses inflammation in asthmatic lungs of BALB/c mice induced with ovalbumin

Asthma is a chronic inflammatory disease that represents high hospitalizations and deaths in world. Copaiba oil (CO) is popularly used for relieving asthma symptoms and has already been shown to be effective in many inflammation models. This study aimed to investigate the immunomodulatory relationship of CO in ovalbumin (OVA)-induced allergic asthma. The composition of CO sample analyzed by GC and GC–MS and the toxicity test was performed in mice at doses of 50 or 100 mg/kg (by gavage). After, the experimental model of allergic asthma was induced with OVA and mice were orally treated with CO in two pre-established doses. The inflammatory infiltrate was evaluated in bronchoalveolar lavage fluid (BALF), while cytokines (IL-4, IL-5, IL-17, IFN-γ, TNF-α), IgE antibody and nitric oxide (NO) production was evaluated in BALF and lung homogenate (LH) of mice, together with the histology and histomorphometry of the lung tissue. CO significantly attenuated the number of inflammatory cells in BALF, suppressing NO production and reducing the response mediated by TH2 and TH17 (T helper) cells in both BALF and LH. Histopathological and histomorphometric analysis confirmed that CO significantly reduced the numbers of inflammatory infiltrate in the lung tissue, including in the parenchyma area. Our results indicate that CO has an effective in vivo antiasthmatic effect.

Lung Tissue Damage Associated with Allergic Asthma in BALB/c Mice Could Be Controlled with a Single Injection of Mesenchymal Stem Cells from Human Bone Marrow up to 14 d After Transplantation

Mesenchymal stem cell (MSC) research has demonstrated the potential of these cells to modulate lung inflammatory processes and tissue repair; however, the underlying mechanisms and treatment durability remain unknown. Here, we investigated the therapeutic potential of human bone marrow-derived MSCs in the inflammatory process and pulmonary remodeling of asthmatic BALB/c mice up to 14 d after transplantation. Our study used ovalbumin to induce allergic asthma in male BALB/c mice. MSCs were injected intratracheally in the asthma groups. Bronchoalveolar lavage fluid (BALF) was collected, and cytology was performed to measure the total protein, hydrogen peroxide (H2O2), and proinflammatory (IL-5, IL-13, and IL-17A) and anti-inflammatory (IL-10) interleukin (IL) levels. The lungs were removed for the histopathological evaluation. On day zero, the eosinophil and lymphochte percentages, total protein concentrations, and IL-13 and IL-17A levels in the BALF were significantly increased in the asthma group, proving the efficacy of the experimental model of allergic asthma. On day 7, the MSC-treated group exhibited significant reductions in the eosinophil, lymphocyte, total protein, H2O2, IL-5, IL-13, and IL-17A levels in the BALF, while the IL-10 levels were significantly increased. On day 14, the total cell numbers and lymphocyte, total protein, IL-13, and IL-17A levels in the BALF in the MSC-treated group were significantly decreased. A significant decrease in airway remodeling was observed on days 7 and 14 in almost all bronchioles, which showed reduced inflammatory infiltration, collagen deposition, muscle and epithelial thickening, and mucus production. These results demonstrate that treatment with a single injection of MSCs reduces the pathophysiological events occurring in an experimental model of allergic asthma by controlling the inflammatory process up to 14 d after transplantation.

Evaluation of Human MSCs Treatment Frequency on Airway Inflammation in a Mouse Model of Acute Asthma.

BackgroundStudies in experimental models of allergic asthma have shown that mesenchymal stem cells (MSCs) have therapeutic potential for T-helper 2 (TH2) cell-mediated inflammation. However, the mechanisms underlying these therapeutic effects are not fully understood and their safety has not been confirmed.MethodsUsing a mouse model of experimental allergic asthma, we investigated the efficacy of human adipose-derived mesenchymal stem cells (hADSCs) or human bone marrow-derived mesenchymal stem cells (hBMSCs) according to treatment frequency and timing.ResultsOvalbumin (OVA)-sensitized and -challenged mice exhibited airway hyperresponsiveness (AHR), airway inflammation, and significant increases in TH2 cytokine levels. Both double and single human mesenchymal stem cell (hMSC) treatments significantly decreased AHR and bronchoalveolar lavage fluid counts. In addition, single treatment with hMSCs showed significant attenuation of allergic airway inflammation. However, double treatment with hMSCs during OVA -sensitization and -challenge further increased inflammatory cell infiltration, and TH2 cytokine levels.ConclusionThe results of treatment with hADSCs or hBMSCs suppresses AHR and airway inflammation. However, double hMSC treatment significantly induces eosinophilic airway inflammation and lung histological changes. Therefore, double hMSC treatment is ineffective against asthma and single injection frequency appears to be more important for the treatment of asthma. These results suggest that hMSC therapy can be used for treatment of asthma patients but that it should be used carefully.

Dysregulation of metabolic pathways in a mouse model of allergic asthma

Asthma is a complex lung disease resulting from the interplay of genetic and environmental factors. To understand the molecular changes that occur during the development of allergic asthma without genetic and environmental confounders, an experimental model of allergic asthma in mice was used. Our goals were to (1) identify changes at the small molecule level due to allergen exposure, (2) determine perturbed pathways due to disease, and (3) determine whether small molecule changes correlate with lung function. In this experimental model of allergic asthma, matched bronchoalveolar lavage (BAL) fluid and plasma were collected from three groups of C57BL6 mice(control vs sensitized and/or challenged with ovalbumin, n=3-5/group) 6 hour, 24 hour, and 48 hour after the last challenge. Samples were analyzed using liquid chromatography-mass spectrometry-based metabolomics. Airway hyper-responsiveness (AHR) measurements and differential cell counts were performed. In total, 398 and 368 dysregulated metabolites in the BAL fluid and plasma of sensitized and challenged mice were identified, respectively. These belonged to four, interconnected pathways relevant to asthma pathogenesis: sphingolipid metabolism (P=6.6×10-5 ), arginine and proline metabolism (P=1.12×10-7 ), glycerophospholipid metabolism (P=1.3×10-10 ), and the neurotrophin signaling pathway (P=7.0×10-6 ). Furthermore, within the arginine and proline metabolism pathway, a positive correlation between urea-1-carboxylate and AHR was observed in plasma metabolites, while ornithine revealed a reciprocal effect. In addition, agmatine positively correlated with lung eosinophilia. These findings point to potential targets and pathways that may be central to asthma pathogenesis and can serve as novel therapeutic targets.

Differential regulation of C5a receptor 1 in innate immune cells during the allergic asthma effector phase.

C5a drives airway constriction and inflammation during the effector phase of allergic asthma, mainly through the activation of C5a receptor 1 (C5aR1). Yet, C5aR1 expression on myeloid and lymphoid cells during the allergic effector phase is ill-defined. Recently, we generated and characterized a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we used this reporter strain to monitor C5aR1 expression in airway, pulmonary and lymph node cells during the effector phase of OVA-driven allergic asthma. C5aR1 reporter and wildtype mice developed a similar allergic phenotype with comparable airway resistance, mucus production, eosinophilic/neutrophilic airway inflammation and Th2/Th17 cytokine production. During the allergic effector phase, C5aR1 expression increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs). Surprisingly, expression in neutrophils was not affected. Of note, moDCs but not CD11b+ cDCs from mediastinal lymph nodes (mLN) expressed less C5aR1 than DCs residing in the lung after OVA challenge. Finally, neither CD103+ cDCs nor cells of the lymphoid lineage such as Th2 or Th17-differentiated CD4+ T cells, B cells or type 2 innate lymphoid cells (ILC2) expressed C5aR1 under allergic conditions. Our findings demonstrate a complex regulation pattern of C5aR1 in the airways, lung tissue and mLN of mice, suggesting that the C5a/C5aR1 axis controls airway constriction and inflammation through activation of myeloid cells in all three compartments in an experimental model of allergic asthma.

Anti-asthmatic activity of osthole in an ovalbumin-induced asthma murine model

Osthole, an active coumarin extracted from the dried fruits of Cnidium monnieri (L.) Cusson, is known to possess a variety of pharmacological activities. In the present study, we investigated and illuminated the mechanisms underlying the protective effects of osthole in an experimental model of allergic asthma. Our results show that osthole treatment significantly reduced the OVA-induced increase in serum IgE and inflammatory cytokines (IL-4, IL-5, IL-13) in bronchoalveolar lavage fluid (BALF), and decreased the recruitment of inflammatory cells in BALF and the lung. It also effectively attenuated goblet cell hyperplasia and mucus overproduction in lung tissue. In addition, western blot analysis demonstrated that osthole blocked NF-κB activation, which may be associated with a reduction in inflammatory cytokine production. These data suggest that osthole attenuated OVA-induced allergic asthma inflammation by inhibiting NF-κB activation. The present study identified the molecular mechanisms of action of osthole, which support the potential pharmaceutical application of osthole treatment for asthma and other airway inflammation disorders.

IL-27 Is Essential for Suppression of Experimental Allergic Asthma by the TLR7/8 Agonist R848 (Resiquimod).

Different models of experimental allergic asthma have shown that the TLR7/8 agonist resiquimod (R848) is a potential inhibitor of type 2 helper cell-driven inflammatory responses. However, the mechanisms mediating its therapeutic effects are not fully understood. Using a model of experimental allergic asthma, we show that induction of IL-27 by R848 is critical for the observed ameliorative effects. R848 significantly inhibited all hallmarks of experimental allergic asthma, including airway hyperreactivity, eosinophilic airway inflammation, mucus hypersecretion, and Ag-specific Ig production. Whereas R848 significantly reduced IL-5, IL-13, and IL-17, it induced IFN-γ and IL-27. Neutralization of IL-27 completely reversed the therapeutic effect of R848 in the experimental asthma model, demonstrating dependence of R848-mediated suppression on IL-27. In vitro, R848 induced production of IL-27 by murine alveolar macrophages and dendritic cells and enhanced expression of programmed death-ligand 1, whose expression on monocytes and dendritic cells has been shown to regulate peripheral tolerance in both murine and human studies. Moreover, in vitro IL-27 enhanced secretion of IFN-γ whereas it inhibited IL-5 and IL-13, demonstrating its direct effect on attenuating Th2 responses. Taken together, our study proves that R848-mediated suppression of experimental asthma is dependent on IL-27. These data provide evidence of a central role of IL-27 for the control of Th2-mediated allergic diseases.

Bronchodilatory, antitussive and anti-inflammatory effect of morin in the setting of experimentally induced allergic asthma.

Using an experimental model of allergic asthma, we evaluated the anti-asthmatic potential of polyphenol flavonol derivate morin after either acute or long-term treatment of male OVA-sensitised guinea pigs. The following methods were used in experiments: the in-vitro tracheal smooth muscle contraction induced by histamine; the changes in specific airway resistance (sRaw) to histamine and the sensitivity of a chemically induced cough reflex both via an in-vivo method; the serum and BALF concentrations' analysis of the inflammatory cytokines interleukin IL-4, IL-5, IL-13; and lung tissue infiltration by eosinophils and mastocytes. Our data show that acute morin (30 mg/kg) and chronic 21-day morin (30 mg/kg/day) administration had a comparable antitussive efficiency with opioid antitussive codeine. Acute morin bronchodilatory activity defined by in-vivo sRaw decline did not reach SABA salbutamol effect. However, bronchodilatory efficiency of morin after long-term administration was by 34% higher as effect of LABA salmeterol. The 21-day morin treatment of OVA-sensitised guinea pigs reduced the serum, BALF levels of IL-4 and IL-13, lung tissue eosinophil and mastocyte infiltration comparable with corticosteroid budesonide. In summary, morin represents very rational target for additional studies as potential substance for control as well as prevention of asthma inflammation and symptoms.

Pharmacodynamic evaluation of RP3128, a novel and potent CRAC channel inhibitor in guinea pig models of allergic asthma

The increase in intracellular Ca2+ levels through the activation of Ca2+ release-activated Ca2+ (CRAC) channels is essential for mediating a wide scale of immune cell responses. Emerging evidence indicates an involvement of abnormal CRAC channel activity in human diseases such as certain types of immunodeficiency, autoimmunity and allergic disorders. This objective of this study was to evaluate the therapeutic potency of a novel CRAC channel inhibitor, RP3128, in experimental models of allergic asthma using guinea pigs.Ovalbumin-induced allergic airway inflammation was determined upon acute and long-term (14 days) oral administration of RP3128. In vivo changes in specific airways resistance (sRaw) and amplitude of isometric contraction (mN) of ASM (in vitro) were estimated to evaluate bronchodilatory effect upon acute and long-term administration of RP3128 or salbutamol. Exhaled nitric oxide (eNO), immunohistochemical and histological analysis of cellular infiltration in airways tissue, and levels of cytokines in plasma as well as bronchoalveolar lavage fluid (BALF), were determined using Bio-Plex® 200 System (BIO-RAD, USA). Ciliary beat frequency (CBF, in Hz) was estimated using a high-speed video camera and LabVIEW™ Software. Additionally, the impact of RP3128 and budesonide on mucociliary clearance was determined.Acute and long-term administration of RP3128 resulted in significant bronchodilation. Long-term administration of RP3128 exceeded the bronchodilatory effect of salbutamol and significantly decreased eNO and cytokine levels in plasma and BALF, which together with histological and immunohistochemical analysis validated its anti-inflammatory effect compared to budesonide. Data demonstrate the therapeutic potential of RP3128 in respiratory diseases causally associated with allergic inflammation.

Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma.

BackgroundCD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.MethodsWe investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.ResultsTreatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs’ ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.ConclusionThese results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.

53: IL-6R blockade: A new personalised treatment for asthma?

Background We recently identified a common genetic variant that increases the levels of the soluble interleukin 6 receptor (sIL-6R) and is a risk factor for asthma. These results suggest that tocilizumab – a drug that blocks IL-6R and is approved to treat rheumatoid arthritis – may have therapeutic potential for asthma. Aims Conduct preclinical studies to validate tocilizumab for the treatment of allergic asthma. Methods The effect of tocilizumab treatment on the development of airway inflammation was tested using mouse models of experimental allergic asthma. We compared drug efficacy between two allergen challenge models that differ in the type of immune cells that infiltrate the lungs after challenge: cockroach (CR) model, that causes mixed granulocytic (neutrophils and eosinophils) infiltration; and house dust mite (HDM) model, which causes a predominant eosinophilic infiltration. Results Tocilizumab significantly attenuated allergen challenge-induced TH2/TH17 responses and associated airway inflammatory infiltration in the CR (mixed granulocytic) model of asthma. This effect was primarily mediated by inhibition of IL-17A expression by γ δ -T cells. Tocilizumab had no effect in the HDM (eosinophilic) model of asthma. To understand the difference in efficacy between the two models, we measured IL-6 and sIL-6R in the airways after allergen challenge. Both allergens caused a significant increase in IL-6 levels, through an LPS/TLR4-dependent mechanism. However, CR but not HDM allergen, caused a significant increase in sIL-6R levels. These results suggest that the IL-6 trans-signalling pathway plays a critical role in CR- but not HDM-induced airway inflammation and likely explain why tocilizumab is effective in the CR but not HDM model of experimental asthma. Conclusions Our results validate tocilizumab as a promising new treatment for allergic asthma and suggest that high airway sIL-6R levels define a group of patients most likely to respond to treatment.

The leucine zipper transcription factor BATF induces IL-10 and IL-3 in allergic asthma (P6061)

Abstract The transcription factor BATF is essentially involved in the differentiation of Th17 and follicular T helper cells. In B cells BATF influences the class-switch recombination of immunoglobulins. In an experimental model of allergic asthma we previously observed that BATF-/- mice do not develop asthma. We found that airway inflammation, hyperresponsiveness and eosinophilia were decreased, concomitant with lower serum IgE levels and reduced Th2 and Th17 cytokine secretion in the lung. Here we show an inhibition of IL-10 secretion in in vitro differentiated Th17 cells lacking BATF, while the expression of IRF4 mRNA was not altered. We confirmed a defect of IL-10 secretion in supernatants of total lung cells isolated from BATF-/- mice sensitized and challenged with OVA. Furthermore, IL-3, an important growth factor for the survival of eosinophils, basophils and mast cells, was also reduced in the supernatants of lung CD4+ T cells isolated from BATF-/- mice. Consistently, in BATF-/- mice treated with OVA, the number of lung mast cells expressing the IL-3 receptor α-chain was decreased. This defect was also confirmed in bone marrow derived mast cells. Taken together, BATF seems to play an important role not only in Th17 and Tfh differentiation but also in mast cell development. Therefore, local inhibition of BATF expression in the lung might be a feasible strategy for treating airway inflammation seen in allergic asthma.

Activation of basophils by the double-stranded RNA poly(A:U) exacerbates allergic inflammation

It is commonly acknowledged that asthma is exacerbated by viral infections. On the other hand, basophil infiltration of lung tissues has been evidenced postmortem in cases of fatal disease, raising the question of a possible link between these two observations. Herein, we addressed the relationship between asthma exacerbation by viral infection and basophil activation and expansion by investigating how stimulation with the dsRNA polyadenylic/polyuridylic acid [poly(A:U)] affected basophil activities and recruitment in an allergic airway inflammation model. The effect of dsRNA on basophils was assessed by measuring the cytokine levels produced upon stimulation. We used an OVA-induced experimental model of allergic asthma. Airway hyperreactivity, recruitment of infiltrating cells, and cytokine production were determined in the lung of mice having received poly(A:U), as compared with untreated controls. The exacerbating effect of basophils was assessed both by adoptive transfer of poly(A:U)-treated basophils and by their in vivo depletion with Ba103 antibody. We found that in vitro treatment with poly(A:U) increased basophil functions by inducing TH 2-type cytokine and histamine production, whereas in vivo treatment increased peripheral basophil recruitment. Furthermore, we provide the first demonstration for increased infiltration of basophils in the lung of mice suffering from airway inflammation. In this model, disease symptoms were clearly exacerbated upon adoptive transfer of basophils exposed to poly(A:U), relative to their unstimulated counterpart. Conversely, in vivo basophil depletion alleviated disease syndromes, thus validating the transfer data. Our findings provide the first evidence for airway inflammation exacerbation by basophils following dsRNA stimulation.

β5i subunit deficiency of the immunoproteasome leads to reduced Th2 response in OVA induced acute asthma.

The immunoproteasome subunit β5i has been shown to play an important role in Th1/Th17 driven models of colitis and arthritis. However, the function of β5i in Th2 dependent diseases remains enigmatic. To study the role of β5i in Th2-driven pathology, β5i knockout (KO) and control mice were tested in different models of experimental allergic asthma. β5i-deficient mice showed reduced OVA/Alum- and subcutaneous/OVA-induced acute asthma with decreased eosinophilia in the bronchoalveolar lavage (BAL), low OVA-specific IgG1 and reduced local and systemic Th2 cytokines. While Th2 cells in the lungs were reduced, Tregs and Th1 cells were not affected. Attenuated asthma in β5i KO mice could not be attributed to defects in OVA uptake or maturation of dendritic cells in the lung. Surprisingly, β5i deficient mice developed HDM asthma which was comparable to control mice. Here, we present novel evidence for the requirement of the β5i immunosubunit to generate a strong Th2 response during OVA- but not HDM-induced acute asthma. The unexpected role of β5i in OVA asthma remains to be clarified.