Toxoplasma gondii Infection Induces Suppression in a Mouse Model of Allergic Airway Inflammation

Allergic asthma is an inflammatory disorder characterized by infiltration of the airway wall with inflammatory cells driven mostly by activation of Th2-lymphocytes, eosinophils and mast cells. There is a link between increased allergy and a reduction of some infections in Western countries. Epidemiological data also show that respiratory allergy is less frequent in people exposed to orofecal and foodborne microbes such as Toxoplasma gondii. We previously showed that both acute and chronic parasite T. gondii infection substantially blocked development of airway inflammation in adult BALB/c mice. Based on the high levels of IFN-γ along with the reduction of Th2 phenotype, we hypothesized that the protective effect might be related to the strong Th1 immune response elicited against the parasite. However, other mechanisms could also be implicated. The possibility that regulatory T cells inhibit allergic diseases has received growing support from both animal and human studies. Here we investigated the cellular mechanisms involved in T. gondii induced protection against allergy. Our results show for the first time that thoracic lymph node cells from mice sensitized during chronic T. gondii infection have suppressor activity. Suppression was detected both in vitro, on allergen specific T cell proliferation and in vivo, on allergic lung inflammation after adoptive transference from infected/sensitized mice to previously sensitized animals. This ability was found to be contact- independent and correlated with high levels of TGF-β and CD4(+)FoxP3(+) cells.     

Porins and lipopolysaccharide of Escherichia coli ATCC 25922 and isogenic rough mutants

Abstract The lipopolysaccharide and porin profile of Escherichia coli ATCC 25922, a smooth strain commonly used in antibiotic susceptibility testing, and five isogenic rough mutants was examined. The lipopolysaccharide of the parent strain had the characteristic ladder pattern on polyacrylamide gels, while that of the mutants appeared similar to chemotypes Ra and Rc of Salmonella typhimurium with some changes in chemical composition. Of the porins, OmpC appeared markedly reduced in the parent strain while OmpF appeared markedly reduced in the mutants. In addition, a new outer-membrane protein of size intermediate to that of OmpC and OmpF was detected in all mutants. Neither parent nor mutants were susceptible to the LPS core-specific P1 phage or the porin-specific PA2 and K20 phages.     

Menadione-catalyzed luminol chemiluminescent assay for the viability of Escherichia coli ATCC 25922.

Escherichia coli ATCC 25922 produced O2- in the presence of menadione, and O2- -dependent luminol chemiluminescence intensity was proportional to colony-forming unit (CFU) in the exponential phase. CFU was determined by using a 96-well plate at a range of 3 X 10(3) to 8 x 10(7) CFU /well (0.1 ml) after a 10-min incubation with menadione, followed by chemiluminescent assay for 5 s. After a 4-hr incubation of E. coli (10(5) CFU/0.1 ml) with menadione and an antimicrobial agent inhibiting the synthesis of peptidoglycan, protein, and DNA, the inhibitory concentration (IC) of the antimicrobial agent determined by menadione-catalyzed luminol chemiluminescent assay was in good agreement with minimal inhibitory concentration (MIC) of the NCCLS (National Committee for Clinical Laboratory Standard) method requiring 18 hr. Menadione-catalyzed luminol chemiluminescent assay is expected to be useful for the rapid determination of cell viability under the conditions of various cell growths and stresses.     

屋尘螨致敏/激发小鼠气道变态反应性炎症模型的构建

目的使用屋尘螨(HDM)提取液致敏和激发C57BL/6小鼠构建气道变态反应性炎症模型的方法。方法模型组和对照组,各8只。模型组以HDM致敏和激发小鼠,分别于第0、7、14、21天腹腔注射HDM,10 d后,连续雾化吸入HDM 3 d。对照组以PBS代替HDM。进行支气管肺泡灌洗液(BALF)细胞总数计数和分类计数,肺组织病理检查,ELISA测定BALF上清中IL-4、IL-5和IFNγ-水平。结果模型组可见明显炎性细胞浸润,以嗜酸性粒细胞为主。而对照组未见明显炎性细胞浸润。BALF中细胞总数计数和嗜酸性粒细胞比例较对照组明显升高(P<0.01);BALF上清中IL-4、IL-5水平较对照组明显升高(P<0.001),IFNγ-水平较对照组降低(P<0.01)。结论使用HDM致敏和激发C57BL/6小鼠成功地建立了小鼠气道变应性炎症模型。     

Effects of Cations and PH on Antimicrobial Activity of Thanatin and s-Thanatin Against Escherichia coli ATCC25922 and B. subtilis ATCC 21332

This study analyzes the in vitro effects of cations and pH on antimicrobial activity of thanatin and s-thanatin against Escherichia coli ATCC25922 and B. subtilis ATCC21332. Thanatin and s-thanatin were synthesized by the solid-phase method using a model 432A synthesizer. The bacterial strains tested included two antibiotic-susceptible strains of Escherichia coli ATCC25922 and B. subtilis ATCC21332. Susceptibility determinations were carried out either in a variety of cation concentrations or in pH conditions from pH 5 to pH 8. NaCl or KCl was added to the media to final concentrations of 0, 10, 50, 100, 200, and 500 mM, whereas CaCl₂ and MgCl₂ were added to the media to final concentrations of 0, 1, 2, 5, 10, and 20 mM. The antimicrobial activity of thanatin and s-thanatin against Escherichia coli ATCC25922 and B. subtilis ATCC21332 decreased, as indicated by the increasing minimal inhibitory concentrations (MICs) of both peptides with increasing concentrations of Na⁺/K⁺/Ca²⁺/Mg²⁺. Both peptides lost their activities at 500 mM Na⁺/K⁺ but retained them at 20 mM Ca²⁺/Mg²⁺. Both peptides have MICs that are not significantly different at a variety of pH levels, with the antimicrobial activity slightly higher in neutral or slightly basic media than under acidic conditions. The antimicrobial peptides thanatin and s-thanatin, which have an anti-parallel β-sheet constrained by disulfide bonds, were salt sensitive against both Gram-positive and Gram-negative pathogens in vitro. Determining the reason why the thanatins are salt sensitive would be useful to provide an understanding of how thanatin and s-thanatin kill bacteria. Futher investigation of the antimicrobial properties of these peptides is warranted. G. Wu and J. Ding contributed equally to this article.     

三七总皂苷对支气管哮喘小鼠气道炎症及气道重建的影响

目的:探讨三七总皂苷对支气管哮喘小鼠气道炎症及气道重建的影响。方法:选择昆明种小鼠24只,将其随机分为三七总皂苷(PNS)治疗组、阳性对照组(哮喘组)和阴性对照组,每组8只。小鼠腹腔内注射氢氧化铝-卵清蛋白悬浊液(OVA-Al(OH)3)进行致敏,致敏完成后用2%卵清蛋白溶液(OVA)雾化激发,在激发前30分钟给药治疗,雾化激发6周。建模完成后,做心肺组织灌流,取左肺固定做HE染色,镜下观察评估气道病理学改变(气道狭窄阻塞率、气道上皮坏死糜烂率、上皮细胞杯状化生率、炎性细胞浸润率及平滑肌增生率);行免疫组织化学染色,用图像分析软件半定量测量转化生长因子-beta1(TGF-β1)的含量;行特殊染色,用图像分析软件测量基底膜厚度;取右肺组织,利用酶联免疫吸附法(ELISA)测定肺组织匀浆白介素-17(IL-17)因子的水平。结果:1各组小鼠经心肺组织灌流后,与哮喘组比较,三七治疗组小鼠的肺部红肿减轻,肺表颜色较正常组略微泛红。2三七治疗组气道狭窄率、上皮细胞坏死率、炎细胞浸润率、平滑肌增生率均显著低于哮喘组(P0.05),但与正常组比较无显著性差异(P0.05)。3三七治疗组气道上皮细胞TGF-β1含量显著低于哮喘组(P0.001),肺组织白介素-17含量降低至哮喘组一半以下(P0.05),与对照组相比均无显著性差异(P=0.94,P=0.23)。4哮喘组、三七组和对照组小鼠的支气管基底膜厚度分别为(0.7893±0.014)、(0.7216±0.016)、(0.5655±0.012)μm,哮喘组显著高于三七组和对照组,均具有显著性差异(P0.05)。结论:三七总皂苷可以有效改善支气管哮喘小鼠的气道重建,并抑制其气道炎性。     

通光散汤对小鼠哮喘模型气道高反应性和气道炎症的影响

目的观察通光散对小鼠哮喘模型气道反应和气道炎症的影响。方法35只6周龄BALB／c小鼠随机分为哮喘模型组、正常对照组和药物实验组。模型组和药物组以鸡卵白蛋白（OVA）致敏、激发;药物组在最后一次致敏后每天灌胃给予通光散汤0．72mL（相当于0．04g生药）;对照组以等体积的Ns代替OVA致敏、激发。末次激发48h后处理小鼠：无创法测定小鼠的气道高反应性,观察气道阻力变化;支气管肺泡灌洗液（BALF）行细胞学分类;观察肺组织的病理变化。结果①药物组小鼠气道阻力的变化与模型组相比明显下降,差异显著（P＜0．05）;②药物组BALF白细胞总数和Eos（％）与模型组相比明显降低（P＜0．05）。③模型组小鼠肺脏组织支气管、血管黏膜下和周围肺组织有明显的炎症细胞浸润,大量炎症细胞向支气管和血管迁移,上皮细胞部分有脱落,部分可见黏液栓,血管壁明显水肿;治疗组小鼠肺组织炎性细胞浸润和管腔黏液分泌情况较模型组明显减轻,气道粘液的分泌量得到明显的控制。结论通光散汤对小鼠哮喘模型气道高反应性和气道炎症有显著抑制作用。     

补肾清肺方对小鼠支气管哮喘模型的影响

目的:探讨中药复方补肾清肺方对小鼠哮喘模型气道炎症及细胞因子的影响。方法:40只BALB/c小鼠随机分为正常对照组(A组)、哮喘模型组(B组)、补肾清肺方低剂量组(C组)和补肾清肺方高剂量组(D组)。B、C、D组采用卵蛋白(OVA)腹腔注射致敏与雾化吸入激发制作哮喘模型。于OVA激发结束后24h收集支气管肺泡灌洗液(BALF)计数炎性细胞总数及嗜酸性粒细胞(EOS)数目,并测定BALF上清液中白细胞介素-4(IL-4)和干扰素-γ(IFN-γ)水平变化。结果:补肾清肺方的干预治疗能显著降低小鼠BALF中炎性细胞总数及EOS数量;BALF上清液中IFN-γ水平明显升高,IL-4水平显著下降。D、C组与A组、B组有显著性差异(P<0.05),C组与D组结果亦存在显著性差异(P<0.05)。结论:补肾清肺方能明显降低哮喘小鼠BALF中炎性细胞数量,影响细胞因子水平变化,从而改善哮喘气道炎症。     

Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma

Levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are increased in lung, sputum, exhaled breath condensate and plasma samples from asthma patients. ADMA is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2. We determined the effect of DDAH1 overexpression on development of allergic inflammation in a mouse model of asthma. The expression of DDAH1 and DDAH2 in mouse lungs was determined by RT-quantitative PCR (qPCR). ADMA levels in bronchoalveolar lavage fluid (BALF) and serum samples were determined by mass spectrometry. Wild type and DDAH1-transgenic mice were intratracheally challenged with PBS or house dust mite (HDM). Airway inflammation was assessed by bronchoalveolar lavage (BAL) total and differential cell counts. The levels of IgE and IgG1 in BALF and serum samples were determined by ELISA. Gene expression in lungs was determined by RNA-Seq and RT-qPCR. Our data showed that the expression of DDAH1 and DDAH2 was decreased in the lungs of mice following HDM exposure, which correlated with increased ADMA levels in BALF and serum. Transgenic overexpression of DDAH1 resulted in decreased BAL total cell and eosinophil numbers following HDM exposure. Total IgE levels in BALF and serum were decreased in HDM-exposed DDAH1-transgenic mice compared to HDM-exposed wild type mice. RNA-Seq results showed downregulation of genes in the inducible nitric oxide synthase (iNOS) signaling pathway in PBS-treated DDAH1-transgenic mice versus PBS-treated wild type mice and downregulation of genes in IL-13/FOXA2 signaling pathway in HDM-treated DDAH1-transgenic mice versus HDM-treated wild type mice. Our findings suggest that decreased expression of DDAH1 and DDAH2 in the lungs may contribute to allergic asthma and overexpression of DDAH1 attenuates allergen-induced airway inflammation through modulation of Th2 responses.     

平喘固本合剂对昆系小鼠急性哮喘模型气道炎症作用影响

目的:探讨平喘固本合剂对昆系小鼠急性哮喘模型气道炎症作用影响及相关机制。方法:昆明系小鼠40只,随机分为对照组(A),哮喘模型组(B),喘固本合剂治疗组(C),布地奈德雾化治疗组(D)及平喘固本合剂联合布地奈德雾化治疗组(E)。用鸡卵蛋白(OVA)致敏建立昆系小鼠哮喘急性气道炎症模型,并给与药物治疗10天。对各组支气管肺泡灌洗液(BALF)中各种细胞进行分类并计数,观察肺组织的病理变化,同时应用ELISA法测定灌洗液中炎症相关因子的水平变化。结果:与B组比较,C组、D组、E组BALF中细胞总数、嗜酸性粒细胞数、巨噬细胞数及IL-4、IL-4/IFN-γ明显降低(P<0.05),IFN-γ明显升高(P<0.05)。对于IL-4、IL-4/IFN-γ水平的比较C组与D组无明显统计学差异,E组与两者具有明显统计学意义(P<0.05)。HE染色显示C组、D组、E组较单纯模型组炎症细胞浸润,平滑肌肥厚及黏膜肺组织水肿等炎症表现明显减轻。结论:平喘固本合剂对哮喘昆系小鼠气道炎症有明显的抑制作用,其作用机制可能与抑制IL-4的表达、炎性细胞聚积及促进IFN-γ的表达有关,并且可能与糖皮质激素有一定协同作用。     

Pulmonary Inflammation and Airway Hyperresponsiveness in a Mouse Model of Asthma Complicated by Acid Aspiration

Several studies have indicated a strong association between asthma and aspiration of stomach contents. However, the complex association between these inflammatory processes has not been studied extensively in animal models. In the present study, we developed an animal model to evaluate the inflammatory cell, chemokine, and airway responses to asthma complicated by aspiration. The model was produced by sensitizing mice to cockroach allergens from house-dust extracts. Mice with asthma-like airway responses then were inoculated intratracheally with either an acidic solution or saline. Acid aspiration increased airway hyperresponsiveness in mice with asthma for at least 8 h. After 6 h, the combined injury caused an additive, not synergistic, increase in airway hyperresponsiveness and neutrophil recruitment to the airways. Although cysteinyl leukotrienes in bronchoalveolar lavage fluid were higher after acid aspiration, treatment with a receptor antagonist before aspiration did not diminish airway hyperresponsiveness. Vagal mechanisms reportedly mediate airway responses in acid aspiration; however, pretreatment with an anticholinergic agent did not reduce airway responses to acid. These results are consistent with an effective model of the acute effects of aspiration on the allergic lung. Further studies could examine how various forms of aspiration influence the severity of asthma.Abbreviations: BAL, bronchoalveolar lavage; MIP, macrophage inflammatory protein; Penh, enhanced pauseAsthma is an escalating public health problem in children and adults.⁴⁹ In patients with asthma, exaggerated immune responses to allergens produce lung inflammation and dysfunction. These responses lead to the characteristic airway hyperresponsiveness, obstructed airflow, and clinical symptoms associated with asthma.⁴⁹ Although several conditions aggravate asthma, much recent attention has focused on the provocative association between asthma and aspiration of stomach acid. The prevalence of gastroesophageal reflux in some asthma patient populations is greater than 50% ²¹ and significantly exceeds the prevalence in nonasthmatic populations.^20,47 This finding suggests an association between the 2 diseases and the possibility that gastroesophageal reflux promotes or aggravates symptoms that lead to the diagnosis asthma. In fact, several studies have shown a decrease in asthma symptoms after medical or surgical treatment of gastroesophageal reflux.^4,11,18,19Stomach acid may exacerbate asthma symptoms through 2 mechanisms. The first is a vagal reflex initiated in response to acid in the esophagus. Clinical studies in humans^20,50 and experimental studies in animals^34,48 have shown that acid in the esophagus promotes neurologic responses leading to bronchoconstriction and impaired airway function. Esophageal acid also may cause substance P- mediated neurogenic inflammation.¹⁶ The second mechanism is due to aspiration into the airways, which also has been documented to occur in asthma patients.²⁵ The presence of acid in the trachea increases airway hyperresponsiveness in anesthetized animals through vagal mechanisms,⁴⁸ particularly in the presence of preexisting lung inflammation.³² In addition to neurologic responses, aspiration of acid induces a pattern of pulmonary inflammation characterized by the release of proinflammatory cytokines and neutrophil recruitment.^26,31 That inflammation may also increase airway responsiveness.⁶Well-established models for both asthma^6,10,14,27 and aspiration^31,39 studies are available currently. However, the patterns of inflammation that occur after sequential insults are complex and may not be predicted by studies of the responses to individual insults.⁸ In addition, the mechanisms for exacerbation of airway hyperresponsiveness by aspiration in asthma have been limited to use of anesthetized animals. A model that allows recovery from the anesthesia after delivery of the aspirate permits the development and evaluation of pulmonary changes under more physiologic conditions. Therefore, the goals of this study were to: (1) describe acute exacerbation of asthma by acid aspiration in mice after recovery from anesthesia; (2) determine the effects of combined insults on airway hyperresponsiveness; and (3) profile the cellular and cytokine responses to the combined insults to assess potential mechanisms for the pulmonary responses to asthma complicated by aspiration.     

Retnla Overexpression Attenuates Allergic Inflammation of the Airway

Resistin-like molecule alpha (Retnla), also known as ‘Found in inflammatory zone 1’, is a secreted protein that has been found in bronchoalveolar lavage (BAL) fluid of ovalbumin (OVA)-induced asthmatic mice and plays a role as a regulator of T helper (Th)2-driven inflammation. However, the role of Retnla in the progress of Th2-driven airway inflammation is not yet clear. To better understand the function of Retnla in Th2-driven airway inflammation, we generated Retnla-overexpressing (Retnla-Tg) mice. Retnla-Tg mice showed increased expression of Retnla protein in BAL fluid and airway epithelial cells. Retnla overexpression itself did not induce any alteration in lung histology or lung function compared to non-Tg controls. However, OVA-sensitized/challenged Retnla-Tg mice had decreased numbers of cells in BAL and inflammatory cells accumulating in the lung. They also showed a reduction in mucus production in the airway epithelium, concomitant with a decreased Muc5ac level. These results were accompanied by reduced levels of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13, with no effect on levels of OVA-specific immunoglobulin isotypes. Furthermore, phosphorylation of ERK was markedly reduced in the lungs of OVA-challenged Retnla-Tg mice. Taken together, these results indicates that Retnla protects against Th2-mediated inflammation in an experimental mouse model of asthma, suggesting that therapeutic approaches to enhance the production of Retnla or Retnla-like molecules could be valuable for preventing allergic lung inflammation.     

Lactoferricin B causes depolarization of the cytoplasmic membrane of Escherichia coli ATCC 25922 and fusion of negatively charged liposomes

Antimicrobial peptides have been extensively studied in order to elucidate their mode of action. Most of these peptides have been shown to exert a bactericidal effect on the cytoplasmic membrane of bacteria. Lactoferricin is an antimicrobial peptide with a net positive charge and an amphipatic structure. In this study we examine the effect of bovine lactoferricin (lactoferricin B; Lfcin B) on bacterial membranes. We show that Lfcin B neither lyses bacteria, nor causes a major leakage from liposomes. Lfcin B depolarizes the membrane of susceptible bacteria, and induces fusion of negatively charged liposomes. Hence, Lfcin B may have additional targets responsible for the antibacterial effect.     

G-Protein-Coupled Estrogen Receptor Agonist Suppresses Airway Inflammation in a Mouse Model of Asthma through IL-10

Estrogen influences the disease severity and sexual dimorphism in asthma, which is caused by complex mechanisms. Besides classical nuclear estrogen receptors (ERαβ), G-protein-coupled estrogen receptor (GPER) was recently established as an estrogen receptor on the cell membrane. Although GPER is associated with immunoregulatory functions of estrogen, the pathophysiological role of GPER in allergic inflammatory lung disease has not been examined. We investigated the effect of GPER-specific agonist G-1 in asthmatic mice. GPER expression in asthmatic lung was confirmed by immunofluorescent staining. OVA-sensitized BALB/c and C57BL/6 mice were treated with G-1 by daily subcutaneous injections during an airway challenge phase, followed by histological and biochemical examination. Strikingly, administration of G-1 attenuated airway hyperresponsiveness, accumulation of inflammatory cells, and levels of Th2 cytokines (IL-5 and IL-13) in BAL fluid. G-1 treatment also decreased serum levels of anti-OVA IgE antibodies. The frequency of splenic Foxp3⁺CD4⁺ regulatory T cells and IL-10-producing GPER⁺CD4⁺ T cells was significantly increased in G-1-treated mice. Additionally, splenocytes isolated from G-1-treated mice showed greater IL-10 production. G-1-induced amelioration of airway inflammation and IgE production were abolished in IL-10-deficient mice. Taken together, these results indicate that extended GPER activation negatively regulates the acute asthmatic condition by altering the IL-10-producing lymphocyte population. The current results have potential importance for understanding the mechanistic aspects of function of estrogen in allergic inflammatory response.     

Compartmental and Temporal Dynamics of Chronic Inflammation and Airway Remodelling in a Chronic Asthma Mouse Model

Background

Allergic asthma is associated with chronic airway inflammation and progressive airway remodelling. However, the dynamics of the development of these features and their spontaneous and pharmacological reversibility are still poorly understood. We have therefore investigated the dynamics of airway remodelling and repair in an experimental asthma model and studied how pharmacological intervention affects these processes.

Methods

Using BALB/c mice, the kinetics of chronic asthma progression and resolution were characterised in absence and presence of inhaled corticosteroid (ICS) treatment. Airway inflammation and remodelling was assessed by the analysis of bronchoalveolar and peribronichal inflammatory cell infiltrate, goblet cell hyperplasia, collagen deposition and smooth muscle thickening.

Results

Chronic allergen exposure resulted in early (goblet cell hyperplasia) and late remodelling (collagen deposition and smooth muscle thickening). After four weeks of allergen cessation eosinophilic inflammation, goblet cell hyperplasia and collagen deposition were resolved, full resolution of lymphocyte inflammation and smooth muscle thickening was only observed after eight weeks. ICS therapy when started before the full establishment of chronic asthma reduced the development of lung inflammation, decreased goblet cell hyperplasia and collagen deposition, but did not affect smooth muscle thickening. These effects of ICS on airway remodelling were maintained for a further four weeks even when therapy was discontinued.

Conclusions

Utilising a chronic model of experimental asthma we have shown that repeated allergen exposure induces reversible airway remodelling and inflammation in mice. Therapeutic intervention with ICS was partially effective in inhibiting the transition from acute to chronic asthma by reducing airway inflammation and remodelling but was ineffective in preventing smooth muscle hypertrophy.     

Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice

Background

The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-κB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be anti-inflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, ragweed pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma.

Methods and Findings

Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on ragweed pollen extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-κB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E₂, IL-6 and IL-8. Further, BALB/c mice were sensitized with endotoxin-free RWE in the absence and presence of AR inhibitor and followed by evaluation of perivascular and peribronchial inflammation, mucin production, eosinophils infiltration and airway hyperresponsiveness. Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice.

Conclusions

These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-κB-dependent mechanism, could be prevented by AR inhibitors. Therefore, inhibition of AR could have clinical implications, especially for the treatment of airway inflammation, a major cause of asthma pathogenesis.     

Temporal Changes in Glutaredoxin 1 and Protein S-Glutathionylation in Allergic Airway Inflammation

Introduction

Asthma is a chronic inflammatory disorder of the airways, involving oxidative stress. Upon oxidative stress, glutathione covalently binds to protein thiols to protect them against irreversible oxidation. This posttranslational modification, known as protein S-glutathionylation, can be reversed by glutaredoxin 1 (Glrx1) under physiological condition. Glrx1 is known to increase in the lung tissues of a murine model of allergic airway inflammation. However, the temporal relationship between levels of Glrx1, protein S-glutathionylation, and glutathione in the lungs with allergic airway inflammation is not clearly understood.

Methods

BALB/c mice received 3 aerosol challenges with ovalbumin (OVA) following sensitization to OVA. They were sacrificed at 6, 24, 48, or 72 h, or 8 days (5 mice per group), and the levels of Glrx1, protein S-glutathionylation, glutathione, and 25 cytokines/chemokines were evaluated in bronchoalveolar lavage fluid (BALF) and/or lung tissue.

Results

Levels of Glrx1 in BALF were significantly elevated in the OVA 6 h (final challenge) group compared to those in the control, with concurrent increases in protein S-glutathionylation levels in the lungs, as well as total glutathione (reduced and oxidized) and oxidized glutathione in BALF. Protein S-glutathionylation levels were attenuated at 24 h, with significant increases in Glrx1 levels in lung tissues at 48 and 72 h. Glrx1 in alveolar macrophages was induced after 6 h. Glrx1 levels concomitantly increased with Th2/NF-κB-related cytokines and chemokines in BALF.

Conclusions

The temporal relationships of Glrx1 with protein S-glutathionylation, glutathione, and cytokines/chemokines were observed as dynamic changes in lungs with allergic airway inflammation, suggesting that Glrx1 and protein–SSG redox status may play important roles in the development of allergic airway inflammation.     

Intranasal Administration of Recombinant Mycobacterium smegmatis Inducing IL-17A Autoantibody Attenuates Airway Inflammation in a Murine Model of Allergic Asthma

Asthma is a chronic inflammatory disorder, previous studies have shown that IL-17A contributes to the development of asthma, and there is a positive correlation between the level of IL-17A and the severity of disease. Here, we constructed recombinant Mycobacterium smegmatis expressing fusion protein Ag85A-IL-17A (rMS-Ag85a-IL-17a) and evaluated whether it could attenuate allergic airway inflammation, and further investigated the underlying mechanism. In this work, the murine model of asthma was established with ovalbumin, and mice were intranasally vaccinated with rMS-Ag85a-IL-17a. Autoantibody of IL-17A in sera was detected, and the airway inflammatory cells infiltration, the local cytokines and chemokines production and the histopathological changes of lung tissue were investigated. We found that the administration of rMS-Ag85a-IL-17a induced the autoantibody of IL-17A in sera. The vaccination of rMS-Ag85a-IL-17a remarkably reduced the infiltration of inflammatory cells and the secretion of mucus in lung tissue and significantly decreased the numbers of the total cells, eosinophils and neutrophils in BALF. Th1 cells count in spleen, Th1 cytokine levels in BALF and supernatant of splenocytes and mediastinal lymph nodes, and T-bet mRNA in lung tissue were significantly increased with rMS-Ag85a-IL-17a administration. Meanwhile, rMS-Ag85a-IL-17a vaccination markedly decreased Th2 cells count, Th2 cytokine and Th17 cytokine levels in BALF and supernatant of splenocytes and mediastinal lymph nodes, and chemokines mRNA expression in lung tissue. These data confirmed that recombinant Mycobacterium smegmatis in vivo could induce autoantibody of IL-17A, which attenuated asthmatic airway inflammation.     

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen^1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response³.DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment⁴. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens⁵. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma^6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.