Neutralisation of Interleukin-13 in Mice Prevents Airway Pathology Caused by Chronic Exposure to House Dust Mite

Background

Repeated exposure to inhaled allergen can cause airway inflammation, remodeling and dysfunction that manifests as the symptoms of allergic asthma. We have investigated the role of the cytokine interleukin-13 (IL-13) in the generation and persistence of airway cellular inflammation, bronchial remodeling and deterioration in airway function in a model of allergic asthma caused by chronic exposure to the aeroallergen House Dust Mite (HDM).

Methodology/Principal Findings

Mice were exposed to HDM via the intranasal route for 4 consecutive days per week for up to 8 consecutive weeks. Mice were treated either prophylactically or therapeutically with a potent neutralising anti-IL-13 monoclonal antibody (mAb) administered subcutaneously (s.c.). Airway cellular inflammation was assessed by flow cytometry, peribronchial collagen deposition by histocytochemistry and airway hyperreactivity (AHR) by invasive measurement of lung resistance (R_L) and dynamic compliance (C_dyn). Both prophylactic and therapeutic treatment with an anti-IL-13 mAb significantly inhibited (P<0.05) the generation and maintenance of chronic HDM-induced airway cellular inflammation, peribronchial collagen deposition, epithelial goblet cell upregulation. AHR to inhaled methacholine was reversed by prophylactic but not therapeutic treatment with anti-IL-13 mAb. Both prophylactic and therapeutic treatment with anti-IL-13 mAb significantly reversed (P<0.05) the increase in baseline R_L and the decrease in baseline C_dyn caused by chronic exposure to inhaled HDM.

Conclusions/Significance

These data demonstrate that in a model of allergic lung disease driven by chronic exposure to a clinically relevant aeroallergen, IL-13 plays a significant role in the generation and persistence of airway inflammation, remodeling and dysfunction.     

Characterization of a Serine Protease Homologous to House Dust Mite Group 3 Allergens from the Scabies Mite Sarcoptes scabiei

The scabies mite, Sarcoptes scabiei var. hominis, infests human skin, causing allergic reactions and facilitating bacterial infection by Streptococcus sp., with serious consequences such as rheumatic fever and rheumatic heart disease. To identify a possible drug target or vaccine candidate protein, we searched for homologues of the group 3 allergen of house dust mites, which we subsequently identified in a cDNA library. The native protein, designated Sar s 3, was shown to be present in the mite gut and excreted in fecal pellets into mite burrows within the upper epidermis. The substrate specificity of proteolytically active recombinant rSar s 3 was elucidated by screening a bacteriophage library. A preference for substrates containing a RS(G/A) sequence at the P1-P2′ positions was revealed. A series of peptides synthesized as internally quenched fluorescent substrates validated the phage display data and high performance liquid chromatography/mass spectrometry analysis of the preferred cleaved substrate and confirmed the predicted cleavage site. Searches of the human proteome using sequence data from the phage display allowed the in silico prediction of putative physiological substrates. Among these were numerous epidermal proteins, with filaggrin being a particularly likely candidate substrate. We showed that recombinant rSar s 3 cleaves human filaggrin in vitro and obtained immunohistological evidence that the filaggrin protein is ingested by the mite. This is the first report elucidating the substrate specificity of Sar s 3 and its potential role in scabies mite biology.     

Alpha-Actinin Is a New Type of House Dust Mite Allergen

Main indoor allergens for humans are from house dust mites. There are more than 30 allergens in Dermatophagoides farinae but only fourteen allergens have been identified from this mite including Der f 1–3, 6, 7, 10, 11, 13–18, and 22. A native allergen protein (Der f 24, 90 kDa) was purified from D. farinae by gel filtration and anionic exchange liquid chromatography combined with IgE immunodetection. Its primary structure was determined by Edman degradation, mass spectrometry analysis and cDNA cloning. Enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, basophil activation test (BAT) and skin prick test (SPT) were performed to evaluate the allergenicity. It was identified as an alpha (α)-actinin containing a CaM-like domain with EF-hand motifs. Der f 24 reacted to sera from 85.4% (35/41) of patients on western blot analysis. It reduced ∼20% sera IgE reactivity to D. farinae extracts on a competitive ELISA. Eighty percent (8/10) of patients with D. farinae allergy showed positive reactions to Der f 24 in skin prick test. The expression of CD63 on basophils from patients was up-regulated by Der f 24 by ∼5.4-fold. Alpha-actinin was identified as a new type of house dust mite allergen. To the best of our knowledge, this is the first report of α-actinin as an allergen.     

Cognitive Factors Mediate Placebo Responses in Patients with House Dust Mite Allergy

Background

Placebo effects have been reported in type I allergic reactions. However the neuropsychological mechanisms steering placebo responses in allergies are largely unknown. The study analyzed whether and to what extend a conditioned placebo response is affecting type I allergic reactions and whether this response can be reproduced at multiple occasions.

Methods

62 patients with house dust mite allergy were randomly allocated to either a conditioned (n = 25), sham-conditioned (n = 25) or natural history (n = 12) group. During the learning phase (acquisition), patients in the conditioned group received the H₁-receptor antagonist desloratadine (5mg) (unconditioned stimulus/US) together with a novel tasting gustatory stimulus (conditioned stimulus/CS). Patients in the sham-conditioned control group received the CS together with a placebo pill. After a wash out time of 9 days patients in the conditioned and sham-conditioned group received placebo pills together with the CS during evocation. Allergic responses documented by wheal size after skin prick test and symptom scores after nasal provocation were analyzed at baseline, after last desloratadine treatment and after the 1^st and 5^th CS re-exposure.

Results

Both conditioned and sham-conditioned patients showed significantly decreased wheal sizes after the 1^st CS-evocation and significantly decreased symptom scores after the 1^st as well as after the 5^th evocation compared to the natural history control group.

Conclusions

These results indicate that placebo responses in type I allergy are not primarily mediated by learning processes, but seemed to be induced by cognitive factors such as patients’ expectation, with these effects not restricted to a single evocation.     

Domestic Mite Antigens in Floor and Airborne Dust at Workplaces in Comparison to Living Areas: A New Immunoassay to Assess Personal Airborne Allergen Exposure

Objectives

Allergens produced by domestic mites (DM) are among the most common allergic sensitizers and risk factors for asthma. To compare exposure levels between workplaces and living areas a new assay able to measure airborne DM antigen concentrations was developed.

Methods

At workplaces and in living areas, 213 floor dust samples and 92 personal inhalable dust samples were collected. For sensitive quantification of DM antigens, a new enzyme immunoassay (EIA) based on polyclonal antibodies to Dermatophagoides farinae extract was developed. Reactivity of five house dust mite and four storage mite species was tested. All dust samples were tested with the new EIA and with the Der f 1 and Der p 1-EIAs (Indoor Biotechnologies, UK) which detect major allergens from D. farinae and D. pteronyssinus by monoclonal antibodies. Samples below the detection limit in the DM-EIA were retested in an assay variant with a fluorogenic substrate (DM-FEIA).

Results

The newly developed DM-EIA detects antigens from all nine tested domestic mite species. It has a lower detection limit of 200 pg/ml of D.farinae protein, compared to 50 pg/ml for the DM-FEIA. DM antigens were detected by DM-EIA/FEIA in all floor dust and 80 (87%) of airborne samples. Der f 1 was found in 133 (62%) floor dust and in only 6 airborne samples, Der p 1 was found in 70 (33%) of floor samples and in one airborne sample. Der f 1 and DM concentrations were highly correlated. DM-antigens were significantly higher in inhalable airborne samples from textile recycling, bed feather filling, feed production, grain storage and cattle stables in comparison to living areas.

Conclusions

A new sensitive EIA directed at DM antigens was developed. DM antigen quantities were well correlated to Der f 1 values and were measurable in the majority (87%) of airborne dust samples. Some workplaces had significantly higher DM antigen concentrations than living areas.     

尘螨变应原Derf1核酸序列测定及其分子进化分析

目的:对尘螨主要变应原Der f1进行核酸序列测定,探讨其系统进化信息。方法:根据Genbank公布的Der f1基因序列设计引物,巢式PCR扩增Der f1的cDNA,纯化、回收、克隆至pMD19-T simple后进行序列测定,序列比对后用Clustal W 1.83构建分子进化树。结果:成功扩增出Der f1的cDNA片段,测序表明该基因含ORF1个,长度966bp,与参考序列同源性达99.9%。该变应原具半胱氨酸蛋白酶活性,与果蝇进化关系最远,与梅氏嗜霉螨进化关系最近。结论:成功获得了尘螨变应原Der f1基因片段,根据其编码的氨基酸序列构建出的系统进化树与形态学分类不一致。     

Asthma,Airway Symptoms and Rhinitis in Office Workers in Malaysia: Associations with House Dust Mite (HDM) Allergy,Cat Allergy and Levels of House Dust Mite Allergens in Office Dust

A prevalence study was conducted among office workers in Malaysia (N= 695). The aim of this study was to examine associations between asthma, airway symptoms, rhinitis and house dust mites (HDM) and cat allergy and HDM levels in office dust. Medical data was collected by a questionnaire. Skin prick tests were performed for HDM allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae) and cat allergen Felis domesticus. Indoor temperature and relative air humidity (RH) were measured in the offices and vacuumed dust samples were analyzed for HDM allergens. The prevalence of D. pteronyssinus, D. farinae and cat allergy were 50.3%, 49.0% and 25.5% respectively. Totally 9.6% had doctor-diagnosed asthma, 15.5% had current wheeze and 53.0% had current rhinitis. The Der p 1 (from D. pteronyssinus) and Der f 1 (from D. farinae) allergens levels in dust were 556 ng/g and 658 ng/g respectively. Statistical analysis was conducted by multilevel logistic regression, adjusting for age, gender, current smoking, HDM or cat allergy, home dampness and recent indoor painting at home. Office workers with HDM allergy had more wheeze (p= 0.035), any airway symptoms (p= 0.032), doctor-diagnosed asthma (p= 0.005), current asthma (p= 0.007), current rhinitis (p= 0.021) and rhinoconjuctivitis (p< 0.001). Cat allergy was associated with wheeze (p= 0.021), wheeze when not having a cold (p= 0.033), any airway symptoms (p= 0.034), doctor-diagnosed asthma (p= 0.010), current asthma (p= 0.020) and nasal allergy medication (p= 0.042). Der f 1 level in dust was associated with daytime breathlessness (p= 0.033) especially among those with HDM allergy. Der f 1 levels were correlated with indoor temperature (p< 0.001) and inversely correlated with RH (p< 0.001). In conclusion, HDM and cat allergies were common and independently associated with asthma, airway symptoms and rhinitis. Der f 1 allergen can be a risk factor for daytime breathlessness.     

House Dust Mite Induced Lung Inflammation Does Not Alter Circulating Vitamin D Levels

Low circulating levels of 25-hydroxyvitamin D [25(OH)D] are associated with chronic lung diseases such as asthma. However, it is unclear whether vitamin D is involved in disease pathogenesis or is modified by the inflammation associated with the disease process. We hypothesized that allergic inflammation decreases the level of circulating 25(OH)D and tested this using a mice model of house dust mite (HDM) induced allergic airway inflammation. Cellular influx was measured in bronchoalvelar lavage (BAL) fluid, and allergic sensitization and 25(OH)D levels were measured in serum. Exposure to HDM caused a robust inflammatory response in the lung that was enhanced by prior influenza infection. These responses were not associated with any change in circulating levels of 25(OH)D. These data suggest that alterations in circulating 25(OH)D levels induced by Th-2 driven inflammation are unlikely to explain the cross-sectional epidemiological association between vitamin D deficiency and asthma.     

尘螨变应原Der f 1核酸序列测定及其分子进化分析

目的：对尘螨主要变应原Der f 1进行核酸序列测定,探讨其系统进化信息。方法：根据Genbank公布的Der f 1基因序列设计引物,巢式PCR扩增Der f 1的cDNA,纯化、回收、克隆至pMD19-T simple后进行序列测定,序列比对后用Clustal W1.83构建分子进化树。结果：成功扩增出Der f 1的cDNA片段,测序表明该基因含ORF1个,长度966bp,与参考序列同源性达99．9％。该变应原具半胱氨酸蛋白酶活性,与果蝇进化关系最远,与梅氏嗜霉螨进化关系最近。结论：成功获得了尘螨变应原Der f 1基因片段．根据其编码的氨基酸序列构建出的系统进化树与形态学分类不一致。     

Regulation of Constitutive Neutrophil Apoptosis Due to House Dust Mite Allergen in Normal and Allergic Rhinitis Subjects

House dust mite (HDM) is a primary allergen in allergic rhinitis (AR) and asthma. Neutrophil apoptosis is associated with allergic diseases and innate immunity to infection. The present study examined how HDM affects constitutive neutrophil apoptosis in normal and AR subjects. Total IgE increased in AR subjects when compared to normal subjects, and patients with AR were HDM-specific IgE positive (+), which is specific IgE to Dermatophagoides pteronissinus and Dermatophagoides farinae. In normal and AR subjects, neutrophil apoptosis was inhibited by extract of Dermatophagoides pteronissinus (DP), but not by extract of Dermatophagoides farina (DF). Aprotinin (serine protease inhibitor) and E64 (cysteine protease inhibitor) have no effect on neutrophil apoptosis due to DP. The anti-apoptotic effect of DP was blocked by TLR4i, an inhibitor of TLR4, rottlerin, an inhibitor of PKCδ, PD98059, an inhibitor of ERK, and BAY-11-7085, an inhibitor of NF-κB. DP induced PKCδ, ERK, and NF-κB activation in a time-dependent manner. DP inhibited the cleavage of procaspase 3 and procaspase 9. The expression of IL-6, IL-8, TNF-α, G-CSF, GM-CSF, and CCL2 increased in the supernatant collected from the normal and AR neutrophils after DP treatment and the supernatant inhibited the apoptosis of normal and AR neutrophils. In summary, DP has anti-apoptotic effects on neutrophils of normal and AR subjects through the TLR4/PKCδ/ERK/NF-κB pathway, and this finding may contribute to solution of the pathogenic mechanism of allergic diseases triggered by DP.     

Diesel Exhaust Particles Induce Cysteine Oxidation and S-Glutathionylation in House Dust Mite Induced Murine Asthma

Background

Diesel exhaust particle (DEP) exposure enhances allergic inflammation and has been linked to the incidence of asthma. Oxidative stress on the thiol molecules cysteine (Cys) and glutathione (GSH) can promote inflammatory host responses. The effect of DEP on the thiol oxidation/reduction (redox) state in the asthmatic lung is unknown.

Objective

To determine if DEP exposure alters the Cys or GSH redox state in the asthmatic airway.

Methods

Bronchoalveolar lavage fluid was obtained from a house dust mite (HDM) induced murine asthma model exposed to DEP. GSH, glutathione disulfide (GSSG), Cys, cystine (CySS), and s-glutathionylated cysteine (CySSG) were determined by high pressure liquid chromatography.

Results

DEP co-administered with HDM, but not DEP or HDM alone, decreased total Cys, increased CySS, and increased CySSG without significantly altering GSH or GSSG.

Conclusions

DEP exposure promotes oxidation and S-glutathionylation of cysteine amino acids in the asthmatic airway, suggesting a novel mechanism by which DEP may enhance allergic inflammatory responses.     

粉尘螨过敏原蛋白Der f 3 的表达纯化及活性鉴定

目的:制备有生物学活性的粉尘螨过敏原蛋白Der f 3。方法:构建带有StrepⅡ标签的原核表达体系Rosetta-p ET44a-Der f 3高效表达目的蛋白,分别用梯度透析法和亲和层析法对其进行复性纯化,通过Western Blot试验验证目的蛋白的有效表达,最后用丝氨酸蛋白酶特异荧光底物及粉尘螨过敏病人血清分别鉴定目的蛋白的酶学活性及免疫学活性。结果:利用原核表达体系成功表达了目的蛋白Der f 3,Western Blot结果显示有目的条带出现。复性纯化后的过敏原蛋白Der f 3能够成功降解丝氨酸蛋白酶特异性荧光底物,ELISA试验结果显示该蛋白血清特异性Ig E在4级以上粉尘螨过敏病人中阳性率为4%。结论:成功获得有酶学活性及免疫学活性的粉尘螨过敏原蛋白Der f 3,为生产高质量的诊断试剂和疫苗奠定了基础。     

House Dust Mite Allergen Regulates Constitutive Apoptosis of Normal and Asthmatic Neutrophils via Toll-Like Receptor 4

House dust mites (HDMs) induce allergic diseases such as asthma. Neutrophil apoptosis is an important process of innate immunity, and its dysregulation is associated with asthma. In this study, we examined the effects of HDM on constitutive apoptosis of normal and asthmatic neutrophils. Extract of Dermatophagoides pteronissinus (DP) inhibited neutrophil apoptosis, but Dermatophagoides farinae extract had no effect. Anti-apoptotic signaling mediated by DP involves in TLR4, Lyn, PI3K, Akt, ERK, and NF-κB in normal neutrophils. DP delayed cleavage of procaspase 9 and procaspase 3 and the decrease in Mcl-1 expression. Supernatant collected from DP-treated normal neutrophils inhibited the constitutive apoptosis of normal neutrophils, and S100A8 and S100A9 were identified as anti-apoptotic proteins in the supernatant. S100A8 and S100A9 transduced the anti-apoptotic signal via TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. DP also suppressed asthmatic neutrophil apoptosis and induced secretion of S100A8 and S100A9, which delayed the constitutive apoptosis. The anti-apoptotic effects of DP, S100A8 and S100A9 in asthmatic neutrophils are associated with TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. The concentrations of S100A8 and S100A9 were significantly elevated in asthmatic bronchoalveolar lavage fluid (BALF) when compared to normal BALF (p<0.01), but not in serum. S100A8 concentration in BALF was positively correlated with the number of BALF neutrophils and negatively correlated with FEV1(%). These findings improve our understanding of the role of HDM in regulation of neutrophil apoptosis in normal individuals and asthmatics and will enable elucidation of asthma pathogenesis.     

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。     

Genetically Engineered Lactococcus lactis Protect against House Dust Mite Allergy in a BALB/c Mouse Model

Background

Mucosal vaccine based on lactic acid bacteria is an attractive concept for the prevention and treatment of allergic diseases, but their mechanisms of action in vivo are poorly understood. Therefore, we sought to investigate how recombinant major dust mite allergen Der p2-expressing Lactococcus lactis as a mucosal vaccine induced the immune tolerance against house dust mite allergy in a mouse model.

Methods

Three strains of recombinant L. lactis producing Der p2 in different cell components (extracellular, intracellular and cell wall) were firstly constructed. Their prophylactic potential was evaluated in a Der p2-sensitised mouse model, and immunomodulation properties at the cellular level were determined by measuring cytokine production in vitro.

Results

Der p2 expressed in the different recombinant L. lactis strains was recognized by a polyclonal anti-Der p2 antibody. Oral treatment with the recombinant L. lactis prior sensitization significantly prevented the development of airway inflammation in the Der p2-sensitized mice, as determined by the attenuation of inflammatory cells infiltration in the lung tissues and decrease of Th2 cytokines IL-4 and IL-5 levels in bronchoalveolar lavage. In addition, the serum allergen-specific IgE levels were significantly reduced, and the levels of IL-4 in the spleen and mesenteric lymph nodes cell cultures were also markedly decreased upon allergen stimulation in the mice fed with the recombinant L. lactis strains. These protective effects correlated with a significant up-regulation of regulatory T cells in the mesenteric lymph nodes.

Conclusion

Oral pretreatment with live recombinant L. lactis prevented the development of allergen-induced airway inflammation primarily by the induction of specific mucosal immune tolerance.     

尘螨变应原Der p1浓度与哮喘患者血清螨特异性抗体的季节消长

目的:探讨哮喘患者血清抗体及患者室内尘螨抗原浓度的季节变化规律。方法:2005年9月、2005年12月、2006年3月、2006年6月用ELISA法检测哮喘患者卧室尘样中Der P 1浓度,同步检测患者外周血总IgE、s-IgE、s-IgG1、s-IgG2和s-IgG4。结果:哮喘患者血清总IgE、s-IgE、s-IgG1、s-IgG2和s-IgG4均高于正常对照组,差异均具显著性(P<0.01);比较患者血清总IgE、s-IgE及s-IgG1、s-IgG2、s-IgG4四次检测值,差异均具显著性(P<0.01),患者血清总IgE、s-IgE在12月份检测值最高、6月份检测值最低,s-IgG1以6月份最高、3月份最低,s-IgG4在3月份最高、6月份最低。统计表明,患者卧室Der P1浓度四次检测值差异具有显著性(P<0.01),以2006年3月和2005年12月为高。结论:哮喘患者血清总IgE、s-IgE、s-IgC1、s-IgG2和s-IgG4和卧室尘螨抗原浓度呈季节变化,IgG1的变化与尘螨抗原浓度的变化趋势相反,IgG4的变化与尘螨抗原浓度的变化趋势一致。