Benzo[a]pyrene aggravates atopic dermatitis-like skin lesions in mice.

Background: Benzo[a]pyrene (BaP) affects the immune system and causes mutagenic and carcinogenic effects. Purpose: We aimed to evaluate the effects of systemic exposure to BaP on mite allergen-induced atopic dermatitis (AD)-like skin lesions in mice. Methods: Mite allergen (Dermatophagoides pteronyssinus; Dp) was injected intradermally into the right ears of NC/Nga male mice on eight occasions every 2-3 days. Benzo[a]pyrene was administered intraperitoneally in the equivalent doses of 0, 2, 20, 200, or 2000 µg/kg/day, once a week on four occasions. Results: AD-like skin inflammation related to mite allergen worsened by BaP exposure at 2, 20 µg/kg/day doses; this was in parallel with eosinophil and mast cell infiltration and mast cell degranulation. A trend was also observed toward increased proinflammatory molecule expression, including macrophage inflammatory protein-1 alpha, interleukin (IL)-4, IL-13, and IL-18, in the ear tissue. However, 200 or 2000 µg/kg/day BaP attenuated the enhancing effects. In the regional lymph nodes, 2 µg/kg/day BaP with Dp enhanced antigen-presenting cell and T cell activation compared with Dp alone. Conclusions: This suggests that BaP exposure can aggravate Dp-induced AD-like skin lesions through TH2-biased responses in the inflamed sites and the activation of regional lymph nodes. Therefore, BaP may be responsible for the recent increase in AD incidence.

Benzo(a)pyrene Enhanced Dermatophagoides Group 1 (Der f 1)-Induced TGFß1 Signaling Activation Through the Aryl Hydrocarbon Receptor-RhoA Axis in Asthma.

We have previously demonstrated that benzo(a)pyrene (BaP) co-exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1-induced airway inflammation. The underlying mechanism, however, remains undetermined. Here we investigated the molecular mechanisms underlying the potentiation of BaP exposure on Der f 1-induced airway inflammation in asthma. We found that BaP co-exposure potentiated Der f 1-induced TGFß1 secretion and signaling activation in human bronchial epithelial cells (HBECs) and the airways of asthma mouse model. Moreover, BaP exposure alone or co-exposure with Der f 1-induced aryl hydrocarbon receptor (AhR) activity was determined by using an AhR-dioxin-responsive element reporter plasmid. The BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation were attenuated by either AhR antagonist CH223191 or AhR knockdown in HBECs. Furthermore, AhR knockdown led to the reduction of BaP and Der f 1 co-exposure-induced active RhoA. Inhibition of RhoA signaling with fasudil, a RhoA/ROCK inhibitor, suppressed BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation. This was further confirmed in HBECs expressing constitutively active RhoA (RhoA-L63) or dominant-negative RhoA (RhoA-N19). Luciferase reporter assays showed prominently increased promoter activities for the AhR binding sites in the promoter region of RhoA. Inhibition of RhoA suppressed BaP and Der f 1 co-exposure-induced airway hyper-responsiveness, Th2-associated airway inflammation, and TGFß1 signaling activation in asthma. Our studies reveal a previously unidentified functional axis of AhR-RhoA in regulating TGFß1 expression and signaling activation, representing a potential therapeutic target for allergic asthma.

Blocking IL-19 Signaling Ameliorates Allergen-Induced Airway Inflammation.

Asthma is a chronic inflammatory disease of the airway. Its major symptoms are reversible breathing problems causing airway narrowing and obstruction. IL-19 is a member of the IL-10 family cytokines. We previously showed that IL-19 induces T-helper 2 (Th2) cytokines and that asthma patients had higher serum IL-19 levels. To further examine whether inhibiting IL-19 and its receptor (IL-20R1) protected rodents against asthma, we used Dermatophagoides pteronyssinus (Der p; house dust mites) to induce chronic airway inflammation in wild-type C57BL/6 and IL-20R1-deficient mice and then analyzed the effect of the IL-20R1 deficiency on the pathogenesis of asthma. We also examined whether inhibiting IL-19 and IL-20R1 ameliorated Der p-induced chronic asthma. Der p induced IL-19 in lung airway epithelial cells, type 2 alveolar cells, and alveolar macrophages. An IL-20R1 deficiency abolished IL-19-induced Th2 cell differentiation in vitro. Th2 cytokine expression, immune cell infiltration in the bronchoalveolar lavage, airway hyperresponsiveness (AHR), and bronchial wall thickening were lower in Der p-challenged IL-20R1-deficient mice. Anti-IL-20R1 monoclonal antibody (mAb) 51D and IL-19 polyclonal antibody (pAb) both ameliorated Der p-induced AHR, lung immune cell infiltration, bronchial wall thickening, and Th2 cytokine expression. Moreover, we confirmed that anti-IL-19 mAb (1BB1) attenuated lung inflammation in a rat ovalbumin-induced asthma model. This is the first report to show that inhibition of IL-19 by targeting IL-19 or IL-20R1 protected rodents from allergic lung inflammation. Our study suggests that targeting IL-19 signaling might be a novel therapeutic strategy for treating allergic asthma.

Effect of PM2.5 mediated oxidative stress on the innate immune cellular response of Der p1 treated human bronchial epithelial cells.

OBJECTIVE: To investigate the effect of stimulation of Human Bronchial Epithelial Cells (HBEC) by Der p1 and PM2.5 on the expression of innate immune cell factors to find new therapeutic targets for treatment of bronchial asthma. MATERIALS AND METHODS: The Der p1 antigen exposure model in the HEBC line, 16HBE-14o, was established in vitro. PM2.5 at a concentration of 50 µM/cm2, was added to these cells for 0.5 h, 1 h, 2 h and 3 h. Cells were treated with the following reagents for the indicated times: 300 ng/mL Der p1 for 21 h, 50 µM/cm2 PM2.5 for 3 h, 10 mM Nac for 3 h and PM2.5 contamination for 3 h. The experiment was divided into five groups: control (group A), Der p1 exposure group (group B), PM2.5 treated group (group C), PM2.5+Der p1 exposure group (group D), Nac+PM2.5+Der p1 exposure group (group E). ELISA method was adopted to test the expression levels of malondialdehyde, IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and Real-time RT-PCT was used to measure IL-25, IL-33 and TSLP mRNA. RESULTS: The protein and mRNA levels of malondialdehyde, IL-25, IL-33 and TSLP in group D were significantly higher than those in the other groups, while the protein and mRNA levels of malondialdehyde, IL-25, IL-33 and TSLP in group E were significantly lower than those in group D (p<0.05). CONCLUSIONS: PM2.5 can enhance the Der p1 antigen-induced HBEC innate immune response through the expression of IL-25, IL-33 and TSLP, which may exacerbate the occurrence rate of bronchial asthma.

A TNFRSF14-FcɛRI-mast cell pathway contributes to development of multiple features of asthma pathology in mice.

Asthma has multiple features, including airway hyperreactivity, inflammation and remodelling. The TNF superfamily member TNFSF14 (LIGHT), via interactions with the receptor TNFRSF14 (HVEM), can support TH2 cell generation and longevity and promote airway remodelling in mouse models of asthma, but the mechanisms by which TNFSF14 functions in this setting are incompletely understood. Here we find that mouse and human mast cells (MCs) express TNFRSF14 and that TNFSF14:TNFRSF14 interactions can enhance IgE-mediated MC signalling and mediator production. In mouse models of asthma, TNFRSF14 blockade with a neutralizing antibody administered after antigen sensitization, or genetic deletion of Tnfrsf14, diminishes plasma levels of antigen-specific IgG1 and IgE antibodies, airway hyperreactivity, airway inflammation and airway remodelling. Finally, by analysing two types of genetically MC-deficient mice after engrafting MCs that either do or do not express TNFRSF14, we show that TNFRSF14 expression on MCs significantly contributes to the development of multiple features of asthma pathology.

Airway epithelial SPDEF integrates goblet cell differentiation and pulmonary Th2 inflammation.

Epithelial cells that line the conducting airways provide the initial barrier and innate immune responses to the abundant particles, microbes, and allergens that are inhaled throughout life. The transcription factors SPDEF and FOXA3 are both selectively expressed in epithelial cells lining the conducting airways, where they regulate goblet cell differentiation and mucus production. Moreover, these transcription factors are upregulated in chronic lung disorders, including asthma. Here, we show that expression of SPDEF or FOXA3 in airway epithelial cells in neonatal mice caused goblet cell differentiation, spontaneous eosinophilic inflammation, and airway hyperresponsiveness to methacholine. SPDEF expression promoted DC recruitment and activation in association with induction of Il33, Csf2, thymic stromal lymphopoietin (Tslp), and Ccl20 transcripts. Increased Il4, Il13, Ccl17, and Il25 expression was accompanied by recruitment of Th2 lymphocytes, group 2 innate lymphoid cells, and eosinophils to the lung. SPDEF was required for goblet cell differentiation and pulmonary Th2 inflammation in response to house dust mite (HDM) extract, as both were decreased in neonatal and adult Spdef(-/-) mice compared with control animals. Together, our results indicate that SPDEF causes goblet cell differentiation and Th2 inflammation during postnatal development and is required for goblet cell metaplasia and normal Th2 inflammatory responses to HDM aeroallergen.

Gene expression in the skin of dogs sensitized to the house dust mite Dermatophagoides farinae.

Atopic dermatitis is a multifactorial allergic skin disease in humans and dogs. Genetic predisposition, immunologic hyperreactivity, a defective skin barrier, and environmental factors play a role in its pathogenesis. The aim of this study was to analyze gene expression in the skin of dogs sensitized to house dust mite antigens. Skin biopsy samples were collected from six sensitized and six nonsensitized Beagle dogs before and 6 hr and 24 hr after challenge using skin patches with allergen or saline as a negative control. Transcriptome analysis was performed by the use of DNA microarrays and expression of selected genes was validated by quantitative real-time RT-PCR. Expression data were compared between groups (unpaired design). After 24 hr, 597 differentially expressed genes were detected, 361 with higher and 226 with lower mRNA concentrations in allergen-treated skin of sensitized dogs compared with their saline-treated skin and compared with the control specimens. Functional annotation clustering and pathway- and co-citation analysis showed that the genes with increased expression were involved in inflammation, wound healing, and immune response. In contrast, genes with decreased expression in sensitized dogs were associated with differentiation and barrier function of the skin. Because the sensitized dogs did not show differences in the untreated skin compared with controls, inflammation after allergen patch test probably led to a decrease in the expression of genes important for barrier formation. Our results further confirm the similar pathophysiology of human and canine atopic dermatitis and revealed genes previously not known to be involved in canine atopic dermatitis.

Assessing the allergenic potential of molds found in water-damaged homes in a mouse model.

Damp/moldy indoor environments, which have resulted from flooding events and may increase as a result of climate change, have been associated with asthma exacerbation. Certain molds found in significantly higher or lower concentrations in asthmatics' homes compared to control homes have been categorized as Group 1 (G1) and Group 2 (G2) molds, respectively. We have compared the allergic potential of selected G1/G2 molds to house dust mite (HDM) in a mouse model. BALB/c mice were exposed to mold (0-80 µg) or HDM (20 µg) extract by intratracheal aspiration either 4X over 4 weeks (allergenicity) or 1X (non-specific responses). Airflow limitation (methacholine challenge) was measured (Day 1) and serum and bronchoalveolar lavage fluid were collected (Day 2) after the final exposure. The G1 molds induced low-to-moderate responses and required higher doses to achieve antigen-specific IgE results similar to those induced by HDM. Compared to HDM responses, the G2 mold in this study required lower doses to induce a similar response. Acute exposure responses suggest some molds may exacerbate asthmatic responses. These studies demonstrate the differing capacities of molds to induce responses associated with allergic asthma, including differences in the threshold dose for allergy induction. Therefore, molds must be evaluated individually for allergic/asthmatic potential. These studies along with our previous studies with G1 (Stachybotrys chartarum)/G2 (Penicillium chrysogenum) molds suggest that the G1/G2 categorization is not indicative of allergic potential but they do not preclude this categorization's utility in determining unhealthy building dampness.

Epithelial NF-κB orchestrates house dust mite-induced airway inflammation, hyperresponsiveness, and fibrotic remodeling.

NF-κB activation within the epithelium has been implicated in the pathogenesis of asthma, yet the exact role of epithelial NF-κB in allergen-induced inflammation and airway remodeling remains unclear. In the current study, we used an intranasal house dust mite (HDM) extract exposure regimen time course in BALB/c mice to evaluate inflammation, NF-κB activation, airway hyperresponsiveness (AHR), and airway remodeling. We used CC10-IκBαSR transgenic mice to evaluate the functional importance of epithelial NF-κB in response to HDM. After a single exposure of HDM, mRNA expression of proinflammatory mediators was significantly elevated in lung tissue of wild-type (WT) mice, in association with increases in nuclear RelA and RelB, components of the classical and alternative NF-κB pathway, respectively, in the bronchiolar epithelium. In contrast, CC10-IκBαSR mice displayed marked decreases in nuclear RelA and RelB and mRNA expression of proinflammatory mediators compared with WT mice. After 15 challenges with HDM, WT mice exhibited increases in inflammation, AHR, mucus metaplasia, and peribronchiolar fibrosis. CC10-IκBαSR transgenic mice displayed marked decreases in neutrophilic infiltration, tissue damping, and elastance parameters, in association will less peribronchiolar fibrosis and decreases in nuclear RelB in lung tissue. However, central airway resistance and mucus metaplasia remained elevated in CC10-IκBαSR transgenic mice, in association with the continued presence of lymphocytes, and partial decreases in eosinophils and IL-13. The current study demonstrates that following airway exposure with an asthma-relevant allergen, activation of classical and alternative NF-κB pathways occurs within the airway epithelium and may coordinately contribute to allergic inflammation, AHR, and fibrotic airway remodeling.

Epigenetic alterations by DNA methylation in house dust mite-induced airway hyperresponsiveness.

Asthma is one of the most prevalent chronic lung diseases, affecting 235 million individuals around the world, with its related morbidity and mortality increasing steadily over the last 20 years. Exposure to the environmental allergen, house dust mite (HDM), results in airway inflammation with a variable degree of airway obstruction. Although there has been much experimental work in the past using HDM challenge models to understand mechanistic details in allergic inflammation and airway hyperresponsiveness (AHR), there has been no study on reprogramming of lung or airways mediated through epigenetic mechanisms in response to an acute HDM exposure. Male mice, 6 weeks of age, were administrated HDM extracts or saline at Days 1, 14, and 21. Exposure of mice to HDM extracts caused significant airway inflammation and increased AHR. These HDM-challenged mice also exhibited a change in global DNA methylation as compared with saline-exposed (control) mice. Next, by employing methylation-sensitive restriction fingerprinting, we identified a set of genes, showing aberrant methylation status, associated with the HDM-induced AHR. These candidate genes are known to be involved in cAMP signaling (pde4 d), Akt-signaling (akt1 s1), ion transport (tm6 sf1, pom121l2, and slc8a3), and fatty acid metabolism (acsl3). Slc8a3 and acsl3 were down-regulated, whereas pde4 d, akt1 s1, tm6 sf1, and pom121l2 were up-regulated in the mice exposed to HDM. Hence, our results suggest that HDM exposure induces a series of aberrant methylated genes that are potentially important for the development of allergic AHR.

Prostaglandin E2 deficiency uncovers a dominant role for thromboxane A2 in house dust mite-induced allergic pulmonary inflammation.

Prostaglandin E(2) (PGE(2)) is an abundant lipid inflammatory mediator with potent but incompletely understood anti-inflammatory actions in the lung. Deficient PGE(2) generation in the lung predisposes to airway hyperresponsiveness and aspirin intolerance in asthmatic individuals. PGE(2)-deficient ptges(-/-) mice develop exaggerated pulmonary eosinophilia and pulmonary arteriolar smooth-muscle hyperplasia compared with PGE(2)-sufficient controls when challenged intranasally with a house dust mite extract. We now demonstrate that both pulmonary eosinophilia and vascular remodeling in the setting of PGE(2) deficiency depend on thromboxane A(2) and signaling through the T prostanoid (TP) receptor. Deletion of TP receptors from ptges(-/-) mice reduces inflammation, vascular remodeling, cytokine generation, and airway reactivity to wild-type levels, with contributions from TP receptors localized to both hematopoietic cells and tissue. TP receptor signaling ex vivo is controlled heterologously by E prostanoid (EP)(1) and EP(2) receptor-dependent signaling pathways coupling to protein kinases C and A, respectively. TP-dependent up-regulation of intracellular adhesion molecule-1 expression is essential for the effects of PGE(2) deficiency. Thus, PGE(2) controls the strength of TP receptor signaling as a major bronchoprotective mechanism, carrying implications for the pathobiology and therapy of asthma.

Ras guanine nucleotide-releasing protein-4 (RasGRP4) involvement in experimental arthritis and colitis.

RasGRP4 (Ras guanine nucleotide-releasing protein-4) is an intracellular, calcium-regulated guanine nucleotide exchange factor and diacylglycerol/phorbol ester receptor expressed in mast cells (MCs) and their progenitors. To study the function of this signaling protein in inflammatory disorders, a homologous recombination approach was used to create a RasGRP4-null C57BL/6 mouse line. The resulting transgenic animals had normal numbers of MCs in their tissues that histochemically and morphologically resembled those in WT C57BL/6 mice. MCs could also be generated from RasGRP4-null mice by culturing their bone marrow cells in IL-3-enriched conditioned medium. Despite these data, the levels of the transcripts that encode the proinflammatory cytokines IL-1ß and TNF-α were reduced in phorbol 12-myristate 13-acetate-treated MCs developed from RasGRP4-null mice. Although inflammation was not diminished in a Dermatophagoides farinae-dependent model of allergic airway disease, dextran sodium sulfate-induced colitis was significantly reduced in RasGRP4-null mice relative to similarly treated WT mice. Furthermore, experimental arthritis could not be induced in RasGRP4-null mice that had received K/BxN mouse serum. The latter findings raise the possibility that the pharmacologic inactivation of this intracellular signaling protein might be an effective treatment for arthritis or inflammatory bowel disease.

Dermatophagoides pteronyssinus 2 regulates nerve growth factor release to induce airway inflammation via a reactive oxygen species-dependent pathway.

Group 2 allergen of Dermatophagoides pteronyssinus 2 (Der p2) induces airway inflammation without protease activity, and elevated nerve growth factor (NGF) levels are also found in this inflammation. How the allergen Der p2 regulates NGF release via reactive oxygen species (ROS) to induce inflammation remains unclear. In the present study, intratracheal administration of Der p2 to mice led to inflammatory cell infiltration, mucus gland hyperplasia, and NGF upregulation in the bronchial epithelium, as well as elevated ROS and NGF production in bronchoalveolar lavage fluids. In addition, Der p2 caused fibrocyte accumulation and mild fibrosis. p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) inhibitors inhibited Der p2-induced NGF release in LA4 lung epithelial cells and MLg lung fibroblasts. Pretreatment with an antioxidant, tiron, reduced the Der p2-induced ROS production, NGF expression and release, p38 MAPK or JNK phosphorylation, and airway inflammation. These results suggest that Der p2 allergen-induced airway inflammation and elevated NGF release were through increasing ROS production and a MAPK-dependent pathway. The use of an antioxidant, tiron, may provide a new therapeutic modality for the treatment of allergic asthma.

[Biological pollution and its relationship with respiratory symptoms indicative of asthma, Bucaramanga, Colombia]. / Contaminación biológica intradomiciliaria y su relación con síntomas respiratorios indicativos de asma bronquial en preescolares de Bucaramanga, Colombia.

INTRODUCTION: Indoorair pollution may play an important role in development and exacerbation of asthma in children. OBJECTIVE: The association between the presence of indoor biological contaminants and respiratory symptoms related to asthma was assessed in preschool children. Materials and methods. This cross-sectional study was undertaken in Bucaramanga, Colombia, and included children <7 years of age living in two urban areas of with different levels of outdoor air pollution. The 678 children were an average of 3.5 years of age. Respiratory symptoms indicative of asthma and indoor air pollutants were assessed by previously validated questionnaires.. Biological samples potentially containing mites and fungi were collected by standardized laboratory methods. The log binomial regression model was used for multivariate analysis, using adjusted prevalence ratios (PR). RESULTS: The prevalence of asthmatic respiratory symptoms was 8.0%; (95% C.I: 5.6-9.6), without significant differences between the two areas. Binomial model analysis showed that asthma symptoms were associated with mites (PR 1.78; 95% C.I. 1.0-3.0), Acremonium sp (PR 6.24; 95 C.I.: 3.8-10.0) and a history of child pneumonia (PR 4.0; 95% C.I. 2.5-6.4), allergic rhinitis (PR 1.9; 95% C.I.: 1.2-3.1), prematurity (PR 3.4; 95% C.I. 1.8-6.5), parents with asthma (PR 2.6; 95% C.I. 1.4-5.0) and pet ownership (PR 0.4; 95% C.I. 0.2-0.9). CONCLUSIONS: The indoor exposure to biological contaminants (dust mites and fungi), history of prematurity, pneumonia, rhinitis and family history of asthma increased the occurence of symptoms suggestive of asthma in young children.

Dust mites living in human lungs--the cause of asthma?

Asthma does not have a clear cause and may represent a cluster of diseases. We propose that asthma in house dust mite sensitive patients may be caused by recurrent inhalation of live dust mites which are able to live for some time in the bronchioles of the lung. To provide themselves with a food source, the mites may purposefully excrete proteolytic enzymes, including Der p1, which increase epithelial shedding by freeing cells from the basement membrane. The mites then feed on the shed respiratory epithelial cells. Consequent loss of an intact respiratory epithelium exposes underlying tissues to dust mite protein and other allergens triggering sensitisation to these proteins. Later, repeated infestation provokes an allergic response which manifests itself as asthma attacks. The evidence for this hypothesis was tested against the Bradford-Hill criteria for causation; consistency, strength, temporal association and dose response. Potential areas for further research were also identified. The association between asthmatic symptoms and pulmonary acariasis was consistent across a number of studies. Determining the strength of the association and any dose response requires more work which is dependent on the development of better tests for the detection of mites in sputum. There was tentative evidence of a temporal association in the published studies identified. Biological plausibility and experimental evidence was available for pulmonary acariasis in primates and arsenic treatment was effective in humans. Better tests for mites in sputum are needed, as is work to assess more modern anti-acaricidal drugs in dust mite sensitive asthmatics.