Effect of Ozone on Allergic Airway Inflammation

Background: Exposure to O3 has been associated with increased risk of exacerbations of asthma, but the underlying mechanisms are not well studied. We hypothesized that O3 exposure would enhance airway inflammatory responses to allergen and the GSTM1 null genotype would modulate this enhancement. Procedures: In a cross-over design, 10 asthmatic subjects (50% with GSTM1 null genotype) who had specific sensitization to Dermatophagoides pteronyssinus (DP) were exposed to 160 ppb O3 or filtered air (FA) control for 4 h with intermittent exercise on two separate days at least three weeks apart. 20 h post-exposure, endobronchial challenge with DP allergen, and sham normal saline (NS) instillation, were performed in two separate lung lobes. Six h later, a second bronchoscopy was performed to collect bronchoalveolar lavage (BAL) fluid from the DP- and NS-challenged lobes for analyses of cellular and biochemical markers of inflammation. Multiple variable regression was used to compare cell and cytokine responses across the four exposure groups (FA-NS, O3-NS, FA-DP, O3-DP). Effect modification by GSTM1 genotype was assessed in stratified regressions. Main Findings: BAL eosinophil and lymphocyte counts were increased in segments challenged with DP compared to segments that received sham challenges (p<0.01). DP challenge compared to sham challenge also caused a significant increase in BAL concentrations of the Th2 cytokines IL-4, IL-5, IL-10, and IL-13 (p<0.03 for all comparisons). O3 exposure did not significantly affect BAL cells or cytokine levels although BAL neutrophil count with DP challenge was non-significantly higher after O3 compared to after FA exposure (p<0.11). Compared to GSTM1-present subjects, GSTM1-null subjects had significantly reduced inflammatory responses including lower eosinophil (p<0.041) and IL-4 (p<0.014) responses to DP challenge after O3 exposure. Conclusions: O3 appears to have mixed effects on allergen-induced airway inflammation. While O3 did not cause a clear differential effect on airway cellular or cytokine responses to allergen challenge, those responses did appear to be modulated by the antioxidant enzyme, GSTM1, as evident by the attenuation of airway inflammatory responses to allergen after O3 exposure in the absence of the gene.

Specific Immunotherapy in a Murine Model of Grass Pollen (Phl p5b)-Induced Airway Inflammation

Background: The use of ovalbumin as a model allergen in murine models of allergic asthma is controversially discussed since it is not an aeroallergen and sensitization can only be achieved by using strong Th2-inducing adjuvants. Therefore, in this study, a murine model of asthma has been established in which sensitization against the major grass pollen allergen Phl p5b was performed without using aluminum hydroxide (alum). We used this model for specific immunotherapy.Methods: Female, 5–6-week-old mice were sensitized by six subcutaneous (s.c.) injections of 20 μg Phl p5b followed by four provocations to induce allergic airway inflammation. For desensitization, 1 mg of Phl p5b was injected subcutaneously during allergen challenge for one to a maximum of four times. Three days after the last challenge, the allergic immune response was analyzed.Results: Sensitized and challenged animals showed a significant infiltration of eosinophils into the airways, and the production of interleukin-5 (IL-5) by in vitro re-stimulated splenocytes could be detected. Furthermore, hyper-responsiveness of the airways was verified by invasive measurement of airway resistance in methacholine-challenged animals. Desensitized animals showed a significant reduction of all parameters.Conclusion: In this study, a murine model of asthma has successfully been established by sensitization against the clinically relevant allergen Phl p5b without using alum. S.c. injection of allergen dose dependently led to desensitization of sensitized mice. We suggest that this model is useful to study adjuvant effects of immune modulatory substances on immunotherapy without the interference of alum.

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.

Airway IL-1β associates with IL-5 production following dust mite allergen inhalation in humans

Background Preclinical studies implicate interleukin (IL)-1β as a key mediator of asthma and have shown the efficacy of IL-1 antagonism for treatment of allergic airway inflammation; human studies in this area are lacking. Objectives Our aim was to study the relationship of airway IL-1β to features of acute allergen-induced asthma exacerbation in humans. Methods Dust mite-allergic adults with mild asthma underwent inhalation challenge with Dermatophagoides farinae. Fractional exhaled nitric oxide (FeNO), induced sputum and peripheral blood samples were obtained pre- and 24 h post-challenge. Spirometry was performed before and throughout the challenge at 10-min intervals, and allergen responsiveness was defined by a 20% fall in Forced Expiratory Volume in 1 s (FEV1). Sputum samples were analyzed for inflammatory cells, cytokines and chemokines. Multiple linear regression was employed to test the association between sputum IL-1β concentration and biomarkers of T helper type 2 (T2)-dominant inflammation. Results Fourteen volunteers underwent inhaled allergen challenge. Allergen responsive volunteers showed a greater positive change in IL-1β in sputum following allergen challenge compared to non-responders. Higher pre-challenge sputum IL-1β was associated with greater increase in sputum IL-5 (p = 0.004), sputum eosinophils (p = 0.001) and blood IL-5 (p = 0.003) following allergen challenge. Allergen-induced sputum IL-1β production was significantly associated with sputum and blood IL-5 (p < 0.001 and p = 0.007, respectively), sputum IL-4 (p = 0.001), IL-13 (p = 0.026), eosinophils (p = 0.008) and FeNO (p = 0.03). Conclusions The positive association between production of IL-1β and biomarkers of T2 inflammation, particularly IL-5, in humans is consistent with work in animal models that demonstrates a link between IL-1β and the pathophysiology of allergic asthma. The role of IL-1β in human asthma warrants further study.

TNFα SIGNALLING IN THE CUTANEOUS IMMUNE NETWORK INSTRUCTS LOCAL Th17 ALLERGEN-SPECIFIC INFLAMMATORY RESPONSES IN ATOPIC DERMATITIS

Accurate regulation of cutaneous immunity is fundamental for human health and quality of life but is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance vs inflammation in human skin, we set up a human in vivo allergen challenge study, exposing patients with atopic dermatitis (AD) to house dust mite (HDM). Analyses of transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of resident and infiltrating immune cells indicated that inflammatory responses to HDM were associated with immune activation in Langerhans cells (LCs) and cutaneous T cells. High basal level of TNFα production by cutaneous Th17 T cells predisposed to an inflammatory reaction and resulted in formation of hub structures where LCs and T cells interacted, leading to loss of functional programming in LCs. Additionally, single nucleotide polymorphisms in MT1X gene associated with enhanced expression of metallothioneins and transcriptional programmes encoding antioxidant defences across skin cell types in non-reactive patients, were protective against T cell mediated inflammation. Our results provide a unique insight into the dynamics of immune regulation in the human skin and define regulatory circuits that can be harnessed to improve skin health and treat disease.

IL-27 Derived From Macrophages Facilitates IL-15 Production and T Cell Maintenance Following Allergic Hypersensitivity Responses

Crosstalk between T cells, dendritic cells, and macrophages in temporal leukocyte clusters within barrier tissues provides a new concept for T cell activation in the skin. Activated T cells from these leukocyte clusters play critical roles in the efferent phase of allergic contact hypersensitivity (CHS). However, the cytokines driving maintenance and survival of pathogenic T cells during and following CHS remain mostly unknown. Upon epicutaneous allergen challenge, we here report that macrophages produce IL-27 which then induces IL-15 production from epidermal keratinocytes and dermal myeloid cells within leukocyte clusters. In agreement with the known role of IL-15 as a T cell survival factor and growth cytokine, this signaling axis enhances BCL2 and survival of skin T cells. Genetic depletion or pharmacological blockade of IL-27 in CHS mice leads to abrogated epidermal IL-15 production resulting in a decrease in BCL2 expression in T cells and a decline in dermal CD8+ T cells and T cell cluster numbers. These findings suggest that the IL-27 pathway is an important cytokine for regulating cutaneous T cell immunity.

Diagnostic Tools in Allergic Rhinitis

Allergic mechanisms account for most cases of chronic rhinitis. This condition is associated with significant impairment of quality of life and high indirect costs. The identification of the allergic triggers of rhinitis has been historically based on the performance of atopy test [skin prick test (SPT) and serum allergen-specific (s)IgE]. Nevertheless, these tests only denote sensitization, and atopy and allergy represent two different phenomena. It is now clear that allergic phenotypes of rhinitis can exist in both atopic (allergic rhinitis, AR) and non-atopic (local allergic rhinitis, LAR) individuals. Moreover, both allergic phenotypes can coexist in the same rhinitis patient (dual allergic rhinitis, DAR). Therefore, a diagnostic approach merely based on atopy tests is associated with a significant rate of misdiagnosis. The confirmation of the allergic etiology of rhinitis requires the performance of in vivo test like the nasal allergen challenge (NAC). NAC is mandatory for the diagnosis of LAR and DAR, and helps decide the best management approach in difficult cases of AR. Nevertheless, NAC is a laborious technique requiring human and technical resources. The basophil activation test (BAT) is a patient-friendly technique that has shown promising results for LAR and DAR diagnosis. In this review, the diagnostic usefulness for chronic rhinitis of SPT, NAC, olfactory tests, serum sIgE, BAT and the quantification of inflammatory mediators in nasal samples will be discussed. The accurate performance of an etiologic diagnosis of rhinitis patients will favor the prescription of specific therapies with disease-modifying potential like allergen immunotherapy.

Lung epithelial CYP1 activity regulates aryl hydrocarbon receptor dependent allergic airway inflammation

The lung epithelial barrier serves as a guardian towards environmental insults and responds to allergen encounter with a cascade of immune reactions that can possibly lead to inflammation. Whether the environmental sensor aryl hydrocarbon receptor (AhR) together with its downstream targets cytochrome P450 (CYP1) family members contribute to the regulation of allergic airway inflammation remains unexplored. By employing knockout mice for AhR and for single CYP1 family members, we found that AhR-/- and CYP1B1-/- but not CYP1A1-/- or CYP1A2-/- animals display enhanced allergic airway inflammation compared to WT. Expression analysis, immunofluorescence staining of murine and human lung sections and bone marrow chimeras suggest an important role of CYP1B1 in non-hematopoietic lung epithelial cells to prevent exacerbation of allergic airway inflammation. Transcriptional analysis of murine and human lung epithelial cells indicates a functional link of AhR to barrier protection/inflammatory mediator signaling upon allergen challenge. In contrast, CYP1B1 deficiency leads to enhanced expression and activity of CYP1A1 in lung epithelial cells and to an increased availability of the AhR ligand kynurenic acid following allergen challenge. Thus, differential CYP1 family member expression and signaling via the AhR in epithelial cells represents an immunoregulatory layer protecting the lung from exacerbation of allergic airway inflammation.