Stefanie Hagner

Expression and distribution of calcitonin receptor-like receptor in human hairy skin.

Calcitonin gene-related peptide and adrenomedullin exert potent effects in skin but their cellular targets are unknown. This study aimed to identify the cellular location of calcitonin receptor-like receptor (CRLR) which is pharmacologically identical to CGRP receptor-1, a putative molecular target of CGRP and adrenomedullin. RT-PCR analysis of human hairy skin revealed the presence of CRLR mRNA and immunohistochemical analysis, employing a previously characterized polyclonal antibody raised to CRLR, provided novel evidence of the cellular distribution of CRLR. Extensive and specific CRLR-immunostaining was detected in arteriolar smooth muscle and venular endothelium and is consistent with CGRPs putative role in neurogenic inflammation. Novel targets for CGRP and/or adrenomedullin were identified, including capillary endothelium, hair follicles and sweat glands.

Tc9 cells, a new subset of CD8(+) T cells, support Th2-mediated airway inflammation.

Similar to T‐helper (Th) cells, CD8+ T cells also differentiate into distinct subpopulations. However, the existence of IL‐9‐producing CD8+ T (Tc9) cells has not been elucidated so far. We show that murine CD8+ T cells activated in the presence of IL‐4 plus TGF‐β develop into transient IL‐9 producers characterized by specific IFN‐γ and IL‐10 expression patterns as well as by low cytotoxic function along with diminished expression of the CTL‐associated transcription factors T‐bet and Eomesodermin. Similarly to the CD4+ counterpart, Tc9 cells required for their differentiation STAT6 and IRF4. Tc9 cells deficient for these master regulators displayed increased levels of Foxp3 that in turn suppressed IL‐9 production. In an allergic airway disease model, Tc9 cells promoted the onset of airway inflammation, mediated by subpathogenic numbers of Th2 cells. This support was specific for Tc9 cells because CTLs failed to exert this function. We detected increased Tc9 frequency in the periphery in mice and humans with atopic dermatitis, a Th2‐associated skin disease that often precedes asthma. Thus, our data point to the existence of Tc9 cells and to their supportive function in Th2‐dependent airway inflammation, suggesting that these cells might be a therapeutic target in allergic disorders.

Immunohistochemical detection of calcitonin gene-related peptide receptor (CGRPR)^1 in the endothelium of human coronary artery and bronchial blood vessels

The potent vasodilatory peptide, calcitonin gene-related peptide (CGRP) is present in the innervation of vascular tissue. The actions of CGRP occur via a receptor, CGRP receptor(R)-1, which is also a target for the cardioprotective peptide adrenomedullin. The human version of the pharmacologically-defined CGRPR-1 has been cloned but its distribution and cellular location is unknown. A rabbit antibody was generated to a synthetic peptide that corresponds to the C-terminus of human CGRPR-1 Immunochemical analysis of the human cell-line, SK-N-MC, which exhibits functional expression of the CGRPR-1 confirmed the antibodys specificity. The antiserum revealed specific staining in the endothelium of human coronary arteries. The vascular smooth muscle and ventricular myocardium were not immunoreactive. In bronchial blood vessels CGRPR-1-immunoreactivity was detected in the endothelium of the venules and not in the arterioles, which is particularly relevant for elucidating the putative role of CGRP in inflammation in this tissue.

Calcitonin receptor-like receptor is expressed on gastrointestinal immune cells.

Background/Aims: Pharmacological and morphological studies suggest that the gut mucosal immune system and local neuropeptide-containing neurones interact. We aimed to determine whether gut immune cells are targets for calcitonin gene-related peptide (CGRP), which has potent immune regulatory properties. Methods: Using density gradient centrifugation, rat lamina propria mononuclear cells (LP-MNCs) and intra-epithelial lymphocytes (IELs) were isolated. RT-PCR was employed for the detection of mRNA of rat calcitonin receptor-like receptor (CRLR), which is considered to represent the pharmacologically defined CGRP receptor-1 subtype, as well as mRNA of the receptor activity-modifying proteins, which are essential for CRLR function and determine ligand specificity. A radioreceptor assay was employed for the detection of specific CGRP binding sites. Results: RT-PCR and DNA sequencing showed that LP-MNCs and IELs express CRLR. Incubation of isolated LP-MNCs with radiolabelled αCGRP revealed the existence of specific binding sites for CGRP. Conclusion: These novel data indicate that mucosal immune cells of the rat gut are a target for CGRP and provide significant evidence that CGRP functions as an immune regulator in the gut mucosa.

Lipid Analysis of Airway Epithelial Cells for Studying Respiratory Diseases

Airway epithelial cells play an important role in the pathogenesis of inflammatory lung diseases such as asthma, cystic fibrosis and COPD. Studies concerning the function of the lipid metabolism of the airway epithelium are so far based only on the detection of lipids by immunohistochemistry but quantitative analyses have not been performed. Although recent advances in mass spectrometry have allowed to identify a variety of lipid classes simultaneously in isolated tissue samples, up until now, these methods were not suitable to analyze lipids in the airway epithelium. To determine all major lipid classes in airway epithelial cells, we used an LC–MS-based approach that can easily be combined with the specific isolation procedure to obtain epithelial cells. We tested the suitability of this method with a mouse model of experimental asthma. In response to allergen challenge, perturbations in the sphingolipids were detected, which led to increased levels of ceramides. We expanded the scope of this approach analysing human bronchus samples without pathological findings of adenocarcinoma patients. For the human lung epithelium an unusual lipid class distribution was found in which ceramide was the predominant sphingolipid. In summary, we show that disease progression and lipid metabolism perturbation can be monitored in animal models and that the method can be used for the analysis of clinical samples.

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.

Suppression of adrenomedullin contributes to vascular leakage and altered epithelial repair during asthma

The anti‐inflammatory peptide, adrenomedullin (AM), and its cognate receptor are expressed in lung tissue, but its pathophysiological significance in airway inflammation is unknown.

House Dust Mite-Specific Sublingual Immunotherapy Prevents the Development of Allergic Inflammation in a Mouse Model of Experimental Asthma

Background: Evidence regarding sublingual immunotherapy (SLIT) efficacy and its good safety profile has been demonstrated with pollen and house dust mite (HDM) allergens in the treatment of airway allergies. In addition, the use of grass pollen presents a SLIT disease-modifying treatment for respiratory allergies. Objectives: The aim of this study was to demonstrate the efficacy of HDM-based SLIT in mouse models of allergic airway inflammation and to gain insights into the involved local immunological mechanisms. Methods: Balb/c mice were sensitized/challenged with Dermatophagoides farinae (Der f) extract and underwent Der f-SLIT in prophylactic and therapeutic settings. The SLIT efficacy was assessed using lung function measurements, analysis of local inflammatory responses by bronchoalveolar lavage cell differentiation and lung histology. Humoral and cellular responses were monitored by ELISA, cytokine bead array and flow cytometry analyses. Results: In a prophylactic setting, Der f-SLIT with 12 development units per dose reduced the eosinophil-dominated inflammatory response in the lung paralleled by a marked reduction in airway hyperresponsiveness. Local Th2 responses were prevented as demonstrated by significantly lower levels of IL-5 and IL-13. Additionally, SLIT-treated mice revealed a lower proportion of CD4-CD8- γδ cells and a higher frequency of CD8+CD25+IFNγ+ T cells in the lungs compared to sham-treated mice. In a therapeutic setting, Der f-SLIT also resulted in reduced inflammatory responses in the lung. Conclusion: The efficacy of Der f-SLIT was demonstrated in prophylactic and therapeutic conditions using experimental mouse models of HDM-induced airway inflammation. A potential role of a so far underestimated lymphocyte subpopulation was also indicated.

Influenza-derived peptides cross-react with allergens and provide asthma protection

Background The hygiene hypothesis is the leading concept to explain the current asthma epidemic, which is built on the observation that a lack of bacterial contact early in life induces allergic TH2 immune responses. Objective Because little is known about the contribution of respiratory tract viruses in this context, we evaluated the effect of prior influenza infection on the development of allergic asthma. Methods Mice were infected with influenza and, once recovered, subjected to an ovalbumin‐ or house dust mite–induced experimental asthma protocol. Influenza‐polarized effector memory T (Tem) cells were transferred adoptively to allergen‐sensitized animals before allergen challenge. A comprehensive in silico analysis assessed homologies between virus‐ and allergen‐derived proteins. Influenza‐polarized Tem cells were stimulated ex vivo with candidate peptides. Mice were immunized with a pool of virus‐derived T‐cell epitopes. Results In 2 murine models we found a long‐lasting preventive effect against experimental asthma features. Protection could be attributed about equally to CD4+ and CD8+ Tem cells from influenza‐infected mice. An in silico bioinformatic analysis identified 4 influenza‐ and 3 allergen‐derived MHC class I and MHC class II candidate T‐cell epitopes with potential antigen‐specific cross‐reactivity between influenza and allergens. Lymphocytes from influenza‐infected mice produced IFN‐&ggr; and IL‐2 but not IL‐5 on stimulation with the aforementioned peptides. Immunization with a mixture of the influenza peptides conferred asthma protection, and peptide‐immunized mice transferred protection through CD4+ and CD8+ Tem cells. Conclusion For the first time, our results illustrate heterologous immunity of virus‐infected animals toward allergens. This finding extends the original hygiene hypothesis. Graphical abstract Figure. No Caption available.

IL-17 and TNF-α Are Key Mediators of Moraxella catarrhalis Triggered Exacerbation of Allergic Airway Inflammation

Alterations of the airway microbiome are often associated with pulmonary diseases. For example, detection of the bacterial pathogen Moraxella catarrhalis in the upper airways is linked with an increased risk to develop or exacerbate asthma. However, the mechanisms by which M. catarrhalis augments allergic airway inflammation (AAI) remain unclear. We here characterized the cellular and soluble mediators of M. catarrhalis triggered excacerbation of AAI in wt and IL-17 deficient as well as in animals treated with TNF-α and IL-6 neutralizing antibodies. We compared the type of inflammatory response in M. catarrhalis infected, house dust mite (HDM)-allergic and animals infected with M. catarrhalis at different time points of HDM sensitization. We found that airway infection of mice with M. catarrhalis triggers a strong inflammatory response with massive neutrophilic infiltrates, high amounts of IL-6 and TNF-α and moderate levels of CD4+ T-cell-derived IFN-γ and IL-17. If bacterial infection occurred during HDM allergen sensitization, the allergic airway response was exacerbated, particularly by the expansion of Th17 cells and increased TNF-α levels. Neutralization of IL-17 or TNF-α but not IL-6 resulted in accelerated clearance of M. catarrhalis and effectively prevented infection-induced exacerbation of AAI. Taken together, our data demonstrate an essential role for TNF-α and IL-17 in infection-triggered exacerbation of AAI.