Mucosal Immunology and Diagnostic
Main Emphasis
Our Group focusses its research on molecular and cellular structures which may serve as markers for the cause, initiation, course and severity of mucosa-associated diseases, especially in the fields of asthma/COPD and allergic disorders. Our goal is to discover such markers, to characterize and to survey them for diagnostic, prognostic and therapeutic purposes.
The mucous membranes constitute the largest surface of the human body that is in contact with the environment and nearly all pathogenic substances and microorganisms invade the body via this route. Nevertheless, the immune defense mechanisms at this site are still poorly investigated. One of the central unsolved questions in mucosal immunology is how mucosal immune reactions are controlled. In contrast to the systemic immune system which may eliminate all foreign antigens indiscriminately, the situation at the mucous membranes is more complex. On one hand benign foreign antigens such as food and the oral, intestinal and genital flora must be tolerated. On the other hand bacterial toxins and hostile microorganisms must be identified and their uptake or active intrusion must be prevented. Misclassification of benign antigens as hostile or of maleficient ones as friendly can result in severe disorders such as chronic inflammatory intestinal or pulmonary diseases, food allergies, autoimmune diseases or in attenuated defense against mucosal pathogens.
Elucidating these antigen classification mechanisms and gaining insight into the pathogenesis of disorders that are caused by antigen misclassification are the main goals of our efforts in basic research.
In accordance with our institution's commitment to translational research we not only aim to understand mucosal immune regulation. We also want to exploit these mechanisms for the good of the patients, that is, for diagnosing mucosal diseases and assessing the risk of acquiring such disorders, for the prevention and therapy of allergic asthma and food allergies or for the development of vaccines against mucosal pathogens. Manipulating a mucosal immune reaction, however, is considerably more difficult than intervening in a systemic one because the mucosal immune system is compartmented so that the immunological microenvironment at the site of antigen/allergen contact tends to govern the type, extent and strength of a mucosal immune reaction. Prophylactic and therapeutic interventions must therefore be carried out with high cell type or tissue specificity. Such a site selective drug delivery requires targeting systems. Usually such systems are target seeking bioconjugates that consist of a ligand which mediates binding to the desired biomarker and a drug molecule or a drug container like a supramolecular complex or a nanoparticle.
The development and evaluation of supramolecular and nanoparticulate targeting systems for the delivery of drugs and diagnostics to certain mucosal sites is a cornerstone of our efforts in applied research.
Uptake and classification of antigens at mucosal surfaces
The prevalence of allergies and allergic asthma has been on the rise for decades in affluent countries so that about 25% of the population are affected by those diseases by now. Allergies and asthma are T-helper-2 (Th2)-cell driven immune defense reactions against food and environmental antigens. Generally, such antigens are either tolerated by the host’s immune system (immune tolerance) or completely ignored (immune ignorance). Immune tolerance is thought to be induced at sites where most antigens enter the body, i.e. the skin and the mucosa, particularly at the gastrointestinal mucosa. In our project we want to investigate why and how tolerance or ignorance founder for certain apathogenic antigens and allergens and whether this failure can be ascribed to erroneous mucosal antigen classification mechanisms.
Analysis of antigen stability in mucosal secretions
The absence of an immune defense reaction as well as the induction of an adverse immune reaction may be due to an early degradation of MHC epitopes in the digestive tract or a formation of certain MHC epitopes due to individual predisposition. Our in-house developed experimental repertoire enables us to determine stabilities of proteinaceous as well as of poly- or oligopeptidic antigens in mucosal secretions under in vivo-like conditions. Hence, we can determine and compare half-lifes and fragmentation patterns of antigens in mucosal secretions. This way, we want to address the question whether intestinal epitope stability affects the decision on ignorance, tolerance or immunity.
Development of ligands for cell-type specific targeting of drugs and molecular imaging agents to distinct mucosal sites
The mucosa is safeguarded by various protective means such as mucus, immunoglobulin A, a glycocalyx and digestive enzymes. Thus, mucosally administered drugs or molecular imaging agents must first overcome these obstacles in order to reach their target cells and exert their desired function. Mucus and immunoglobulin A act as traps by crosslinking, engulfing or adsorbing hazardous agents. The glycocalyx presents a porous filter that prevents pathogens or particulate noxae to access the cell membranes. And digestive enzymes directly attack hazardous invaders aiming to destroy them. For mastering these hurdles mucosal targeting systems should be "mucophobic" and free of immunogenic structures in order to escape the mucus and not to induce an immunoglobulin trap. They should be scalable in size in order to penetrate the glyxocalyx filter or to exploit it as a barrier. And finally they should be degradation resistant in order to escape premature destruction. Classical targeting ligands such as monoclonal antibodies, lectins or microbial toxoids do not meet these requirements satisfactorily as they are susceptible to proteolysis, immunogenic and difficult to size. In order to realize drug targeting and molecular imaging at mucosal surfaces we are establishing a technology for the identification of peptidic or peptoid ligands in the 2 kDa range. The technology allows peptidic or peptidomimetic ligands to be tailored such that they evade the abovementioned barriers of mucosal surfaces. Once fully operational, we want to use our platform for the design of peptide-based mucosal targeting systems.
Interactions of nanoparticles with mucosal secretions and surfaces
Actual and envisioned fields of application for nanoparticles range from semiconductors over dirt repellant coatings to suntan lotions and new drugs. With such an omnipresence, involuntary exposure to engineered nanoscale particles is inevitable. This calls for exposure assessments and a thorough investigation of the medical consequences of nanoparticle incorporation. The mucosa represents the first site of contact for ingested or inhaled nanoparticles. Here, it is decided whether, in which form and via which route nanoparticles enter the body. In this project we investigate to which extent nanoparticles are modified by mucosal secretions and how nanoparticles interact with or cross the mucosal surface.
The intestinal glycocalyx as a biomarker
Apart from the mucus layer the glycocalyx – a complex coat of carbohydrates that is linked to the epithelial cell surface – represents an outer barrier of the mucosa. Displaying a height of several nanometers, this carbohydrate fleece is important for the protection of the mucosa but also for nutrient uptake. Food components as well as intestinal proteases can adsorb to and deeply penetrate into this network. Physical or chemical impacts can easily impair the glycocalyx’ integrity, which may lead to a loss of mucosal protection and impaired nutrient uptake. In this project, we want to use electron microscopic techniques in order to investigate whether – in addition to exogenous factors – pathological factors such as cancer or ischemia can lead to alterations in the glycocalyx.
The projects are supported in part by grants from the Federal Ministry of Education and Science, the Volkswagen Foundation and the Chiesi Group
Deutsches Zentrum für Lungenforschung (DZL)
- Projektbereiche "Asthma und Allergie" und "COPD"
BMBF-Research Focus "Photonik-Forschung Deutschland"
- Project " On-site exacerbation diagnostics for asthma & COPD: samples, models, analytes & capture systems " in conjunction with EXASENS
Förderinitiative "Experiment!" der VolkswagenStiftung
- A Hitch-Hiker's Ride to Immunity: Antigen backpacks as vaccines soliciting their own transportation by the mucosal immune system to induce protection against pathogens and allergens
Immunological methods:
- Experimental immunizations
- Extraction of immunoglobulins from secretions
- ELISA
- Epitope mapping
- Immunohistochemistry
- Production of monoclonal antibodies
Cell biological & histological methods:
- Cell culture
- In vivo and ex vivo transport studies at mucosal tissues
- Light and electron microscopy
Biochemical & chemical methods:
- Synthesis of crosslinkers and tags
- Generation of protein and peptide conjugates
- Solid phase synthesis of peptide libraries
- Immobilization of proteins and peptides on solid surfaces
- Screening of synthetic peptide libraries
- Preparation of mucosal secretions and secreted proteins
- Proteolysis assays
Bioinformatic methods:
- Evolutionary optimization of peptide ligands
Molecular biological methods:
- Molecular cloning in E. coli
- Expression and purification of recombinant proteins
- Screening of microbial peptide libraries
2024
Röckendorf, N, Ramaker, K, Gaede, K, Tappertzhofen, K, Lunding, L, Wegmann, M, Horbert, P, Weber, K & Frey, A 2024, 'Parallel detection of multiple biomarkers in a point-of-care-competent device for the prediction of exacerbations in chronic inflammatory lung disease', Scientific Reports, Jg. 14, Nr. 1, S. 12830. https://doi.org/10.1038/s41598-024-62784-8
2022
Kozlowski, PA, Mantis, NJ & Frey, A 2022, 'Editorial: Mucosal Vaccination: Strategies to Induce and Evaluate Mucosal Immunity', FRONTIERS IN IMMUNOLOGY, Jg. 13, S. 905150. https://doi.org/10.3389/fimmu.2022.905150
Röckendorf, N, Ramaker, K & Frey, A 2022, Artificial Evolutionary Optimization Process to Improve the Functionality of Cell Penetrating Peptides. in Methods in Molecular Biology . Bd. 2383, Methods in Molecular Biology, S. 45-61. https://doi.org/10.1007/978-1-0716-1752-6_3
Roeckendorf, N, Nehls, C & Gutsmann, T 2022, 'Design of Membrane Active Peptides Considering Multi-Objective Optimization for Biomedical Application', Membranes, Jg. 12, Nr. 2, 12020180. https://doi.org/10.3390/membranes12020180
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