Chloride Channel Regulators, Calcium Activated (CLCA) Proteins in Equine Asthma - Mechanistic Relevance as Immunomodulators and Potential Mucus Biomarkers?

• Applicant: Privatdozent Dr. Lars Mundhenk
• Subject Area: Veterinary Medical Science
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 508761433

Equine asthma is among the most important pulmonary diseases of horses with many current challenges regarding therapy and diagnostics. In addition to the incomplete knowledge on its development and sustainable therapeutic options of this distressing disease, there is still a lack of suitable biomarkers for target-oriented diagnostics. The project proposed here aims to link research resources in a networked, interdisciplinary approach with a cross-regional strategy to provide impetus for improved treatment of this common disease. Our preliminary work and the contributions of other research groups suggest that members of the chloride channel regulator, calcium-activated (CLCA) protein family may play a central role in the transmigration of neutrophil granulocytes into the airways in humans with asthma and corresponding mouse models via stimulation of alveolar macrophages. Furthermore, some representatives are overexpressed in airway diseases with mucus overproduction. Except for one representative, CLCA1, which is secreted by airway mucin-producing cells, the CLCA family is unknown in the horse to date. Translation of findings from other species is impeded by several inter-species differences known for this family. The goals of this project include (1) the characterization of the CLCA proteins in the horse and their occurrence in the airways, (2) deciphering their role as signaling molecules for neutrophil influx, (3) the identification of CLCA1 interaction partners and regulatory networks in the mucus that play a role in immune cell influx, and (4) the exploration of their potential as biomarkers in the mucus. First, we aim to identify CLCA members expressed in the airways of healthy and asthmatic horses. Equine alveolar macrophages will be stimulated with recombinant CLCA proteins or their functional domains to determine their response using global RNA and protein technologies and to uncover protein structure-activity relationships. Furthermore, we will test whether the immunostimulatory response of macrophages results in neutrophil influx in vitro. For the remaining objectives, proteomics technologies will be used to comparatively quantify the total proteome specifically in the mucus of bronchoalveolar lavage fluid between healthy horses and those with equine asthma. Here, CLCA proteins and other proteins will be differentially quantified in the landscaping approach in the context of the clinical picture to assess their biomarker potential and discover protein-protein interaction networks. The results will help to (1) better understand the development of neutrophilic bronchiolitis in equine asthma, (2) identify suitable mucus proteins as biomarkers for improved diagnostics, and (3) potentially explore new therapeutic approaches.

• DFG Programme: Research Grants