Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study.
Abstract: Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses. Proteins from () were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses' sera (n=5 per group). Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics' sera on four but higher binding with healthy horses' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA. Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.
Copyright © 2023 Schnabel, Jentsch, Lübke, Kaiser-Thom, Gerber, Vrtala, Huang, Rhyner, Wagner, Hoffmann and Volke.
Publication Date: 2023-12-15 PubMed ID: 38162673PubMed Central: PMC10754955DOI: 10.3389/fimmu.2023.1293684Google Scholar: Lookup
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Summary
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Overview
- This study investigated specific protein antigens that trigger immune responses in severe equine asthma (SEA), focusing on the types of antibodies that bind to these antigens.
- Using immunoproteomics, the researchers identified increased serum IgG3/5 antibody binding to proteins from dust mites and yeast in horses with SEA compared to healthy horses, suggesting these antigens contribute to the disease’s immune response.
Background and Significance
- Severe equine asthma (SEA) is a chronic respiratory condition in horses characterized by heightened sensitivity to agents found in hay dust.
- SEA shares similarities with severe neutrophilic asthma in humans, especially in immune system involvement and inflammation types.
- Previous knowledge suggested molds and mites as possible antigen sources stimulating SEA but comprehensive identification of these antigens was lacking.
- Immunoproteomics enables detailed analysis of antibody-antigen interactions, allowing identification of specific proteins that provoke immune responses.
- Understanding the antigens involved and the antibody types that bind to them can aid in understanding disease mechanisms and potential diagnostics or treatments.
Methodology
- Protein samples were prepared from a mixed culture associated with SEA (including potential allergens from mites and yeast) and separated by two-dimensional gel electrophoresis.
- Resulting gels showed approximately 440 protein spots representing different proteins or isoforms.
- Immunoblotting (specifically 2D immunoblots) was performed: serum from 5 SEA-affected and 5 healthy horses was applied to detect antibodies binding to specific protein spots.
- Antibody detection focused on:
- Pan-Ig (all immunoglobulins)
- IgG isotypes linked to type-2 immune responses: IgG3/5
- IgG isotypes linked to type-1 immune responses: IgG4/7
- Differences in antibody binding between groups were identified, with 30 spots showing statistically significant differences.
- Subsequent extraction and mass spectrometry (liquid chromatography MS) identified proteins at these spots, revealing allergens such as Der p proteins from mites and several yeast proteins.
- Eleven antigen candidates were selected for further testing using ELISA assays on serum samples, validating the binding differences for five proteins.
Key Findings
- Ig binding to several antigens was increased in SEA horses compared to healthy controls:
- Pan-Ig was increased on 4 spots
- IgG3/5 (type-2 associated antibodies) showed increased binding on 18 spots – the most prominent difference
- IgG4/7 (type-1 associated antibodies) showed mixed results with smaller differences
- Important allergen proteins identified included:
- Mite allergens Der p 1 and Der p 11, Group 15 allergens, and myosin heavy chain
- Yeast proteins such as enolase, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins
- ELISA confirmed higher serum antibody binding in SEA horses to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, PGK).
- This indicates that antigens from both mites and yeast provoke a stronger immune response in SEA-affected horses.
- IgG3/5 binding increase suggests SEA may involve a stronger type-2 immunity component, which is relevant because type-2 immune responses are typically associated with allergic inflammation.
- This is the first study to suggest yeast antigens as important contributors to SEA.
Implications and Future Directions
- The identification of specific allergens in SEA can improve targeted diagnostics and may guide the development of immunotherapies or avoidance strategies.
- Yeast proteins emerging as new antigen candidates highlight the need to consider microbial components in the environment affecting SEA.
- Increased IgG3/5 response implies a skewed type-2 immune response, but further research is necessary to confirm if corresponding cellular immune responses (such as T helper 2 cell activity) support this finding.
- Future work may focus on validating these findings in larger populations, assessing causal roles of these antigens in disease progression, and exploring therapeutic interventions targeting these immune responses.
Cite This Article
APA
Schnabel CL, Jentsch MC, Lübke S, Kaiser-Thom S, Gerber V, Vrtala S, Huang HJ, Rhyner C, Wagner B, Hoffmann R, Volke D.
(2023).
Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study.
Front Immunol, 14, 1293684.
https://doi.org/10.3389/fimmu.2023.1293684 Publication
Researcher Affiliations
- Institute of Immunology, Faculty of Veterinary Medicine, and Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, Germany.
- Institute of Immunology, Faculty of Veterinary Medicine, and Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, Germany.
- Institute of Immunology, Faculty of Veterinary Medicine, and Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, Germany.
- Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
- Swiss Institute of Equine Medicine (ISME), Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
- Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
- Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
- Christine Kühne Center for Allergy, Research, and Education (CK-CARE), Davos, Switzerland.
- Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland.
- Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.
- Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, Germany.
- Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Center for Biotechnology and Biomedicine, Leipzig University, Leipzig, Germany.
MeSH Terms
- Pulmonary Disease, Chronic Obstructive
- Saccharomyces cerevisiae
- Asthma
- Antigens, Dermatophagoides
- Pyroglyphidae
- Allergens
- Immunoglobulin G
- Horses
- Immunoglobulin E
- Animals
- Humans
- Fungal Proteins
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Citations
This article has been cited 3 times.- Jentsch MC, Keilhaue A, Wagner B, Rhyner C, Lübke S, Karagulyan M, Arnold C, Lohmann KL, Schnabel CL. Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma.. Front Immunol 2024;15:1406794.
- Cao Z, Li Q, Li Y, Wu J. Identification of plasma protein markers of allergic disease risk: a mendelian randomization approach to proteomic analysis.. BMC Genomics 2024 May 22;25(1):503.
- Jentsch MC, Lübke S, Schrödl W, Volke D, Krizsan A, Hoffmann R, Kaiser-Thom S, Gerber V, Marti E, Wagner B, Schnabel CL. Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.. Front Immunol 2024;15:1347164.
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