Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study

Abstract

IntroductionSevere 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.MethodsProteins from Dermatophagoides pteronyssinus (Der p) 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).ResultsIg 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 Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p 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.DiscussionAntigens 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.

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma

“…However, this polarization was not very clear regarding the total serum Ig isotype contents and was not reflected in Aspergillus- binding Ig in MEA. Additionally, the previously reported serum IgG3/5 bias in SEA for A. fumigatus antigen binding on immunoblots ( 24 , 70 ) could not be corroborated in this study, which included a larger and different cohort of horses. The previous study on horses with SEA compared to HE analyzed serum Ig binding to A. fumigatus on immunoblots ( 24 ) and different methods likely contribute to the different results compared to ELISA analyses here.…”

“…With our analysis focused on one mold species, it is not clear if A. fumigatus particularly or several fungi have provoked the Ig binding responses to the antigens observed. Increased serum Ig binding to yeast antigens was also demonstrated in SEA before ( 70 ). Such shared or homologous antigens may pose a challenge to identify the specificity of Ig responses in EA but could also constitute an opportunity for broadly applicable serological diagnostic tools or immune-therapeutic approaches in the future.…”

Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma

“…With this, these proteins could support IgG3/5 binding predominance and the indication of a shift to specific type 2 responses in SEA. Globally, this was not indicated as total serum IgG3/5 was similar between healthy and asthmatic horse sera and the direct comparison of T cell responses was not possible in the present study ( 37 ).…”

“…These serum samples were stored at -80°C for 5 years and kept at 4°C during the experimental series, over three months. In addition, aliquots of the same samples were used to detect antibody binding against mite proteins on immunoblots ( 37 ).…”

Identification of plasma protein markers of allergic disease risk: a mendelian randomization approach to proteomic analysis

“…The human plasma proteome participates in inter-tissue communications via metabolic, signaling, and physiological pathways and includes potential drug targets [ 9 – 13 ]. The link between levels of specific plasma proteins and allergic disease risk has been reported previously but residual confounders and reverse causation mean that observational studies do not demonstrate causality [ 14 , 15 ]. Mendelian randomization (MR) uses genetic variation as an instrumental variable (IV) to explore the causal effect of exposure on outcomes.…”