Sage, Sophie Elena (2023). Analysis of bronchoalveolar lavage transcriptome profiles of asthmatic horses by single-cell mRNA sequencing. (Thesis). Universität Bern, Bern
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Abstract
Severe equine asthma (SEA) is a common respiratory condition of horses, whose underlying immune mechanisms remain to be elucidated. In this thesis project, we took advantage of the recently developed single-cell mRNA (scRNA-seq) technology to investigate the immunological landscape of equine bronchoalveolar lavage fluid (BALF) cells in both health and disease. Initially, we conducted a pilot experiment involving three horses to demonstrate the feasibility of scRNA-seq on cryopreserved equine BALF samples. Although the experiment was successful, the proportion of reads aligning to the annotated equine reference transcriptome was suboptimal. To address this, we generated a custom equine BALF transcriptome using long-read sequencing, aiming to improve the quality of 3'-UTR annotation and document BALF-specific isoforms. While we identified several novel isoforms, the read mapping percentage did not improve when aligning our scRNA-seq transcripts to the custom transcriptome. By extending the 3'-UTRs of the existing reference annotation, we achieved a satisfactory read mapping percentage, enabling subsequent qualitative downstream analysis. Our scRNA-seq dataset encompassed six major cell populations: monocytes-macrophages, neutrophils, T cells, B cells and dendritic cells. Within the monocyte-macrophage and T cell groups, we identified previously uncharacterized cell subtypes. Encouraged by these findings, we applied our optimized experimental protocol and analysis pipeline to study SEA. ScRNA-seq analysis of cryopreserved BALF cells from 6 asthmatic and 5 healthy controls revealed the same major cell populations as observed in the pilot study. In addition to T cells and monocytes-macrophages, we characterized several cell subtypes within the B cell, dendritic cell and neutrophil populations. Differential gene expression analysis revealed a strong T helper (Th)17 signature in SEA, primarily driven by monocytes-macrophages and T cells. Notably, BALF from SEA horses was enriched in B cells, with a lower proportion of activated plasma cells. Neutrophils in the SEA group displayed increased migratory capacity and a heightened propensity to form neutrophil extracellular traps (NETs). An intriguing finding in both scRNA-seq experiments was the detection of a dual monocyte-lymphocyte population, potentially representing genuine cellular complexes engaged in an immunological synapse. In summary, this thesis project represents pioneering work employing scRNA-seq in the field of equine pulmonology. Our findings support a predominant Th17 immune pathway in SEA, necessitating further investigation to improve diagnostic tools and therapeutic management of severely asthmatic horses.
| Item Type: | Thesis |
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| Dissertation Type: | Cumulative |
| Date of Defense: | 22 September 2023 |
| Subjects: | 500 Science > 590 Animals (Zoology) 600 Technology > 630 Agriculture |
| Institute / Center: | 05 Veterinary Medicine > Department of Clinical Veterinary Medicine (DKV) > Equine Clinic |
| Depositing User: | Hammer Igor |
| Date Deposited: | 03 Oct 2023 15:33 |
| Last Modified: | 22 Sep 2024 22:25 |
| URI: | https://boristheses.unibe.ch/id/eprint/4565 |
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