Analysis of bronchoalveolar lavage transcriptome profiles of asthmatic horses by single-cell mRNA sequencing
BORIS DOI
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.
Date of Publication
2023
Year of graduation
2023
Theses Type
dissertation
Subject(s)
Language(s)
en
Author(s)
Faculty/Graduate School
Institute
Access(Rights)
open.access
Primary OA Publication
true