Using single-cell RNA sequencing to elucidate the biological basis of IBD and response to therapy

Abstract

Inflammatory bowel disease (IBD) is an umbrella term for disorders involving chronic inflammation of the gastrointestinal tract, with over 6 million cases reported worldwide. 320 IBD risk loci have been identified for this disease, however, the role of many of these risk loci in the pathogenesis of IBD is largely unknown. While there is no cure for IBD, biologic treatments including anti-TNF have improved the quality of life for many IBD patients. Unfortunately, many patients will fail to respond or lose response to anti-TNF treatment over time. In this thesis, single-cell RNA sequencing (scRNA-seq) was utilised to understand the biological basis of IBD and response to anti-TNF therapy. The first project utilised peripheral blood mononuclear cells (PBMCs) to investigate the effects of IBD-associated genetic variants in an ex vivo setting, while the second project utilised a co-culture model to understand the immune-cell response to anti-TNF therapy. In the first research chapter of this thesis, scRNA-seq was performed on resting PBMCs from healthy donors recruited with carriage of risk variants in the adenylate cyclase (ADCY) genes ADCY3 and ADCY7, to understand the role of these IBD risk variants in immune cells. A trans-eQTL analysis performed in the immune cells of donors with carriage of the rare coding ADCY7 variant rs78534766 identified 65 unique trans-genes and enriched pathways related to cytokine signalling and the citric acid cycle. Colocalisation analysis using updated CD genome-wide association summary statistics from our lab showed that the disease-associated variant likely decreased the expression of ADCY3 in immune cell subsets including CD4+ T cells. Only a single trans-eQTL effect was detected for the ADCY3 CD-associated variant, suggesting the study was not appropriately powered to detect trans effects of modest effect regulatory variants. The second and third research chapters utilise a co-culture model of CD4+ memory T cells and macrophages to investigate the immune consequences of anti-TNF treatment. Cells were treated with infliximab (anti-TNF), or left untreated and scRNA-seq performed at baseline before co-culture, and 40 hours and five days after co-culture. Firstly, Chapter 3 focused on the annotation of CD4+ memory T cells in this dataset. Overall, 10 unique T cell populations were identified over the three time points. T cells co-cultured in the control condition were also compared to another CD4+ T cell dataset activated in the absence of antigen-presenting cells, to better understand the transcriptional variation between these two cellular models which are often used to explore disease biology. Finally, the third research chapter focuses on the effect of anti-TNF on T cells and macrophages. Effects of anti-TNF on the composition of T cells and macrophages revealed that drug-treated cells were depleted in several proliferating T cell populations. This was supported by flow cytometry of CD4+ memory T cells showing reduced proliferation in comparison to control cells re-stimulated in the absence of drug. Finally, differential gene expression analysis in macrophages revealed upregulation of MRC1 and downregulation of IDO1, suggesting an anti-inflammatory phenotype consistent with previous findings. Gene set enrichment analysis identified pathways downregulated in several T cell subsets related to cytokine signalling including TNF receptor 2 signalling and interleukin-1 (IL-1) signalling, and pathways related to downstream TCR signalling, antigen presentation and cell cycle. The use of scRNA-seq in this thesis has illuminated the roles of T cells and macrophages in modulating responses to treatment, indicating a shift towards an anti-inflammatory phenotype in macrophages and identifying pathways in T cells that enhance our understanding of how IBD therapies may perturb immune responses. Investigation into the risk variant ADCY7 has uncovered potential insights into its role in the immune response, laying the groundwork for subsequent research to delve deeper into its mechanisms of action in IBD. Continued research, including functional studies such as CRISPR-Cas9 gene editing and cytokine profiling, will further our understanding of how anti-TNF therapies affect the immune response and the role of these genetic variants in immune-mediated diseases.