Abstract
Heritable variation in immune system function alters our risks for inflammatory bowel disease (IBD) and colorectal cancers (CRC). Antagonists of interleukin 6 (IL-6) are potential therapies in both settings, and responsiveness to treatments may vary between individuals due to genetic differences. IL-6 is predominantly produced by macrophages (Mφ) and alters their polarisation: Pro-inflammatory (M1) Mφ produce high IL-6, whilst anti-inflammatory alternatively-activated Mφ (M2) are promoted by IL-6. A single nucleotide polymorphism (SNP) called rs1800795 within the IL6 gene promoter alters our IL-6 expression, and this thesis proposes that rs1800795 alters IBD and CRC risks by altering Mφ polarisation and in turn, alters responsiveness to IL-6 blockade.
To enable testing of this, CRISPR-edited cell lines representing both homozygous versions of the SNP were generated and differences in IL-6 expression and Mφ polarisation were measured. In a macrophage cell line, this showed increased M1 polarisation and IL-6 expression for rs1800795:CC macrophages, and in a cancer cell line, the rs1800795 G allele led to increased IL-6 expression.
Genetically modified mice were used to model the human rs1800795 variant. When colitis was induced chemically in these animals, those with a rs1800795:CC genotype developed more severe IBD symptoms than those with rs1800795:GG, including greater weight loss, increased incidence of bleeding, together with elevated expression of Il6 and other inflammation genes in colonic immune cells (CD45+). In contrast, ex vivo differentiation of bone marrow cells from rs1800795:GG mice showed an increased Il6 expression compared to rs1800795:CC mice, which is proposed to favour M2 polarisation.
When IL-6 trans-signalling was therapeutically blocked with an inhibitor called sgp130Fc, mice with rs1800795:CC showed a better response than their rs1800795:GG siblings. Treatment with a Janus kinase (JAK) inhibitor showed favourable response in rs1800795:GG mice. Single-cell RNA sequencing of colonic CD45+ cells supported these findings, as genes differentially regulated included those contributing to epithelial barrier integrity, the activity of Treg and Th17 cells, IL-6 signalling, and JAK/STAT activation.
These results substantiate the rs1800795 SNP as a potential biomarker for personalising and facilitating the use of emerging drug treatments, and demonstrate mice with the introduced rs1800795 SNP can be used as preclinical model for further research.