Understanding type 1 diabetes development

: current risk factors and novel targets

Abstract

Type 1 diabetes (T1DM) is a chronic autoimmune pathology driven by loss of pancreatic beta cells as a consequence of T-cell mediated inflammatory responses and/or dedifferentiation of beta cells. Numerous genetic markers have been identified that carry risk for T1D development, worse secondary complications, and poor beta cell function. However, the mechanism by which risk is conferred is poorly understood. This thesis aimed to unravel the molecular basis for T1D risk by examining candidate markers identified in microarray and Genome Wide Association Studies (GWAS) and to identify and explore novel pathways of interest in beta cell function and survival.

Our initial focus for T1D risk wasTNF Alpha Induced Protein 3 (TNFAIP3/A20), a negative regulator of the NF-kB pathway. TNFAIP3 has been widely cited as the most highly regulated antiapoptotic gene in the pancreatic islet and a risk locus for T1D development (in addition to other autoimmune conditions). We have shown that silencing of TNFAIP3 in human pancreatic islets led to increased levels of apoptosis, reduced insulin secretion in response to glucose stimulation and a reduction in the expression of beta cell markers and transcription factors. These effects were driven by cytokine production and found to be partly, although not exclusively, dependent on the P65 subunit of NF-kB. We next focused on two variants- rs10517086 located at chr4:26083889 and rs1534422 located at chr2:12500615 - identified in The Environmental Determinants of Diabetes in the Young (TEDDY) study as being associated with T1D risk, worse outcomes for residual beta cell function and strong correlations with islet autoantibodies. Using clinical data from the DARE (Diabetes Alliance for Research in England) study, we confirmed the association of rs10517086, but not rs1534422, with low c-peptide concentrations in T1D. The variants were introduced into the BRIN-BD11 beta cell line using CRISPR/Cas9 and consistently showed effects on glucose-stimulated insulin secretion, gene expression and cytokine secretion that were similar to the impact of TNFAIP3 deficiency. However, there was one notable exception in gene expression: HNF1A expression was significantly upregulated in the presence of either variant, and we did not find a role for pro -inflammatory cytokines in this process. Subsequent silencing ofHNF1A in variant-expressing cells largely normalised gene expression and we propose that these variants inflict a double insult on the beta cell through increased cytokine production and excessive upregulation of HNF1A.

These data provide insights into the mechanisms by which T1D risk occurswith low levels of TNFAIP3 or the presence of rs10517086 and rs1534422, which may be clinically useful in the prediction of outcomes in T1D. Although these markers have prognostic value, they are not readily targetable. Pathway analysis on microarray data from TNFAIP3 deficient beta cells identified tumor necrosis factor-like weak inducer of apoptosis (TWEAK) signalling as novel pathway that could be targeted for therapy. TWEAK signalling appears to be largely protective in the beta cell since TWEAK silencing reduced glucose-stimulated insulin secretion and expression of beta cell markers and transcription factors. Again, there was one notable exception inNGN3,which was significantly upregulated upon TWEAK silencing. Overexpression of NGN3 is associated with hormone negative beta cells and shunting towards an alpha cell phenotype early in embryogenesis. We propose that novel therapies targeting TWEAK and its receptors may prove useful in the context of diabetes and other chronic inflammatory pathologies.

Overall, the work presented in this thesis sheds light on how established risk factors for T1D development act within the beta cell and has identified a novel and targetable pathway that regulates beta cell function warranting further attention.

Date of Award	May 2022
Original language	English
Sponsors	European Union Regional Development Fund (ERDF) EU Sustainable Competitiveness Programme for N. Ireland, Northern Ireland Public Health Agency (HSC R&D) & Department for the Economy
Supervisor	Sarah Atkinson (Supervisor) & Catriona Kelly (Supervisor)