Partial CRISPR/Cas9 IL1R1 & IFNGR1 Knock-Down Improves β-cell Survival And Function Under Cytokine-Induced Inflammation

Abstract

Background/Aims: Type 1 diabetes (T1D) is a disease characterized by the autoimmune destruction of pancreatic β cells. This destruction is mediated by lymphocytes T helper and cytotoxic, and by the action of the pro-inflammatory cytokines IL1β and IFNγ inside the islets of Langerhans. We propose a new approach to alleviate islet inflammation by targeting pro-inflammatory cytokine receptors. Our hypothesis is that the downregulation of inflammatory pathways may improve β cell survival in the context of inflammation after T1D onset. Methods: We knocked-down IL1R1 or IFNGR1 receptors in the MIN6 β-cell line by using the CRISPR/Cas9 gene editing system. Results: The knockdown efficiency was evaluated by immunostaining and ranged from 12 to 40%. Naive MIN6 or CRISPR-KnockedOut MIN6 cells were then treated with IL1β or IFNγ during 48 h at various concentrations (5 or 10 ng/mL). Cell viability of CRISPR-IFNGR1 and CRISPR-IL1R1 cell lines was improved after cytokine exposure compared to naive MIN6 (117 ± 16 vs 84 ± 19%; p=0.015 and 134 ± 20 vs 71 ± 4%; p=0.016). The assessment of insulin secretion capacities of CRISPR-IFNGR1 and CRISPR-IL1R1 cells showed higher secretion rates (1.24 ± 0.21 vs 0.35 ± 0.14 I.A.; p= 0.006 and 0.91 ± 0.22 vs 0.32 ± 0.09; p=0.014), after cytokine treatment, as compared to naive MIN6. Gene expression of the pro-apoptotic receptor Fas was decreased inside the CRISPR-MIN6 cell lines and the expression of the pro-inflammatory cytokine Il6 gene was decreased inside the CRISPR-IL1R1 cell line, as compared to MIN6 controls. Similarly, gene expression of ER stress markers Atf4 and Chop decreased inside the CRISPR-IL1R1 and CRISPR-IFNGR1 cell lines, respectively, as compared to controls. Our results show that the targeting of IL1R1 or IFNGR1 could protect pancreatic β cells from the inflammatory attack found in T1D by decreasing apoptosis, inflammation and ER stress. Conclusion: Our results show the feasibility of the CRISPR technique to protect β cells are encouraging and require the development of the three-cytokine receptor (IL1R1, IFNGR1 and TNFR1) knockdown to fully address the potential of this system to be translated into clinical research protocols. The possibility of a translational perspective of our knockdown system is suggested by the ongoing clinical trial using the CRISPR/Cas9 system to evaluate the safety of PD-1 knockout engineered T cells in treating metastatic non-small cell lung cancer (NCT02793856)