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

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)

Nasobiliary drainage prior to surgical biliary diversion in patients with progressive familial intrahepatic cholestasis type II.

Objectives and study: Progressive familial intrahepatic cholestasis (PFIC) can cause intense pruritus refractory to medical therapy. Surgical biliary diversion techniques, including partial internal biliary diversion (PIBD), have been developed over the years to relieve pruritus without requiring liver transplantation. Despite the efficacy and safety of PIBD procedure, some patients do not respond to surgery and there is currently no possibility to predict pruritus response according to genotype or other clinical parameters. Our aim was to evaluate nasobiliary drainage (NBD) as a method to predict pruritus response to PIBD, in order to avoid unnecessary surgery. Methods: We present two PFIC 2 patients who underwent two and three transient endoscopic NBD prior to PIBD surgery, which was performed at the age of 25 and 28 years old respectively. Both patients suffered from invalidating pruritus refractory to medical therapy. Liver transplantation was not performed since both patients had normal liver function and normal liver histology (patient 1) or only very mild fibrosis (patient 2). Pruritus was assessed according to the following score: 0 = none, 1 = rubbing or mild scratching when undistracted, 2 = active scratching without evident skin abrasions, 3 = abrasions evident, 4 = cutaneous mutilation and scarring evident, impaired quality of life. Results: Both patients repeatedly responded to NBD (pruritus score 4 to 0-1) and had complete pruritus resolution after subsequent PIBD (score 4 to 0). Serum total biliary acids (TBA) decreased from 282 µmol/L and 314.4 µmol/L before PIBD to 40.7 µmol/L and 220.9 µmol/L after PIBD respectively (normal TBA levels < 10 µmol/L). Follow-up duration after surgery was seven years for patient 1 and one month for patient 2. Pruritus did not recur after PIBD in our patients despite important variations in TBA levels during follow-up. Mild post-endoscopic biological pancreatitis occurred in 1/5 NBD procedures, and resolved spontaneously. The only adverse effect observed within 7 years post-PIBD was very mild transient osmotic diarrhoea, easily treated with cholestyramine. Conclusion: Our data suggest that NBD is a safe and effective way to predict pruritus response before performing a permanent biliary diversion surgery in PFIC patients

Phage therapy to allow liver transplantation in a toddler infected by an extensively-drug resistant Pseudomonas aeruginosa

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