Speaker
Description
Diabetes mellitus is a chronic disease characterised by high blood glucose, primarily due to the body’s inability to produce enough insulin or the body’s failure to use the produced insulin. High blood glucose has been reported to suppress the type 1 interferon (IFN-1) response, an innate immune response pathway that prevents infections and cancers. However, the effect of high glucose on the nuclear translocation of IFN-stimulated gene factor 3 (ISGF3) complex required for IFN-1 response signalling is largely unknown. Thus, we investigated the effect of high glucose on the nuclear localisation of IFN regulatory factor 9 (IRF9), an essential component of the ISGF3 complex. Human hepatoma (HepG2) cells, surrogating hepatocytes, were treated without or with 1000 IU/ml recombinant human IFN-α and glucose (5.5, 25 and 100 mM) for 24 hours. The cells were fixed using 4% paraformaldehyde, permeabilised using 0.2% Triton X-100, non-specific binding sites blocked with 2% bovine serum albumin and labelled using rabbit anti-IRF9 antibody and Alexa Fluor 647-labelled donkey anti-rabbit antibody. The cytoplasm and nuclei were defined using Alexa Fluor 488-labelled wheat germ agglutinin and DAPI. Optically sectioned images were acquired using a Zeiss LSM 980 laser scanning confocal microscope with a 63X 1.4 NA objective lens. Image analysis was performed using a custom ImageJ and CellPose plugin to measure the Alexa Fluor 647 signal (IRF9 localisation) within each cell. There was a decrease in IRF9 nuclear localisation in the presence of glucose in a dose-dependent manner, which was statistically significant at 100 mM glucose (blood glucose in uncontrolled diabetes mellitus). Our results support previous studies and demonstrate for the first time the suppression of the type 1 interferon response during diabetic hyperglycaemia at the intracellular level.