Redox Regulation of m6A Methyltransferase METTL3 in β-Cells Controls the Innate Immune Response in Type 1 Diabetes
Year: 2024
Type: article
Abstract: Over-activation of the innate immune system, leading to autoimmunity, has been shown to be one key element responsible for the development of type 1 diabetes (T1D). The purpose of this study was to determine the role of a common messenger RNA methylator, N6-methyladenosine (m6A), regulated by the enzyme methyltransferase 3 (METTL3), during the onset of T1D by modifying antiviral innate immune responses, specifically 2′−5′-oligoadenylate synthetase (OAS) gene expression, within insulin-producing pancreatic β-cells.This study looked to examine the mediators of pancreatic β-cell responses to innate immunity. As such, no human subjects were involved. Instead, the use of human-derived islet cells, human β-cell line (EndoC-βH1), and a nonobese diabetic (NOD) mouse model were used.Human islets derived from T1D patients and the EndoC-βH1 cell line were primarily used in this study. Cells were treated with cytokines ,including IL1β, IFN-α, IFN-γ or in combination for 24 hours, followed by RNA and protein isolation. For reactive oxidative species (ROS) experiments, cells were treated with H2O2 alone or with NAC, followed by protein isolation. For knock-down experiments, cells were transfected with siRNA for targeted messenger RNAs. For mice experiments, nonobese diabetic female mice were used and considered diabetic if blood glucose levels exceeded 250 mg dl−1. Islet cells were harvested and sorted using FACS for immune cell profiling. For in vivo METTL3 overexpression, mice were injected with adeno-associated virus serotype 8 and monitored for 20 weeks. Finally, RNA and m6A-seq data were analyzed for differential gene expression and methylation using various bioinformatics tools.METTL3, an enzyme involved in methylation of mRNA by m6A, is upregulated at T1D onset but declines in both mice and human-isolated pancreatic β-cells as the disease progresses. In addition, stimulation of these cells with innate immune mediators IL1β and IFN-α induced upregulation of METTL3. RNA sequencing and m6A methylome analysis of islets from 15 human donors stimulated with IL1β and IFN-α revealed both upregulation and hypermethylation of numerous antiviral innate immune system pathways, including adenosine deaminase acting on RNA 1 and OAS. Additionally, β-cells with METTL3 knockdown treated with IL1β and IFN-α had increased OAS protein levels. Treatment with a transcription inhibitor (actinomycin D) also led to increased mRNA stability of OAS genes. Importantly, overexpression of METTL3 in mice led to decreased immune infiltration of islets and decreased insulitis scores compared with control, suggesting that prolonged upregulation of METTL3 protects β-cells and delays T1D onset. ROS-induced oxidative stress led to downregulation of METTL3. Finally, post-translational S-nitrosylation of METTL3 controlled its enzymatic activity, decreasing OAS production via mRNA decay.The study suggests that m6A-dependent mRNA methylation via METTL3 acts as a protective mechanism in pancreatic β-cells, controlling excessive OAS-induced antiviral innate immune responses at the onset of T1D in both mice and human β-cells. This protective mechanism is dependent on β-cell redox state, the balance between ROS and antioxidants. Increased m6A led to faster decay of T1D-promoting OAS mRNA in β-cells. Additionally, S-nitrosylation is identified as a new mechanism that can upregulate METTL3 protein function and increase OAS mRNA decay, delaying T1D disease progression.T1D is a chronic autoimmune disease that affects ∼1 in 400 pediatric patients under 20 years of age. The development of T1D has been shown to be multifactorial, but viral infections with resulting induction of innate immunity has been shown to be a main contributor. In this study, the authors show the convergence of innate immunity, oxidative stress, and T1D onset via mRNA methylation and cysteine oxidative modification. This study allowed for a clearer understanding of the immune events involved in the onset of T1D, including a novel mechanism that protects β-cells from destruction. Modifying mRNA and controlling enzymatic activity may offer new T1D treatment modalities in the future.
Source: PEDIATRICS
Authors: Isabel Vallecillo-Viejo, Stephanie Kubala
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4404924469