Neutrophil-enriched circulating miRNAs driving NETosis identify individuals at risk for Stage 3 Type 1 Diabetes

Type 1 diabetes (T1D), a chronic autoimmune disease characterized by immune-mediated destruction of pancreatic β-cells, progresses from islet autoantibody (AAb) positivity with normoglycaemia (pre-Stage 1/Stage 1) to dysglycaemia (Stage 2) and overt hyperglycaemia (Stage 3). While AAbs are essential for identifying at-risk individuals, their ability to accurately predict the timing of progression to Stage 3 remains limited. Here, we aimed to identify an integrated biochemical–miRNA signature capable of predicting progression to clinical T1D within a defined timeframe, while investigating its link with innate immune activation, particularly neutrophils. Plasma samples were collected at baseline from n=65 AAb-positive individuals (age: 15.3 ± 11.1 years; follow-up: 18.5 ± 10.2 months) enrolled in the INNODIA consortium at pre-Stage 1, Stage 1, or Stage 2 and prospectively followed for up to 4 years. Participants were stratified into n=21 progressors (T1D-P) and n=44 non-progressors (T1D-NP). Circulating miRNAs were profiled by small RNA sequencing, and differential expression analysis was performed using DESeq2. A LASSO regression model integrated miRNA expression with biochemical parameters. Cell-type enrichment analyses were conducted using isomiRdb and miR-Blood to investigate the cellular origin of candidate miRNAs. Functional experiments in primary neutrophils were performed to assess miRNA dynamics, release, and downstream effects on inflammatory pathways and NETosis. Nineteen circulating miRNAs were differentially expressed between T1D-P and T1D-NP (adjusted p < 0.05) and validated by ddPCR (p < 0.05). The LASSO-derived predictive signature included HbA1c, OGTT glucose AUC, OGTT C-peptide AUC, and circulating miR-1294, miR-628-3p, miR-425-3p, and miR-328-3p, improving the ROC AUC from 0.78 (clinical model) to 0.95 (combined model). Enrichment analyses identified miR-425-3p and miR-628-3p as neutrophil-enriched miRNAs. In primary neutrophils, both miRNAs were significantly upregulated upon activation (180 min, p < 0.001), with miR-425-3p also increased in the extracellular fraction (p = 0.003), indicating active release and contribution to circulating miRNA levels. Functionally, modulation of these miRNAs altered neutrophil behavior, as their overexpression enhanced NETosis, while inhibition reduced NETs formation. Mechanistically, these effects were associated with suppression of key negative regulators of inflammatory signaling, as miR-425-3p and miR-628-3p reduced the expression of DUSP1 and PTPN22, respectively, supporting a role in amplifying inflammatory responses. In conclusion, we identified a neutrophil-associated circulating miRNA signature that predicts progression to Stage 3 T1D within 15 months in at-risk individuals. This integrated biomarker represents a minimally invasive liquid biopsy approach that improves temporal risk stratification while providing mechanistic insight into disease progression, linking circulating miRNAs to neutrophil activation, NETosis, and dysregulation of inflammatory signaling pathways.