Syntaxin 4 Enrichment in ß-Cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice

Syntaxin 4 (STX4), a plasma membrane–localized SNARE protein, regulates human islet ß-cell insulin secretion and preservation of ß-cell mass. We found that human type 1 diabetes (T1D) and NOD mouse islets show reduced ß-cell STX4 expression, consistent with decreased STX4 expression, as a potential driver of T1D phenotypes. To test this hypothesis, we generated inducible ß-cell–specific STX4-expressing NOD mice (NOD-ißSTX4). Of NOD-ißSTX4 mice, 73% had sustained normoglycemia vs. <20% of control NOD (NOD-Ctrl) mice by 25 weeks of age. At 12 weeks of age, before diabetes conversion, NOD-ißSTX4 mice demonstrated superior whole-body glucose tolerance and ß-cell glucose responsiveness than NOD-Ctrl mice. Higher ß-cell mass and reduced ß-cell apoptosis were also detected in NOD-ißSTX4 pancreata compared with pancreata of NOD-Ctrl mice. Single-cell RNA sequencing revealed that islets from NOD-ißSTX4 had markedly reduced interferon-? signaling and tumor necrosis factor-a signaling via nuclear factor-?B in islet ß-cells, including reduced expression of the chemokine CCL5; CD4+ regulatory T cells were also enriched in NOD-ißSTX4 islets. These results provide a deeper mechanistic understanding of STX4 function in ß-cell protection and warrant further investigation of STX4 enrichment as a strategy to reverse or prevent T1D in humans or protect ß-cell grafts. Overall design: Islets were isolated from female NOD-Ctrl or NOD-ißSTX4 transgenic mice and dispersed into single cells with TrypLE (Invitrogen, Carlsbad, CA). Cell numbers and viability were measured using a TC20 Automated Cell Counter (Bio-Rad); only samples showing at least 70% viability were used. Cells were then loaded onto a chromium controller (10x Genomics, Pleasanton, CA) to generate single-cell Gel Bead-In Emulsions (GEMs) captured in droplets at a targeted cell recovery of 3,000 cells. GEM reverse transcriptions were performed in a Veriti 96-Well Thermal Cycler (Thermo Fisher Scientific). The Chromium Single Cell 3' Reagent Kit V2 (10x Genomics) was used to process samples into single-cell RNA sequencing (scRNA-seq) libraries according to the manufacturer's protocol. The libraries were sequenced with the paired-end setting of 28 cycles of read 1, 101 cycles of read 2, and 8 cycles of the index read on the Illumina NovaSeq 6000 platform at the Translational Genomics Research Institute (TGen) (Phoenix, AZ).