Background and aims: Type 1 diabetes (T1D) is a chronic autoimmune disease, characterized by T-cell mediated destruction of the pancreatic insulin-producing beta-cells. Neutrophils, cells of the innate immune system, have been shown to infiltrate the pancreas and undergo neutrophil extracellular trap (NET) formation (NETosis). However, little is known about the involvement of neutrophils and specifically the role of NETosis in the pathophysiology of the disease. Our aim was to study the NET proteome and bio-energetic profile of neutrophils from people with established T1D in response to stimuli such as phorbol 12-myristate 13-acetate (PMA) and ionomycin.
Material and methods: Peripheral blood neutrophils isolated from people with established T1D (14±7 years at T1D onset; 12±10 years disease duration; 133±37 mg/ml random glycaemia) and sex- and age-matched healthy controls (HC) were stimulated with PMA (100 nM) or ionomycin (20 μM) for 3 hours. The NETomes were studied by LC-MS/MS analysis, while metabolic changes during NETosis were explored by Seahorse extracellular flux analysis.
Results: Levels of PMA- and ionomycin-stimulated NETosis were comparable in HC and T1D neutrophils (PMA: 85% vs 90%; ionomycin: 63% vs 77% respectively), as well as plasma levels of NET markers. However, the NETome of T1D neutrophils was distinct from that of HC subjects in response to PMA and ionomycin. Upon quantification with Progenesis QI software, a total of 44 proteins were differentially expressed in the NETomes of HC and T1D subjects when stimulated with PMA. Ionomycin-induced NETomes contained 27 differentially expressed proteins (1% FDR, P<0.05). Reactome analysis revealed that the proteins enriched in HC NETomes in PMA- and ionomycin- stimulated conditions were involved in neutrophil degranulation and innate immunity (i.e., neutrophil elastase [ELANE], azurocidin [AZU1]). In both stimulated conditions, proteins enriched in T1D NETomes were involved in glucose metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK1), fructose-bisphosphate aldolase A (ALDOA), and UTP-glucose-1- phosphate uridylyltransferase (UGP2). Interestingly, metabolic profiling revealed that the rate of extracellular acidification, an approximate measure for glycolysis, was similar between T1D and HC neutrophils, in response to both PMA and ionomycin. Lactate levels in cell supernatants of PMA- and ionomycin-stimulated neutrophils were also comparable in T1D and HC subjects. Despite a lack of response to ionomycin, PMA induced a comparable increase in mitochondrial respiration in T1D and HC subjects.
Conclusion: Our results showed that the T1D NET proteome was enriched in proteins involved in glucose and glycogen metabolism. Interestingly, T1D neutrophils did not have an aberrant bioenergetic profile as determined by Seahorse extracellular flux analysis compared to HC neutrophils. These results suggest that T1D neutrophils, when activated, may alter their NET proteome to avoid impaired glycolysis and dysfunctional NETosis.