NETosis in Long-Term Type 1 Diabetes Mellitus and Its Link to Coronary Artery Disease

Abstract

BackgroundNeutrophil extracellular traps NETs have been linked to glucose and the pathogenesis of type 1 diabetes mellitus (T1DM). NETs also play a role in vascular inflammation and the development of coronary artery disease (CAD). The role of NETs in CAD progression in patients with long-term T1DM is unclear. We aimed to 1) investigate whether levels of circulating NETs markers were elevated in long-term T1DM subjects compared to controls, and 2) explore whether levels of NETs were related to the presence of CAD.Material and Methods102 patients with > 45 years of T1DM and 75 age-matched controls were enrolled in a cross-sectional study. Median age was 62 years. Computed tomography coronary angiography (CTCA) was performed in 148 subjects without established coronary heart disease. For the current study, CAD was defined as a coronary artery stenosis >50%. Double-stranded deoxyribonucleic acid (dsDNA) was measured by a nucleic acid stain, myeloperoxidase-DNA (MPO-DNA), citrullinated histone 3 (H3Cit) and peptidylarginine deiminase 4 (PAD4) by ELISAs, while gene expression of PAD4 was measured in leukocytes from PAXgene tubes.ResultsCirculating MPO-DNA levels were significantly lower in patients with T1DM than in controls (0.17 vs 0.29 OD, p<0.001), while dsDNA, H3Cit, PAD4 and gene expression of PAD4 did not differ with respect to the presence of T1DM. There were no significant associations between NETs markers and HbA1c in the T1DM group. None of the NETs markers differed according to the presence of CAD in patients with T1DM. While all circulating NETs markers correlated significantly with circulating neutrophils in the control group (r=0.292-393, p<0.014), only H3Cit and PAD4 correlated with neutrophils in the T1DM group (r= 0.330-0.449, p ≤ 0.001).ConclusionsIn this cross-sectional study of patients with long-term T1DM and age-matched controls, circulating NETs levels were not consistently associated with the presence of T1DM or glycemic status, and did not differ according to the presence of CAD in patients with T1DM. Our results entail the possibility of altered neutrophil function and reduced NETosis in T1DM. This warrants further investigation.

High-Throughput Analysis of Neutrophil Extracellular Trap Levels in Subtypes of People with Type 1 Diabetes

“…In the case of T1D, most reports employ quantification of soluble NET remnants such as MPO, PR3, NE, and citrullinated histones (CitH3), as end-point surrogate markers of NETosis and do not objectively quantify the sequential dynamics and morphological changes of NETing neutrophils overtime [ 18 , 19 , 20 , 21 , 22 ]. Moreover, T1D is a heterogeneous disease in which younger age is associated with a higher risk and rate of progression through the developmental stages of the disease, which could lead to different NETosis patterns [ 23 ].…”

“…Moreover, absolute neutrophil counts and peripheral levels of the soluble NET markers, MPO and NE, were comparable between the different groups. In contrast, others observed a reduction in circulating levels of PR3 and cell-free MPO-DNA complexes in established T1D patients [ 21 , 22 ]. However, in the former study, the difference in peripheral NET markers between the established T1D group and HC was lost after adjusting for absolute neutrophil counts.…”

“…This was also the case in the pediatric population, where increased levels of MPO and NE, as well as other NET-associated biomarkers such as PR3, PAD4, cell-free DNA-histone complexes, and extracellular DNA were observed at the time of T1D onset [ 19 , 20 ]. However, other studies have reported lower MPO-DNA markers as well as reduced NE and PR3 levels in adults with established T1D compared to healthy control (HC) donors [ 21 , 22 ]. The heterogeneity of NETosis levels in T1D could be explained by the influence of different disease stages on NET formation and the lack of a standardized technique to measure NETosis.…”

“…As such, the various T1D stages might have an impact on the reported NETosis levels in the aforementioned studies. As for the techniques used to determine levels of NETosis, the studies described either indirect measurements of NET formation (i.e., serum or plasma levels of NET markers such as MPO and NE or extracellular DNA) or employed error-prone, laborious, and relatively subjective methods to measure NETosis levels (i.e., manual counting) [ 18 , 19 , 20 , 21 , 22 ]. It could be argued that these discrepancies might affect the reported NETosis levels.…”

Editorial: The emerging role of metabolism and metabolic-related receptors on neutrophil extracellular traps (NET) formation

“…Evidence suggests that the hyperglycaemia observed in patients with type II diabetes could increase release of NET ( 14 ); however, this does not seem to be to the same extent in patients with type I diabetes mellitus (T1DM). Aukrust et al.…”