Elevated Biomarkers of NETosis in the Serum of Pediatric Patients With Type 1 Diabetes and Their First-Degree Relatives

Abstract

Type 1 diabetes (T1D) is an autoimmune disorder with unambiguous involvement of both innate and adaptive immune mechanisms in the destruction of pancreatic beta cells. Recent evidence demonstrated that neutrophils infiltrate the pancreas prior to disease onset and therein extrude neutrophil extracellular traps (NETs), web-like structures of DNA and nuclear proteins with a strong pro-inflammatory biologic activity. Our previous work showed that T1D NETs activate dendritic cells, which consequently induce IFNγ-producing Th1 lymphocytes. The aim of this study was to assess direct ex vivo biomarkers of NETosis in the serum of recent onset and long-term pediatric T1D patients, their first-degree relatives and healthy controls. To this end we evaluated serum levels of myeloperoxidase (MPO), neutrophil elastase (NE), proteinase 3 (PR3), protein arginine deiminase 4 (PAD4), LL37 and cell-free DNA-histone complexes in sex- and age-matched cohorts of T1D first-degree relatives, recent-onset T1D patients, and in patients 12 months after clinical manifestation of the disease. Our data shows that disease onset is accompanied by peripheral neutrophilia and significant elevation of MPO, NE, PR3, PAD4 and cell-free DNA-histone complexes. Most biomarkers subsequently decrease but do not always normalize in long-term patients. First-degree relatives displayed an intermediate phenotype, except for remarkably high levels of LL37. Together, this report provides evidence for the presence of ongoing NETosis in pediatric patients with T1D at time of clinical manifestation of the disease, which partly subsides in subsequent years.

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

“…In adult autoantibody-positive and newly diagnosed T1D individuals, serum levels of MPO and NE were shown to be increased, suggesting enhanced NETosis [ 18 ]. 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 ].…”

“…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.…”

Platelet–Neutrophil Interaction and Thromboinflammation in Diabetes: Considerations for Novel Therapeutic Approaches

“…Levels of neutrophils and biomarkers of NETosis, including neutrophil elastase and cfDNA, were elevated in the serum of pediatric patients with recent‐onset T1D, suggesting that altered neutrophil activity leading to increased NETosis at disease onset may be involved in the inflammatory trigger of T1D. 60 In fact, inhibition of peptidylarginine deiminases 4, primarily expressed in neutrophils and critical in the NETs generation, by cl‐amidine decreased T1D incidence and delayed its onset in nonobese diabetic mice. 61 Furthermore, proteomic characteristics, determined by gene ontology analysis, of plasma microparticles from patients with T2D showed that levels of S100A8/A9 were upregulated 26 (Table 1 ).…”

PADs and NETs in digestive system: From physiology to pathology

“…Type 1 diabetes (T1D), closely related to islets in pancreas, is an autoimmune disease with a remarkable immune correlation with the destruction of β cells. Evidence suggests that neutrophil infiltration and the formation of NETs are observed before the onset of T1D ( 75 ). A recent study also found high expression of PAD2 mRNA in islet cells, instead of neutrophils, most likely occurring at an earlier stage before inflammatory infiltration.…”