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.
Highlights
Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-producing beta cells in the pancreas, which involves both innate and adaptive immunity
Since the process of NETosis has previously been implicated in the pathogenesis of T1D and our patients displayed a discrete transient neutrophilia upon reaching clinical onset of T1D, we hypothesized that neutrophil extracellular traps (NETs)-associated biomarkers should be elevated in the serum of T1D patients, especially at the time of clinical manifestation of the disease
Residual beta-cell activity was not directly quantified through the measurement of fasting or stimulated C-peptide, there was no correlation between the metabolic control of T1D measured as glycated hemoglobin fraction HbA1c (Supplementary Figure 6) and NET-associated biomarkers in either recent onset or long-term patients. In this brief report we demonstrate the elevation of neutrophils and indirect serum biomarkers of neutrophil NETosis – the enzymes myeloperoxidase (MPO), proteinase 3 (PR3), neutrophil elastase (NE) and protein arginine deiminase 4 (PAD4), the active form of the antimicrobial peptide cathelicidin, LL37, and cell-free DNA-histone complexes – in the blood of pediatric patients with recent onset type 1 diabetes
Summary
Type 1 diabetes (T1D) is an autoimmune disease resulting from the destruction of insulin-producing beta cells in the pancreas, which involves both innate and adaptive immunity. While it has been shown that neutrophils can infiltrate the pancreas and initiate the autoimmune response driving its destruction in mice [6], their precise role in the pathogenesis of T1D is still under debate. Neutrophils are able to extrude web-like structures called neutrophil extracellular traps (NET) in a process of specific cell death called NETosis [7]. We and others have shown the involvement of NETs in the pathogenesis of autoimmune diabetes [6, 8,9,10], where neutrophils and NETrelated products act as drivers of inflammation [11], Th1 polarization and type II interferon production [10]. The inhibition of NET formation is able to attenuate the development of T1D [6]
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