Background and Objectives: Type 1 diabetes (T1D) is an autoimmune disease that results from the immune-mediated destruction of insulin-producing β cells in the islets of Langerhans within the pancreas. A combination of genetic and environmental triggers has been acknowledged to contribute to the development of T1D. However, the detailed mechanisms underlying the initiation and progression of autoimmune diabetes still remain poorly understood. Recent studies have found that the reduction of circulating neutrophils is accompanied by neutrophil infiltration in the pancreas at the onset of T1D, suggesting that neutrophils may be causally involved in the pathogenesis of this disorder. However, further investigations are needed to clarify the precise roles of neutrophils and their cellular components in autoimmune destruction of pancreatic β cells. The objective of this study was to investigate whether neutrophil elastase (NE) and proteinase 3 (PR3), both neutrophil serine proteases stored in neutrophil primary granules, and NETosis, a unique form of cell death of neutrophils characterized by the release of decondensed chromatin and granular contents to the extracellular space, were involved in the pathogenesis of T1D. Key findings: 1) We developed several in-house immunoassays for the measurement of circulating levels of NE, PR3 and their endogenous inhibitor alpha-1 antitrypsin (A1AT), and validated the specificity, precision and sensitivity of these assays in clinical samples; 2) We provided the first clinical evidence demonstrating that both circulating protein levels and enzymatic activities of NE and PR3 were dramatically increased in patients with T1D, especially in those with disease duration less than one year. On the contrary, circulating concentrations of A1AT were significantly decreased in these patients; 3) By measuring circulating levels of myeloperoxidase (MPO)-DNA complexes, we demonstrated that NETosis was evidently increased in T1D patients, and positively correlated with the circulating protein levels as well as enzymatic activities of NE and PR3, suggesting that increased circulating NE and PR3 at least in part attributed to augmented NETosis; 4) Circulating NE and PR3 levels increased progressively with the increase in the positive numbers and titers of autoantibodies against pancreatic β cell antigens, but no significant correlation of NE or PR3 with fasting blood glucose levels was observed, suggesting that elevated NE and PR3 might be causally associated with β-cell autoimmunity, but not glycaemic status, in T1D patients. Furthermore, an obvious elevation of NE and PR3 was detected even in those autoantibody-negative patients, suggesting that circulating NE and PR3 may serve as a novel class of biomarkers for the early diagnosis of T1D. Conclusions: Our present study demonstrated that the drastic elevation of NE and PR3, accompanied by a decrease in the endogenous inhibitor A1AT and the enhancement of NETosis, are closely associated with the β-cell autoimmunity in patients with T1D. Measurement of circulating protein levels of neutrophil serine proteases and/or their enzymatic activities can be used to assist the differential diagnosis of autoimmune diabetes. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Mucosal immune system comprises not only the major compartment of the immune system but also important interface with the outer environment. It is responsible in maintaining an intricate balance with the danger and non-danger stimuli of the outer world by employing specific anatomical features and unique functional mechanisms. Mucosal immune system has been long understudied, perhaps due to the limited accessibility, and its biological importance is thus still underevaluated. However, it has become evident that it is important to study mucosal immune system not only in local mucosal affections but also when uncovering pathogenic mechanisms and novel prevention strategies of organ specific autoimmune diseases such as type 1 diabetes. Thus, the first, more clinically oriented part of this thesis is focused on mucosal immune system of the upper respiratory tract in disease conditions - in nasal polyposis (NP). Because there is a substantial accumulation of eosinophils and neutrophils in the most frequent type of NP, we investigated and described increased expression of chemokine receptors CCR1 and CCR3 in NP versus nasal mucosa. Both innate immune mechanisms as well as homeostasis of epithelial cells may participate in NP. We have documented increased numbers of iNOS-positive and insulin-like growth...

Mucosal immune system comprises not only the major compartment of the immune system but also important interface with the outer environment. It is responsible in maintaining an intricate balance with the danger and non-danger stimuli of the outer world by employing specific anatomical features and unique functional mechanisms. Mucosal immune system has been long understudied, perhaps due to the limited accessibility, and its biological importance is thus still underevaluated. However, it has become evident that it is important to study mucosal immune system not only in local mucosal affections but also when uncovering pathogenic mechanisms and novel prevention strategies of organ specific autoimmune diseases such as type 1 diabetes. Thus, the first, more clinically oriented part of this thesis is focused on mucosal immune system of the upper respiratory tract in disease conditions - in nasal polyposis (NP). Because there is a substantial accumulation of eosinophils and neutrophils in the most frequent type of NP, we investigated and described increased expression of chemokine receptors CCR1 and CCR3 in NP versus nasal mucosa. Both innate immune mechanisms as well as homeostasis of epithelial cells may participate in NP. We have documented increased numbers of iNOS-positive and insulin-like growth...