Neutrophil elastase as an early mediator in autoimmune diabetes

Abstract

Type 1 diabetes (T1D) is an autoimmune disease resulted from self-destruction of insulin-producing pancreatic β-cells. Recently, neutrophil has been implicated in both the onset and progression of T1D. Reduced neutrophil counts have been observed in patients with T1D and the neutrophil infiltration and neutrophil extracellular trap (NET) formation in pancreatic islets were observed as early as two weeks after birth in non-obese diabetic (NOD) mice. Our previous clinical studies demonstrated that augmented NETosis and increased circulating neutrophil elastase (NE), a major member of neutrophil serine proteases (NSPs) stored in azurophilic granules within neutrophils, are closely associated with β-cell autoimmunity in patients with T1D. In this study, we used the NOD mice, a well-established T1D animal model, to further investigate how NE participates in the pathogenesis of autoimmune diabetes.

By adopting enzyme-linked immunosorbent assay (ELISA), immuno-histochemistry (IHC) and in vivo non-invasive imaging analysis, we observed a dynamic change in the infiltration and activation of neutrophils in pancreas of neonatal NOD mice during the weaning period, leading to a transient elevation of local NE activity. Therapeutic intervention of pancreatic NE activity was carried out by supplementing NE-specific endogenous (pre-elafin/elafin) or pharmaceutical (sivelestat) inhibitors through multiple strategies, including: 1) systemic administration of recombinant pre-elafin protein, 2) pancreatic islet-specific over-expression of elafin via adeno-associated virus (AAV) gene delivery system, and 3) intraperitoneal injection of sivelestat. It was found that supplementation of NE-specific inhibitors rectified the elevated pancreatic NE activity, improved insulitis and largely prevented the development and onset of T1D in NOD mice. IHC, quantitative real-time PCR and flow cytometry analysis demonstrated that inhibition of NE activity significantly attenuated the infiltration of pro-inflammatory macrophages into pancreas, reduced the production of inflammatory cytokines, alleviated islet inflammation and β-cell damage and profoundly compromised the pathogenic responses of Th1 and Th17 cells in both spleen and pancreatic draining lymph nodes (PLNs) of NOD mice. In vitro studies showed that NE directly induced inflammatory responses in both min6 β-cells and RAW264.7 macrophages through the activation of TLR4-IκB-NFκB pathway. In addition, exaggerated expression of inflammatory cytokines was observed in NE-treated co-culture system containing min6 and RAW264.7 cells, suggesting an NE-exacerbated inflammatory interaction between these two types of cells. Moreover, RAW264.7 macrophages were effectively attracted by the inflammatory signals produced by NE-treated min6 cells or NE-treated co-culture system containing min6 and RAW264.7 cells, indicating a promoting role of NE in macrophage recruitment.

Collectively, our results demonstrate that NE-facilitated crosstalk between β-cell and macrophages forms a vicious feedback loop, resulting in the exaggerated β-cell damage as well as an inflammatory milieu favored by the diabetogenic adaptive responses, which in turn contribute to the β-cell autoimmunity in NOD mice.