Neutrophils are major cell type of innate immunity, that can eliminate pathogens by different mechanisms. One of these mechanisms is called NETosis, which leads to release of decondensed chromatin and citrullinated histone proteins. Citrullination is post-translational modification catalysed by peptidylarginine deiminase (PAD) and causing transformation of possitively charged arginin to neutral citrullin and can change expression of cytokine genes. Concetrations of pro-inflammatory cytokines (IL-8, TNF, IL-1) were measured after activation of PAD4 and induction of citrullination. Calcium ionophore was used to induce citrullinaton, Cl-amidine and TDFA were used as inhibitors. Production of cytokines was assessed by ELISA on protein level and by qPCR on mRNA level. It was found that induction of citrullination led to increased concentrations of IL-8 and IL-1. Elevated gene expression of IL-8 was confirmed on mRNA level. Both inhibitors were able to decrease level of histone H3 citrullination and IL-8 and IL-1 concentrations. Expression of TNF was not detected on protein and mRNA level.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23773号 / 医博第4819号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 金子 新, 教授 杉田 昌彦, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The phagocytic cells circulating in the bloodstream play a key role in both the defense of the body and the pathology of inflammatory diseases. Thus, targeting their functions has potential to modulate an immune response, especially during the inflammatory phase. This master's thesis was focused on the influence of electric pulses on the most abundant phagocyte population in human peripheral blood, namely neutrophils. The theoretical part describes the role of neutrophils in the development of the immune response and the effects of the electric field on various cells. Consequent part of the thesis was the optimization of the electrical stimulation of neutrophils using a unique platform with a network of gold electrodes. In stimulated cells by electrical pulses, activation of selected signaling pathways, degranulation, ROS production, citrullination of histone H3 and expression of surface markers were monitored. Overall, electrical stimulation was observed to induce neutrophil activation but only electrical pulses of size 1 V were found to be statistically significant in the case of ROS production and 10 mV and 100 mV electrical pulses in the case of metalloproteinase MMP8 degranulation. The absence of significant effects in the most observed parameters was probably due to unwanted activation of neutrophils in control samples.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple sclerosis (MS) is a disabling disease that afflicts an estimated two million people worldwide. The disease is characterized by degradation of the myelin sheath that insulates neurons of the central nervous system manifesting as a heterogeneous collection of symptoms. Two enzymes, protein arginine deaminases type 2 and 4 (PAD2 and PAD4) have been implicated to play an etiologic role in demyelination and neurodegeneration by catalyzing a post-translational modification of arginine peptide residues to citrulline. The pathogenesis of MS is poorly understood, though vitamin D deficiency is a well-associated risk factor for developing the disorder. Using the experimental autoimmune encephalomyelitis (EAE) model of MS we demonstrate vitamin D treatment to attenuate over-expression of PAD 2 and 4 in the brain and spine during EAE. In addition, we identify two molecules produced by peripheral immune cells, IFNɣ and IL-6, as candidate signaling molecules that induce PAD expression in the brain. We demonstrate vitamin D treatment to inhibit IFNɣ mediated up regulation of PAD2 and PAD4 both directly within the brain and by modulating PAD-inducing cytokine production by infiltrating immune cells. These results provide neuroprotective rational for the supplementation of vitamin D in MS patients. More importantly, these results imply an epigenetic link between vitamin D deficiency and the pathogenesis of MS that merits further investigation.