In 2010 we continued our accrual of patients into our blister study protocol. The protocol involves the formation of blisters on the forearms of human volunteers to study the inflammatory response in vivo. This year, we compared the transcriptomes of peripheral blood neutrophils with matched inflammatory exudate (blister) neutrophils from several normal donors. For this project, we have teamed with the Rocky Mountain Laboraty Research Technologies Branch (RML) who have provided expert microarray analysis of these samples. We are also studying the biological differences of exudate PMN in comparison with peripheral PMN. In addition to 21 normals, we have also studied three patients with chronic graft-versus-host disease and six patients with Job's Syndrome. Results to date indicate that Job's patients have elevated basal TNF alpha and decreased IL-17 in the neat fluid (extracellular fluid) compared to normal subjects. Interestingly IL17 is secreted at normal levels in Job's blisters over time, a surprising finding given the reported STAT3 dependence of IL17 production in peripheral blood cells and the fact that Job's patients have a mutation of STAT3 gene. This finding underscores that in vivo findings may differ strikingly from in vitro studies of isolated immune cells. Further finctional studies of exudate PMN vs peripheral blood PMN will be performed.(Kol Zarember, 20% effort). This past year we initiated a project to better understand the possible roles of microRNA in the regulation of phagocyte function, including regulation of production of cytokines. MicroRNAs comprise a class of over 500 small, non-coding RNA that regulate gene expression by inhibiting RNA translation. They are felt to regulate the expression of over 30% of the genome and specific microRNA species have been shown to be active in neutrophils. A complete survey of the microRNA species in neutrophils and monocytes has not been performed. We have been collaborating with the Rocky Mountain Laboratory to perform RNA sequencing of microRNA isolated from purified neutrophils preparations from normal controls and patients with both X-linked (gp91phox-deficient) and autosomal recessive (p47phox-deficient) CGD patients. The expression of several individual miRNA were found to be significantly different between healthy volunteers and CGD patients. The potential targets of the differentially expressed miRNA were probed in silico and pathway analysis identified that several of the potentially regulated proteins were involved in apoptosis. To determine if the observed differences result from differences in the oxidative state between normal and CGD neutrophils, we plan to manipulate both noraml and CGD neutrophils using well established models of increased (addition of xanthine/xanthine oxidase or hydrogen peroxide) and decreased (addition of catalase or superoxide dismutase) oxidative stress to determine the effect on miRNA expression. We believe that delayed death of neutrophils may contribute to excessive inflammation and granuloma formation in CGD. Our hope is that in the future specific miRNAs will be identified that will modulate apoptosis and be useful in management of the excessive inflammation in CGD patients.
