Thapsigargin and flavin adenine dinucleotide ex vivo treatment improves leukocyte microbiocidal activity through rescuing trafficking-defective gp91-phox in chronic granulomatous disease (134.37)

Abstract

Mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in white blood cells and cause chronic granulomatous diseases (CGD) in affected patients. The most common form of CGD is caused by mutations in the membrane-bound component of gp91-phox, which is encoded by the CYBB gene located on the X chromosome. We previously reported a patient with CYBB mutation (H338 to Y) which prevents the intracellular trafficking and expression of gp91-phox on the leukocyte surface. By treating the leukocytes with thapsigargin and flavin adenine dinucleotide (FAD), we rescued the capacity of the leukocytes to produce reactive oxygen species (ROS). We visualized the enhanced cell surface expression of the mutant protein after thapsigargin and FAD treatment with GFP-tagged gp91-phox expression with the CGD mutation. Protein and cellular analyses showed that treated cells expressed more endo-H resistant gp91-phox protein and are more effective in killing bacteria. Thapsigargin and FAD-treated CGD leukocytes had enhanced activity in protecting mice from S. aureus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin and FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients.