Effects of apocynin on production of inflammatory cytokines by CD8+ T cells

Abstract

Apocynin is a well-known chemical inhibitor of NADPH oxidase which is enzyme complex generating reactive oxygen species (ROS), specifically phagocytic NADPH oxidase (PHOX or NOX2). Given the pro-inflammatory effects of ROS, apocynin has been studied for its use as a therapeutic agent in various disease models. While the effects of apocynin on neutrophils and monocytes have been investigated, it remains to be elucidated whether apocynin modulates the effector function of T cells even though T cells also express functional phagocytic NADPH oxidase. Thus, in the present study we examined the effect of apocynin upon, and investigated its mechanism of action in, T cells. We found that apocynin directly inhibited the production of cytokines such as TNF-α, IFN-γ, and IL-2 in anti-CD3/anti-CD28-stimulated CD8+ and CD4+ T cells. We then attempted to delineate the mechanism of action of apocynin specifically in CD8+ T cells. To this end, electrophoretic mobility shift assays revealed that apocynin attenuates anti-CD3/anti-CD28-induced NF-κB activation in CD8+ T cells. TNF-α mRNA transcription induced by anti-CD3/anti-CD28-stimulation also inhibited by apocynin treatment. Interestingly, the inhibitory effect of apocynin on TNF-α production by CD8+ T cells was also observed in CD8+ T cells from NOX2-deficient mice, suggesting that apocynin acts in a NOX2-independent manner. Apocynin could not inhibit the production of TNF-α from CD8+ T cells stimulated by phorbol 12-myristate 13-acetate (PMA) and ionomycin suggesting that the action is upstream of protein kinase C. We also systematically studied NOX2-dependency of apocynin in CD8+ T cells by gene expression profiling studies and demonstrated that gene regulation in response to apocynin can occur via both NOX2-independent and -dependent mechanism. In functional annotation clustering of genes, cytokine activity-related genes were significantly enriched among NOX2-independently downregulated genes by apocynin treatment. Amon...